[go: up one dir, main page]

WO2024227030A1 - Bcl11b sustains multipotency and restricts effector programs of intestinal resident memory cd8+ t cells - Google Patents

Bcl11b sustains multipotency and restricts effector programs of intestinal resident memory cd8+ t cells Download PDF

Info

Publication number
WO2024227030A1
WO2024227030A1 PCT/US2024/026583 US2024026583W WO2024227030A1 WO 2024227030 A1 WO2024227030 A1 WO 2024227030A1 US 2024026583 W US2024026583 W US 2024026583W WO 2024227030 A1 WO2024227030 A1 WO 2024227030A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
bcl11b
virus
species
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/026583
Other languages
French (fr)
Inventor
Dorina AVRAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
H Lee Moffitt Cancer Center and Research Institute Inc
Original Assignee
H Lee Moffitt Cancer Center and Research Institute Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by H Lee Moffitt Cancer Center and Research Institute Inc filed Critical H Lee Moffitt Cancer Center and Research Institute Inc
Publication of WO2024227030A1 publication Critical patent/WO2024227030A1/en
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • CD8+ T cells are critical in the immune response to intracellular pathogens and tumors and provide long-lasting protection from reinfection. Following infection, antigen-specific CD8+ T cells undergo robust clonal expansion and differentiate into Klrg1hiCD127lo terminal effector cells (TEs) which die after pathogen clearance, and CD127hiKlrg1lo memory precursor cells (MPs), which have potential to form long-lived memory cells.
  • TEs Klrg1hiCD127lo terminal effector cells
  • MPs CD127hiKlrg1lo memory precursor cells
  • TRM T central memory
  • TEM T effector memory
  • TRM cells are seeded in non-lymphoid tissues early during infection and do not exit into the circulation, except during reinfection. TRM cells provide an important first line of defense upon antigen reencounter due to their localization at common sites of infection including at key barrier surfaces, ability to directly kill infected cells and rapidly induce an innate-like immune response, proliferate, and trigger the recruitment of circulating memory and other immune cells to the site of reinfection.
  • TRM cells have been shown to provide protection against numerous pathogens, including herpes simplex, vaccinia and influenza viruses, as well as Listeria monocytogenes (Lm). However, in tumors exhausted cells are generated, which are inefficient in Attorney Docket Number 10110-443WO1 controlling the anti-tumor immune response. What are needed are methods and compositions for increasing the number of TRM at sites of infection and tumor microenvironments. II. SUMMARY 2. Disclosed are methods and compositions related to inhibiting expression of Bcl11b. 3.
  • a disease or condition or a recurrent disease or condition such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease) (including, but not limited to diseases or conditions with T cell mediated immune responses) in a subject
  • administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b); disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by CRISPR/cas9); or by administering to the subject T cells that have reduced Bcl
  • the T cells comprise tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (MILs), chimeric antigen receptor (CAR) T cells, CD4 T cells, CD8 T cells, effector memory T cells (TEM), central memory T cells (TCM), peripheral memory T cells (T PM ), or tissue-resident memory T cells (T RM ). 4. Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a disease or condition or a recurrent disease or condition of any preceding aspect, further comprising obtaining T cells from a donor source and disrupting Bcl11b expression in the T cells prior to administering the T cells to the subject. 5.
  • TILs tumor infiltrating lymphocytes
  • MILs marrow infiltrating lymphocytes
  • CAR chimeric antigen receptor
  • CD4 T cells CD8 T cells
  • TEM effector memory T cells
  • TCM central memory T cells
  • TPM peripheral memory T cells
  • T RM tissue-resident
  • cytotoxicity i.e., increasing expression of effector molecules such as, for example, granzyme b, perforin, or cytokines including, but not limited to, IFN- ⁇ and/or TNF- ⁇
  • T cells specific for a disease or condition such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease) (including, but not limited to diseases or conditions with T cell mediated immune responses)
  • said methods comprising contacting T cell mediated disease specific T cells with an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b)
  • the guide RNA for the CRISPR/cas9 are set forth in SEQ ID NO:1 and SEQ ID NO: 2. 6. Also disclosed herein are methods of reducing exhaustion of T cells specific for a disease or condition (such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease)( including, but not limited to diseases or conditions with T cell mediated immune responses), said method comprising contacting T cell mediated disease specific T cells with an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b) or disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by C
  • the guide RNA for the CRISPR/cas9 are set forth in SEQ ID NO:1 and SEQ ID NO: 2. 7.
  • a disease or condition or a recurrent disease or condition of any preceding aspect methods of reducing exhaustion of T cells specific for a disease or condition of any preceding aspect; or methods of increasing the cytotoxicity of T cells specific for a disease or condition of any preceding aspect; wherein the infectious disease is caused by a viral infection (such as, for example, a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus
  • a viral infection such as, for example, a virus selected from the group consisting of Herpes Simple
  • a bacterial infection such as, for example, an infection with a bacteria selected from the group consisting of Bacillus anthracis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Mycobacterium chimaera, Nocardia as
  • methods of increasing resident memory T cells at the site of infection or in a tumor microenvironment comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity.
  • methods of increasing NK cell activation in a tumor microenvironment or site of infection comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity.
  • Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, and 1K show Bcl11b deletion post oral infection with Lm-Ova InlAM results in reduced circulating memory cells, but excessive SI T RM cells.
  • Figure 1A shows contour plots and quantification Attorney Docket Number 10110-443WO1 of splenic Bcl11b -/- (CD45.2) and WT (CD45.1/2) cells.
  • Figure 1B shows contour plots and quantification of Klrg1 versus CD127 within Bcl11b -/- (CD45.2) and WT (CD45.1/2) CD8 + CD44 + splenic population.
  • Figure 1C shows Bcl11b -/- and WT cells within the siIEL CD8 + CD44 + CD45.2 + population.
  • Figure 1D shows contour plots and quantification of CD69 versus CD103 Bcl11b -/- (KO) and WT within the siIEL CD8 + CD44 + CD45.2 + population.
  • Figure 1E shows contour plots and quantification of splenic Bcl11b -/- and WT cells 30 and 90 DPI.
  • Figure 1F shows contour plots and quantification of Bcl11b -/- and WT cells within the splenic TCM (CD62L + ) and TEM (CD62L-) compartments at 30 DPI.
  • Figure 1G shows contour plots and quantification of Bcl11b -/- and WT cells in the siIEL of co-transferred mice at 30 and 90 DPI.
  • Figure 1H shows percentages of Bcl11b -/- and WT CD69 + CD103 + , CD69 + CD103- and CD69- CD103- subsets within the siIEL CD8 + CD44 + CD45.2 + cells at 30 DPI. (I-K) Separate transfers.
  • Figures 1I and 1J show contour plots and quantification of splenic (1I) and siIEL (1J) Bcl11b -/- and WT CD8 + T cells at 30 DPI. P values by Mann-Whitney-Wilcoxon test.
  • Figure 1K shows the percentages of Bcl11b -/- and WT CD69 + CD103 + , CD69 + CD103- and CD69-CD103- subsets within siIEL CD8 + CD44 + CD45.2 + cells at 30 DPI.
  • Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I show Bcl11b -/- memory cells have an impaired recall response to oral Lm-Ova InlA M infection.
  • Figures 2A-2F show separate transfers.
  • Figure 2A shows Lm-Ova InlA M CFU on Day 3 post-reinfection in the liver.
  • Figures 2B and 2C show contour plots and quantification of transferred cells in spleen and siIEL post reinfection (Day 3).
  • Figure 2D shows contour plots and quantification of Bcl11b -/- and WT CD8 + CD44 + siIEL cells based on CD69 and CD103.
  • Figure 2E shows Ifn ⁇ and Tnf ⁇ following reinfection.
  • Figure 2F shows the percentages and numbers of recipient CD45.1 + CD45.2-NK1.1 + in the siIEL following reinfection.
  • Figure 2G shows contour plots and quantification of CD45.2 (KO) and CD45.1/2 (WT) cells in spleen and siIEL in co-transferred experiments post reinfection.
  • Figure 2H shows liver Lm-Ova InlA M CFU on Day 3 post-reinfection in a sex mismatched transfer.
  • Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, and 3M show loss of Bcl11b in the effector and memory phases of oral Lm-Ova InlA M infection has a major impact on T RM program.
  • Figure 3A shows a histogram and quantification of Bcl11b Mean Fluorescence Attorney Docket Number 10110-443WO1 Intensity (MFI) in WT siIEL T RMPs , TEs and MPs 9 DPI.
  • MFI Mean Fluorescence
  • Figure 3B shows PCA of RNA-seq data on all high fold-changed genes.
  • Figure 3C shows hierarchical clustering on Euclidean distances of rlog normalized values of RNA-seq data derived from three independent samples.
  • Figure 3D shows differentially expressed gene numbers in TEs, MPs and T RMPs . Genes increasing and decreasing in expression in Bcl11b -/- (KO) are indicated in red and blue, respectively.
  • Figure 3E shows a heatmap clustering of genes differentially expressed in T RMP s , TEs, MPs. Heatmap shows relative log2 fold change. Select genes of the multipotent/multifunctional (MP/MF) and effector T RM programs are indicated.
  • MP/MF multipotent/multifunctional
  • Figure 3F shows gene set enrichment analysis (GSEA) of the MP/MF and effector T RM programs in Bcl11b -/- (KO) and WT T RMPs .
  • Figure 3G shows GSEA of a Tcf1 gene signature of lung T RMs in Bcl11b -/- and WT T RMPs .
  • Figure 3H shows GSEA of an Ahr gene signature in intestinal of ILC2s in Bcl11b -/- (KO) and WT T RMPs .
  • Figure 3I shows Tcf1, Blimp1 and Ahr histograms and MFIs in siIEL T RMPs 9 DPI.
  • Negative controls are endogenous splenic CD44 - CD8 + T cells or isotype controls.
  • Figure 3J shows GSEA of the MP/MF and effector T RM programs in Bcl11b-/- and WT siIEL T RM cells at 30 DPI.
  • Figure 3K shows volcano plot showing genes with upregulated (red) or downregulated (blue) expression in Bcl11b -/- versus WT siIEL T RM cells 30 DPI.
  • Horizontal line represents P value level 0.05 as a threshold of significance. Eight outliers are not shown. Selected genes are indicated.
  • Figure 3L shows GSEA of the MP/MF and effector T RM programs in siIEL recall Bcl11b -/- (KO) and WT cells 2 days after reinfection.
  • Figure 3M shows volcano plot showing genes with upregulated (red) or downregulated (blue) expression in Bcl11b -/- versus WT siIEL recall CD8 + T cells 2 days after reinfection. Fifteen outliers are not shown. Selected genes are indicated.
  • 3F and 3L “*” indicates moderately differentially expressed genes (p ⁇ 0.05) after batch correction. Representative of 2-3 independent experiments.
  • K and M DESeq2 P values are shown. 15.
  • Figures 4A, 4B, 4C, 4D, 4E, 4F, and 4G show the impact of Bcl11b deletion on chromatin accessibility, H3K27ac and H3K4me3 genome wide.
  • Figure 4A shows the number of differentially accessible regions (P ⁇ 0.05) and H3K27ac ChIP-seq peaks with decreased (blue) or increased (red) signal in Bcl11b -/- T RMPs 9 DPI and T RM -like cells, respectively.
  • Figure 4B shows LEFT: Peaks with increased/decreased chromatin accessibility in Bcl11b -/- cells predominantly associated with peaks with increased/decreased (green/orange) H3K27ac signal.
  • MIDDLE Correlation of ATAC-seq and H3K27ac ChIP-seq peaks, differential in Bcl11b -/- cells versus WT.
  • RIGHT Peaks with increased/decreased chromatin accessibility and H3K27ac in Attorney Docket Number 10110-443WO1 Bcl11b -/- cells were predominantly associated with TSS of genes ( ⁇ 10 kb) with decreased/increased (green/orange) expression in Bcl11b -/- cells. Filtered peaks were used for the analysis (P ⁇ 0.05;
  • Figure 4C shows the correlation of differential ATAC-seq and H3K27ac ChIP-seq signal in relation to Bcl11b binding (shape) and changes in gene expression (color).
  • Figure 4D shows the proportion of ATAC-seq, H3K27ac- and Bcl11b ChIP- seq peaks annotated to different genomic elements.
  • Figure 4E shows a heat map for H3K4me3 CUT&RUN signal visualized for TSS ( ⁇ 3 kb) of genes with Bcl11b binding at their promoter (TSS -1000 bp to +200 bp).
  • Figure 5F shwos the profile plot for data displayed in 4E.
  • Figure 4G shows enrichment of motifs at differential ATAC-seq and H3K27ac peaks.
  • Color gradient represents row-scaled relative enrichment (z- score) based on the motif ln(P) value calculated by Homer. 1 6.
  • Figures 5A, 5B, 5C, 5D, 5E, 5F, and 5G show that Bcl11b binds at Tcf7 to control its expression and siIEL TRMP cell distribution.
  • Figure 5A shows genomic tracks at Tcf7 locus from Bcl11b ChIP-seq analysis in WT murine TRM-like cells; Tcf1 CUT&RUN, H3K27ac ChIP-seq, H3K4me3 CUT&RUN in Bcl11b -/- and WT murine TRM-like cells and ATAC-seq in Bcl11b -/- and WT murine siIEL TRMPs.
  • Figure 5B shows BCL11B ChIP-seq in human memory CD3 + CD8 + CD45RA + CD57 + CCR7- T cells at the TCF7 locus. Data are from two donors.
  • FIG. 5A-5B shows a heat map for Tcf1 CUT&RUN signal visualized for TSS ( ⁇ 3 kb) with or without Bcl11b binding at gene promoter (TSS -1000 bp to +200 bp); further categorized for genes with decreased or increased expression in Bcl11b -/- cells.
  • Figure 5D shows a heat map for Tcf1 CUT&RUN signal visualized for Bcl11b peaks (scaled to 1000 bp, ⁇ 3 kb), associated with regions with either significantly increased or decreased chromatin accessibility (P ⁇ 0.05).
  • Figure 5E shows representative histograms (left) and quantification (right) of Tcf1 expression in the siIEL for indicated groups.
  • Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J, and 6K show that Bcl11b binds at Prdm1 and Ahr loci to control their expression and siIEL TRMP and TRM cell distribution.
  • Figures 6A and 6C show genomic tracks at Prdm1 and Ahr loci for Bcl11b ChIP- seq in WT murine T RM -like cells; H3K27ac ChIP-seq and H3K4me3 CUT&RUN in Bcl11b -/- and WT murine T RM -like cells and ATAC-seq in Bcl11b -/- and WT murine siIEL T RMPs .
  • Figures 6B Attorney Docket Number 10110-443WO1 and 6D show BCL11B ChIP-seq in human memory CD3 + CD8 + CD45RA + CD57 + CCR7- cells at the PRDM1 and AHR loci. Data is from two donors.
  • FIG. 6E shows a heat map for Ahr ChIP-seq signal visualized for TSS ( ⁇ 3 kb) of genes with or without Bcl11b binding at their promoter (TSS -1000 bp to +200 bp); further categorized for genes with decreased or increased expression in Bcl11b -/- cells.
  • Figure 6F shows a heat map for Ahr ChIP- seq signal visualized for Bcl11b peaks (scaled to 1000 bp, ⁇ 3 kb), associated with regions with either significantly increased or decreased chromatin accessibility (P ⁇ 0.05).
  • Figures 6G and 6H show quantification of transferred Bcl11b -/- /WT ratios siIEL CD69 + CD103 + cells (6G) or splenic cells (6H) in mice on regular diet (7912) or Ahr-ligand deficient diet (AIN76a).
  • Figure 6I shows the percentages and numbers of CD45.1 + CD8 + T cells of recipient mice in the indicated groups.
  • Figure 6J shows Ahr MFIs in transferred Bcl11b -/- and WT siIEL CD69 + CD103 + cells in the indicated groups.
  • Figures 7A, 7B, 7C, 7D, 7E, and 7F show that Bcl11b represses genes of the NK and myeloid programs across memory and effector CD8 + T cells.
  • Figure 7A shows a heat map of NK receptor and effector genes, and myeloid genes upregulated in Bcl11b-/- Day 9 TEs, MPs and siIEL TRMPs, Day 30 siIEL TRMs and recall siIEL CD8 + T cells 2 days post-infection. Heatmap shows relative log2 expression fold change.
  • Figures 7B, 7C, and 7D show genomic tracks at NK cell receptor, Itgam, Itgax and Fcer1g loci from Bcl11b ChIP-seq analysis in WT murine T RM -like cells, H3K27ac ChIP-seq and H3K4me3 CUT&RUN in Bcl11b -/- and WT T RM -like cells and ATAC-seq in Bcl11b -/- and WT siIEL T RMPs .
  • Figures 7E and 7F show BCL11B ChIP-seq in human memory CD3 + CD8 + CD45RA + CD57 + CCR7- T cells at the NK cell receptor and ITGAM and ITGAX loci. Data is from two donors. 19.
  • Figures 8A, 8B, and 8C show depletion of BCL11B in human memory CD8 + T cells or TRM-like cells causes changes in expression of markers of the TRM and NK program.
  • Figure 8A shows quantification of Bcl11b knockout efficiency and representative histograms.
  • Figures 8B and 8C show flow cytometry plots and quantification of molecules associated with residency program (8B) and NK receptors (8C) in WT and BCL11B -/- T RM -like cells following Attorney Docket Number 10110-443WO1 CRISPR/Cas9 mediated knockout with the indicated guides. Points represent individual donors. P values by two-way ANOVA followed by Tukey's test. 20.
  • Figures 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, and 9I show characteristics of CD8 + T cells derived from Bcl11b f/f GzmbCre + R26REYFP + OT- I and WT OT-I mice after infection with Lm-Ova InlA M .
  • CD8 + T cells from Bcl11b f/f GzmbCre + R26REYFP + OT-I CD45.2 and Bcl11b f/f OT-I CD45.1/2 mice were co- transferred into CD45.1 mice. Recipient mice were infected orally the following day with Lm-Ova InlA M .
  • Figures 9A, 9B, and 9C show mLNs were evaluated at 3 DPI.
  • Figure 9A shows histograms of YFP in transferred CD45.2 and CD45.1/2 CD8 + T cells.
  • Figure 9B shows contour plots and quantification of transferred CD45.2 and CD45.1/2 CD8 + T cells.
  • Figure 9C shows histograms and quantification of CD69 in transferred CD45.2 and CD45.1/2 CD8 + T cells.
  • Figures 9D, 9E, 9F, 9G, and 9H show siIEL and splenic transferred cells were evaluated at 9 DPI.
  • Figure 9D shows histograms of YFP in transferred CD45.2 and CD45.1/2 siIEL T RMP (CD69 + CD103 + ) and splenic MPs (CD127 + Klrg1 - ) and TEs (Klrg1 + CD127 + ) CD45.2 + CD8 + at 9 DPI.
  • Figures 9E and 9F shows that on Day 8 post-infection, transferred mice as above, were injected i.p. with EdU, and further euthanized next day when EdU incorporation in Bcl11b -/- (CD45.2) and WT (CD45.1/2) transferred CD8 + T cells was evaluated in the spleen (9E) or siIEL (9F).
  • Figure 9G shows a histogram and quantification of AnnexinV in Bcl11b -/- and WT siIEL TRMPs 9 DPI.
  • Figure 9H shows post-sort purity of populations for RNA-seq at 9 DPI.
  • siIEL T RMPs CD69 + CD103 +
  • splenic MPs CD127 + Klrg1-
  • TEs Klrg1 + CD127-
  • FIG. 10A and 10B show antigen-specific Bcl11b -/- CD8 + T cells are reduced in the spleen and elevated in siIEL in a polyclonal response.
  • Bcl11b f/f GzmbCre + and WT mice were orally infected with Lm- Ova InlA M .
  • Spleen and siIEL H2K b -SIINFEKL (SEQ ID NO: 3) tetramer + CD8 + CD44 + cells were evaluated 21 days post infection.
  • Figures 12A, 12B, 12C, and 12D show that Bcl11b regulates gut homing early in differentiation of memory cells following oral Lm-Ova InlA M infection.
  • Figure 12A shows DESeq2 normalized counts of Itga4, Itgb7 and Ccr9 in Bcl11b -/- and WT MPs and TEs. FDR adjusted P values by DESeq2 are shown.
  • Figures 12B and 12C show representative histograms and quantification of ⁇ 4 ⁇ 7 (12B) and Ccr9 (12C) in splenic Bcl11b -/- and WT MPs and TEs.
  • Figure 12D shows ⁇ 4 ⁇ 7 blockade in infected mice. Mice were adoptively transferred, infected as above and injected with either IgG or anti- ⁇ 4 ⁇ 7 (Datk32) antibodies 5-8 DPI. Ratio of transferred Bcl11b -/- /WT in spleen (left) and siIEL (middle) and percentages of total transferred cells in siIEL (right) following the blockade.
  • FIG. 13A, 13B, and 13C show differential gene expression analysis of TRM- like cells differentiated ex vivo and comparison with siIEL TRMPs and TRMs.
  • Figure 13A shows a volcano plot showing genes with upregulated (red) or downregulated (blue) expression in Bcl11b -/- TRM-like cells versus WT. Genes associated with the MP/MF or effector TRM programs are depicted.
  • Figures 13B and 13C show differentially expressed genes in Bcl11b -/- T RM -like cells versus WT, examined for enrichment in T RMPs on 9 DPI (13B) and in T RMs on 30 DPI (13C) by GSEA. Core genes upregulated or downregulated are highlighted with a red or blue square, respectively. 25.
  • Figures 14A, 14B, 14C, and 14D show genome wide integrative analysis of Bcl11b ChIP-seq, H3K27ac ChIP-seq and ATAC-seq.
  • Figure 14A shows a panel related to Fig. 4B, showing all significant peaks (P ⁇ 0.05; no fold change filtering).
  • FIG. 14C shows a panel related to Fig.4G. Enrichment of motifs at differential ATAC-seq and H3K27ac peaks, categorized based on Bcl11b binding. Selected motifs which were significant (P ⁇ 0.001) in at least one dataset are depicted, including their motif family annotation. Color gradient represents row-scaled relative enrichment (z-score) based on the motif ln(P) value calculated by Homer.
  • Figure 14D shows motifs enriched in Bcl11b ChIP-seq peaks.
  • FIG. 15A, 15B, 15C, 15D, and 15E show Bcl11b binding, H3K27ac, H3K4me3 and chromatin accessibility at genes of the MP/MF program in T RM -like cells and TRMPs.
  • Figures 15A, 15C, 15D, and 15E show genomic tracks at Ccr7 (15A), Id3 (15C), Klf2 (15D) and S1pr1 (15E) loci from Bcl11b ChIP-seq analysis in WT murine T RM -like cells, Tcf1 CUT&RUN, H3K27ac ChIP-seq and H3K4me3 CUT&RUN analyses in Bcl11b -/- and WT murine T RM -like cells and ATAC-seq analysis in Bcl11b -/- and WT murine siIEL T RMPs .
  • Figure 15B shows genomic tracks at the CCR7 locus from BCL11B ChIP-seq analysis in human memory CD8 + T cells. Data is from two donors. 27.
  • Figures 16A, 16B, 16C, 16D, 16E, and 16F show Bcl11b binding, H3K27ac, H3K4me3 and chromatin accessibility at additional genes of the MP/MF program in TRM-like cells and T RMPs .
  • Figures 17A, 17B, 17C, 17D, 17E, 17F, 17G, and 17H show Bcl11b binding, H3K27ac, H3K4me3 and chromatin accessibility at genes of effector program and Ahr-bound genes in T RM -like cells and T RMPs .
  • FIG. 18A shows that Bcl11b KO TILs display enhanced anti-melanoma activity in adoptive cell therapy. Attorney Docket Number 10110-443WO1 30.
  • Figures 18B and 18C show that Bcl11b KO TILs accumulate in the tumor through elevated CD69. 31.
  • Figure 18D show that Bcl11b KO TILs have increased Gzmb. 32.
  • Figure 19A shows human melanoma CD8+ TILs depleted in BCL11B upregulate the essential NK receptor CD56, but leave intact CD3 and CD8. 33.
  • Figure 19B shows BCL11B depletion in TILs with double guides.
  • Figure 19C shows BCL11B depletion in TILs with double guides – upregulation of CD56 but intact CD8.
  • Figure 20 shows BCL11B depletion in human TILs increases cytotoxicity in a manner dependent on MHC I.
  • Figure 21 shows Bcl11b-deficient CD8+ T cells confer elevated anti-tumor activity in ovarian tumors implanted intraperitoneally.
  • Figures 22A, 22B, 22C, and 22D show BCL11B-deficient TILs have elevated cytotoxic genes, NK receptor and stemness program genes, while inhibitory receptor genes remain unchanged.
  • Figure 22A shows expression of stemness program genes in CD8+ TILs.
  • Figure 22B shows the expression of genes associated with dysfunction/exhaustion in CD8+ TILs.
  • Figure 22C shows the expression of genes for NK receptors and cytotoxicity in CD8+ TILs.
  • Figure 22D shows a plot of the gene expression of all genes in BCL11B-deficient TILs. 38.
  • Figures 23A, 23B, 23C, and 23D show expression of gene subsets in various clusters of TILs in WT and Bcl11b -/- (knockout) mice with peritoneal tumors.
  • Figure 23A shows a scRNA-seq UMAP plot of TILs following adoptive transfer.
  • FIGs 23B and 23C that the expression of Stemness genes (23B) and Gzmc and Gzmf genes (23C) are spread across clusters in BCL11B KO TILs.
  • Figure 23D shows the expression of exhaustion genes is decreased in Ttex clusters in BCL11B KO TILs. 39.
  • Figurer 24A, 24B, 24C, 24D, and 24E show that Gzmb HI (Granzyme B hi) CD8+ TILs from Bcl11b -/- mice continue to express stemness transcription factor TCF1 (24A and 24B), expand more (24C), have reduced levels of the exhaustion transcription factor Tox, as well as, exhaustion markers CD39, PD1, and Tim3 (24D), and have increased metabolic potential and mass and thus greater fitness (24E).
  • Figures 25 shows the experimental model for Bcl11b -/- (knockout) CD4+ TILs. 41.
  • Figures 26A and 26B shows that Bcl11b -/- (KO) CD4+ TILs display enhanced anti-tumor activity in adoptive cell therapy (26A) and result in tumor eradication (26B) in ovarian tumor models.
  • Figure 27 shows that Bcl11b-deficient CD4+ T cells confer elevated anti-tumor activity in ovarian and colon cancer tumors implanted intraperitoneally. 43.
  • Figures 28A, 28B, 28C, and 28D show human melanoma specific CD8+ TILs that have been depleted for Bcl11b have elevatored perforin together with stemness TF TCF1, as well as the NK receptor CD56, but do not downregulate CD3 and CD8, thus maintaining CD8 TIL identity.
  • Figure 28A and 28B shows a brightfield image (28A) and fluorescent staining (28B) of tumor cells, TILs, and killed cancer cells.
  • Figure 28C shows that the number of dead tumor cells is greatly increased in Bcl11b depleted TILs.
  • Figure 28D shows a comparison of various markers between WT and Bcl11b depleted TILs.
  • An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant. 50.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the Attorney Docket Number 10110-443WO1 native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. 52. By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth).
  • an event or characteristic e.g., tumor growth
  • tumor growth means reducing the rate of growth of a tumor relative to a standard or a control. 53.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented.
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • the term “patient” refers to a subject under the treatment of a clinician, e.g., physician. 55.
  • the term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and Attorney Docket Number 10110-443WO1 supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • Attorney Docket Number 10110-443WO1 supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • Biocompatible generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
  • compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of'' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. 59. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive” or "negative.” 60.
  • Effective amount of an agent refers to a sufficient amount of an agent to provide a desired effect.
  • the amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject.
  • Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. 61.
  • a "pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a Attorney Docket Number 10110-443WO1 deleterious manner with any of the other components of the formulation in which it is contained.
  • “Pharmaceutically acceptable carrier” means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein. 63.
  • “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. 64.
  • “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • therapeutic agent e.g., sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium stea, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate
  • a composition comprising an agent refers to an amount that is effective to achieve a desired therapeutic result.
  • a desired therapeutic result is the control of type I diabetes.
  • a desired therapeutic result is the control of obesity.
  • Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the Attorney Docket Number 10110-443WO1 age, gender, and weight of the subject.
  • the term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief.
  • a desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
  • “Primers” are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • “Probes” are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. 68. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains.
  • CD8+ T cells are critical in the immune response to intracellular pathogens and tumors and provide long-lasting protection from reinfection. Following infection, antigen-specific CD8+ T cells undergo robust clonal expansion and differentiate into Klrg1 hi CD127 lo terminal effector cells (TEs) which die after pathogen clearance, and CD127 hi Klrg1 lo memory precursor cells (MPs), which have potential to form long-lived memory cells.
  • TEs CD127 hi CD127 lo terminal effector cells
  • MPs CD127 hi Klrg1 lo memory precursor cells
  • Memory CD8+ T cells are heterogeneous and can be divided into circulating memory and tissue resident memory cells.
  • T central memory (TCM) cells survey lymphoid tissue for antigen
  • T effector memory (T EM ) cells predominantly survey the blood.
  • T RM cells are seeded in non-lymphoid tissues early during infection and do not exit into the circulation, except during reinfection.
  • T RM cells provide an important first line of defense upon Attorney Docket Number 10110-443WO1 antigen reencounter due to their localization at common sites of infection including at key barrier surfaces, ability to directly kill infected cells and rapidly induce an innate-like immune response, proliferate, and trigger the recruitment of circulating memory and other immune cells to the site of reinfection.
  • TRM cells have been shown to provide protection against numerous pathogens, including herpes simplex, vaccinia and influenza viruses, as well as Listeria monocytogenes (Lm). However, in tumors and some chronic infections, exhausted T cells are generated, which are inefficient in controlling the anti-tumor immune response. 70. Commitment to memory CD8 + T cell subsets occurs early during infection. Multiple transcription factors (TFs) coordinate MP versus TE cell differentiation and regulate circulating versus resident memory cell fates. TFs critical for MP differentiation include Eomes, Stat3, Tcf1, Bcl6 and Id3, while those driving TE differentiation include Runx3, Zeb2, Blimp1, Id2 and Tbet.
  • TFs critical for MP differentiation include Eomes, Stat3, Tcf1, Bcl6 and Id3, while those driving TE differentiation include Runx3, Zeb2, Blimp1, Id2 and Tbet.
  • Runx3 Hobit and Blimp1 initiate the TRM program in the gut and skin. Runx3 controls expression of multiple components of the residency program, including Itgae (CD103) and CD69, to promote tissue retention. Hobit and Blimp1 dampen expression of Klf2, and its target S1pr1, to ultimately block tissue egress. Hobit and Blimp1 also repress expression of Tcf7 (encoding Tcf1) and Ccr7, recently found to promote tissue egress, whose expression is controlled by Tcf1.
  • Tcf7 is expressed in a subset of small intestine (SI) TRM cells, with high expression of Id3 and low expression of Blimp1, which have pronounced memory potential and increased polyfunctionality, expressing Ifn ⁇ and Tnf ⁇ .
  • SI small intestine
  • Blimp1 hi Id3 lo effector-like subset was also identified, which in addition to Prdm1, expresses mRNAs for Id2, Tbet, Granzymes, Prf1 and Klrg1.
  • the Id3 hi subset also expresses Il7ra and Cd28, as well as elevated Ifng, Tnf and Klf2 transcripts.
  • TRM cells This is likely related to the multipotency and ability of TRM cells not only to expand and generate secondary TRM cells following reinfection, but to egress from tissue and form secondary circulating effector and memory cells.
  • An additional TF aryl hydrocarbon receptor (Ahr)
  • the transcription factor Bcl11b acts both as a transcriptional repressor and as a transcriptional activator. It has essential roles in thymocyte development at multiple stages and also controls mature T cell differentiation and function.
  • Bcl11b deficient regulatory T (Treg) cells fail to maintain their identity and function.
  • Teg regulatory T
  • Bcl11b In mucosal-associated invariant T (MAIT) cells, Bcl11b sustains the MAIT17 program, while in iNKT cells, it represses the iNKT17 program and sustains the iNKT1 and iNKT2 cell programs. Removal of Bcl11b in na ⁇ ve CD8 + T cells leads to a failure in antigen-specific clonal expansion and effector function following infection. 72. In this study we investigated the role of Bcl11b in intestinal TRM cells and circulating memory CD8 + T cells after oral infection with Listeria monocytogenes (Lm) or cancer challenge. We found that absence of Bcl11b results in increased gut T RM cells and their precursors.
  • Lm Listeria monocytogenes
  • the T cells are responsive and able to exercise their full assortment of cytotoxic activity including, but not limited to the secretion of granzyme B, perforin, and cytokines (such as, for example, IFN- ⁇ and TNF- ⁇ ). 74.
  • a or condition or a recurrent disease or condition such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease (including, but not limited to diseases or conditions with T cell mediated immune responses)) in a subject
  • administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b); disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by CRISPR/cas9); or by administering to the subject T cells that have reduced Bcl11
  • a cancer such as, for example, a primary or recurrent cancer including, but not limited to
  • the T cells comprise tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (MILs), chimeric antigen receptor (CAR) T cells, CD4 T cells, CD8 T cells, effector memory T cells (T EM ), central memory T cells (T CM ), peripheral memory T cells (TPM), or tissue-resident memory T cells (TRM).
  • TILs tumor infiltrating lymphocytes
  • MILs marrow infiltrating lymphocytes
  • CAR chimeric antigen receptor
  • CD4 T cells CD4 T cells
  • CD8 T cells effector memory T cells
  • T EM effector memory T cells
  • T CM central memory T cells
  • TPM peripheral memory T cells
  • TRM tissue-resident memory T cells
  • a decrease, reduction, ablation, or disruption of Bcl11b activity or Attorney Docket Number 10110-443WO1 transcription or expression increases the cytotoxicity of disease or condition specific effector T cells (including, but not limited to the increased expression of perforin, granzyme b and effector cytokines, such as, IFN- ⁇ and TNF- ⁇ ) at the site of infection or tumor microenvironment (such as, for example, tumor infiltrating lymphocytes (TILs) and/or can reduce exhaustion of disease or condition specific effector T cells (including, but not limited to tissue resident memory T cells (TRM) or T cells that have been become exhausted or turned off (such as, for example, chronically stimulated T cells and T cells that have been inhibiting by an immune checkpoint such as PD1, TIM3, LAG-3, and/or CTLA-4).
  • TRM tissue resident memory T cells
  • Such rescued cells can exhibit a reduction in exhaustion hallmarks (such as, for example, a reduction in the exhaustion transcription factor Tox, as well as, exhaustion markers CD39, PD1, and Tim3).
  • the disease or condition is a T cell mediated disease or condition.
  • T cell mediated it is meant that T cells play a significant role in the immune response to disease or condition. 75.
  • T cells including chimeric antigen receptor (CAR) T cells and other adoptively transferred to cells such as tumor infiltrating lymphocytes (TILs) to the subject.
  • a disease or condition or a recurrent disease or condition including, but not limited to T cell mediated diseases or conditions
  • the disease is an infectious disease caused by a viral infection (such as, for example, a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein- Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (
  • a bacterial infection such as, for example, an infection with a bacteria selected from the group consisting of Bacillus anthracis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Mycobacterium chimaera, Nocardia as
  • compositions and methods can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers.
  • lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin’s Disease; myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and/or chronic myeloid leukemia (CML)); bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer; lung cancers such as small cell lung cancer, non-small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma/glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer including, but not limited to triple negative breast cancer
  • the treatment of the cancer can include ovarian cancer, colon cancer, or melanoma.
  • a T cell mediated disease or condition or a recurrent disease or condition such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease) in a subject comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense Attorney Docket Number 10110-443WO1 oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b); disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by
  • the T cells comprise tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (MILs), chimeric antigen receptor (CAR) T cells, CD4 T cells, CD8 T cells, effector memory T cells (TEM), central memory T cells (TCM), peripheral memory T cells (TPM), or tissue-resident memory T cells (TRM).
  • TILs tumor infiltrating lymphocytes
  • MILs marrow infiltrating lymphocytes
  • CAR chimeric antigen receptor
  • CD4 T cells CD8 T cells
  • TEM effector memory T cells
  • TCM central memory T cells
  • TPM peripheral memory T cells
  • TRM tissue-resident memory T cells
  • any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC- T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, AL
  • the treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT-011, MK- 3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS- 936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA- 4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as,
  • compositions 82 are methods of increasing resident memory T cells at the site of infection or in a tumor microenvironment comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity.
  • methods of increasing of increasing NK cell activation in a tumor microenvironment or site of infection comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity.
  • Antibodies (1) Antibodies Generally 83.
  • the term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with Bcl11b.
  • the antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.
  • IgA immunoglobulins
  • IgD immunoglobulins
  • IgE immunoglobulins
  • IgG immunoglobulins
  • IgG immunoglobulins
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. 84.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.
  • the disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No.5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al. 87.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec.22, 1994 and U.S. Pat. No.4,342,566.
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, sFv, scFv, and the like, including hybrid fragments.
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided.
  • antibody or fragment thereof fragments of antibodies which maintain Bcl11b binding activity are included within the meaning of the term “antibody or fragment thereof.”
  • Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
  • 89 Also included within the meaning of “antibody or fragments thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies). 90.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive Attorney Docket Number 10110-443WO1 property, such as specific binding to its cognate antigen.
  • Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • antibody or “antibodies” can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • Human antibodies 92.
  • the disclosed human antibodies can be prepared using any technique.
  • the disclosed human antibodies can also be obtained from transgenic animals.
  • transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).
  • the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge.
  • Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.
  • Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
  • a humanized form of a non-human antibody is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.
  • a humanized antibody residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues Attorney Docket Number 10110-443WO1 from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen).
  • Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues.
  • Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).
  • Fc antibody constant region
  • humanized antibodies can be generated according to the methods of Winter and co-workers, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Methods that can be used to produce humanized antibodies are also described in U.S. Patent No.4,816,567 (Cabilly et al.), U.S. Patent No.5,565,332 (Hoogenboom et al.), U.S. Patent No.5,721,367 (Kay et al.), U.S. Patent No.5,837,243 (Deo et al.), U.S. Patent No.5, 939,598 (Kucherlapati et al.), U.S.
  • Patent No.6,130,364 Jakobovits et al.
  • U.S. Patent No.6,180,377 Morgan et al.
  • (4) Administration of antibodies 96 Administration of the antibodies can be done as disclosed herein.
  • Nucleic acid approaches for antibody delivery also exist.
  • the broadly neutralizing anti Bcl11b antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment.
  • the delivery of the nucleic acid can be by any means, as disclosed herein, for example. 2.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical Attorney Docket Number 10110-443WO1 composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. 98.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. 99.
  • Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting Attorney Docket Number 10110-443WO1 of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced.
  • receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration.
  • compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. 103. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like. 105.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. 107.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.. 109.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch.22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365-389.
  • a typical daily dosage of the antibody used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. 1 12.
  • CD8 + T cells derived from Bcl11b f/f GzmbCre + R26REYFP + OT-I (CD45.2) mice showed no expression of YFP reporter related to Gzmb-Cre activity (Fig.9A), and their percentages were within the same range to those derived from WT (CD45.1/2) mice (Fig. 9B).
  • the transferred CD8 + T cells from Bcl11b f/f GzmbCre + R26REYFP + OT-I mice were YFP + both in the siIEL and spleen (Fig.9D).
  • a four-fold decrease in the percentages of transferred Bcl11b -/- CD44 + CD8 + T cells versus WT was observed in the spleen (Fig.1A), and no difference in the splenic CD127 hi Klrg1 lo (MPs) or Klrg1 hi CD127 lo (TEs) was observed. Rather, the absolute numbers of both populations were reduced, related to the overall reduction of Bcl11b- 115.
  • CD69 + CD103 + siIEL Bcl11b -/- CD8 + T cells remained elevated both in the co-transfer model, as well as in separate transfers (Fig.1G-H and J-K).
  • CD69 + CD103- cells (which at 30 DPI represent a very small fraction of the siIEL transferred CD8 + T cells) were reduced in the absence of Bcl11b in both transfer systems (Fig. 1H and 1K).
  • transferred siIEL and splenic CD8 + T cells derived from Bcl11b f/f GzmbCre + OT-I mice were mainly YFP + at 30 DPI (Fig.9I).
  • Bcl11b -/- memory CD8 + T have altered response to reinfection 118.
  • recipient CD45.1 mice were separately transferred and infected as above, and then re-infected at 30 DPI. Mice harboring Bcl11b -/- OT-I cells had significantly elevated bacterial burden in the liver following reinfection compared to WT (Fig.2A).
  • siIEL recipient CD45.1 + NK1.1 + cells were in lower percentages and numbers in the reinfected mice bearing Bcl11b -/- CD8 + T cells versus WT (Fig.2F), Attorney Docket Number 10110-443WO1 indicating defective ability of Bcl11b -/- CD8 + T cells in recruiting cells from circulation, likely related to defective Ifn ⁇ and Tnf ⁇ production. 120.
  • a sex-mismatched adoptive transfer system male cells transferred into female mice
  • Bcl11b -/- CD8 + T cells arise in the effector phase (9 DPI)
  • RNA-seq on splenic MPs (CD127 hi Klrg1 lo ), TEs (CD127 lo Klrg1 hi ) and siIEL CD69 + CD103 + T RMPs on 9 DPI, sorted to over 90% purity for the specific markers (Fig.9H, 9D).
  • Bcl11b expression was the highest in WT siIEL T RMPs compared to splenic MPs and TEs (Fig.3A).
  • Id3 hi Blimp1 lo MP/MF TRM program Numerous genes of the Id3 hi Blimp1 lo MP/MF TRM program were found to be downregulated in Bcl11b -/- TRMPs, including Id3 and Tcf7 (Fig.3E-F). In addition to Tcf7 downregulation at the mRNA and protein level (Fig.3I), a Tcf1-dependent gene signature was also reduced in Bcl11b -/- TRMPs (Fig.3G). 123. Genes of the Blimp1 hi Id3 lo effector-like TRM program were upregulated, including Prdm1 (encoding Blimp1) and effector genes (Fig.3E-F).
  • Ahr and its target Ahrr, were also upregulated in Bcl11b -/- TRMPs, as well as Ahr-dependent genes (Fig.3E, H). Protein levels of Ahr and Blimp1 were also upregulated in Bcl11b -/- siIEL TRMPs (Fig.3I). Attorney Docket Number 10110-443WO1 124. RNA-seq analysis Bcl11b -/- and WT siIEL TRMs during the memory phase at 30 DPI further revealed that numerous genes of the MP/MF TRM program remained reduced in Bcl11b -/- siIEL TRMs (Fig.3J). Conversely, effector TRM program genes were elevated (Fig. 3J).
  • Results show that genes encoding several TFs driving TE cell differentiation, including Runx3, Egr2, Zeb2 and Prdm1 remained unchanged, while Id2 and Tbx21 (encoding T-bet) were modestly upregulated in Bcl11b -/- TEs (Fig.11A).
  • MP program TF genes including Eomes, Stat3 and Klf2 were also unaltered, while Tcf7 was modestly upregulated in Bcl11b -/- MPs (Fig.11B).
  • Ahr was not expressed in MPs or TEs (Fig.11C). 1 27.
  • ⁇ 4 ⁇ 7 and Ccr9 proteins were significantly increased in both Bcl11b -/- populations (Fig.12B-C), indicating that increased intestinal homing of circulating Bcl11b -/- cells can lead at least in part to their selective loss in the spleen.
  • genes with decreased and/or increased expression in Bcl11b -/- TRMP and TRM cells were consistently deregulated in Bcl11b -/- TRM-like cells (Fig.13A-C), resembling the phenotype of the primary TRMP and TRM cells (Fig.3).
  • Fig.13A-C Bcl11b-dependent differences were observed for 9,874 peaks in chromatin accessibility, 27,445 in H3K27ac and 12,485 in H3K4me3, respectively.
  • Differential H3K27ac peaks with increased or decreased signal were overall equally distributed, however peaks with increased chromatin accessibility were enriched in Bcl11b -/- cells (Fig.4A).
  • TF DNA binding motif analysis in the differential ATAC-seq and H3K27ac peaks show that Blimp1 and Ahr motifs were enriched in peaks with increased signal in Bcl11b -/- Attorney Docket Number 10110-443WO1 cells (Fig.4G), while Tcf1 and Lef1 motifs were enriched in peaks with decreased signal in Bcl11b -/- cells (Fig.4G).
  • Detailed motif enrichment analysis separately at differential ATAC-seq and H3K27ac peaks, and correlated with Bcl11b binding, grouped TF motifs into several categories (Fig.14C).
  • ETS family motifs were mostly enriched in differential peaks not bound by Bcl11b, indicating ETS TF activity is independent of Bcl11b binding.
  • motifs for Egr1, Egr2, Irf4, and NF- ⁇ B were enriched at ATAC-seq and H3K27ac peaks with increased signal in Bcl11b -/- cells at sites bound by Bcl11b in WT cells (Fig.14C), indicating a competitive mechanism of action by Bcl11b with these TFs.
  • Fig.14C the most enriched motifs were for ETS, bZIP and Runx family (Fig.14D), similarly to what we observed before in MAIT cells and Treg cells.
  • Binding motifs for Tcf1/Lef1, Blimp1 and Ahr were enriched at differential ATAC-seq and H3K27ac peaks, irrespective of Bcl11b binding, however numerous Bcl11b peaks also contained significant enrichment of Tcf1/Lef1, Blimp1 and Ahr motifs (Fig.14C-D), supporting a functional intersection between Bcl11b and these TFs related to the MP/MF and effector TRM programs.
  • Bcl11b binds at the Tcf7 locus, Tcf1-dependent genes and genes of the MP/MF program 132. We next sought to determine whether Bcl11b is directly implicated in regulation of Tcf7 and other genes of the MP/MF TRM program.
  • Bcl11b binding at multiple intronic, promoter, and upstream regions of Tcf7 gene (Fig.5A).
  • Deletion of Bcl11b in TRMPs resulted in decreased chromatin accessibility at upstream and intronic sites bound by Bcl11b, as well as reduced H3K27ac and H3K4me3 at an upstream site and at Tcf7 promoter (Fig.5A), indicating that Bcl11b sustains H3K27ac, H3K4me3 and chromatin accessibility at Tcf7 to drive its expression.
  • BCL11B In human memory CD8 + T cells, BCL11B also bound at two upstream regions of TCF7 gene, indicating common modules of regulation in mice and humans, dependent on BCL11B (Fig.5B).
  • Tcf7 expression was only downregulated in siIEL TRMPs and TRMs, but not in splenic MPs or TEs (Fig.3; Fig.11), indicating a specific role for Bcl11b in regulating its expression only in siIEL TRM cells and precursors. 133.
  • the Tcf1 signal at promoters was almost exclusively found at promoters bound by Bcl11b (Fig.5C).
  • Bcl11b also bound at several other genes of the MP/MF program, and remarkably, its binding sites at Id3, Lef1, Klf2, S1pr1, Cd5 and Btla were associated with decreased H3K27ac, H3K4me3 and/or chromatin accessibility in its absence.
  • binding of Bcl11b occurred at regions with decreased Tcf1 binding in Bcl11b -/- cells (Fig.15C, 16A-C).
  • some other genes from the program such as Lef1, Cd5 and mostly Tcf7 itself displayed increased Tcf1 binding in the absence of Bcl11b (Fig.16A-B and Fig.5A), indicating a compensatory mechanism of regulation.
  • Bcl11b also bound and regulated H3K27ac deposition at Ifng and Tnf loci, as well as at Cd28, associated with regulation of chromatin accessibility (Fig. 16D-F), indicating direct regulation of expression of these genes.
  • Fig. 16D-F chromatin accessibility
  • Ex vivo activated Bcl11b -/- and WT OT-I CD8 + T cells were transduced with Tcf7- retroviruses and adoptively transferred into Lm-Ova InlA M infected recipient mice.
  • Tcf7 transduction led to similar levels of Tcf1 between siIEL Bcl11b -/- and WT cells at 9 DPI (Fig.5E) and restored Bcl11b -/- /WT ratio within the siIEL CD69 + CD103 + TRMPs (Fig.5F).
  • Bcl11b binds at Prdm1, effector genes and Ahr loci to control their expression in TRM cells 135.
  • Bcl11b bound at several sites surrounding the Prdm1 locus, including at the promoter.
  • Bcl11b -/- cells these sites had elevated H3K4me3, while Attorney Docket Number 10110-443WO1 other sites had increased H3K27ac and chromatin accessibility (Fig.6A).
  • BCL11B also bound at several sites at the human PRDM1 locus (Fig.6B). 1 36.
  • Bcl11b bound at the loci of Gzma and Prf1, genes of the T RM effector program, and at Gzmc, another effector gene, at several locations (Fig.17A-C).
  • Bcl11b bound at genes associated with exhaustion, including Havcr2 and Entpd1 (Fig.17D-E).
  • BCL11B bound AHR in an intronic region (Fig.6D).
  • regulation of Ahr transcription by Bcl11b occurred solely in siIEL Bcl11b -/- TRMPs and TRMs, but not in MPs or TEs (Fig.3E and 11C).
  • Fig.3E and 11C a binding preference for Ahr related to Bcl11b.
  • ChIP-seq analysis in Bcl11b -/- and WT T RM -like cells and integrated them with the other datasets. Similar to Tcf1, Ahr co-binding with Bcl11b was mostly located at gene promoters (Fig.6E).
  • Ahr signal in Bcl11b -/- cells was slightly increased, in line with the overall increased Ahr protein. Moreover, Ahr binding was enriched and increased in Bcl11b -/- cells at sites corresponding to Bcl11b binding in WT cells, and associated with increased chromatin accessibility, whereas no changes in Ahr binding in the Bcl11b -/- cells were observed for Bcl11b peaks associated with decreased accessibility (Fig.6F). Note that these observations were in agreement with the motif analysis (Fig.4G). We further focused the analysis on genes bound by Ahr and with elevated expression in Bcl11b -/- cells.
  • Bcl11b bound at several NK genes, located in the NK cell receptor locus in regions associated with broad H3K27ac in NK cells, including at Klrb1.
  • Bcl11b -/- T RM -like cells we observed increased H3K27ac and chromatin accessibility at NK cell receptor enhancers (Fig.7B).
  • Bcl11b binding at Itgam, Itgax (encoding CD11b and CD11c) and Fcer1g loci was associated with increased H3K27ac, H3K4me3 and/or chromatin accessibility (Fig.7C- D).
  • BCL11B also bound at human KLRB1 (Fig. 7E), as well as at ITGAM and ITGAX (Fig. 7F), indicating a common role of BCL11B between mice and humans in repressing innate gene expression in CD8 + T cells.
  • BCL11B regulates the TRM program in human CD8 + T cells ex vivo 1 41.
  • NK receptors including CD56, CD161 (KLRB1), CD117 and NKP46/NCR1, were upregulated in BCL11B-deficient human T RM -like cells (Fig.8C), indicating a common role of BCL11B in mice and humans.
  • BCL11B bound at numerous genes of the T RM program, also bound and regulated by Bcl11b in mouse (Fig. 5-7, Fig. 15), indicating that BCL11B regulates aspects of the CD8 + T cell residency program in human and mouse in a similar fashion.
  • Bcl11b Depletion of Bcl11b also increases cytotoxicity of TILs in human cancer models 143. As shown in Figure 20, BCL11B depletion in human TILs increases cytotoxicity in a manner dependent on MHC I. In fact, Bcl11b-deficient CD8+ T cells confer elevated anti- tumor activity in ovarian tumors implanted intraperitoneally ( Figure 21) including elevated cytotoxic genes and NK receptor and stemness program genes, while inhibitory receptor genes remain unchanged ( Figure 22). Looking at peritoneal tumors, we showed that expression of gene subsets in TILs clusters in WT and Bcl11b -/- (knockout) mice with peritoneal tumors following adoptive transfer.
  • Figurer 24 shows that CD8+ TILs from Bcl11b -/- mice continue to express stemness transcription factor TCF1 (24A and 24B), expand more (24C), have reduced levels of the exhaustion transcription factor Tox, as well as, exhaustion markers CD39, PD1, and Tim3 (24D), and have increased metabolic potential and mass and thus greater fitness (24E).
  • This paradigm also holds for CD4 T cells as shown in Figures 25 and 26 showing that Bcl11b -/- (KO) CD4+ TILs display enhanced anti-tumor activity in adoptive cell therapy and result in tumor eradication in ovarian tumor models.
  • Bcl11b-deficient CD4+ T cells confer elevated anti-tumor activity in ovarian and colon cancer tumors implanted intraperitoneally (Figure 27).
  • Figure 28A and 28B shows a brightfield image ( Figure 28A) and fluorescent staining ( Figure 28B) of tumor cells, TILs, and killed cancer cells.
  • Figure 28C shows that the number of dead tumor cells is greatly increased in Bcl11b depleted TILs.
  • Figure 28D shows a comparison of various markers between WT and Bcl11b depleted TILs.
  • Bcl11b was depleted using CRISPR/cas9 targeting Bcl11b using the guide Hs.Cas9.Bcl11b.AA (SEQ ID NO: 1) in combination with Hs.Cas9.Bcl11b.AE (SEQ ID NO: 2).
  • Hs.Cas9.Bcl11b.AE SEQ ID NO: 2. Discussion
  • Bcl11b plays a critical role in SI CD8 + TRM cells, including in their precursors, promoting the MP/MF program and suppressing the Attorney Docket Number 10110-443WO1 effector program, thus expanding our current knowledge on transcriptional control of intestinal TRM cell differentiation and divergent programs.
  • Bcl11b exerts its role by binding, regulating the epigenetic landscape and directly controlling expression of essential genes of the MP/MF TRM program, including of Tcf7, Id3, Lef1, Ifng , Tnf, Cd5, Btla, and additionally Klf2, S1pr1 and Ccr7.
  • Bcl11b also bound and repressed the expression of essential genes of the effector program, including the lead TF Blimp1, Granzyme genes and Prf1.
  • recall response was poor, with elevated bacterial burden and reduced numbers of recall Bcl11b -/- CD8 + T cells in the SI.
  • SI recall Bcl11b -/- CD8 + T cells Although the paucity of circulating memory cells is likely to contribute to the reduced numbers of SI recall Bcl11b -/- CD8 + T cells, the sex mismatch experiment demonstrated that SI Bcl11b -/- TRM cells have intrinsic alterations. In line with this, recall SI Bcl11b -/- CD8 + T cells continue to show reduced MP/MF program gene expression and altered ability to produce Ifn ⁇ , important for recruitment not only of circulating memory CD8 + T cells during recall, but also of other immune populations, including of NK cells.
  • Id3 hi Blimp1 lo TRM cells also expressing Tcf1 are able to provide a more efficient secondary response during recall compared to the Id3 lo Blimp1 hi counterparts, are multifunctional and multipotent and generate both secondary resident and circulating memory cells.
  • Id3 hi Blimp1 lo MP/MF subset a memory phase CD28 hi cell cluster (with high Id3), has been identified, with elevated Ifng and Tnf transcripts, as well as high Klf2, likely to provide support for egress from tissue into the circulation following reinfection.
  • Bcl11b regulates TRM multipotency by controlling both the recruitment of cells from circulation through Ifn ⁇ , as well the egress from the tissue, through Klf2, S1pr1 and Ccr7.
  • the impaired MP/MF signature in the absence of Bcl11b explains the deficient response in the recall.
  • Bcl11b -/- TRM cells which have high Blimp1, but low Id3 and Tcf1, are long-lived TRM cells, but their protective recall response is impaired, despite elevated expression of effector genes for granzymes and Prf1.
  • Bcl11b -/- CD8 + T cells can provide enhanced protection in response to other pathogens and at other location or in cancer, particularly given their elevated expression of cytolytic molecules, is of particular interest.
  • restoration of Tcf1 expression in Bcl11b- /- CD8 + T cells early in the response can restrict TRM cell differentiation.
  • Bcl11b seems to restrict Ahr activity at the Il2rb promoter (IL15 receptor), and thus possibly responsiveness to IL15, known to control at some extent together with Tgf ⁇ TRM cell fate. It remains to be established where Ahr is positioned in regulation of MP/MF versus effector TRM cell programs. 150.
  • ablation of Bcl11b causes a major reduction of circulating memory cells mainly due to their enhanced intestinal homing with minimal impact on program specific TFs of memory precursors and effector cells.
  • Bcl11b restricted expression of the innate program, including of NK and myeloid receptors, but not in MAIT cells.
  • Bcl11b -/- memory cells were investigated in secondary infections with Lm-Ova InlA M , including a sex-mismatched system for siIEL T RM only response. All data are representative of at least 2 experiments. Statistics were conducted mostly with two-tailed paired or unpaired Student’s t-test. Samples were unblinded.
  • Bcl11b F/F mice bread on C57BL/6 background, were crossed to B6-Tg(GZMB- cre)1Jcb/J (GzmbCre) on C57BL/6 background, provided by Barbara Kee, (University of Chicago).
  • B6.129X1-Gt(ROSA)26Sor tm1(EYFP)Cos /J (R26REYFP) and C57BL/6- Tg(TcraTcrb)1100Mjb/J (OT-I) mice were from Jackson laboratory.
  • B6.SJL-Ptprc a Pepc b /BoyJ (CD45.1) (wild type) recipient mice were purchased from Taconic and bred in our colony.
  • CD8 + T cells were enriched from spleens of OT-I mice using the CD8 + T cell negative selection kit (Stem Cell, 19853), followed by sorting of na ⁇ ve CD62L hi CD44 lo CD8 + T cells (BD Aria II). Mixed 1:1 ratio (for co-transfers) 5 ⁇ 10 4 cells were transferred intravenously into WT CD45.1 mice, 1 day prior to infection. Donor cells were subsequently assessed at indicated time points post-infection in different experiments. 157.
  • mice were food starved for several hours during the day and infected late afternoon by feeding infected bread containing 2 ⁇ 10 9 (primary) or 2 ⁇ 10 10 (reinfection) colony forming units (CFUs) of Lm-Ova InlA M .
  • CFUs colony forming units
  • CD8 + T cells were purified from na ⁇ ve Bcl11b f/f GzmbCre + R26REYFP + OT-I and WT OT-I mice as above and then activated with anti-CD3/anti-CD28 beads (Invitrogen, 114525D) at 2.5 ⁇ 10 5 cells/ml in 10% FBS MEM alpha supplemented with 100 U/ml IL-2 (Shenandoah Biotechnology, 100-12) for 48 hours at 37°C, 5% CO 2 , when cells were split 1:3 in fresh media supplemented with 10 ng/ml TGF ⁇ 1 (Shenandoah Biotechnologies, 100-39) and 100 ng/ml IL-33 (Shenandoah Biotechnologies, 200-36), for an additional 48 hours.
  • FACS sorted na ⁇ ve CD8 + CD45RA + CD62L + CD27 + CD57 – T cells were pre- activated with 10 ⁇ l/ml anti-CD3/28 immunocomplexes (StemCell Technologies) and 100 U/ml IL-2 (Peprotech) and incubated in complete medium at 37°C. After 48 hours, pre-activated CD8 + T cells were washed twice with RPMI supplemented with 10% FBS and cultured for 14 days with anti-CD3/28 immunocomplexes, 100 U/ml IL-2 (Peprotech), 10 ng/ml TGF- ⁇ (Peprotech) and 100 ng/ml IL- 33 (Peprotech).
  • ChIP-seq for murine Bcl11b, H3K27ac and Ahr 1 61. T RM -like cells were differentiated as above.
  • Bcl11b ChIP-seq was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003), with the following changes to the protocol: (i) 10 7 cells were used, (ii) formaldehyde fixation was performed in 1 ml, 162.
  • ChIP was performed using the ChIP- IT High Sensitivity Kit (Active Motif, 53040), following the manufacturer's protocol with the following modifications: (i) the nuclear pellet of 10 6 cells was lysed in 500 ⁇ l ChIP buffer and chromatin was sheared by the Q125 Sonicator (QSonica); (ii) the immunoprecipitation was performed with anti-H3K27ac (D5E4, CST) or anti-Ahr antibodies (Enzo Life Sciences, BML- SA210–0100).
  • CUT&RUN was performed on 0.35 ⁇ 10 6 T RM -like cells using the ChIC/CUT&RUN Kit (EpiCypher, 14-1048) with 1 ⁇ l anti-H3K4me3 (EpiCypher, 13-0041) or 1.5 ⁇ l anti-Tcf1 (C46C7, CST) antibodies, following the manufacturer's protocol, except that 1 ⁇ eBioscience Perm Buffer (Invitrogen, 00-8333) supplemented with Spermidine and cOmpleteTMEDTA-free Protease Inhibitor (Roche, 11836170001) was used during the incubation step with pAG-MNase and 0.5 ⁇ eBioscience Perm Buffer during the wash steps.
  • 1 ⁇ eBioscience Perm Buffer Invitrogen, 00-8333
  • Spermidine and cOmpleteTMEDTA-free Protease Inhibitor
  • the intestine was cut into 1-2 cm pieces and washed 3-4 times in RPMI supplemented with 1% L- glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 168. 0.01 M HEPES, 220 ⁇ M 2-mercaptoethanol, and 1% FBS (1% RPMI).
  • Intestinal pieces were incubated in 20 ml 1 ⁇ HBSS containing 1 mM dithioerythritol and 10% FBS with shaking at 37°C twice for 20 minutes, followed by filtration on 70 ⁇ m cell strainer to isolate IELs. 46.25 ml Percoll (GE Healthcare, Cat.
  • No.17-0891-01 were mixed with 3.6 ml 10 ⁇ HBSS (Corning, Cat. No.20- 023-CV) and 0.6 ml 7.5% sodium bicarbonate and further diluted to 44% and 66% isotonic Percoll with 1 ⁇ HBSS.
  • IEL were resuspended in 8 ml 44% Percoll and underlayed with 2 ml 66% Percoll using a glass Pasteur pipette. Cells were centrifuged for 20 minutes 1600 ⁇ g at room temperature and the interface was collected with Pasteur pipette. Cells were washed twice with threefold volume of 1% FBS RPMI. n) In vivo treatments 169.
  • mice were injected intraperitoneal with 100 mg SIINFEKL (SEQ ID NO: 3) peptide and 0.3 mg brefeldin A (BFA) (Sigma Aldrich, Cat. No. B7651) 5 hours prior to organ harvest. Spleen and IEL were processed with additional supplementation of 0.02 mg BFA in all solutions until cells were fixed. o) Human cells isolation and culture 170. Buffy coats from healthy donors were collected from the Karolinska University Hospital blood bank. PBMCs were isolated by density gradient centrifugation, thereafter bulk CD8 + T cells were enriched from PBMCs using negative magnetic selection (StemCell Techologies; Miltenyi Biotech).
  • SIINFEKL SEQ ID NO: 3
  • BFA brefeldin A
  • Platinum-E (Plat E) retroviral packaging cells (Cell Biolabs, Cat. No. RV-101) were grown in DMEM supplemented with 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 ⁇ M 2-mercaptoethanol, and 10% FBS (10% DMEM). Retroviral particles were produced.3 ⁇ 10 6 Plat E cells were seeded on 10-cm cell cultures plates such that 80% confluence was reached overnight.
  • the DNA-Lipofectamine 2000 complexes in a total volume of 900 ⁇ l were added dropwise to Plat E cells.
  • the transfected Plat E cells were incubated overnight at 37°C, 5% CO 2 .
  • the medium was aspirated from transfected Plat E cells; then 5.5 ml of pre-warmed (37°C) RPMI supplemented with 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 220 ⁇ M 2- mercaptoethanol, and 10% FBS (10% RPMI) was added to the edge of the plate slowly.
  • Retroviral particles in 10% RPMI were collected at 48 and 72 hours post-transfection and filtered through a 0.45- ⁇ m syringe filter (Corning, Cat. No.431220). Retrovirus was further concentrated 5-fold with Lenti-X Concentrator (Takara, Cat. No.631232) according to manufacturer’s instructions and resuspended in 10% FBS RPMI. Concentrated retrovirus was stored at ⁇ 80°C until use. r) Transduction with retroviruses 173.
  • Spleen and peripheral lymph node Bcl11b -/- or WT OT-I CD8 + T cells were isolated using the mouse CD8 + T cell isolation kit (Stem Cell, Cat. No.19853). Purified cells were activated with 5 ⁇ g/ml plate-bound anti-CD3 (BioXCell, Cat. No. BE0002) and 2 ⁇ g/ml soluble anti-CD28 (BioXCell, Cat. No. BE0015-1) at 1 x 10 6 cells/ml in 10% RPMI supplemented with 100 U/ml IL- 2 (Shenandoah Biotechnology, Cat. No.100-12) for 24 hours at Attorney Docket Number 10110-443WO1 37°C, 5% CO 2 .
  • Retrovirus containing 10% RPMI and 8 ⁇ g/ml Polybrene (1 ml) was added to a 24-well plate, and 1-3 x 10 6 activated cells (1 ml) were immediately added to the retrovirus containing media. The cells were then centrifuged at 2000 x g at 30°C for 1 hour, then incubated at 37°C and 5% CO 2 for 24 hours. The transduced cells were subsequently harvested, washed twice with PBS and 5 x 10 4 cells were co-transferred into Lm-Ova InlA M infected mice. s) Flow cytometry 174. Single cell suspensions from spleen and intestine were isolated at indicated time points.
  • Sonicated cells were spun at 10,000 g for 5 mins, and supernatants taken and diluted with ChIP dilution buffer (100mM NaCl, 100mM Tris HCl pH 8.6, 5mM EDTA, 0.2% NaN3, 1% Triton X ⁇ 100) and incubated overnight with 40 ⁇ l Protein G Dynabeads (ThermoFisher) pre-loaded with 10 ⁇ g antibody (Bcl11b, clone 25B6, AbCam).
  • ChIP dilution buffer 100mM NaCl, 100mM Tris HCl pH 8.6, 5mM EDTA, 0.2% NaN3, 1% Triton X ⁇ 100
  • 40 ⁇ l Protein G Dynabeads pre-loaded with 10 ⁇ g antibody (Bcl11b, clone 25B6, AbCam).
  • Chromatin was washed twice each with low salt buffer (50 mM HEPES, 1% Triton, 140 mM NaCl), high salt buffer (salt increased to 500 mM NaCl) then TE buffer (10 mM Tris-HCl, 1 Attorney Docket Number 10110-443WO1 mM EDTA), then reverse cross-linked in 100 ⁇ l ChIP SDS buffer with 200 mM NaCl and Proteinase K at 65°C for 15 hours followed by RNaseA (ThermoFisher) treatment for 1 hour. DNA was purified (ChIP DNA Clean & Concentrate, Zymo), before sequencing libraries prepared (Thruplex DNA-seq kit, TakaraBio).
  • CD8 + T cells were purified from na ⁇ ve Bcl11b -/- and WT OT-I mice as above. Purified cells were incubated with 10% RPMI supplemented with 5 ng/ml recombinant mouse IL-7 (R&D Systems, Cat.
  • No.402-ML-020 at 1 ⁇ 10 6 cells/ml and preincubated for 24 hours before transfection at 37°C, 5% CO 2.
  • sgRNA design the top 3 ranked sgRNAs as determined by Synthego’s CRISPR Design Tool (Synthego) against Prdm1 and Ahr were chosen.
  • Precomplexing of Cas9/RNP was performed in a PCR tube by the addition of 0.2-0.4 ⁇ l of each 100 pmol sgRNAs (for a final volume of all sgRNAs 1.2 ⁇ l), 0.66 ⁇ l Cas9 (ThermoFisher, Cat.
  • RNAs targeting BCL11B gene were formed by hybridizing tracrRNA (200 ⁇ M; IDT) and crRNA (200 ⁇ M; IDT) for 5 minutes at 95°C.
  • Recombinant Cas9 (40 ⁇ M; MacroLab) was incubated with gRNA duplex for 10 minutes at 37°C at a 2:1 ratio to form CAS9 ribonucleoprotein complexes (RNPs).
  • Cells were washed twice with PBS and resuspended in electroporation buffer (1 M; 5 mM KCl, 15 mM MgCl 2, 120 mM Na 2 HPO 4 pH7.2 and 50 mM mannitol) at a concentration of 10 ⁇ 10 6 cells/ml.
  • RNA-seq data was processed using a previously established pipeline. In brief, fastq files were trimmed using seqtk and quality was assessed using FastQC. Trimmed reads were aligned to the mouse genome (mm10) using Hisat2 and transcript counts were obtained using FeatureCounts.
  • DESeq2 was used for differential expression analysis.
  • GSEA was performed using DRPPM-EASY with genes ranked based on signal-to-noise.
  • the GSEA function was run under default parameters implemented by the clusterProfile package.
  • Rlog values of all differentially expressed genes (padj ⁇ 0.05) calculated by DESeq2 were used for principal component analysis and to calculate Euclidean distances for hierarchical clustering using Ward’s method.
  • cells were lysed in 50 ⁇ l cold lysis buffer (10mM Tris Cl, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% v/v IGEPAL CA-630) and centrifuged immediately at 500 ⁇ g for 10 minutes.
  • DNA was transposed using a Nextera Tn5 kit (Illumina, FC-121-1030) at 37°C for 30 minutes.
  • DNA was purified using a MiniElute PCR Purification kit (Qiagen, Cat. No. 28004) and amplified using barcoded PCR primers. DNA was purified using SPRIselect magnetic beads (Beckman Coulter, Cat. No. B23318).
  • Reads were aligned to the mouse genome (mm10) using Bowtie2 (v.2.2.5) with the default parameters for ATAC-seq and ChIP-seq data and with the following parameters for CUT&RUN: --dovetail --local --very- sensitive-local --no-unal --no- mixed --no-discordant --phred33 -I 10 -X 700.
  • the resulting SAM files were pruned (Q > 30), converted to BAM format, and sorted using SAMtools. Duplicate reads were removed using Picard tools.
  • MACS2 was used for peak calling, with the following parameters for ATAC-seq, Bcl11b and H3K27ac ChIP-seq data: macs2 callpeak --call-summits --nomodel -g mm -p 0.01 -f BAM --SPMR -B.
  • -q 0.05 was used with no subsequent IDR filtering as described below.
  • the following parameters were used for CUT&RUN: macs2 callpeak --call- summits -g mm -p 0.00001 -f BAMPE --SPMR -B.
  • Bedgraph files produced by MACS2 were lambda control subtracted.
  • Hs.Cas9.BCL11B.1.AA SEQ ID NO: 1 CATGACTCGGTCGAAGGCAC combined in a single reaction with: Hs.Cas9.BCL11B.1.AE (SEQ ID NO: 2) GTGTCAAATGAACTTCCCCT F.
  • T RM integrins CD103 and CD49a differentially support adherence and motility after resolution of influenza virus infection. Proceedings of the National Academy of Sciences of the United States of America 117, (2020). E. J. Wherry, V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, R. Ahmed, Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol 4, 225-234 (2003). F. Cruz-Guilloty, M.
  • T-box transcription factors combine with the cytokines TGF- ⁇ and IL-15 to control tissue-resident memory T cell fate. Immunity 43, 1101-1111 (2015). L. K. Mackay, M. Minnich, N. A. Kragten, Y. Liao, B. Nota, C. Seillet, A. Zaid, K. Man, S. Preston, D. Freestone, A. Braun, E. Wynne-Jones, F. M. Behr, R. Stark, D. G. Pellicci, D. I.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Disclosed are compositions and methods for treating, inhibiting, reducing, decreasing, and/or preventing a cancer, microbial infection, recurrent cancer, or recurrent infection via Bcl11b inhibition.

Description

Attorney Docket Number 10110-443WO1 BCL11B SUSTAINS MULTIPOTENCY AND RESTRICTS EFFECTOR PROGRAMS OF INTESTINAL RESIDENT MEMORY CD8+ T CELLS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit to U.S. Provisional Application No.63/499,203, filed on April 28, 2023, which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant No. AI067846 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via Patent Center encoded as XML in UTF-8 text. The electronic document, created on April 26, 2024, is entitled “10110-443WO1_ST26.xml”, and is 4,868 bytes in size. I. BACKGROUND 1. CD8+ T cells are critical in the immune response to intracellular pathogens and tumors and provide long-lasting protection from reinfection. Following infection, antigen- specific CD8+ T cells undergo robust clonal expansion and differentiate into Klrg1hiCD127lo terminal effector cells (TEs) which die after pathogen clearance, and CD127hiKlrg1lo memory precursor cells (MPs), which have potential to form long-lived memory cells. Memory CD8+ T cells are heterogeneous and can be divided into circulating memory and tissue resident memory cells. Of the circulating subsets, T central memory (TCM) cells survey lymphoid tissue for antigen, and T effector memory (TEM) cells predominantly survey the blood. In contrast, TRM cells are seeded in non-lymphoid tissues early during infection and do not exit into the circulation, except during reinfection. TRM cells provide an important first line of defense upon antigen reencounter due to their localization at common sites of infection including at key barrier surfaces, ability to directly kill infected cells and rapidly induce an innate-like immune response, proliferate, and trigger the recruitment of circulating memory and other immune cells to the site of reinfection. TRM cells have been shown to provide protection against numerous pathogens, including herpes simplex, vaccinia and influenza viruses, as well as Listeria monocytogenes (Lm). However, in tumors exhausted cells are generated, which are inefficient in Attorney Docket Number 10110-443WO1 controlling the anti-tumor immune response. What are needed are methods and compositions for increasing the number of TRM at sites of infection and tumor microenvironments. II. SUMMARY 2. Disclosed are methods and compositions related to inhibiting expression of Bcl11b. 3. In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a disease or condition or a recurrent disease or condition (such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease) (including, but not limited to diseases or conditions with T cell mediated immune responses) in a subject comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b); disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by CRISPR/cas9); or by administering to the subject T cells that have reduced Bcl11b activity. In some aspects the T cells comprise tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (MILs), chimeric antigen receptor (CAR) T cells, CD4 T cells, CD8 T cells, effector memory T cells (TEM), central memory T cells (TCM), peripheral memory T cells (TPM), or tissue-resident memory T cells (TRM). 4. Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a disease or condition or a recurrent disease or condition of any preceding aspect, further comprising obtaining T cells from a donor source and disrupting Bcl11b expression in the T cells prior to administering the T cells to the subject. 5. In one aspect, disclosed herein are methods of increasing the cytotoxicity (i.e., increasing expression of effector molecules such as, for example, granzyme b, perforin, or cytokines including, but not limited to, IFN-γ and/or TNF-α) of T cells specific for a disease or condition (such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease) (including, but not limited to diseases or conditions with T cell mediated immune responses), said methods comprising contacting T cell mediated disease specific T cells with an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b) or disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by CRISPR/cas9) whereby a decrease, reduction, ablation, or disruption of Bcl11b Attorney Docket Number 10110-443WO1 activity or transcription or expression increases the cytotoxicity of disease or condition specific T cells. In some aspects the guide RNA for the CRISPR/cas9 are set forth in SEQ ID NO:1 and SEQ ID NO: 2. 6. Also disclosed herein are methods of reducing exhaustion of T cells specific for a disease or condition (such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease)( including, but not limited to diseases or conditions with T cell mediated immune responses), said method comprising contacting T cell mediated disease specific T cells with an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b) or disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by CRISPR/cas9); whereby a decrease, reduction, ablation, or disruption of Bcl11b activity or transcription or expression rescues the disease or condition specific T cells returning them to an active functional state (such as, for example, a reduction in the exhaustion transcription factor Tox, as well as, exhaustion markers CD39, PD1, and Tim3). In some aspects the guide RNA for the CRISPR/cas9 are set forth in SEQ ID NO:1 and SEQ ID NO: 2. 7. In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a disease or condition or a recurrent disease or condition of any preceding aspect; methods of reducing exhaustion of T cells specific for a disease or condition of any preceding aspect; or methods of increasing the cytotoxicity of T cells specific for a disease or condition of any preceding aspect; wherein the infectious disease is caused by a viral infection (such as, for example, a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS- CoV-2) (including, but not limited to the B1.351 variant, B.1.1.7 variant, and P.1 variant), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous Attorney Docket Number 10110-443WO1 sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2); a bacterial infection (such as, for example, an infection with a bacteria selected from the group consisting of Bacillus anthracis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Mycobacterium chimaera, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species, and Mycoplasma species); a fungal infection (such as, for example, an infection with a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata); or a a parasitic infection (such as, for example, an infection with a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius Attorney Docket Number 10110-443WO1 gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica). 8. Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a T cell mediated disease or condition or a recurrent disease or condition of any preceding aspect; methods of reducing exhaustion of T cells specific for a T cell mediated disease or condition of any preceding aspect; or methods of increasing the cytotoxicity of T cells specific for a T cell mediated disease or condition of any preceding aspect; further comprising administering to the subject an agent that blocks MHC class 1 expression. 9. In one aspect, disclosed herein are methods of increasing resident memory T cells at the site of infection or in a tumor microenvironment comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity. 10. Also disclosed herein are methods of increasing NK cell activation in a tumor microenvironment or site of infection comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity. III. BRIEF DESCRIPTION OF THE DRAWINGS 11. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods. 12. Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, and 1K show Bcl11b deletion post oral infection with Lm-Ova InlAM results in reduced circulating memory cells, but excessive SI TRM cells. (1A-1H) Co-transfers. Figure 1A shows contour plots and quantification Attorney Docket Number 10110-443WO1 of splenic Bcl11b-/- (CD45.2) and WT (CD45.1/2) cells. Figure 1B shows contour plots and quantification of Klrg1 versus CD127 within Bcl11b-/- (CD45.2) and WT (CD45.1/2) CD8+CD44+ splenic population. Figure 1C shows Bcl11b-/- and WT cells within the siIEL CD8+CD44+CD45.2+ population. Figure 1D shows contour plots and quantification of CD69 versus CD103 Bcl11b-/- (KO) and WT within the siIEL CD8+CD44+CD45.2+ population. Figure 1E shows contour plots and quantification of splenic Bcl11b-/- and WT cells 30 and 90 DPI. Figure 1F shows contour plots and quantification of Bcl11b-/- and WT cells within the splenic TCM (CD62L+) and TEM (CD62L-) compartments at 30 DPI. Figure 1G shows contour plots and quantification of Bcl11b-/- and WT cells in the siIEL of co-transferred mice at 30 and 90 DPI. Figure 1H shows percentages of Bcl11b-/- and WT CD69+CD103+, CD69+CD103- and CD69- CD103- subsets within the siIEL CD8+CD44+CD45.2+ cells at 30 DPI. (I-K) Separate transfers. Figures 1I and 1J show contour plots and quantification of splenic (1I) and siIEL (1J) Bcl11b-/- and WT CD8+ T cells at 30 DPI. P values by Mann-Whitney-Wilcoxon test. Figure 1K shows the percentages of Bcl11b-/- and WT CD69+CD103+, CD69+CD103- and CD69-CD103- subsets within siIEL CD8+CD44+CD45.2+ cells at 30 DPI. In all graphs data are mean ± SEM with n=3- 14. Each mouse is represented by an individual point. P values by two- tailed paired (co-transfer) or unpaired (separate transfer) Student’s t-test. Representative of 2-3 independent experiments. 13. Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I show Bcl11b-/- memory cells have an impaired recall response to oral Lm-Ova InlAM infection. Figures 2A-2F show separate transfers. Figure 2A shows Lm-Ova InlAM CFU on Day 3 post-reinfection in the liver. Figures 2B and 2C show contour plots and quantification of transferred cells in spleen and siIEL post reinfection (Day 3). Figure 2D shows contour plots and quantification of Bcl11b-/- and WT CD8+CD44+ siIEL cells based on CD69 and CD103. Figure 2E shows Ifnγ and Tnfα following reinfection. Figure 2F shows the percentages and numbers of recipient CD45.1+CD45.2-NK1.1+ in the siIEL following reinfection. Figure 2G shows contour plots and quantification of CD45.2 (KO) and CD45.1/2 (WT) cells in spleen and siIEL in co-transferred experiments post reinfection. Figure 2H shows liver Lm-Ova InlAM CFU on Day 3 post-reinfection in a sex mismatched transfer. Figure 2I shows contour plots and quantification of CD45.2+ (Bcl11b-/- or WT) in sex mismatched transfer. In all graphs data are mean ± SEM with n=4-16. Each individual point represents one mouse. P values by two-tailed paired (co-transfer) or unpaired (separate transfer) Student’s t-test. Representative of 2-3 independent experiments. 14. Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, and 3M show loss of Bcl11b in the effector and memory phases of oral Lm-Ova InlAM infection has a major impact on TRM program. Figure 3A shows a histogram and quantification of Bcl11b Mean Fluorescence Attorney Docket Number 10110-443WO1 Intensity (MFI) in WT siIEL TRMPs, TEs and MPs 9 DPI. Figure 3B shows PCA of RNA-seq data on all high fold-changed genes. Figure 3C shows hierarchical clustering on Euclidean distances of rlog normalized values of RNA-seq data derived from three independent samples. Figure 3D shows differentially expressed gene numbers in TEs, MPs and T RMPs . Genes increasing and decreasing in expression in Bcl11b -/- (KO) are indicated in red and blue, respectively. Figure 3E shows a heatmap clustering of genes differentially expressed in T RMPs, TEs, MPs. Heatmap shows relative log2 fold change. Select genes of the multipotent/multifunctional (MP/MF) and effector TRM programs are indicated. Figure 3F shows gene set enrichment analysis (GSEA) of the MP/MF and effector TRM programs in Bcl11b-/- (KO) and WT TRMPs. Figure 3G shows GSEA of a Tcf1 gene signature of lung TRMs in Bcl11b-/- and WT TRMPs. Figure 3H shows GSEA of an Ahr gene signature in intestinal of ILC2s in Bcl11b-/- (KO) and WT TRMPs. Figure 3I shows Tcf1, Blimp1 and Ahr histograms and MFIs in siIEL T RMPs 9 DPI. Negative controls are endogenous splenic CD44-CD8 + T cells or isotype controls. Each individual point represents one mouse. Figure 3J shows GSEA of the MP/MF and effector T RM programs in Bcl11b-/- and WT siIEL T RM cells at 30 DPI. Figure 3K shows volcano plot showing genes with upregulated (red) or downregulated (blue) expression in Bcl11b-/- versus WT siIEL TRM cells 30 DPI. Horizontal line represents P value level 0.05 as a threshold of significance. Eight outliers are not shown. Selected genes are indicated. Figure 3L shows GSEA of the MP/MF and effector TRM programs in siIEL recall Bcl11b-/- (KO) and WT cells 2 days after reinfection. Figure 3M shows volcano plot showing genes with upregulated (red) or downregulated (blue) expression in Bcl11b-/- versus WT siIEL recall CD8+ T cells 2 days after reinfection. Fifteen outliers are not shown. Selected genes are indicated. In 3A, 3I, data are mean ± SEM with n=4-8; P values by two-tailed paired Student’s t-test. In 3F and 3L “*” indicates moderately differentially expressed genes (p < 0.05) after batch correction. Representative of 2-3 independent experiments. In K and M, DESeq2 P values are shown. 15. Figures 4A, 4B, 4C, 4D, 4E, 4F, and 4G show the impact of Bcl11b deletion on chromatin accessibility, H3K27ac and H3K4me3 genome wide. Figure 4A shows the number of differentially accessible regions (P < 0.05) and H3K27ac ChIP-seq peaks with decreased (blue) or increased (red) signal in Bcl11b-/- TRMPs 9 DPI and TRM-like cells, respectively. Figure 4B shows LEFT: Peaks with increased/decreased chromatin accessibility in Bcl11b-/- cells predominantly associated with peaks with increased/decreased (green/orange) H3K27ac signal. MIDDLE: Correlation of ATAC-seq and H3K27ac ChIP-seq peaks, differential in Bcl11b-/- cells versus WT. RIGHT: Peaks with increased/decreased chromatin accessibility and H3K27ac in Attorney Docket Number 10110-443WO1 Bcl11b-/- cells were predominantly associated with TSS of genes (±10 kb) with decreased/increased (green/orange) expression in Bcl11b-/- cells. Filtered peaks were used for the analysis (P < 0.05; |log2 fold change|>1). P values by the chi-square test of independence in R. Figure 4C shows the correlation of differential ATAC-seq and H3K27ac ChIP-seq signal in relation to Bcl11b binding (shape) and changes in gene expression (color). Figure 4D shows the proportion of ATAC-seq, H3K27ac- and Bcl11b ChIP- seq peaks annotated to different genomic elements. Figure 4E shows a heat map for H3K4me3 CUT&RUN signal visualized for TSS (±3 kb) of genes with Bcl11b binding at their promoter (TSS -1000 bp to +200 bp). Figure 5F shwos the profile plot for data displayed in 4E. Figure 4G shows enrichment of motifs at differential ATAC-seq and H3K27ac peaks. Color gradient represents row-scaled relative enrichment (z- score) based on the motif ln(P) value calculated by Homer. 16. Figures 5A, 5B, 5C, 5D, 5E, 5F, and 5G show that Bcl11b binds at Tcf7 to control its expression and siIEL TRMP cell distribution. Figure 5A shows genomic tracks at Tcf7 locus from Bcl11b ChIP-seq analysis in WT murine TRM-like cells; Tcf1 CUT&RUN, H3K27ac ChIP-seq, H3K4me3 CUT&RUN in Bcl11b-/- and WT murine TRM-like cells and ATAC-seq in Bcl11b-/- and WT murine siIEL TRMPs. Figure 5B shows BCL11B ChIP-seq in human memory CD3+CD8+CD45RA+CD57+CCR7- T cells at the TCF7 locus. Data are from two donors. For (5A-5B), the rectangles below Bcl11b tracks represent reproducible (based on IDR) ChIP-seq peaks. The rectangles below all other tracks represent significantly differential peaks between WT and Bcl11b-/- cells. Figure 5C shows a heat map for Tcf1 CUT&RUN signal visualized for TSS (±3 kb) with or without Bcl11b binding at gene promoter (TSS -1000 bp to +200 bp); further categorized for genes with decreased or increased expression in Bcl11b-/- cells. Figure 5D shows a heat map for Tcf1 CUT&RUN signal visualized for Bcl11b peaks (scaled to 1000 bp, ±3 kb), associated with regions with either significantly increased or decreased chromatin accessibility (P < 0.05). Figure 5E shows representative histograms (left) and quantification (right) of Tcf1 expression in the siIEL for indicated groups. Figures 5F and 5G show quantification of EV or Tcf7-transduced (GFP+) Bcl11b-/- or WT CD44+ CD8+ T cells in siIEL (5F) or spleen (5G). P values by paired Student’s t-test. Data are mean ± SEM with n=5-7. Representative of 3 independent experiments. 17. Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J, and 6K show that Bcl11b binds at Prdm1 and Ahr loci to control their expression and siIEL TRMP and TRM cell distribution. Figures 6A and 6C show genomic tracks at Prdm1 and Ahr loci for Bcl11b ChIP- seq in WT murine TRM-like cells; H3K27ac ChIP-seq and H3K4me3 CUT&RUN in Bcl11b-/- and WT murine TRM-like cells and ATAC-seq in Bcl11b-/- and WT murine siIEL TRMPs. Figures 6B Attorney Docket Number 10110-443WO1 and 6D show BCL11B ChIP-seq in human memory CD3+CD8+CD45RA+CD57+CCR7- cells at the PRDM1 and AHR loci. Data is from two donors. For (6A-6D), the rectangles below Bcl11b tracks represent reproducible (based on IDR) ChIP-seq peaks. The rectangles below all other tracks represent significantly differential peaks between WT and Bcl11b-/- cells. Figure 6E shows a heat map for Ahr ChIP-seq signal visualized for TSS (±3 kb) of genes with or without Bcl11b binding at their promoter (TSS -1000 bp to +200 bp); further categorized for genes with decreased or increased expression in Bcl11b-/- cells. Figure 6F shows a heat map for Ahr ChIP- seq signal visualized for Bcl11b peaks (scaled to 1000 bp, ±3 kb), associated with regions with either significantly increased or decreased chromatin accessibility (P < 0.05). Figures 6G and 6H show quantification of transferred Bcl11b-/-/WT ratios siIEL CD69+CD103+ cells (6G) or splenic cells (6H) in mice on regular diet (7912) or Ahr-ligand deficient diet (AIN76a). Figure 6I shows the percentages and numbers of CD45.1+ CD8+ T cells of recipient mice in the indicated groups. Figure 6J shows Ahr MFIs in transferred Bcl11b-/- and WT siIEL CD69+CD103+ cells in the indicated groups. Figure 6K shows the percentages of CD69+CD103+ Bcl11b-/- and WT TRM- like CD8+ T cells transduced with CRISPR gAhr alone or gAhr+gPrdm1. Data are mean ± SEM with n=3-7. Representative of 2-3 independent experiments. P values by two-tailed unpaired Student’s t-test; in 6K adjusted for multiple comparisons by the FDR method. 18. Figures 7A, 7B, 7C, 7D, 7E, and 7F show that Bcl11b represses genes of the NK and myeloid programs across memory and effector CD8+ T cells. Figure 7A shows a heat map of NK receptor and effector genes, and myeloid genes upregulated in Bcl11b-/- Day 9 TEs, MPs and siIEL TRMPs, Day 30 siIEL TRMs and recall siIEL CD8+ T cells 2 days post-infection. Heatmap shows relative log2 expression fold change. Figures 7B, 7C, and 7D show genomic tracks at NK cell receptor, Itgam, Itgax and Fcer1g loci from Bcl11b ChIP-seq analysis in WT murine TRM-like cells, H3K27ac ChIP-seq and H3K4me3 CUT&RUN in Bcl11b-/- and WT TRM-like cells and ATAC-seq in Bcl11b-/- and WT siIEL TRMPs. H3K27ac ChIP-seq for the NK receptor locus from. Figures 7E and 7F show BCL11B ChIP-seq in human memory CD3+CD8+CD45RA+CD57+CCR7- T cells at the NK cell receptor and ITGAM and ITGAX loci. Data is from two donors. 19. Figures 8A, 8B, and 8C show depletion of BCL11B in human memory CD8+ T cells or TRM-like cells causes changes in expression of markers of the TRM and NK program. Figure 8A shows quantification of Bcl11b knockout efficiency and representative histograms. Figures 8B and 8C show flow cytometry plots and quantification of molecules associated with residency program (8B) and NK receptors (8C) in WT and BCL11B-/- TRM-like cells following Attorney Docket Number 10110-443WO1 CRISPR/Cas9 mediated knockout with the indicated guides. Points represent individual donors. P values by two-way ANOVA followed by Tukey's test. 20. Figures 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, and 9I show characteristics of CD8+ T cells derived from Bcl11bf/fGzmbCre+R26REYFP+ OT- I and WT OT-I mice after infection with Lm-Ova InlAM. CD8+ T cells from Bcl11bf/fGzmbCre+R26REYFP+OT-I CD45.2 and Bcl11bf/fOT-I CD45.1/2 mice were co- transferred into CD45.1 mice. Recipient mice were infected orally the following day with Lm-Ova InlAM. Figures 9A, 9B, and 9C show mLNs were evaluated at 3 DPI. Figure 9A shows histograms of YFP in transferred CD45.2 and CD45.1/2 CD8+ T cells. Figure 9B shows contour plots and quantification of transferred CD45.2 and CD45.1/2 CD8+ T cells. Figure 9C shows histograms and quantification of CD69 in transferred CD45.2 and CD45.1/2 CD8+ T cells. Figures 9D, 9E, 9F, 9G, and 9H show siIEL and splenic transferred cells were evaluated at 9 DPI. Figure 9D shows histograms of YFP in transferred CD45.2 and CD45.1/2 siIEL TRMP (CD69+CD103+) and splenic MPs (CD127 + Klrg1-) and TEs (Klrg1+CD127+) CD45.2+CD8+ at 9 DPI. Figures 9E and 9F shows that on Day 8 post-infection, transferred mice as above, were injected i.p. with EdU, and further euthanized next day when EdU incorporation in Bcl11b-/- (CD45.2) and WT (CD45.1/2) transferred CD8+ T cells was evaluated in the spleen (9E) or siIEL (9F). Figure 9G shows a histogram and quantification of AnnexinV in Bcl11b-/- and WT siIEL TRMPs 9 DPI. Figure 9H shows post-sort purity of populations for RNA-seq at 9 DPI. siIEL TRMPs (CD69+CD103+), and splenic MPs (CD127+Klrg1-) and TEs (Klrg1+CD127-) were obtained from transferred mice at 9 DPI as described above and sorted for the indicated markers. Figure 9I show histograms of YFP in transferred CD45.2 and CD45.1/2 in siIEL and spleen at 30 DPI. Data are mean ± SEM with n=4-7. Representative of 2-3 independent experiments. Each individual point represents one mouse. P values by paired Student’s t-test. 21. Figures 10A and 10B show antigen-specific Bcl11b-/- CD8+ T cells are reduced in the spleen and elevated in siIEL in a polyclonal response. Bcl11bf/fGzmbCre+ and WT mice were orally infected with Lm- Ova InlAM. Spleen and siIEL H2Kb-SIINFEKL (SEQ ID NO: 3) tetramer+ CD8+CD44+ cells were evaluated 21 days post infection. Representative contour plots (10A) and quantification (10B) of H2Kb-SIINFEKL (SEQ ID NO: 3) tetramer+ CD8+CD44+ cells. Data are mean ± SEM with n=3-6. Each individual point represents one mouse. P values by unpaired Student’s t-test assuming unequal variance. 22. Figures 11A, 11B, and 11C show Bcl11b ablation post-infection has little impact on MP and TE transcription factors. Figures 11A, 11B and 11C show DESeq2 Normalized Attorney Docket Number 10110-443WO1 counts for the indicated genes derived from RNA-seq analysis in transferred Bcl11b-/- and WT splenic TEs (11A) MPs (11B), and MPs, TEs and siIEL TRMPs (11C) at 9 DPI following Lm- Ova InlAM infection. Data are mean ± SEM with n=3 (RNA- seq). FDR adjusted P values by DESeq2 are shown. 23. Figures 12A, 12B, 12C, and 12D show that Bcl11b regulates gut homing early in differentiation of memory cells following oral Lm-Ova InlAM infection. CD8+ T cells from Bcl11bf/fGzmbCre+R26REYFP+OT-I CD45.2 and Bcl11bf/fOT-I CD45.1/2 mice were co- transferred into recipient mice which were infected orally the following day with Lm-Ova InlAM and mice were euthanized at 9 DPI. Figure 12A shows DESeq2 normalized counts of Itga4, Itgb7 and Ccr9 in Bcl11b-/- and WT MPs and TEs. FDR adjusted P values by DESeq2 are shown. Figures 12B and 12C show representative histograms and quantification of α4β7 (12B) and Ccr9 (12C) in splenic Bcl11b-/- and WT MPs and TEs. Figure 12D shows α4β7 blockade in infected mice. Mice were adoptively transferred, infected as above and injected with either IgG or anti- α4β7 (Datk32) antibodies 5-8 DPI. Ratio of transferred Bcl11b-/-/WT in spleen (left) and siIEL (middle) and percentages of total transferred cells in siIEL (right) following the blockade. Data are mean ± SEM with n=3 (RNA-seq) or 3-6 (FACS). Representative of 2 independent experiments. P values by paired (12B and 12C) or unpaired Student’s t-test assuming unequal variance (12D). 24. Figures 13A, 13B, and 13C show differential gene expression analysis of TRM- like cells differentiated ex vivo and comparison with siIEL TRMPs and TRMs. Figure 13A shows a volcano plot showing genes with upregulated (red) or downregulated (blue) expression in Bcl11b-/- TRM-like cells versus WT. Genes associated with the MP/MF or effector TRM programs are depicted. Figures 13B and 13C show differentially expressed genes in Bcl11b-/- TRM-like cells versus WT, examined for enrichment in TRMPs on 9 DPI (13B) and in TRMs on 30 DPI (13C) by GSEA. Core genes upregulated or downregulated are highlighted with a red or blue square, respectively. 25. Figures 14A, 14B, 14C, and 14D show genome wide integrative analysis of Bcl11b ChIP-seq, H3K27ac ChIP-seq and ATAC-seq. Figure 14A shows a panel related to Fig. 4B, showing all significant peaks (P < 0.05; no fold change filtering). LEFT: Peaks with increased/decreased chromatin accessibility in Bcl11b-/- cells were predominantly associated with peaks with increased/decreased H3K27ac signal. MIDDLE: Correlation of ATAC-seq and H3K27ac ChIP-seq peaks which were differential in Bcl11b-/- cells. RIGHT: Peaks with increased/decreased chromatin accessibility and H3K27ac in Bcl11b-/- cells predominantly associated with TSS of genes (±10 kb) with decreased/increased expression. P values by the chi- Attorney Docket Number 10110-443WO1 square test of independence in R. Figure 14B shows a Venn diagram showing overlap betweendifferentially expressed genes (Padj. < 0.05) and gene-annotated peaks for ATAC-seq and Bcl11b- and H3K27ac ChIP-seq (localized within TSS ±10 kb). Figure 14C shows a panel related to Fig.4G. Enrichment of motifs at differential ATAC-seq and H3K27ac peaks, categorized based on Bcl11b binding. Selected motifs which were significant (P < 0.001) in at least one dataset are depicted, including their motif family annotation. Color gradient represents row-scaled relative enrichment (z-score) based on the motif ln(P) value calculated by Homer. Figure 14D shows motifs enriched in Bcl11b ChIP-seq peaks. Representative motifs from the most abundant motif families are depicted, alongside with motifs for factors associated with the effector and MP/MF TRM programs (Prdm1, Tcf1, Lef1 and Ahr). 26. Figures 15A, 15B, 15C, 15D, and 15E show Bcl11b binding, H3K27ac, H3K4me3 and chromatin accessibility at genes of the MP/MF program in T RM -like cells and TRMPs. Figures 15A, 15C, 15D, and 15E show genomic tracks at Ccr7 (15A), Id3 (15C), Klf2 (15D) and S1pr1 (15E) loci from Bcl11b ChIP-seq analysis in WT murine TRM-like cells, Tcf1 CUT&RUN, H3K27ac ChIP-seq and H3K4me3 CUT&RUN analyses in Bcl11b-/- and WT murine TRM-like cells and ATAC-seq analysis in Bcl11b-/- and WT murine siIEL TRMPs. Figure 15B shows genomic tracks at the CCR7 locus from BCL11B ChIP-seq analysis in human memory CD8+ T cells. Data is from two donors. 27. Figures 16A, 16B, 16C, 16D, 16E, and 16F show Bcl11b binding, H3K27ac, H3K4me3 and chromatin accessibility at additional genes of the MP/MF program in TRM-like cells and T RMPs . Genomic tracks at Lef1 (16A), Cd5 (16B), Btla (16C), Ifng (16D) Tnf (16E) and Cd28 (16F) loci from Bcl11b ChIP-seq analysis in WT murine TRM-like cells, Tcf1 CUT&RUN, H3K27ac ChIP-seq and H3K4me3 CUT&RUN analyses in Bcl11b-/- and WT murine TRM-like cells and ATAC-seq analysis in Bcl11b-/- and WT murine siIEL TRMPs. 28. Figures 17A, 17B, 17C, 17D, 17E, 17F, 17G, and 17H show Bcl11b binding, H3K27ac, H3K4me3 and chromatin accessibility at genes of effector program and Ahr-bound genes in T RM -like cells and T RMPs . Genomic tracks at Gzma(17A), Gzmc (17B), Prf1 (17C), Havcr2 (17D) Entpd1 (17E), Ahrr (17F), Il2rb (17G) and Aldh7a1 (17H) loci from Bcl11b ChIP-seq analysis in WT murine TRM-like cells, H3K27ac ChIP-seq and H3K4me3 CUT&RUN analyses in Bcl11b-/- and WT murine TRM-like cells and ATAC-seq analysis in Bcl11b-/- and WT murine siIEL TRMPs. (F-H) Ahrr, Il2rb and Aldh7a1 loci have also tracks from Ahr ChIP- seq. 29. Figure 18A shows that Bcl11b KO TILs display enhanced anti-melanoma activity in adoptive cell therapy. Attorney Docket Number 10110-443WO1 30. Figures 18B and 18C show that Bcl11b KO TILs accumulate in the tumor through elevated CD69. 31. Figure 18D show that Bcl11b KO TILs have increased Gzmb. 32. Figure 19A shows human melanoma CD8+ TILs depleted in BCL11B upregulate the essential NK receptor CD56, but leave intact CD3 and CD8. 33. Figure 19B shows BCL11B depletion in TILs with double guides. 34. Figure 19C shows BCL11B depletion in TILs with double guides – upregulation of CD56 but intact CD8. 35. Figure 20 shows BCL11B depletion in human TILs increases cytotoxicity in a manner dependent on MHC I. 36. Figure 21 shows Bcl11b-deficient CD8+ T cells confer elevated anti-tumor activity in ovarian tumors implanted intraperitoneally. 37. Figures 22A, 22B, 22C, and 22D show BCL11B-deficient TILs have elevated cytotoxic genes, NK receptor and stemness program genes, while inhibitory receptor genes remain unchanged. Figure 22A shows expression of stemness program genes in CD8+ TILs. Figure 22B shows the expression of genes associated with dysfunction/exhaustion in CD8+ TILs. Figure 22C shows the expression of genes for NK receptors and cytotoxicity in CD8+ TILs. Figure 22D shows a plot of the gene expression of all genes in BCL11B-deficient TILs. 38. Figures 23A, 23B, 23C, and 23D show expression of gene subsets in various clusters of TILs in WT and Bcl11b-/- (knockout) mice with peritoneal tumors. Figure 23A shows a scRNA-seq UMAP plot of TILs following adoptive transfer. Figures 23B and 23C that the expression of Stemness genes (23B) and Gzmc and Gzmf genes (23C) are spread across clusters in BCL11B KO TILs. Figure 23D shows the expression of exhaustion genes is decreased in Ttex clusters in BCL11B KO TILs. 39. Figurer 24A, 24B, 24C, 24D, and 24E show that GzmbHI (Granzyme B hi) CD8+ TILs from Bcl11b-/- mice continue to express stemness transcription factor TCF1 (24A and 24B), expand more (24C), have reduced levels of the exhaustion transcription factor Tox, as well as, exhaustion markers CD39, PD1, and Tim3 (24D), and have increased metabolic potential and mass and thus greater fitness (24E). 40. Figures 25 shows the experimental model for Bcl11b-/- (knockout) CD4+ TILs. 41. Figures 26A and 26B shows that Bcl11b-/- (KO) CD4+ TILs display enhanced anti-tumor activity in adoptive cell therapy (26A) and result in tumor eradication (26B) in ovarian tumor models. Attorney Docket Number 10110-443WO1 42. Figure 27 shows that Bcl11b-deficient CD4+ T cells confer elevated anti-tumor activity in ovarian and colon cancer tumors implanted intraperitoneally. 43. Figures 28A, 28B, 28C, and 28D show human melanoma specific CD8+ TILs that have been depleted for Bcl11b have elevatored perforin together with stemness TF TCF1, as well as the NK receptor CD56, but do not downregulate CD3 and CD8, thus maintaining CD8 TIL identity. Figure 28A and 28B shows a brightfield image (28A) and fluorescent staining (28B) of tumor cells, TILs, and killed cancer cells. Figure 28C shows that the number of dead tumor cells is greatly increased in Bcl11b depleted TILs. Figure 28D shows a comparison of various markers between WT and Bcl11b depleted TILs. Bcl11b was depleted using CRISPR/cas9 targeting Bcl11b using the guide Hs.Cas9.Bcl11b.AA (SEQ ID NO: 1) in combination with Hs.Cas9.Bcl11b.AE (SEQ ID NO: 2). IV. DETAILED DESCRIPTION 44. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. A. Definitions 45. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like. 46. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or Attorney Docket Number 10110-443WO1 equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. 47. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: 48. “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. 49. An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant. 50. A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant. 51. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the Attorney Docket Number 10110-443WO1 native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. 52. By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. 53. By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. 54. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. 55. The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. 56. The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and Attorney Docket Number 10110-443WO1 supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. 57. "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject. 58. "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of'' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of'' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. 59. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." 60. “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. 61. A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a Attorney Docket Number 10110-443WO1 deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration. 62. "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein. 63. “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. 64. “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. 65. “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the Attorney Docket Number 10110-443WO1 age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. 66. “Primers” are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation. 67. “Probes” are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. 68. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. B. Method of treating cancer or infectious disease 69. CD8+ T cells are critical in the immune response to intracellular pathogens and tumors and provide long-lasting protection from reinfection. Following infection, antigen- specific CD8+ T cells undergo robust clonal expansion and differentiate into Klrg1hiCD127lo terminal effector cells (TEs) which die after pathogen clearance, and CD127hiKlrg1lo memory precursor cells (MPs), which have potential to form long-lived memory cells. Memory CD8+ T cells are heterogeneous and can be divided into circulating memory and tissue resident memory cells. Of the circulating subsets, T central memory (TCM) cells survey lymphoid tissue for antigen, and T effector memory (TEM) cells predominantly survey the blood. In contrast, TRM cells are seeded in non-lymphoid tissues early during infection and do not exit into the circulation, except during reinfection. TRM cells provide an important first line of defense upon Attorney Docket Number 10110-443WO1 antigen reencounter due to their localization at common sites of infection including at key barrier surfaces, ability to directly kill infected cells and rapidly induce an innate-like immune response, proliferate, and trigger the recruitment of circulating memory and other immune cells to the site of reinfection. TRM cells have been shown to provide protection against numerous pathogens, including herpes simplex, vaccinia and influenza viruses, as well as Listeria monocytogenes (Lm). However, in tumors and some chronic infections, exhausted T cells are generated, which are inefficient in controlling the anti-tumor immune response. 70. Commitment to memory CD8+ T cell subsets occurs early during infection. Multiple transcription factors (TFs) coordinate MP versus TE cell differentiation and regulate circulating versus resident memory cell fates. TFs critical for MP differentiation include Eomes, Stat3, Tcf1, Bcl6 and Id3, while those driving TE differentiation include Runx3, Zeb2, Blimp1, Id2 and Tbet. Runx3, Hobit and Blimp1 initiate the TRM program in the gut and skin. Runx3 controls expression of multiple components of the residency program, including Itgae (CD103) and CD69, to promote tissue retention. Hobit and Blimp1 dampen expression of Klf2, and its target S1pr1, to ultimately block tissue egress. Hobit and Blimp1 also repress expression of Tcf7 (encoding Tcf1) and Ccr7, recently found to promote tissue egress, whose expression is controlled by Tcf1. Recent evidence identified that Tcf7 is expressed in a subset of small intestine (SI) TRM cells, with high expression of Id3 and low expression of Blimp1, which have pronounced memory potential and increased polyfunctionality, expressing Ifnγ and Tnfα. A more terminally differentiated Blimp1hiId3lo effector-like subset was also identified, which in addition to Prdm1, expresses mRNAs for Id2, Tbet, Granzymes, Prf1 and Klrg1. During the late memory phase, the Id3hi subset also expresses Il7ra and Cd28, as well as elevated Ifng, Tnf and Klf2 transcripts. This is likely related to the multipotency and ability of TRM cells not only to expand and generate secondary TRM cells following reinfection, but to egress from tissue and form secondary circulating effector and memory cells. An additional TF, aryl hydrocarbon receptor (Ahr), has also been implicated in intestinal CD8+ TRM differentiation, in maintaining CD8+ T cells in the intraepithelial lymphocytes (IEL) compartment at steady state, as well as in long-term in skin TRM cells. 71. The transcription factor Bcl11b acts both as a transcriptional repressor and as a transcriptional activator. It has essential roles in thymocyte development at multiple stages and also controls mature T cell differentiation and function. Deletion of Bcl11b in Th17 cells leads to de-repression of the Th2 program, whilst in Th2 cells absence of Bcl11b impairs their differentiation and the response to asthma and helminth infection. Furthermore, Bcl11b deficient regulatory T (Treg) cells fail to maintain their identity and function. Bcl11b is essential for Attorney Docket Number 10110-443WO1 development of ILC2s and maintenance of their identity, and in NK cells is required for both canonical and adaptive differentiation. In mucosal-associated invariant T (MAIT) cells, Bcl11b sustains the MAIT17 program, while in iNKT cells, it represses the iNKT17 program and sustains the iNKT1 and iNKT2 cell programs. Removal of Bcl11b in naïve CD8+ T cells leads to a failure in antigen-specific clonal expansion and effector function following infection. 72. In this study we investigated the role of Bcl11b in intestinal TRM cells and circulating memory CD8+ T cells after oral infection with Listeria monocytogenes (Lm) or cancer challenge. We found that absence of Bcl11b results in increased gut TRM cells and their precursors. Major alterations were found in the gut TRM cell and their precursor programs, with reduction in the MP/MF gene signature and upregulation of the effector gene signature. We found that Bcl11b levels inversely correlated with effector residency program gene expression, and positively with multipotency gene expression. Thus, our results show an essential role of Bcl11b in controlling MP/MF versus effector residency programs through regulation of program essential genes. 73. Accordingly, it is understood and herein contemplated that reduction of Bcl11b can have beneficial effects in restoring, recovering, or rescuing T cell responses that have been functionally exhausted as well as increasing the cytotoxicity of T cells at the site of infection or in a tumor microenvironment. That is, the T cells are responsive and able to exercise their full assortment of cytotoxic activity including, but not limited to the secretion of granzyme B, perforin, and cytokines (such as, for example, IFN-γ and TNF-α). 74. In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a or condition or a recurrent disease or condition (such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease (including, but not limited to diseases or conditions with T cell mediated immune responses)) in a subject comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b); disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by CRISPR/cas9); or by administering to the subject T cells that have reduced Bcl11b activity. In some aspects the T cells comprise tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (MILs), chimeric antigen receptor (CAR) T cells, CD4 T cells, CD8 T cells, effector memory T cells (TEM), central memory T cells (TCM), peripheral memory T cells (TPM), or tissue-resident memory T cells (TRM). It is understood and herein contemplated that a decrease, reduction, ablation, or disruption of Bcl11b activity or Attorney Docket Number 10110-443WO1 transcription or expression increases the cytotoxicity of disease or condition specific effector T cells (including, but not limited to the increased expression of perforin, granzyme b and effector cytokines, such as, IFN-γ and TNF-α) at the site of infection or tumor microenvironment (such as, for example, tumor infiltrating lymphocytes (TILs) and/or can reduce exhaustion of disease or condition specific effector T cells (including, but not limited to tissue resident memory T cells (TRM) or T cells that have been become exhausted or turned off (such as, for example, chronically stimulated T cells and T cells that have been inhibiting by an immune checkpoint such as PD1, TIM3, LAG-3, and/or CTLA-4). Such rescued cells can exhibit a reduction in exhaustion hallmarks (such as, for example, a reduction in the exhaustion transcription factor Tox, as well as, exhaustion markers CD39, PD1, and Tim3). In some aspects, it is understood that the disease or condition is a T cell mediated disease or condition. By “T cell mediated” it is meant that T cells play a significant role in the immune response to disease or condition. 75. Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a disease or condition or a recurrent disease or condition, further comprising obtaining T cells from a donor source and disrupting Bcl11b expression in the T cells prior to administering the T cells (including chimeric antigen receptor (CAR) T cells and other adoptively transferred to cells such as tumor infiltrating lymphocytes (TILs) to the subject. 76. In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a disease or condition or a recurrent disease or condition (including, but not limited to T cell mediated diseases or conditions) or increasing the cytotoxicity of T cells specific for said disease or condition, wherein the disease is an infectious disease caused by a viral infection (such as, for example, a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein- Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to the B1.351 variant, B.1.1.7 variant, and P.1 variant), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow Attorney Docket Number 10110-443WO1 fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2); a bacterial infection (such as, for example, an infection with a bacteria selected from the group consisting of Bacillus anthracis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Mycobacterium chimaera, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species, and Mycoplasma species); a fungal infection (such as, for example, an infection with a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata); or a a parasitic infection (such as, for example, an infection with a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma Attorney Docket Number 10110-443WO1 duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica). 77. Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a T cell mediated disease or condition or a recurrent disease or condition, further comprising administering to the subject an agent that blocks MHC class 1 expression. 78. The disclosed compositions and methods can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin’s Disease; myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and/or chronic myeloid leukemia (CML)); bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer; lung cancers such as small cell lung cancer, non-small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma/glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer including, but not limited to triple negative breast cancer; genitourinary cancer; pulmonary cancer; esophageal carcinoma; head and neck carcinoma; large bowel cancer; hematopoietic cancers; testicular cancer; and colon and rectal cancers. In one aspect, the treatment of the cancer can include ovarian cancer, colon cancer, or melanoma. For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing, and/or preventing a T cell mediated disease or condition or a recurrent disease or condition (such as, for example, a cancer (such as, for example, a primary or recurrent cancer including, but not limited to ovarian cancer, colon cancer, or melanoma) or an infectious disease) in a subject comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells (such as, for example a small molecule, antibody, nanobody, diabody, antibody fragment, antisense Attorney Docket Number 10110-443WO1 oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA that targets Bcl11b); disrupting Bcl11b transcription or expression (including targeted disruption of Bcl11b by CRISPR/cas9); or by administering to the subject T cells that have reduced Bcl11b activity. In some aspects the T cells comprise tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (MILs), chimeric antigen receptor (CAR) T cells, CD4 T cells, CD8 T cells, effector memory T cells (TEM), central memory T cells (TCM), peripheral memory T cells (TPM), or tissue-resident memory T cells (TRM). 79..It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC- T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil--Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, Attorney Docket Number 10110-443WO1 CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S- Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR- U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil--Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EVACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil--Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil- -Topical), Fluorouracil Injection, Fluorouracil--Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI- CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Attorney Docket Number 10110-443WO1 Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado-Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® Attorney Docket Number 10110-443WO1 (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R- EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R-ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Attorney Docket Number 10110-443WO1 Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil--Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VeIP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and/or ZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT-011, MK- 3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS- 936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA- 4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS-986207, OMP- 313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T- lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, Attorney Docket Number 10110-443WO1 for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep). 80. In one aspect, disclosed herein are methods of increasing resident memory T cells at the site of infection or in a tumor microenvironment comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity. 81. Also disclosed herein are methods of increasing of increasing NK cell activation in a tumor microenvironment or site of infection comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity. C. Compositions 82. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular inhibitor of Bcl11b is disclosed and discussed and a number of modifications that can be made to a number of molecules including the inhibitor of Bcl11b are discussed, specifically contemplated is each and every combination and permutation of inhibitor of Bcl11b and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be Attorney Docket Number 10110-443WO1 performed with any specific embodiment or combination of embodiments of the disclosed methods. 1. Antibodies (1) Antibodies Generally 83. The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with Bcl11b. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. 84. The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity. 85. The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. Attorney Docket Number 10110-443WO1 86. The monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No.5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al. 87. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec.22, 1994 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen. 88. As used herein, the term “antibody or fragments thereof” encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, sFv, scFv, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain Bcl11b binding activity are included within the meaning of the term “antibody or fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)). 89. Also included within the meaning of “antibody or fragments thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies). 90. The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive Attorney Docket Number 10110-443WO1 property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol.3:348-354, 1992). 91. As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response. (2) Human antibodies 92. The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein. (3) Humanized antibodies 93. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody. 94. To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues Attorney Docket Number 10110-443WO1 from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)). 95. Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No.4,816,567 (Cabilly et al.), U.S. Patent No.5,565,332 (Hoogenboom et al.), U.S. Patent No.5,721,367 (Kay et al.), U.S. Patent No.5,837,243 (Deo et al.), U.S. Patent No.5, 939,598 (Kucherlapati et al.), U.S. Patent No.6,130,364 (Jakobovits et al.), and U.S. Patent No.6,180,377 (Morgan et al.). (4) Administration of antibodies 96. Administration of the antibodies can be done as disclosed herein. Nucleic acid approaches for antibody delivery also exist. The broadly neutralizing anti Bcl11b antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment. The delivery of the nucleic acid can be by any means, as disclosed herein, for example. 2. Pharmaceutical carriers/Delivery of pharmaceutical products 97. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical Attorney Docket Number 10110-443WO1 composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. 98. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. 99. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No.3,610,795, which is incorporated by reference herein. 100. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting Attorney Docket Number 10110-443WO1 of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). a) Pharmaceutically Acceptable Carriers 101. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier. 102. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. 103. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art. Attorney Docket Number 10110-443WO1 104. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like. 105. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. 106. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. 107. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. 108. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.. 109. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. Attorney Docket Number 10110-443WO1 b) Therapeutic Uses 110. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch.22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365-389. A typical daily dosage of the antibody used alone might range from about 1 µg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above. D. Examples 111. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. 112. In this study we investigated the role of Bcl11b in intestinal TRM cells and circulating memory CD8+ T cells after oral infection with Listeria monocytogenes (Lm) or cancer challenge. We found that absence of Bcl11b results in increased gut T RM cells and their precursors, but reduced effector and circulating memory cells. Despite the reduction in circulating MPs and TEs, no major alterations in their transcriptional programs were identified, except upregulation of gut homing markers. However, major alterations were found in the gut T RM cell and their precursor programs, with reduction in the MP/MF gene signature and upregulation of the effector gene signature. Employing transcriptomics and genomics approaches Attorney Docket Number 10110-443WO1 and rescue experiments, we positioned Bcl11b upstream of essential TFs of the gut T RM cell programs. These investigations were also extended to humans, where we found that Bcl11b levels inversely correlated with effector residency program gene expression, and positively with multipotency gene expression. Thus, our results show an essential role of Bcl11b in controlling MP/MF versus effector residency programs through regulation of program essential genes. 1. Results a) Loss of Bcl11b following Lm-Ova infection results in reduction of circulating memory cells and accumulation of T RM cells in the siIEL 113. To investigate the role of Bcl11b in memory CD8+ T cells, we utilized adoptive transfers (separate or co-transfers) of naïve CD8+ T cells from Bcl11bf/fGzmbCre+R26REYFP+ OT-I and wild type (WT) OT-I mice. GzmbCre acts post-activation, thus allowing to bypass the previously observed defects in expansion of CD8+ T cells associated with Bcl11b removal prior to activation. Following transfer, recipient mice were infected with oral Lm-Ova InlAM, using a natural route of foodborne infection, which induces a robust memory CD8+ T cell response. 114. In the draining mesenteric lymph nodes (mLNs), during the priming phase (3 days post infection (DPI)), CD8+ T cells derived from Bcl11bf/fGzmbCre+R26REYFP+ OT-I (CD45.2) mice showed no expression of YFP reporter related to Gzmb-Cre activity (Fig.9A), and their percentages were within the same range to those derived from WT (CD45.1/2) mice (Fig. 9B). CD69 expression, indicative of activation at priming phase, was equivalent between the two groups (Fig.9C). Thus, we extended the analysis to the peak of the CD8+ T cell response in this experimental system, at 9 DPI. At 9 DPI, the transferred CD8+ T cells from Bcl11bf/fGzmbCre+R26REYFP+ OT-I mice were YFP+ both in the siIEL and spleen (Fig.9D). A four-fold decrease in the percentages of transferred Bcl11b-/- CD44+CD8+ T cells versus WT was observed in the spleen (Fig.1A), and no difference in the splenic CD127hiKlrg1lo (MPs) or Klrg1hiCD127lo (TEs) was observed. Rather, the absolute numbers of both populations were reduced, related to the overall reduction of Bcl11b- 115. /- CD44+CD8+ T cells (Fig.1A-B). In contrast, there was a significant increase of transferred siIEL Bcl11b-/- CD44+CD8+ T cells, including the TRM precursors (TRMPs), defined as CD69+CD103+ (Fig.1C-D), while the Bcl11b-/- CD69-CD103- cell percentages and numbers were markedly reduced (Fig.1D). Analysis of proliferation by EdU incorporation showed no significant difference between the two groups in the spleen or siIEL (Fig.9E-F). There was however a modest reduction in Annexin V staining in Bcl11b-/- siIEL TRMPs versus WT (Fig. 9G), indicating that decreased apoptosis rate can contribute at some extent to increased numbers of siIEL Bcl11b- /- CD8+ T cells. Attorney Docket Number 10110-443WO1 116. Evaluation of Bcl11b-/- and WT co-transferred cells in the memory phase, at 30 DPI, further showed a reduction in percentages and absolute numbers of splenic Bcl11b-/- CD8+ T cells, which occurred both in the CD62L+ TCM and CD62L- TEM cell subsets (Fig.1E-F). Similarly, percentages of splenic Bcl11b-/- CD8+ T cells were reduced at 30 DPI in a separate transfer system (Fig.1I). Conversely, total and CD69+CD103+ siIEL Bcl11b-/- CD8+ T cells remained elevated both in the co-transfer model, as well as in separate transfers (Fig.1G-H and J-K). CD69+CD103- cells (which at 30 DPI represent a very small fraction of the siIEL transferred CD8+ T cells) were reduced in the absence of Bcl11b in both transfer systems (Fig. 1H and 1K). Of note, transferred siIEL and splenic CD8+ T cells derived from Bcl11bf/fGzmbCre+ OT-I mice were mainly YFP+ at 30 DPI (Fig.9I). Further analysis of long- term memory cells at 90 DPI showed similar ratios of Bcl11b-/-/WT cells in the spleen and siIEL, as at 30 DPI (Fig.1E and G). In a polyclonal response, Bcl11bf/fGzmbCre+ mice showed defective accumulation of splenic antigen-specific, SIINFEKL (SEQ ID NO: 3) tetramer+ memory CD8+ T cells, and increased presence in the siIEL (Fig.10A-B). 117. Taken together, these results indicate that Bcl11b controls the balance of siIEL TRMs. and their precursors versus circulating splenic memory and effector cells. b) Bcl11b -/- memory CD8 + T have altered response to reinfection 118. We next evaluated Bcl11b-/- memory CD8+ T cell response to reinfection. To determine bacterial burden, recipient CD45.1 mice were separately transferred and infected as above, and then re-infected at 30 DPI. Mice harboring Bcl11b-/- OT-I cells had significantly elevated bacterial burden in the liver following reinfection compared to WT (Fig.2A). Percentages of Bcl11b-/- CD8+ T cells were decreased not only in the spleen 3 days following the reinfection, but also in the siIEL (Fig.2B-C), and the same was observed in the co-transfer experiments (Fig.2G). WT CD69+CD103- and CD69-CD103- CD8+ T cells were present within the siIEL at 3 days post- reinfection, likely reflecting newly recruited cells from circulation. However, these cells were in much smaller percentages in the absence of Bcl11b (Fig.2D), likely associated with the reduced Bcl11b-/- memory CD8+ T cells in the circulation (Fig.2B). In addition, the percentages of siIEL Bcl11b-/- Ifnγ+Tnfα+ CD8+ T were reduced compared to WT at 3 days post-reinfection (Fig. 2E). Ifnγ and Tnfα produced by T RM cells have been implicated in activation and recruitment of circulating memory cells and other immune populations, including NK cells, to the site of infection. 119. . Related to this, siIEL recipient CD45.1+NK1.1+ cells were in lower percentages and numbers in the reinfected mice bearing Bcl11b-/- CD8+ T cells versus WT (Fig.2F), Attorney Docket Number 10110-443WO1 indicating defective ability of Bcl11b-/- CD8+ T cells in recruiting cells from circulation, likely related to defective Ifnγ and Tnfα production. 120. To further determine to what extent the defect in the recall was due to intrinsic alterations of Bcl11b-/- TRM cells, we utilized a sex-mismatched adoptive transfer system (male cells transferred into female mice) to selectively deplete cells in circulation, but not in tissues. In this system, bacterial burden post reinfection remained higher in female mice transferred with male Bcl11b-/- CD8+ T cells compared to WT (Fig.2H). siIEL Bcl11b-/- CD8+ T cells were slightly, but not significantly increased, while transferred splenic cells were barely detectable in both groups (Fig.2I). Thus, these results show that in the absence of cells recruited from circulation, mice bearing memory Bcl11b-/- CD8+ T cells still have increased bacterial burden, suggesting an impaired recall response of intestinal Bcl11b-/- TRM cells. c) Loss of Bcl11b in the effector phase causes greater transcriptional changes in siIEL TRMPs compared to TEs and MPs 121. Given that the defects in memory Bcl11b-/- CD8+ T cells arise in the effector phase (9 DPI), we first conducted RNA-seq on splenic MPs (CD127hiKlrg1lo), TEs (CD127loKlrg1hi) and siIEL CD69+CD103+ TRMPs on 9 DPI, sorted to over 90% purity for the specific markers (Fig.9H, 9D). Notably, Bcl11b expression was the highest in WT siIEL TRMPs compared to splenic MPs and TEs (Fig.3A). Splenic MPs and TEs clustered more closely relative to siIEL TRMPs, and all Bcl11b-/- cells were transcriptionally distinct from WT counterparts (Fig.3B-C). Notably, loss of Bcl11b resulted in greater transcriptional changes in TRMPs compared to TEs and MPs (Fig.3D). d) Bcl11b controls the multipotent/multifunctional (MP/MF) program and restricts the effector program in TRMPs and TRM cells 122. Given the greater transcriptional deregulation in siIEL Bcl11b-/- TRMPs compared to TEs and MPs, we first focused on this group, specifically on genes which had modest or no changes in TEs and MPs. Numerous genes of the Id3hiBlimp1lo MP/MF TRM program were found to be downregulated in Bcl11b-/- TRMPs, including Id3 and Tcf7 (Fig.3E-F). In addition to Tcf7 downregulation at the mRNA and protein level (Fig.3I), a Tcf1-dependent gene signature was also reduced in Bcl11b-/- TRMPs (Fig.3G). 123. Genes of the Blimp1hiId3lo effector-like TRM program were upregulated, including Prdm1 (encoding Blimp1) and effector genes (Fig.3E-F). In addition, Ahr, and its target Ahrr, were also upregulated in Bcl11b-/- TRMPs, as well as Ahr-dependent genes (Fig.3E, H). Protein levels of Ahr and Blimp1 were also upregulated in Bcl11b-/- siIEL TRMPs (Fig.3I). Attorney Docket Number 10110-443WO1 124. RNA-seq analysis Bcl11b-/- and WT siIEL TRMs during the memory phase at 30 DPI further revealed that numerous genes of the MP/MF TRM program remained reduced in Bcl11b-/- siIEL TRMs (Fig.3J). Conversely, effector TRM program genes were elevated (Fig. 3J). In addition, Ahr mRNA remained elevated in Bcl11b-/- siIEL TRMs (Fig.3K). Klf2, known to control tissue egress and recently found to be expressed in a multifunctional memory phase SI CD28hi TRM cluster, was downregulated in Bcl11b-/- siIEL TRMs, together in with its target S1pr1, and Cd28 itself (Fig.3K). Ccr7, also known to play a role in tissue egress and to be a target of Tcf1 regulation, was also downregulated (Fig.3K). RNA-seq analysis of the siIEL recall Bcl11b-/- and WT CD8+ T cells 2 days after reinfection further revealed decreased expression of the most MP/MF program genes and increased expression of effector program genes (Fig. 3L). Klf2, S1pr1 and Cd28 remained reduced in the recall Bcl11b-/- cells, but Ccr7 and Ahr were equivalent to WT (Fig.3M). 125. Taken together, these results show that Bcl11b regulates expression of essential genes of both MP/MF and effector TRM programs early in differentiation, in the memory phase and during recall. These results also indicate that Bcl11b restricts expression of Ahr and implicate Ahr in regulation of gut TRM cells and their precursors, as recently reported. e) Bcl11b-deficient MP and TE cells have unaltered program- specific transcription factors, while intestinal homing program is upregulated 126. Given the marked reduction of MPs and TEs in the absence of Bcl11b, we next assessed the canonical TFs of the circulating MP and TE cell programs. Results show that genes encoding several TFs driving TE cell differentiation, including Runx3, Egr2, Zeb2 and Prdm1 remained unchanged, while Id2 and Tbx21 (encoding T-bet) were modestly upregulated in Bcl11b-/- TEs (Fig.11A). MP program TF genes, including Eomes, Stat3 and Klf2 were also unaltered, while Tcf7 was modestly upregulated in Bcl11b-/- MPs (Fig.11B). As expected, Ahr was not expressed in MPs or TEs (Fig.11C). 127. Homing to the SI is essential for the generation of gut TRM cells and is mediated through the integrin α4β7 (encoded by Itga4 and Itgb7 genes), as well as by the chemokine receptor Ccr9. Both Itga4 and Itgb7 mRNAs were increased in Bcl11b-/- MPs and TEs (Itga4 only significant in TEs, while Itgb7 only significant in MPs). Ccr9 mRNA was significantly increased in both Bcl11b-/- MPs and TEs (Fig.12A). Furthermore, α4β7 and Ccr9 proteins were significantly increased in both Bcl11b-/- populations (Fig.12B-C), indicating that increased intestinal homing of circulating Bcl11b-/- cells can lead at least in part to their selective loss in the spleen. Treatment of recipient mice co-transferred with Bcl11b-/- and WT OT-I cells, with the Attorney Docket Number 10110-443WO1 anti-α4β7 antibody DATK-32, resulted in significant improvement of the splenic Bcl11b-/-/WT ratio. However the siIEL Bcl11b-/-/WT distribution remained unchanged, despite an overall reduction of the total transferred CD8+ T cells in the siIEL (Fig.12D). These results indicate that the reduction of the Bcl11b-/- MPs and TEs in the spleen is likely to be caused at least in part by their migration to the SI without major changes in their transcriptional programs. However additional factors contribute to the increased numbers of Bcl11b-/- cells in the siIEL. f) Bcl11b controls H3K27ac, H3K4me3 and chromatin accessibility genome wide 128. To investigate the mechanisms of Bcl11b-dependent regulation of the TRM program, we conducted ATAC-seq on siIEL TRMPs and Bcl11b ChIP-seq, H3K27ac ChIP-seq and H3K4me3 CUT&RUN on TRM-like cells. To generate TRM-like cells, following activation, CD8+ T cells were grown in the presence of TGFβ1 and IL-33, allowing their differentiation toward a TRM-like phenotype, allowing for generation of sufficient numbers of cells for genomics studies. Importantly, in these conditions around 60% of the WT cells become CD69+CD103+, and this was increased when Bcl11b was deleted (Fig.6K). Additionally, genes with decreased and/or increased expression in Bcl11b-/- TRMP and TRM cells were consistently deregulated in Bcl11b-/- TRM-like cells (Fig.13A-C), resembling the phenotype of the primary TRMP and TRM cells (Fig.3). 129. Bcl11b-dependent differences were observed for 9,874 peaks in chromatin accessibility, 27,445 in H3K27ac and 12,485 in H3K4me3, respectively. Differential H3K27ac peaks with increased or decreased signal were overall equally distributed, however peaks with increased chromatin accessibility were enriched in Bcl11b-/- cells (Fig.4A). Differential ATAC- seq and H3K27ac ChIP-seq positively correlated with each other (Fig.14A), which was further intensified upon filtering peaks with high differential scores (Fig.4B). Peaks located within ± 10kb of the transcription start site (TSS) were used to integrate Bcl11b binding, H3K27ac and chromatin accessibility with changes in gene expression. First, differential H3K27ac and ATAC- seq peaks positively correlated with changes in gene expression (Fig.4B-C; Fig.14A), and most of them colocalized with Bcl11b binding (Fig.14B). Differential ATAC-seq and H3K27ac peaks and Bcl11b binding peaks occurred predominantly at intronic regions (Fig.4D). Gene promoters were the second most abundant genomic feature bound by Bcl11b (Fig.4D), and H3K4me3 signal displayed overall reduction in Bcl11b-/- cells at promoters bound by Bcl11b in WT cells (Fig.4E- F). 130. TF DNA binding motif analysis in the differential ATAC-seq and H3K27ac peaks show that Blimp1 and Ahr motifs were enriched in peaks with increased signal in Bcl11b-/- Attorney Docket Number 10110-443WO1 cells (Fig.4G), while Tcf1 and Lef1 motifs were enriched in peaks with decreased signal in Bcl11b-/- cells (Fig.4G). Detailed motif enrichment analysis, separately at differential ATAC-seq and H3K27ac peaks, and correlated with Bcl11b binding, grouped TF motifs into several categories (Fig.14C). ETS family motifs were mostly enriched in differential peaks not bound by Bcl11b, indicating ETS TF activity is independent of Bcl11b binding. Conversely, motifs for Egr1, Egr2, Irf4, and NF-κB were enriched at ATAC-seq and H3K27ac peaks with increased signal in Bcl11b-/- cells at sites bound by Bcl11b in WT cells (Fig.14C), indicating a competitive mechanism of action by Bcl11b with these TFs. 131. In terms of Bcl11b binding, the most enriched motifs were for ETS, bZIP and Runx family (Fig.14D), similarly to what we observed before in MAIT cells and Treg cells. Binding motifs for Tcf1/Lef1, Blimp1 and Ahr were enriched at differential ATAC-seq and H3K27ac peaks, irrespective of Bcl11b binding, however numerous Bcl11b peaks also contained significant enrichment of Tcf1/Lef1, Blimp1 and Ahr motifs (Fig.14C-D), supporting a functional intersection between Bcl11b and these TFs related to the MP/MF and effector TRM programs. g) Bcl11b binds at the Tcf7 locus, Tcf1-dependent genes and genes of the MP/MF program 132. We next sought to determine whether Bcl11b is directly implicated in regulation of Tcf7 and other genes of the MP/MF TRM program. We detected Bcl11b binding at multiple intronic, promoter, and upstream regions of Tcf7 gene (Fig.5A). Deletion of Bcl11b in TRMPs resulted in decreased chromatin accessibility at upstream and intronic sites bound by Bcl11b, as well as reduced H3K27ac and H3K4me3 at an upstream site and at Tcf7 promoter (Fig.5A), indicating that Bcl11b sustains H3K27ac, H3K4me3 and chromatin accessibility at Tcf7 to drive its expression. In human memory CD8+ T cells, BCL11B also bound at two upstream regions of TCF7 gene, indicating common modules of regulation in mice and humans, dependent on BCL11B (Fig.5B). Of note, Tcf7 expression was only downregulated in siIEL TRMPs and TRMs, but not in splenic MPs or TEs (Fig.3; Fig.11), indicating a specific role for Bcl11b in regulating its expression only in siIEL TRM cells and precursors. 133. We next investigated Bcl11b-dependent Tcf1 occupancy genome-wide by performing Tcf1 CUT&RUN in Bcl11b-/- and WT TRM-like cells. Notably, the Tcf1 signal at promoters was almost exclusively found at promoters bound by Bcl11b (Fig.5C). Nevertheless, no difference was found in Tcf1 binding preference at promoters of genes with decreased or increased expression in Bcl11b-/- cells and the overall Tcf1 signal was similar between Bcl11b-/- and WT cells (Fig.5C). This could, however be attributed to the experimental setup, as the TRM- Attorney Docket Number 10110-443WO1 like cells generated ex vivo may not have had enough time to fully manifest the genome-wide changes related to the decreased Tcf7 expression and the expected underlying changes in Tcf1 binding in Bcl11b-/- cells. Interestingly, Tcf1 binding was enriched at Bcl11b-bound regions associated with decreased chromatin accessibility (Fig.5C-D). This observation is in line with the motif enrichment analysis (Fig.4G). Furthermore, in keeping with the overall decreased MP/MF program gene expression, including of Tcf7 itself, we detected decreased Tcf1 binding at several loci, including at its target Ccr7, where Bcl11b was also bound abundantly (Fig.15A). Common binding of Bcl11b and Tcf1 was noticeable at the Ccr7 promoter, as well as at several sites located upstream and downstream, which correlated with decreased chromatin accessibility and/or H3K27ac and which displayed reduced Tcf1 binding in Bcl11b-/- cells (Fig.15A). Bcl11b also bound at several other genes of the MP/MF program, and remarkably, its binding sites at Id3, Lef1, Klf2, S1pr1, Cd5 and Btla were associated with decreased H3K27ac, H3K4me3 and/or chromatin accessibility in its absence. At Id3 and Klf2 loci, binding of Bcl11b occurred at regions with decreased Tcf1 binding in Bcl11b-/- cells (Fig.15C, 16A-C). However, some other genes from the program such as Lef1, Cd5 and mostly Tcf7 itself displayed increased Tcf1 binding in the absence of Bcl11b (Fig.16A-B and Fig.5A), indicating a compensatory mechanism of regulation. In addition, Bcl11b also bound and regulated H3K27ac deposition at Ifng and Tnf loci, as well as at Cd28, associated with regulation of chromatin accessibility (Fig. 16D-F), indicating direct regulation of expression of these genes. These results indicate that Bcl11b contributes to regulation of the MP/MF program by controlling Tcf7 expression, as well as by direct regulation of essential genes of the MP/MF program. 134. We next assessed whether forced expression of Tcf7 compensates for Bcl11b deficiency. Ex vivo activated Bcl11b-/- and WT OT-I CD8+ T cells were transduced with Tcf7- retroviruses and adoptively transferred into Lm-Ova InlAM infected recipient mice. Tcf7 transduction led to similar levels of Tcf1 between siIEL Bcl11b-/- and WT cells at 9 DPI (Fig.5E) and restored Bcl11b-/-/WT ratio within the siIEL CD69+CD103+ TRMPs (Fig.5F). However, in the spleen, Tcf7 transduction only partially corrected the reduction in Bcl11b-/- cell percentages compared to EV transduced Bcl11b-/- cells (Fig.5G), indicating implication of additional factors. h) Bcl11b binds at Prdm1, effector genes and Ahr loci to control their expression in TRM cells 135. We further investigated the mechanisms involved in Bcl11b-dependent regulation of genes of the effector TRM program. Bcl11b bound at several sites surrounding the Prdm1 locus, including at the promoter. In Bcl11b-/- cells these sites had elevated H3K4me3, while Attorney Docket Number 10110-443WO1 other sites had increased H3K27ac and chromatin accessibility (Fig.6A). BCL11B also bound at several sites at the human PRDM1 locus (Fig.6B). 136. Bcl11b bound at the loci of Gzma and Prf1, genes of the TRM effector program, and at Gzmc, another effector gene, at several locations (Fig.17A-C). In addition, Bcl11b bound at genes associated with exhaustion, including Havcr2 and Entpd1 (Fig.17D-E). The majority of these binding sites were associated with increased chromatin accessibility and changes in histone modifications in Bcl11b-/- TRM-like cells (Fig.17A-E). 137. In addition to upregulation of Blimp1 and effector T RM program genes, Ahr was upregulated together with genes of its program (Fig. 3E, H, I). This was of particular interest, given Ahr’s roles in TRM differentiation, as well as in general maintenance of IELs, and thus contribution to increased accumulation of Bcl11b-/- TRM cells in the gut. Bcl11b bound at two regions upstream of the Ahr locus, as well as at the promoter and in its absence caused elevated H3K4me3 (Fig.6C). In human memory CD8+ T cells, BCL11B bound AHR in an intronic region (Fig.6D). Of note, regulation of Ahr transcription by Bcl11b occurred solely in siIEL Bcl11b-/- TRMPs and TRMs, but not in MPs or TEs (Fig.3E and 11C). 138. To further delineate binding preferences of Ahr related to Bcl11b, we performed ChIP-seq analysis in Bcl11b-/- and WT TRM-like cells and integrated them with the other datasets. Similar to Tcf1, Ahr co-binding with Bcl11b was mostly located at gene promoters (Fig.6E). Ahr signal in Bcl11b-/- cells was slightly increased, in line with the overall increased Ahr protein. Moreover, Ahr binding was enriched and increased in Bcl11b-/- cells at sites corresponding to Bcl11b binding in WT cells, and associated with increased chromatin accessibility, whereas no changes in Ahr binding in the Bcl11b-/- cells were observed for Bcl11b peaks associated with decreased accessibility (Fig.6F). Note that these observations were in agreement with the motif analysis (Fig.4G). We further focused the analysis on genes bound by Ahr and with elevated expression in Bcl11b-/- cells. Several of these genes, including Ahrr, Il2rb and Aldh7a1, not only had increased expression (Fig.3E) and elevated Ahr binding in Bcl11b-/- cells, but also elevated levels of H3K4me3, H3K27ac and/or chromatin accessibility at regions of Bcl11b binding (Fig.17F-H). 139. To determine the relevance of Ahr elevation in Bcl11b-/- TRMPs, we next eliminated diet- derived Ahr ligands to block Ahr activity. Specifically, mice were placed on Ahr ligand-deficient diet (AIN-76a) two weeks prior to adoptive transfer, and for the duration of the whole experiment. Absence of diet derived Ahr ligands normalized Bcl11b-/-/WT ratio in the siIEL at 9 DPI, however only partially at 30 DPI (Fig. 6G), indicating contribution of additional factors in the dysregulation later in memory phase. Frequencies in the spleen Attorney Docket Number 10110-443WO1 remained unaltered (Fig.6H), indicating mechanisms independent of Ahr. Importantly, the AIN76a diet led to a reduction in siIEL recipient CD45.1+CD8+ T cells, demonstrating the efficacy of the diet even on recipient CD8+ T cells (Fig.6I). Ahr protein remained elevated (Fig. 6J), demonstrating that the effects are not due to a reduction in Ahr levels, but due to its ligand- dependent activity. Given that recently Prdm1 was identified as a target of Ahr, we next used sgRNAs/CRISPR/CAS9 to deplete Ahr and Prdm1 in WT and Bcl11b-/- T RM -like cells. In WT T RM -like cells, Ahr depletion alone reduced CD69 + CD103 + population, while deletion of both Ahr and Prdm1 did not show a further reduction (Fig. 6K). However, deletion of both Ahr and Prdm1 in Bcl11b-/- T RM -like cells, further reduced even more the percentages of CD69 + CD103 + cells compared to Ahr deletion alone (Fig. 6K). Taken together, these results indicate that Bcl11b restricts TRM cell differentiation through control of both Ahr and Prdm1. i) Bcl11b commonly restrains an innate program in all CD8+ T cell subsets 140. We further addressed the question on what genes are commonly regulated by Bcl11b in siIEL TRMs and their precursors, in TEs and MPs and recall siIEL CD8+ T cells. Our results show that the commonly regulated genes comprise important NK and myeloid genes (Fig. 7A). Importantly, Itgam and Itgax genes have also been included in the effector TRM program. Bcl11b bound at several NK genes, located in the NK cell receptor locus in regions associated with broad H3K27ac in NK cells, including at Klrb1. Notably, in Bcl11b-/- TRM-like cells we observed increased H3K27ac and chromatin accessibility at NK cell receptor enhancers (Fig.7B). Moreover, Bcl11b binding at Itgam, Itgax (encoding CD11b and CD11c) and Fcer1g loci was associated with increased H3K27ac, H3K4me3 and/or chromatin accessibility (Fig.7C- D). BCL11B also bound at human KLRB1 (Fig. 7E), as well as at ITGAM and ITGAX (Fig. 7F), indicating a common role of BCL11B between mice and humans in repressing innate gene expression in CD8+ T cells. j) BCL11B regulates the TRM program in human CD8+ T cells ex vivo 141. We further determined the impact of BCL11B on human TRM-like CD8+ T cells differentiated ex vivo. Depletion of BCL11B by CRISPR/CAS9 gene editing resulted in upregulation of several residency program proteins, including CD69 and CD49a, and downregulation of CD62L and CCR7 (Fig.8A-B), part of the TRM cell program. Additionally, several NK receptors, including CD56, CD161 (KLRB1), CD117 and NKP46/NCR1, were upregulated in BCL11B-deficient human TRM-like cells (Fig.8C), indicating a common role of BCL11B in mice and humans. Attorney Docket Number 10110-443WO1 142. In primary human memory CD3+CD8+CD45RA+CD57+CCR7- T cells, BCL11B bound at numerous genes of the TRM program, also bound and regulated by Bcl11b in mouse (Fig. 5-7, Fig.15), indicating that BCL11B regulates aspects of the CD8+ T cell residency program in human and mouse in a similar fashion. k) Depletion of Bcl11b also increases cytotoxicity of TILs in human cancer models 143. As shown in Figure 20, BCL11B depletion in human TILs increases cytotoxicity in a manner dependent on MHC I. In fact, Bcl11b-deficient CD8+ T cells confer elevated anti- tumor activity in ovarian tumors implanted intraperitoneally (Figure 21) including elevated cytotoxic genes and NK receptor and stemness program genes, while inhibitory receptor genes remain unchanged (Figure 22). Looking at peritoneal tumors, we showed that expression of gene subsets in TILs clusters in WT and Bcl11b-/- (knockout) mice with peritoneal tumors following adoptive transfer. Figurer 24 shows that CD8+ TILs from Bcl11b-/- mice continue to express stemness transcription factor TCF1 (24A and 24B), expand more (24C), have reduced levels of the exhaustion transcription factor Tox, as well as, exhaustion markers CD39, PD1, and Tim3 (24D), and have increased metabolic potential and mass and thus greater fitness (24E). This paradigm also holds for CD4 T cells as shown in Figures 25 and 26 showing that Bcl11b-/- (KO) CD4+ TILs display enhanced anti-tumor activity in adoptive cell therapy and result in tumor eradication in ovarian tumor models. Also Bcl11b-deficient CD4+ T cells confer elevated anti-tumor activity in ovarian and colon cancer tumors implanted intraperitoneally (Figure 27). 144. As shown in Figure 28, human melanoma specific CD8+ TILs that have been depleted for Bcl11b have elevatored perforin together with stemness TF TCF1, as well as the NK receptor CD56, but do not downregulate CD3 and CD8, thus maintaining CD8 TIL identity. Figure 28A and 28B shows a brightfield image (Figure 28A) and fluorescent staining (Figure 28B) of tumor cells, TILs, and killed cancer cells. Figure 28C shows that the number of dead tumor cells is greatly increased in Bcl11b depleted TILs. Figure 28D shows a comparison of various markers between WT and Bcl11b depleted TILs. Bcl11b was depleted using CRISPR/cas9 targeting Bcl11b using the guide Hs.Cas9.Bcl11b.AA (SEQ ID NO: 1) in combination with Hs.Cas9.Bcl11b.AE (SEQ ID NO: 2). 145. 2. Discussion 146. In this study we provide evidence that Bcl11b plays a critical role in SI CD8+ TRM cells, including in their precursors, promoting the MP/MF program and suppressing the Attorney Docket Number 10110-443WO1 effector program, thus expanding our current knowledge on transcriptional control of intestinal TRM cell differentiation and divergent programs. 147. Bcl11b exerts its role by binding, regulating the epigenetic landscape and directly controlling expression of essential genes of the MP/MF TRM program, including of Tcf7, Id3, Lef1, Ifng , Tnf, Cd5, Btla, and additionally Klf2, S1pr1 and Ccr7. Bcl11b also bound and repressed the expression of essential genes of the effector program, including the lead TF Blimp1, Granzyme genes and Prf1. Despite accumulation of Bcl11b-/- TRM cells in the intestine, recall response was poor, with elevated bacterial burden and reduced numbers of recall Bcl11b-/- CD8+ T cells in the SI. Though the paucity of circulating memory cells is likely to contribute to the reduced numbers of SI recall Bcl11b-/- CD8+ T cells, the sex mismatch experiment demonstrated that SI Bcl11b-/- TRM cells have intrinsic alterations. In line with this, recall SI Bcl11b-/- CD8+ T cells continue to show reduced MP/MF program gene expression and altered ability to produce Ifnγ, important for recruitment not only of circulating memory CD8+ T cells during recall, but also of other immune populations, including of NK cells. Our results are in line with recent findings that Id3hiBlimp1lo TRM cells (also expressing Tcf1) are able to provide a more efficient secondary response during recall compared to the Id3loBlimp1hi counterparts, are multifunctional and multipotent and generate both secondary resident and circulating memory cells. In addition to the Id3hiBlimp1lo MP/MF subset, a memory phase CD28hi cell cluster (with high Id3), has been identified, with elevated Ifng and Tnf transcripts, as well as high Klf2, likely to provide support for egress from tissue into the circulation following reinfection. In line with this, not only Tcf7 and Id3 expression was reduced in Bcl11b-/- TRM cells, but also Klf2 and its target S1pr1, as well as of Ccr7. Thus, Bcl11b regulates TRM multipotency by controlling both the recruitment of cells from circulation through Ifnγ, as well the egress from the tissue, through Klf2, S1pr1 and Ccr7. As such, the impaired MP/MF signature in the absence of Bcl11b explains the deficient response in the recall. Intriguingly, our findings show that Bcl11b-/- TRM cells, which have high Blimp1, but low Id3 and Tcf1, are long-lived TRM cells, but their protective recall response is impaired, despite elevated expression of effector genes for granzymes and Prf1. Whether Bcl11b-/- CD8+ T cells can provide enhanced protection in response to other pathogens and at other location or in cancer, particularly given their elevated expression of cytolytic molecules, is of particular interest. 148. From rescue experiments, it is clear that restoration of Tcf1 expression in Bcl11b- /- CD8+ T cells early in the response can restrict TRM cell differentiation. But the precise mechanisms and contributions of Tcf1 in the multipotency/multifunctionality of intestinal Id3hiBlimp1lo TRM cells, and further in their maintenance and recall response remains unclear. Attorney Docket Number 10110-443WO1 149. Our results also show that Bcl11b restricts expression of Ahr, similarly to what we found in ILC2 cells. Ahr has been shown recently to drive SI TRM cell differentiation, supporting expression of Prdm1, Hic1, Gzmb and Itgae. Our results indicate that Bcl11b represses expression of Prdm1 independent of Ahr. However, Bcl11b seems to restrict Ahr activity at the Il2rb promoter (IL15 receptor), and thus possibly responsiveness to IL15, known to control at some extent together with Tgfβ TRM cell fate. It remains to be established where Ahr is positioned in regulation of MP/MF versus effector TRM cell programs. 150. We also show that ablation of Bcl11b causes a major reduction of circulating memory cells mainly due to their enhanced intestinal homing with minimal impact on program specific TFs of memory precursors and effector cells. However both in circulating memory cells, effector cells and TRM cells and precursors, as well as in recall CD8+ T cells, Bcl11b restricted expression of the innate program, including of NK and myeloid receptors, but not in MAIT cells. Conversely, both in human and mouse NK cells, Bcl11b sustained expression of NK receptors and both canonical and adaptive NK cell differentiation. 151. One of the limitations of the current study is the use of ex vivo differentiated TRM- like cells for genomics studies. Although we have demonstrated a comparable transcriptional deregulation of TRM programs in the absence of Bcl11b, as well as highly correlating ATAC- seq and H3K27ac ChIP-seq data in Bcl11b-/- primary TRMPs and TRM-like cells, respectively, we acknowledge that certain aspects of molecular regulation could be different. Whether our findings in SI TRMs are generalizable to different organs and different pathogens remains of high interest. 152. In summary, in the present work we investigated mechanisms governing SI TRM cell differentiation and established Bcl11b as a frontrunner in resident memory cell programs, acting in lineage decision, upstream of essential TFs to promote TRM multipotency and restrict effector programs. Many questions remain to be addressed including detailed characterization of spatiotemporal relationships of regulatory networks, understanding of tissue-specific regulomes and connection of characterized programs to pathophysiological states. 3. Materials and Methods a) Study Design 153. This study investigated the mechanisms by which Bcl11b regulates memory CD8+ T cell fates. We used an oral infection model with Lm-Ova InlAM in conjunction with adoptive co-transfer or separate transfer experiments of CD8+ T cells from Bcl11bf/fGzmbCre+R26REYFP+ OT-I (CD45.2) and WT OT-I (CD45.1/2) mice into CD45.1 recipients. Memory cells and their precursors were evaluated in spleen and siIEL by FACS and Attorney Docket Number 10110-443WO1 transcriptional programs by RNA- seq. ATAC-seq on siIEL T RMPs Bcl11b ChIP-seq, H3K27ac ChIP-seq and H3K4me3 CUT&RUN on TRM-like cells, together with Ahr ChIP-seq and Tcf1 CUT&RUN experiments were conducted for genomic mechanistic studies. BCL11B ChIP-seq in human memory CD8 + T cells and CRISPR BCL11B knockout in human T RM -like cells were conducted to establish commonalities between humans and mice. Results were validated by rescue experiments with: retrovirus-mediated overexpression of Tcf7, Ahr ligand-deficient diet, and CRISPR-mediated knockout of Ahr and Prdm1. Bcl11b-/- memory cells were investigated in secondary infections with Lm-Ova InlA M , including a sex-mismatched system for siIEL T RM only response. All data are representative of at least 2 experiments. Statistics were conducted mostly with two-tailed paired or unpaired Student’s t-test. Samples were unblinded. b) Approval for human and mouse studies. 154. Studies were approved by the Swedish Ethical Review Authority. Peripheral blood was obtained from healthy volunteers from the Karolinska University Hospital blood bank with informed consent. All mouse studies were approved by the Institutional Animal Care and Use Committees of University of Florida, University of South Florida and Moffitt Cancer Center. c) Mice 155. Bcl11bF/F mice, bread on C57BL/6 background, were crossed to B6-Tg(GZMB- cre)1Jcb/J (GzmbCre) on C57BL/6 background, provided by Barbara Kee, (University of Chicago). B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (R26REYFP) and C57BL/6- Tg(TcraTcrb)1100Mjb/J (OT-I) mice were from Jackson laboratory. B6.SJL-PtprcaPepcb/BoyJ (CD45.1) (wild type) recipient mice were purchased from Taconic and bred in our colony. Mice of both sexes were used at age between 7-12 weeks. Mice were bred under specific pathogen-free conditions. d) Adoptive Cell Transfer and Infection studies 156. For adoptive transfers CD8+ T cells were enriched from spleens of OT-I mice using the CD8+ T cell negative selection kit (Stem Cell, 19853), followed by sorting of naïve CD62Lhi CD44lo CD8+ T cells (BD Aria II). Mixed 1:1 ratio (for co-transfers) 5×104 cells were transferred intravenously into WT CD45.1 mice, 1 day prior to infection. Donor cells were subsequently assessed at indicated time points post-infection in different experiments. 157. For Lm-Ova InlAM experiments, mice were food starved for several hours during the day and infected late afternoon by feeding infected bread containing 2×109 (primary) or 2×1010 (reinfection) colony forming units (CFUs) of Lm-Ova InlAM. Attorney Docket Number 10110-443WO1 e) Murine TRM-like cell differentiation 158. CD8+ T cells were purified from naïve Bcl11bf/fGzmbCre+ R26REYFP+ OT-I and WT OT-I mice as above and then activated with anti-CD3/anti-CD28 beads (Invitrogen, 114525D) at 2.5×105 cells/ml in 10% FBS MEM alpha supplemented with 100 U/ml IL-2 (Shenandoah Biotechnology, 100-12) for 48 hours at 37°C, 5% CO2, when cells were split 1:3 in fresh media supplemented with 10 ng/ml TGFβ1 (Shenandoah Biotechnologies, 100-39) and 100 ng/ml IL-33 (Shenandoah Biotechnologies, 200-36), for an additional 48 hours. This protocol was adapted from. f) Human CD8+ T cells 159. Enriched CD8+ T lymphocytes were labelled with fluorochrome-conjugated antibodies to surface epitopes and a fixable dead cell stain for 20 minutes at room temperature. Cells were washed twice in PBS with 0.5% BSA and 2 mM EDTA and specific subsets were sorted using a FACSAria (BD Bioscience) in complete medium for cell culture. The purity of specific sorted human or mouse subsets was evaluated by flow cytometry and was more than 95%. g) Human TRM-like cell differentiation 160. FACS sorted naïve CD8+CD45RA+CD62L+CD27+CD57– T cells were pre- activated with 10 µl/ml anti-CD3/28 immunocomplexes (StemCell Technologies) and 100 U/ml IL-2 (Peprotech) and incubated in complete medium at 37°C. After 48 hours, pre-activated CD8+ T cells were washed twice with RPMI supplemented with 10% FBS and cultured for 14 days with anti-CD3/28 immunocomplexes, 100 U/ml IL-2 (Peprotech), 10 ng/ml TGF-β (Peprotech) and 100 ng/ml IL- 33 (Peprotech). Prior to phenotypic analysis cells were washed twice and rested 24 hours in complete medium at 37°C. h) ChIP-seq for murine Bcl11b, H3K27ac and Ahr 161. TRM-like cells were differentiated as above. For Bcl11b, ChIP-seq was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003), with the following changes to the protocol: (i) 107 cells were used, (ii) formaldehyde fixation was performed in 1 ml, 162. (iii) buffer A and B steps were all performed in 1 ml, (iv) cells were sheared in ChIP buffer to an average of 200 to 1000 bp using a Bioruptor Pico (Diagenode, B01060002), (v) a cocktail of three anti-Bcl11b antibodies was used (4 µg of Abcam, ab18465; 4 µg of CST, 12120; and 4 µg of Bethyl, A300-385), and (vi) final DNA purification was performed by PCI extraction and MaXtract high-density columns (Qiagen, 129046). Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, E7645S) and sequenced as Attorney Docket Number 10110-443WO1 PE100 on Illumina NovaSeq 6000. For Ahr ChIP-seq, TRM-like cells were treated with the Ahr ligand 6- formylindolo[3,2-b]carbazole (FICZ, final concentration 200 nM; Enzo Life Sciences, BML- GR206) for 4 hours before harvesting. For H3K27ac and Ahr, ChIP was performed using the ChIP- IT High Sensitivity Kit (Active Motif, 53040), following the manufacturer's protocol with the following modifications: (i) the nuclear pellet of 106 cells was lysed in 500 µl ChIP buffer and chromatin was sheared by the Q125 Sonicator (QSonica); (ii) the immunoprecipitation was performed with anti-H3K27ac (D5E4, CST) or anti-Ahr antibodies (Enzo Life Sciences, BML- SA210–0100). Libraries were prepared using the Next Gen DNA Library kit (Active Motif, 53216) and Next Gen Indexing Kit (Active Motif, 53264) and sequenced as PE150 on Illumina NovaSeq 6000. i) Cleavage Under Targets & Release Using Nuclease (CUT&RUN) 163. CUT&RUN was performed on 0.35×106 TRM-like cells using the ChIC/CUT&RUN Kit (EpiCypher, 14-1048) with 1 µl anti-H3K4me3 (EpiCypher, 13-0041) or 1.5 µl anti-Tcf1 (C46C7, CST) antibodies, following the manufacturer's protocol, except that 1× eBioscience Perm Buffer (Invitrogen, 00-8333) supplemented with Spermidine and cOmplete™EDTA-free Protease Inhibitor (Roche, 11836170001) was used during the incubation step with pAG-MNase and 0.5× eBioscience Perm Buffer during the wash steps. Libraries were prepared with the NEBNext Ultra II Library Prep Kit (New England Biolabs, E7645) and sequenced as PE150 on Illumina NovaSeq 6000. j) Statistics 164. For non-sequencing experiments, statistical analyses were conducted between two groups by a two-tailed paired (co-transfer) or unpaired (separate transfer) Student’s t-test assuming unequal (ratio > 4) or equal variance (ratio < 4), unless otherwise specified. For RNA- seq experiments, significance was calculated by DESeq2 with an FDR cutoff of 0.05. For ATAC-seq, significantly differential regions of chromatin accessibility were calculated using an optimized MANorm script, with a minimum fold change of 2 and significance of P < 0.05 following Benjamini- Hochberg adjustment. For determination of reproducible peaks for ChIP- seq and ATAC-seq, an IDR ≤ 0.05 was used. k) Enumerating bacterial burden 165. For tissue burden, livers were harvested at the indicated timepoint and homogenized in 5 ml of sterile distilled water through a 70 µm cell strainer. Serial dilutions of each sample were prepared and plated on BHI agar plates with 200 µg/ml Streptomycin. Colonies were counted after 48 hours of growth at 37°C. Attorney Docket Number 10110-443WO1 l) AIN76a Diet studies 166. Mice were either kept on 7912 (control) diet or placed on Ahr ligand-free AIN76a (Envigo) diet two weeks prior to adoptive transfer and infection, and further for the duration of the experiment, to deplete diet-derived Ahr ligands, while allowing normal immune cell development. m) Tissue preparation 167. Single cell suspensions of spleen were generated by mashing the organs through a 40 µm cell strainer. For isolation of siIEL, excess fat and Peyer’s patches were removed, intestinal contents and mucus were removed by gentle scraping and the intestine was cut open longitudinally. The intestine was cut into 1-2 cm pieces and washed 3-4 times in RPMI supplemented with 1% L- glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 168. 0.01 M HEPES, 220 µM 2-mercaptoethanol, and 1% FBS (1% RPMI). Intestinal pieces were incubated in 20 ml 1× HBSS containing 1 mM dithioerythritol and 10% FBS with shaking at 37°C twice for 20 minutes, followed by filtration on 70 µm cell strainer to isolate IELs. 46.25 ml Percoll (GE Healthcare, Cat. No.17-0891-01) were mixed with 3.6 ml 10× HBSS (Corning, Cat. No.20- 023-CV) and 0.6 ml 7.5% sodium bicarbonate and further diluted to 44% and 66% isotonic Percoll with 1× HBSS. IEL were resuspended in 8 ml 44% Percoll and underlayed with 2 ml 66% Percoll using a glass Pasteur pipette. Cells were centrifuged for 20 minutes 1600× g at room temperature and the interface was collected with Pasteur pipette. Cells were washed twice with threefold volume of 1% FBS RPMI. n) In vivo treatments 169. For in situ cytokine-production, mice were injected intraperitoneal with 100 mg SIINFEKL (SEQ ID NO: 3) peptide and 0.3 mg brefeldin A (BFA) (Sigma Aldrich, Cat. No. B7651) 5 hours prior to organ harvest. Spleen and IEL were processed with additional supplementation of 0.02 mg BFA in all solutions until cells were fixed. o) Human cells isolation and culture 170. Buffy coats from healthy donors were collected from the Karolinska University Hospital blood bank. PBMCs were isolated by density gradient centrifugation, thereafter bulk CD8+ T cells were enriched from PBMCs using negative magnetic selection (StemCell Techologies; Miltenyi Biotech). All cells were cultured in complete medium (RPMI 1640 supplemented with Glutamax [Gibco] and 10% fetal bovine serum [Hyclone]). Attorney Docket Number 10110-443WO1 p) Vectors 171. pVPack-VSV-G (Cat. No.217567) and pVPack-GP (Cat. No.217566) were from Agilent Technologies. MIGR1 encoding p45 Tcf1 (MIGR1-Tcf7) was a gift from Dr. Hai-Hui Xue. Qiagen Plasmid Plus Midi kit (Cat. No.12943) was used for DNA preparation and retroviruses and transduction were conducted as described in materials and methods. q) Cells and Generation of retrovirus 172. Platinum-E (Plat E) retroviral packaging cells (Cell Biolabs, Cat. No. RV-101) were grown in DMEM supplemented with 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 µM 2-mercaptoethanol, and 10% FBS (10% DMEM). Retroviral particles were produced.3×106 Plat E cells were seeded on 10-cm cell cultures plates such that 80% confluence was reached overnight. Media was aspirated off Plat E cells and 5 ml of fresh 10% DMEM was added; 9 µg MIGR1 or MIGR1-Tcf7 vectors were added into serum-free DMEM along with 4.5 µg pVPack-VSV-G and 9 µg pVPack- GP to a total volume of 450 µl, then incubated at room temperature for 5 minutes. Lipofectamine 2000 (36 µl) was added to 414 µl of serum-free DMEM and incubated at room temperature for 5 minutes. The Lipofectamine solution was added to the DNA, mixed by flicking and then incubated at room temperature for 20 min. The DNA-Lipofectamine 2000 complexes in a total volume of 900 µl were added dropwise to Plat E cells. The transfected Plat E cells were incubated overnight at 37°C, 5% CO2. The medium was aspirated from transfected Plat E cells; then 5.5 ml of pre-warmed (37°C) RPMI supplemented with 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 220 µM 2- mercaptoethanol, and 10% FBS (10% RPMI) was added to the edge of the plate slowly. The transfected Plat E cells in 10% RPMI were incubated overnight at 37°C, 5% CO2. Retroviral particles in 10% RPMI were collected at 48 and 72 hours post-transfection and filtered through a 0.45-μm syringe filter (Corning, Cat. No.431220). Retrovirus was further concentrated 5-fold with Lenti-X Concentrator (Takara, Cat. No.631232) according to manufacturer’s instructions and resuspended in 10% FBS RPMI. Concentrated retrovirus was stored at −80°C until use. r) Transduction with retroviruses 173. Spleen and peripheral lymph node Bcl11b-/- or WT OT-I CD8+ T cells were isolated using the mouse CD8+ T cell isolation kit (Stem Cell, Cat. No.19853). Purified cells were activated with 5 µg/ml plate-bound anti-CD3 (BioXCell, Cat. No. BE0002) and 2 µg/ml soluble anti-CD28 (BioXCell, Cat. No. BE0015-1) at 1 x 106 cells/ml in 10% RPMI supplemented with 100 U/ml IL- 2 (Shenandoah Biotechnology, Cat. No.100-12) for 24 hours at Attorney Docket Number 10110-443WO1 37°C, 5% CO2. Retrovirus containing 10% RPMI and 8 µg/ml Polybrene (1 ml) was added to a 24-well plate, and 1-3 x 106 activated cells (1 ml) were immediately added to the retrovirus containing media. The cells were then centrifuged at 2000 x g at 30°C for 1 hour, then incubated at 37°C and 5% CO2 for 24 hours. The transduced cells were subsequently harvested, washed twice with PBS and 5 x 104 cells were co-transferred into Lm-Ova InlAM infected mice. s) Flow cytometry 174. Single cell suspensions from spleen and intestine were isolated at indicated time points. Cells were incubated with surface antibodies in the dark at 4°C for 30 minutes. For SIINFEKL (SEQ ID NO: 3) tetramer staining, cells were first stained with tetramer for 1 hour at room temperature (RT) in the dark. Exclusion of dead cells was conducted with a Fixable Viability Dye (eBioscience, 65-0865-14). If necessary, to maintain YFP or GFP expression with intracellular staining, cells were pre-fixed with 1% paraformaldehyde for 10 minutes at RT and subsequently fixed with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, 00- 5523-00) for 30 minutes at RT. Foxp3/Transcription Factor Staining Buffer Set was used for staining of all TFs. Samples were acquired on an LSR II (BD Biosciences) or an Aurora Spectral Cytometer (Cytek). Data were analyzed using FlowJo (TreeStar). t) Human ChIP-seq for Bcl11b 175. PBMC from two donor buffy coats were gradient separated (Lymphoprep, Axis Shield) and CD8+ T cells enriched by negative magnetic isolation (Miltenyi biotech). Cells were washed in PBS and fixed with 2 mM disuccinimidyl glutarate (DSG) (ThermoFisher) for 40 mins. During the last 10 mins, 1% formaldehyde was added before quenching with 1.25 M glycine. Cells were FACS sorted (BD Symphony) as CD3+CD8+CD45RA+CD57+CCR7– (T EMRA ) cells purified and stored at -80°C with 5-10×10 6 purified T cells per condition. Thawed cells were resuspended in 1 ml of cold nuclear lysis buffer (10 mM Tris–HCl, pH 7.5 1% NP-400.5% sodium deoxycholate 0.1% SDS) with protease inhibitor (Roche) for 10 mins on ice then pelleted at 2000 g at 4°C for 3 mins, resuspended in 150 µl ChIP SDS buffer (100 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 0.2% NaN 3 , 0.5% SDS) with protease inhibitor (Roche) for 15 mins on ice, sonicated in fibre containing AFA tubes (Covaris) for 6 minutes at peak power of 140W (Covaris S220, Covaris). Sonicated cells were spun at 10,000 g for 5 mins, and supernatants taken and diluted with ChIP dilution buffer (100mM NaCl, 100mM Tris HCl pH 8.6, 5mM EDTA, 0.2% NaN3, 1% Triton X‐100) and incubated overnight with 40 µl Protein G Dynabeads (ThermoFisher) pre-loaded with 10 µg antibody (Bcl11b, clone 25B6, AbCam). Chromatin was washed twice each with low salt buffer (50 mM HEPES, 1% Triton, 140 mM NaCl), high salt buffer (salt increased to 500 mM NaCl) then TE buffer (10 mM Tris-HCl, 1 Attorney Docket Number 10110-443WO1 mM EDTA), then reverse cross-linked in 100 µl ChIP SDS buffer with 200 mM NaCl and Proteinase K at 65°C for 15 hours followed by RNaseA (ThermoFisher) treatment for 1 hour. DNA was purified (ChIP DNA Clean & Concentrate, Zymo), before sequencing libraries prepared (Thruplex DNA-seq kit, TakaraBio). Libraries were purified (AMPure XP, Beckman Coulter), quantified on a bioanalyzer (Agilent) and spectrometer (Qubit, ThermoFisher). Pooled libraries were run on a NextSeq instrument (Illumina) at the Norwegian Sequencing Centre, Oslo, Norway. u) Murine CRISPR/Cas9 gene editing 176. CD8+ T cells were purified from naïve Bcl11b-/- and WT OT-I mice as above. Purified cells were incubated with 10% RPMI supplemented with 5 ng/ml recombinant mouse IL-7 (R&D Systems, Cat. No.402-ML-020) at 1×106 cells/ml and preincubated for 24 hours before transfection at 37°C, 5% CO 2. For sgRNA design, the top 3 ranked sgRNAs as determined by Synthego’s CRISPR Design Tool (Synthego) against Prdm1 and Ahr were chosen. Precomplexing of Cas9/RNP was performed in a PCR tube by the addition of 0.2-0.4 µl of each 100 pmol sgRNAs (for a final volume of all sgRNAs 1.2 µl), 0.66 µl Cas9 (ThermoFisher, Cat. No, A36498), and 10.94 µl of the nucleofector P2 primary solution and 2.4 µl of supplement from the P2 Primary Cell 4D- Nucleofector X Kit S (Lonza, V4XP-2032) and the solution was incubated for 10 minutes at room temperature. Preincubated cells were subsequently spun down and resuspended in 20 µl of precomplexed Cas9/RNP and transferred to Nucleofection strips (Lonza, V4XP-2032). Cells were electroporated using a 4D nucleofector (Lonza, AAF-1002B, AAF-1002X) and pulsed using the DS137 setting. Following nucleofection, prewarmed 10% RPMI with 5 ng/ml IL-7 was used to transfer transfected cells to 96 well plates, for a final concentration of 1 x 106 cells per well in 200 µl for 72 hours. Cells subsequently underwent TRM-like differentiation as described above. v) Human CRISPR/Cas9 gene editing 177. After sorting, naïve CD8+ T cells were cultured overnight in complete medium at 37°C. Guide RNAs (gRNA) targeting BCL11B gene were formed by hybridizing tracrRNA (200 µM; IDT) and crRNA (200 µM; IDT) for 5 minutes at 95°C. Recombinant Cas9 (40 µM; MacroLab) was incubated with gRNA duplex for 10 minutes at 37°C at a 2:1 ratio to form CAS9 ribonucleoprotein complexes (RNPs). Cells were washed twice with PBS and resuspended in electroporation buffer (1 M; 5 mM KCl, 15 mM MgCl2, 120 mM Na2HPO4 pH7.2 and 50 mM mannitol) at a concentration of 10×106 cells/ml. Subsequently, 3 µl RNPs were added to 20 µl cell suspension per well and cells were electroporated in a 16 well cuvette (LONZA) using the Attorney Docket Number 10110-443WO1 pulse code CM137 (Amaxa). Immediately after electroporation, 200 µl of pre-warmed medium was added to each well and cells were transferred from the cuvette to a 96-U well plate. Cells were rested for 5 hours at 37°C in the incubator and subsequently stimulated with 100 U/ml IL-2 (Peprotech) and 10 µl/ml anti-CD3/28 immunocomplexes (StemCell Technologies) for 48 hours at 37°C. Cells were then prepared for T RM -like cell differentiation and cultured for 2 weeks with 10 µl/ml anti-CD3/20 immunocomplexes, 100 U/ml IL-2, 5 ng/ml TGF-β and 100 ng/ml IL-33 in RPMI 10% FCS, as described above. w) Murine RNA-seq library preparation and data processing 178. RNA was extracted from 2-5×104 sort-purified Bcl11b-/- (YFP+) or WT TE (Klrg1hiCD127lo), MP (Klrg1loCD127hi) or TRMP (CD69+CD103+) 9DPI using a RNeasy Micro Kit (Qiagen, 74034). Each sample represents 2-4 mice. rRNA was depleted using the NEBNext rRNA Depletion Kit (NEB, E63102) and cDNA libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (NEB, E7770S). Quality was assessed on a Bioanalyzer (Agilent, #5067-4626) and sequenced on a NextSeq 500 or NovaSeq 6000 to approximately 50 million reads per sample. RNA-seq data was processed using a previously established pipeline. In brief, fastq files were trimmed using seqtk and quality was assessed using FastQC. Trimmed reads were aligned to the mouse genome (mm10) using Hisat2 and transcript counts were obtained using FeatureCounts. DESeq2 was used for differential expression analysis. GSEA was performed using DRPPM-EASY with genes ranked based on signal-to-noise. The GSEA function was run under default parameters implemented by the clusterProfile package. Rlog values of all differentially expressed genes (padj < 0.05) calculated by DESeq2 were used for principal component analysis and to calculate Euclidean distances for hierarchical clustering using Ward’s method. x) Assay for transposase-accessible chromatin sequencing 179. ATAC-seq was performed on 30,000 sort-purified Bcl11b-/- (YFP+) and WT TRMPs 9 DPI. Briefly, cells were lysed in 50 µl cold lysis buffer (10mM Tris Cl, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% v/v IGEPAL CA-630) and centrifuged immediately at 500× g for 10 minutes. DNA was transposed using a Nextera Tn5 kit (Illumina, FC-121-1030) at 37°C for 30 minutes. DNA was purified using a MiniElute PCR Purification kit (Qiagen, Cat. No. 28004) and amplified using barcoded PCR primers. DNA was purified using SPRIselect magnetic beads (Beckman Coulter, Cat. No. B23318). Library quality and adapter dimer contamination were assessed on a Bioanalyzer (Agilent, Cat. No.5067-4626). Libraries were sequenced as PE100 on Illumina NovaSeq 6000. Attorney Docket Number 10110-443WO1 y) ATAC-seq, ChIP-seq and CUT&RUN data processing 180. Data analysis for ATAC-seq, ChIP-seq and CUT&RUN was performed using a customized pipeline. Two biological replicates were performed for each experiment and processed individually. Briefly, fastq files were first trimmed using Trimmomatic and read quality was assessed using FastQC. Reads were aligned to the mouse genome (mm10) using Bowtie2 (v.2.2.5) with the default parameters for ATAC-seq and ChIP-seq data and with the following parameters for CUT&RUN: --dovetail --local --very- sensitive-local --no-unal --no- mixed --no-discordant --phred33 -I 10 -X 700. The resulting SAM files were pruned (Q > 30), converted to BAM format, and sorted using SAMtools. Duplicate reads were removed using Picard tools. MACS2 was used for peak calling, with the following parameters for ATAC-seq, Bcl11b and H3K27ac ChIP-seq data: macs2 callpeak --call-summits --nomodel -g mm -p 0.01 -f BAM --SPMR -B. For Ahr ChIP-seq, -q 0.05 was used with no subsequent IDR filtering as described below. The following parameters were used for CUT&RUN: macs2 callpeak --call- summits -g mm -p 0.00001 -f BAMPE --SPMR -B. Bedgraph files produced by MACS2 were lambda control subtracted. A single representative file for each experiment was created as a mean of two biological replicates for ATAC and ChIP-seq experiments, whereas only one replicate of suitable quality was used for CUT&RUN samples. Resulting files were converted to bigWig format and used for visualization by GenomePaint. For ATAC-seq and ChIP-seq, irreproducible discovery rate (IDR) was performed on each pair of replicates and peaks with an IDR value ≤ 0.05 were kept. Due to technical challenges of Ahr ChIP-seq, no IDR filtering was performed for this dataset. A single replicate of suitable quality was used for CUT&RUN samples. Differential analysis was conducted using MAnorm with default parameters for ATAC and Bcl11b and H3K27ac ChIP-seq experiments and following parameters: --pe -w 1000; --pe -w 1000; and --pe -w 2000 for Ahr, Tcf1 and H3K4me3 experiments, respectively. Only peaks with P < 0.05 were used for downstream analyses and these were in some instances additionally filtered for |log2FC|>1. P value was adjusted for the FDR by the Benjamini-Hochberg adjustment. For additional analysis, peaks were annotated using annotatePeaks script from HOMER. Peaks were gene-annotated when they resided within ±10kb from its TSS and these were used for correlation and overlap analyses. Differential peaks determined by MAnorm were analyzed for known motif enrichment by HOMER, using the findMotifsGenome function with the following parameters: -size given -len 6,8,10,12 -mset vertebrates -mask. Only motifs which were enriched with P ≤ 0.001 in at least one dataset were considered for the analysis. Genome-wide analyses of binding patterns were performed using deepTools. Attorney Docket Number 10110-443WO1 E. SEQUENCES To CRISPR BCL11B in human melanoma TILs we used the following guides: Hs.Cas9.BCL11B.1.AA (SEQ ID NO: 1) CATGACTCGGTCGAAGGCAC combined in a single reaction with: Hs.Cas9.BCL11B.1.AE (SEQ ID NO: 2) GTGTCAAATGAACTTCCCCT F. References A. Banerjee, S. M. Gordon, A. M. Intlekofer, M. A. Paley, E. C. Mooney, T. Lindsten, E. J. Wherry, S. L. Reiner, Cutting edge: The transcription factor eomesodermin enables CD8+ T cells to compete for the memory cell niche. J Immunol 185, 4988-4992 (2010). A. C. Dolens, K. Durinck, M. Lavaert, J. Van der Meulen, I. Velghe, J. De Medts, K. Weening, J. Roels, K. De Mulder, P. J. Volders, K. De Preter, T. Kerre, B. Vandekerckhove, G. Leclercq, J. Vandesompele, P. Mestdagh, P. Van Vlierberghe, F. Speleman, T. Taghon, Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development. EMBO reports 21, (2020). A. Kallies, A. Xin, G. T. Belz, S. L. Nutt, Blimp-1 transcription factor is required for the differentiation of effector CD8(+) T cells and memory responses. Immunity 31, 283-295 (2009). A. M. Bolger, M. Lohse, B. Usadel, Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120 (2014). A. Obermayer, L. Dong, Q. Hu, M. Golden, J. D. Noble, P. Rodriguez, T. J. Robinson, M. Teng, A. C. Tan, T. I. Shaw, DRPPM-EASY: A Web-Based Framework for Integrative Analysis of Multi-Omics Cancer Datasets. Biology (Basel) 11, (2022). A. Xin, F. Masson, Y. Liao, S. Preston, T. Guan, R. Gloury, M. Olshansky, J. X. Lin, P. Li, T. P. Speed, G. K. Smyth, M. Ernst, W. J. Leonard, M. Pellegrini, S. M. Kaech, S. L. Nutt, W. Shi, G. T. Belz, A. Kallies, A molecular threshold for effector CD8(+) T cell differentiation controlled by transcription factors Blimp-1 and T-bet. Nat Immunol 17, 422-432 (2016). Attorney Docket Number 10110-443WO1 A. Zaid, L. K. Mackay, A. Rahimpour, A. Braun, M. Veldhoen, F. R. Carbone, J. H. Manton, W. R. Heath, S. N. Mueller, Persistence of skin-resident memory T cells within an epidermal niche. Proceedings of the National Academy of Sciences of the United States of America 111, (2014). B. A. Zabel, W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, E. C. Ebert, N. Kassam, S. Qin, M. Zovko, G. J. LaRosa, L. L. Yang, D. Soler, E. C. Butcher, P. D. Ponath, C. M. Parker, D. P. Andrew, Human G protein-coupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis. The Journal of experimental medicine 190, (1999). B. Langmead, S. L. Salzberg, Fast gapped-read alignment with Bowtie 2. Nature Methods 9, 357- 359 (2012). B. S. Sheridan, Q.-M. Pham, Y.-T. Lee, L. S. Cauley, L. Puddington, L. Lefrançois, Oral infection drives a distinct population of intestinal resident memory CD8(+) T cells with enhanced protective function. Immunity 40, 747-757 (2014). B. Slütter, N. Van Braeckel-Budimir, G. Abboud, S. M. Varga, S. Salek-Ardakani, J. T. Harty, Dynamics of influenza-induced lung-resident memory T cells underlie waning heterosubtypic immunity. Science Immunology 2, eaag2031-eaag2031 (2017). C. Berlin, E. L. Berg, M. J. Briskin, D. P. Andrew, P. J. Kilshaw, B. Holzmann, I. L. Weissman, A. Hamann, E. C. Butcher, Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74, (1993). C. N. Skon, J. Y. Lee, K. G. Anderson, D. Masopust, K. A. Hogquist, S. C. Jameson, Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells. Nat Immunol 14, 1285-1293 (2013). C. X. Dominguez, R. A. Amezquita, T. Guan, H. D. Marshall, N. S. Joshi, S. H. Kleinstein, S. M. Kaech, The transcription factors ZEB2 and T-bet cooperate to program cytotoxic T cell terminal differentiation in response to LCMV viral infection. J Exp Med 212, 2041-2056 (2015). D. Avram, A. Fields, K. Pretty On Top, D. Nevrivy, J. Ishmael, M. Leid, Isolation of a novel family of C(2)H(2) zinc finger proteins implicated in transcriptional repression mediated by Attorney Docket Number 10110-443WO1 chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan nuclear receptors. J Biol Chem 275, 10315-10322 (2000). D. Avram, A. Fields, T. Senawong, A. Topark-Ngarm, M. Leid, COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein. Biochem J 368, 555-563 (2002). D. Avram, D. Califano, The multifaceted roles of Bcl11b in thymic and peripheral T cells: impact on immune diseases. J Immunol 193, 2059-2065 (2014). D. Califano, J. J. Cho, M. N. Uddin, K. J. Lorentsen, Q. Yang, A. Bhandoola, H. Li, D. Avram, Transcription Factor Bcl11b Controls Identity and Function of Mature Type 2 Innate Lymphoid Cells. Immunity 43, 354-368 (2015). D. Califano, K. J. Sweeney, H. Le, J. VanValkenburgh, E. Yager, W. O'Connor, Jr., J. S. Kennedy, D. M. Jones, D. Avram, Diverting T helper cell trafficking through increased plasticity attenuates autoimmune encephalomyelitis. J Clin Invest 124, 174-187 (2014). D. I. Albu, D. Feng, D. Bhattacharya, N. A. Jenkins, N. G. Copeland, P. Liu, D. Avram, BCL11B is required for positive selection and survival of double-positive thymocytes. J Exp Med 204, 3003- 3015 (2007). D. I. Albu, J. Vanvalkenburgh, N. Morin, D. Califano, N. A. Jenkins, N. G. Copeland, P. Liu, D. Avram, Transcription factor Bcl11b controls selection of invariant natural killer T-cells by regulating glycolipid presentation in double-positive thymocytes. Proc Natl Acad Sci U S A 108, 6211-6216 (2011). D. J. Topham, E. C. Reilly, Tissue-Resident Memory CD8+ T Cells: From Phenotype to Function. Frontiers in Immunology 9, 515-515 (2018). D. Kim, J. M. Paggi, C. Park, C. Bennett, S. L. Salzberg, Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37, 907-915 (2019). D. Masopust, D. Choo, V. Vezys, E. J. Wherry, J. Duraiswamy, R. Akondy, J. Wang, K. A. Casey, D. L. Barber, K. S. Kawamura, K. A. Fraser, R. J. Webby, V. Brinkmann, E. C. Butcher, K. A. Newell, R. Ahmed, Dynamic T cell migration program provides resident memory within intestinal epithelium. The Journal of experimental medicine 207, 553-564 (2010). Attorney Docket Number 10110-443WO1 D. Masopust, V. Vezys, A. L. Marzo, L. Lefrancois, Preferential localization of effector memory cells in nonlymphoid tissue. Science 291, 2413-2417 (2001). E. C. Reilly, K. Lambert Emo, P. M. Buckley, N. S. Reilly, I. Smith, F. A. Chaves, H. Yang, P. W. Oakes, D. J. Topham, T RM integrins CD103 and CD49a differentially support adherence and motility after resolution of influenza virus infection. Proceedings of the National Academy of Sciences of the United States of America 117, (2020). E. J. Wherry, V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, R. Ahmed, Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol 4, 225-234 (2003). F. Cruz-Guilloty, M. E. Pipkin, I. M. Djuretic, D. Levanon, J. Lotem, M. G. Lichtenheld, Y. Groner, Rao, Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs. J Exp Med 206, 51-59 (2009). F. M. Behr, L. Parga-Vidal, N. A. M. Kragten, T. J. P. van Dam, T. H. Wesselink, B. S. Sheridan, R. Arens, R. A. W. van Lier, R. Stark, K. van Gisbergen, Tissue-resident memory CD8(+) T cells shape local and systemic secondary T cell responses. Nat Immunol 21, 1070- 1081 (2020). F. Ramirez, D. P. Ryan, B. Gruning, V. Bhardwaj, F. Kilpert, A. S. Richter, S. Heyne, F. Dundar, T. Manke, deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res 44, W160-165 (2016). F. Sallusto, D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia, Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708-712 (1999). G. Abboud, J. Stanfield, V. Tahiliani, P. Desai, T. E. Hutchinson, K. J. Lorentsen, J. J. Cho, D. Avram, S. Salek-Ardakani, Transcription Factor Bcl11b Controls Effector and Memory CD8 T cell Fate Decision and Function during Poxvirus Infection. Frontiers in immunology 7, (2016). G. Jeannet, C. Boudousquie, N. Gardiol, J. Kang, J. Huelsken, W. Held, Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory. Proc Natl Acad Sci U S A 107, 9777-9782 (2010). Attorney Docket Number 10110-443WO1 H. Hosokawa, M. Romero-Wolf, Q. Yang, Y. Motomura, D. Levanon, Y. Groner, K. Moro, T. Tanaka, E. V. Rothenberg, Cell type-specific actions of Bcl11b in early T- lineage and group 2 innate lymphoid cells. J Exp Med 217, (2020). H. Li, B. Handsaker, A. Wysoker, T. Fennell, J. Ruan, N. Homer, G. Marth, G. Abecasis, R. Durbin, The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079 (2009). H. Y. Shih, G. Sciume, Y. Mikami, L. Guo, H. W. Sun, S. R. Brooks, J. F. Urban, Jr., F. P. Davis, Y. Kanno, J. J. O'Shea, Developmental Acquisition of Regulomes Underlies Innate Lymphoid Cell Functionality. Cell 165, 1120-1133 (2016). J. A. Walker, C. J. Oliphant, A. Englezakis, Y. Yu, S. Clare, H. R. Rodewald, G. Belz, P. Liu, P. G. Fallon, A. N. McKenzie, Bcl11b is essential for group 2 innate lymphoid cell development. J Exp Med 212, 875-882 (2015). J. D. Buenrostro, B. Wu, H. Y. Chang, W. J. Greenleaf, ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol 109, 21.29.21-29 (2015). J. J. Cho, Z. Xu, U. Parthasarathy, T. T. Drashansky, E. Y. Helm, A. N. Zuniga, K. J. Lorentsen, S. Mansouri, J. Y. Cho, M. J. Edelmann, D. M. Duong, T. Gehring, T. Seeholzer, D. Krappmann, M. N. Uddin, D. Califano, R. L. Wang, L. Jin, H. Li, D. Lv, D. Zhou, L. Zhou, D. Avram, Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation. Nature communications 10, (2019). J. J. Milner, C. Toma, B. Yu, K. Zhang, K. Omilusik, A. T. Phan, D. Wang, A. J. Getzler, T. Nguyen, S. Crotty, W. Wang, M. E. Pipkin, A. W. Goldrath, Runx3 programs CD8(+) T cell residency in non- lymphoid tissues and tumours. Nature 552, 253-257 (2017). J. J. Milner, C. Toma, Z. He, N. S. Kurd, Q. P. Nguyen, B. McDonald, L. Quezada, C. E. Widjaja, D. Witherden, J. T. Crowl, L. A. Shaw, G. W. Yeo, J. T. Chang, K. D. Omilusik, A. W. Goldrath, Heterogenous Populations of Tissue-Resident CD8(+) T Cells Are Generated in Response to Infection and Malignancy. Immunity 52, 808-824 e807 (2020). J. Jacob, D. Baltimore, Modelling T-cell memory by genetic marking of memory T cells in vivo. Nature 399, 593-597 (1999). Attorney Docket Number 10110-443WO1 J. M. Schenkel, K. A. Fraser, L. K. Beura, K. E. Pauken, V. Vezys, D. Masopust, T cell memory. Resident memory CD8 T cells trigger protective innate and adaptive immune responses. Science 346, 98-101 (2014). J. Roels, A. Kuchmiy, M. De Decker, S. Strubbe, M. Lavaert, K. L. Liang, G. Leclercq, B. Vandekerckhove, F. Van Nieuwerburgh, P. Van Vlierberghe, T. Taghon, Distinct and temporary- restricted epigenetic mechanisms regulate human αβ and γδ T cell development. Nature immunology 21, (2020). J. W. Dean, E. Y. Helm, Z. Fu, L. Xiong, N. Sun, K. N. Oliff, M. Muehlbauer, D. Avram, L. Zhou, The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8+ T cell differentiation and function. Cell Reports 42, 111963 (2023). J. Wu, A. Madi, A. Mieg, A. Hotz-Wagenblatt, N. Weisshaar, S. Ma, K. Mohr, T. Schlimbach, M. Hering, H. Borgers, G. Cui, T Cell Factor 1 Suppresses CD103+ Lung Tissue-Resident Memory T Cell Development. Cell reports 31, (2020). K. A. Casey, K. A. Fraser, J. M. Schenkel, A. Moran, M. C. Abt, L. K. Beura, P. J. Lucas, D. Artis, E. J. Wherry, K. Hogquist, V. Vezys, D. Masopust, Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues. J Immunol 188, 4866- 4875 (2012). K. D. Klonowski, K. J. Williams, A. L. Marzo, D. A. Blair, E. G. Lingenheld, L. Lefrançois, Dynamics of Blood-Borne CD8 Memory T Cell Migration In Vivo. Immunity 20, 551-562 (2004). K. D. Omilusik, J. A. Best, B. Yu, S. Goossens, A. Weidemann, J. V. Nguyen, E. Seuntjens, A. Stryjewska, C. Zweier, R. Roychoudhuri, L. Gattinoni, L. M. Bird, Y. Higashi, H. Kondoh, D. Huylebroeck, J. Haigh, A. W. Goldrath, Transcriptional repressor ZEB2 promotes terminal differentiation of CD8+ effector and memory T cell populations during infection. J Exp Med 212, 2027-2039 (2015). K. J. Lorentsen, J. J. Cho, X. Luo, A. N. Zuniga, J. F. Urban, Jr., L. Zhou, R. Gharaibeh, C. Jobin, M. P. Kladde, D. Avram, Bcl11b is essential for licensing Th2 differentiation during helminth infection and allergic asthma. Nat Commun 9, 1679 (2018). L. K. Beura, J. S. Mitchell, E. A. Thompson, J. M. Schenkel, J. Mohammed, S. Wijeyesinghe, R. Fonseca, B. J. Burbach, H. D. Hickman, V. Vezys, B. T. Fife, D. Attorney Docket Number 10110-443WO1 Masopust, Intravital mucosal imaging of CD8+ resident memory T cells shows tissue- autonomous recall responses that amplify secondary memory. Nature Immunology 19, 173-182 (2018). L. K. Mackay, E. Wynne-Jones, D. Freestone, D. G. Pellicci, L. A. Mielke, D. M. Newman, A. Braun, F. Masson, A. Kallies, G. T. Belz, F. R. Carbone, T-box transcription factors combine with the cytokines TGF-β and IL-15 to control tissue-resident memory T cell fate. Immunity 43, 1101-1111 (2015). L. K. Mackay, M. Minnich, N. A. Kragten, Y. Liao, B. Nota, C. Seillet, A. Zaid, K. Man, S. Preston, D. Freestone, A. Braun, E. Wynne-Jones, F. M. Behr, R. Stark, D. G. Pellicci, D. I. Godfrey, G. T. Belz, M. Pellegrini, T. Gebhardt, M. Busslinger, W. Shi, F. R. Carbone, R. A. van Lier, A. Kallies, K. P. van Gisbergen, Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes. Science 352, 459-463 (2016). L. Li, M. Leid, E. V. Rothenberg, An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b. Science (New York, N.Y.) 329, 89-93 (2010). M. I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA- seq data with DESeq2. Genome Biology 15, 550 (2014). M. N. Uddin, D. A. Sultana, K. J. Lorentsen, J. J. Cho, M. E. Kirst, M. L. Brantly, D. Califano, D. B. Sant'Angelo, D. Avram, Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival. Proc Natl Acad Sci U S A 113, 7608- 7613 (2016). N. S. Joshi, W. Cui, A. Chandele, H. K. Lee, D. R. Urso, J. Hagman, L. Gapin, S. M. Kaech, Inflammation directs memory precursor and short-lived effector CD8(+) T cell fates via the graded expression of T-bet transcription factor. Immunity 27, 281-295 (2007). N. S. Kurd, Z. He, T. L. Louis, J. J. Milner, K. D. Omilusik, W. Jin, M. S. Tsai, C. E. Widjaja, J. N. Kanbar, J. G. Olvera, T. Tysl, L. K. Quezada, B. S. Boland, W. J. Huang, C. Murre, A. W. Goldrath, G. W. Yeo, J. T. Chang, Early precursors and molecular determinants of tissue- resident memory CD8(+) T lymphocytes revealed by single-cell RNA sequencing. Sci Immunol 5, (2020). Attorney Docket Number 10110-443WO1 N. Wagner, J. Löhler, E. J. Kunkel, K. Ley, E. Leung, G. Krissansen, K. Rajewsky, W. Müller, Critical role for beta7 integrins in formation of the gut-associated lymphoid tissue. Nature 382, (1996). P. Li, S. Burke, J. Wang, X. Chen, M. Ortiz, S. C. Lee, D. Lu, L. Campos, D. Goulding, B. L. Ng, G. Dougan, B. Huntly, B. Gottgens, N. A. Jenkins, N. G. Copeland, F. Colucci, P. Liu, Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion. Science 329, 85- 89 (2010). Q. Li, J. B. Brown, H. Huang, P. J. Bickel, Measuring reproducibility of high- throughput experiments. The Annals of Applied Statistics 5, 1752-1779 (2011). Q. Shan, X. Li, X. Chen, Z. Zeng, S. Zhu, K. Gai, W. Peng, H. H. Xue, Tcf1 and Lef1 provide constant supervision to mature CD8(+) T cell identity and function by organizing genomic architecture. Nat Commun 12, 5863 (2021). R. Fonseca, L. K. Beura, C. F. Quarnstrom, H. E. Ghoneim, Y. Fan, C. C. Zebley, M. C. Scott, N. J. Fares-Frederickson, S. Wijeyesinghe, E. A. Thompson, H. Borges da Silva, V. Vezys, B. Youngblood, D. Masopust, Developmental plasticity allows outside-in immune responses by resident memory T cells. Nat Immunol, (2020). R. L. Rutishauser, G. A. Martins, S. Kalachikov, A. Chandele, I. A. Parish, E. Meffre, J. Jacob, K. Calame, S. M. Kaech, Transcriptional repressor Blimp-1 promotes CD8(+) T cell terminal differentiation and represses the acquisition of central memory T cell properties. Immunity 31, 296-308 (2009). S. Ariotti, M. A. Hogenbirk, F. E. Dijkgraaf, L. L. Visser, M. E. Hoekstra, J. Y. Song, H. Jacobs, J. B. Haanen, T. N. Schumacher, T cell memory. Skin-resident memory CD8(+) T cells trigger a state of tissue-wide pathogen alert. Science 346, 101-105 (2014). S. C. Jameson, D. Masopust, Understanding Subset Diversity in T Cell Memory. Immunity 48, 214-226 (2018). S. Heinz, C. Benner, N. Spann, E. Bertolino, Y. C. Lin, P. Laslo, J. X. Cheng, C. Murre, H. Singh, C. K. Glass, Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Molecular cell 38, (2010). Attorney Docket Number 10110-443WO1 S. K. Bromley, S. Y. Thomas, A. D. Luster, Chemokine receptor CCR7 guides T cell exit from peripheral tissues and entry into afferent lymphatics. Nat Immunol 6, 895-901 (2005). S. Li, J. W. Bostick, J. Ye, J. Qiu, B. Zhang, J. F. Urban, Jr., D. Avram, L. Zhou, Aryl Hydrocarbon Receptor Signaling Cell Intrinsically Inhibits Intestinal Group 2 Innate Lymphoid Cell Function. Immunity 49, 915-928.e915 (2018). S. N. Hasan, A. Sharma, S. Ghosh, S. W. Hong, S. Roy-Chowdhuri, S. H. Im, K. Kang, D. Rudra, Bcl11b prevents catastrophic autoimmunity by controlling multiple aspects of a regulatory T cell gene expression program. Sci Adv 5, eaaw0706 (2019). S. Zhang, M. Rozell, R. K. Verma, D. I. Albu, D. Califano, J. VanValkenburgh, A. Merchant, J. Rangel-Moreno, T. D. Randall, N. A. Jenkins, N. G. Copeland, P. Liu, D. Avram, Antigen-specific clonal expansion and cytolytic effector function of CD8+ T lymphocytes depend on the transcription factor Bcl11b. J Exp Med 207, 1687-1699 (2010). T. D. Holmes, R. V. Pandey, E. Y. Helm, H. Schlums, H. Han, T. M. Campbell, T. T. Drashansky, S. Chiang, C. Y. Wu, C. Tao, M. Shoukier, E. Tolosa, S. Von Hardenberg, M. Sun, C. Klemann, R. A. Marsh, C. M. Lau, Y. Lin, J. C. Sun, R. Månsson, F. Cichocki, D. Avram, Y. T. Bryceson, The transcription factor Bcl11b promotes both canonical and adaptive NK cell differentiation. Science immunology 6, (2021). T. Gebhardt, P. G. Whitney, A. Zaid, L. K. Mackay, A. G. Brooks, W. R. Heath, F. R. Carbone, S. N. Mueller, Different patterns of peripheral migration by memory CD4+ and CD8+ T cells. Nature 477, 216-219 (2011). T. T. Drashansky, E. Helm, Z. Huo, N. Curkovic, P. Kumar, X. Luo, U. Parthasarathy, A. Zuniga, J. J. Cho, K. J. Lorentsen, Z. Xu, M. Uddin, S. Moshkani, L. Zhou, D. Avram, Bcl11b prevents fatal autoimmunity by promoting Treg cell program and constraining innate lineages in Treg cells. Sci Adv 5, eaaw0480 (2019). T. T. Drashansky, E. Y. Helm, N. Curkovic, J. Cooper, P. Cheng, X. Chen, N. Gautam, L. Meng, A. J. Kwiatkowski, W. O. Collins, B. G. Keselowsky, D. Sant'Angelo, Z. Huo, W. Zhang, L. Zhou, D. Avram, BCL11B is positioned upstream of PLZF and RORγt to control thymic development of mucosal-associated invariant T cells and MAIT17 program. iScience 24, (2021). Attorney Docket Number 10110-443WO1 V. Cismasiu, K. Adamo, J. Gecewicz, J. Duque, Q. Lin, D. Avram, BCL11B functionally associates with the NuRD complex in T lymphocytes to repress targeted promoter. Oncogene 24, 6753- 6764 (2005). V. Cismasiu, S. Ghanta, J. Duque, D. Albu, H. Chen, R. Kasturi, D. Avram, BCL11B participates in the activation of IL2 gene expression in CD4+ T lymphocytes. Blood 108, 2695-2702 (2006). W. Cui, Y. Liu, J. S. Weinstein, J. Craft, S. M. Kaech, An interleukin-21-interleukin-10- STAT3 pathway is critical for functional maturation of memory CD8+ T cells. Immunity 35, 792-805 (2011). X. Jiang, R. A. Clark, L. Liu, A. J. Wagers, R. C. Fuhlbrigge, T. S. Kupper, Skin infection generates non-migratory memory CD8+ TRM cells providing global skin immunity. Nature 483, 227-231 (2012). X. Zhou, J. Wang, J. Patel, M. Valentine, Y. Shao, S. Newman, E. Sioson, L. Tian, Y. Liu, S. W. Brady, D. Flasch, X. Ma, Y. Liu, R. Paul, M. N. Edmonson, M. C. Rusch, C. Li, S. J. Baker, J. Easton, J. Zhang, Exploration of Coding and Non-coding Variants in Cancer Using GenomePaint. Cancer Cell 39, 83-95 e84 (2021). Y. Li, S. Innocentin, D. R. Withers, N. A. Roberts, A. R. Gallagher, E. F. Grigorieva, C. Wilhelm, M. Veldhoen, Exogenous stimuli maintain intraepithelial lymphocytes via aryl hydrocarbon receptor activation. Cell 147, (2011). Y. Wakabayashi, H. Watanabe, J. Inoue, N. Takeda, J. Sakata, Y. Mishima, J. Hitomi, T. Yamamoto, M. Utsuyama, O. Niwa, S. Aizawa, R. Kominami, Bcl11b is required for differentiation and survival of alphabeta T lymphocytes. Nat Immunol 4, 533-539 (2003). Y. Zhang, T. Liu, C. A. Meyer, J. Eeckhoute, D. S. Johnson, B. E. Bernstein, C. Nusbaum, R. M. Myers, M. Brown, W. Li, X. S. Liu, Model-based analysis of ChIP-Seq (MACS). Genome Biol 9, R137 (2008). Z. Qiu, C. Khairallah, G. Romanov, B. S. Sheridan, Cutting Edge: Batf3 Expression by CD8 T Cells Critically Regulates the Development of Memory Populations. Journal of immunology (Baltimore, Md. : 1950) 205, (2020). Attorney Docket Number 10110-443WO1 Z. Shao, Y. Zhang, G. C. Yuan, S. H. Orkin, D. J. Waxman, MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets. Genome biology 13, (2012).

Claims

Attorney Docket Number 10110-443WO1 V. CLAIMS What is claimed is: 1. A method of treating a T cell mediated disease or condition in a subject comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity. 2. The method of claim 1, wherein the T cells comprise tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (MILs), chimeric antigen receptor (CAR) T cells, CD4 T cells, CD8 T cells, effector memory T cells (TEM), central memory T cells (TCM), peripheral memory T cells (TPM), or tissue-resident memory T cells (TRM). 3. The method of claim 1 or 2, wherein the agent that, reduces, decreases, or disrupts Bcl11b activity comprises a small molecule, antibody, nanobody, diabody, antibody fragment, antisense oligonucleotide, peptide, protein, siRNA, shRNA, lncRNA, mRNA, or miRNA. 4. The method of claim 1 or 2, wherein the Bcl11b expression is disrupted through CRISPR/cas9 mediated disruption of Bcl11b. 5. The method of any of claims 1-4, further comprising obtaining T cells from a donor source and disrupting Bcl11b expression in the T cells prior to administering the T cells to the subject. 6. The method of any of claims 1-5, wherein the T cell mediated disease comprises a cancer or an infectious disease. 7. The method of claim 6, wherein the cancer comprises ovarian cancer, colon cancer, or melanoma. 8. The method of claim 6, wherein the infectious disease is caused by a viral infection. 9. The method of claim 8, wherein the viral infection is an infection with a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Attorney Docket Number 10110-443WO1 Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2. 10. The method of claim 6, wherein the infectious disease is caused by a bacterial infection. 11. The method of claim 10, wherein the bacterial infection is an infection with a bacteria selected from the group consisting of Bacillus anthracis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Mycobacterium chimaera, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species, and Mycoplasma species. 12. The method of claim 6, wherein the infectious disease is caused by a fungal infection. 13. The method of claim 12, wherein the fungal infection is an infection with a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata. 14. The method of claim 6, wherein the infectious disease is caused by a parasitic infection. 15. The method of claim 14, wherein the parasitic infection is an infection with a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria Attorney Docket Number 10110-443WO1 fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica. 16. The method of any of claims 1-15, further comprising administering to the subject an agent that blocks MHC class 1 expression. 17. A method of increasing resident memory T cells at the site of infection or in a tumor microenvironment comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity. 18. A method of increasing of increasing NK cell activation in a tumor microenvironment or site of infection comprising administering to the subject an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in T cells; disrupting Bcl11b transcription or expression; or by administering to the subject T cells that have reduced Bcl11b activity. 19. A method of increasing the cytotoxicity of T cells specific for a disease or condition comprising contacting disease or condition specific T cells with an agent that inhibits, reduces, decreases, ablates, or disrupts Bcl11b activity in the T cells or disrupting Bcl11b transcription or expression; whereby a decrease, reduction, ablation, or disruption of Bcl11b activity or transcription or expression increases the cytotoxicity of disease or condition specific T cells. 20. A method of reducing exhaustion of T cells specific for a or condition comprising contacting disease specific T cells with an agent that inhibits, reduces, decreases, or disrupts Bcl11b activity in the T cells or disrupting Bcl11b transcription or expression; whereby a decrease, reduction, ablation, or disruption of Bcl11b activity or transcription or expression increases the cytotoxicity of disease or condition specific T cells.
PCT/US2024/026583 2023-04-28 2024-04-26 Bcl11b sustains multipotency and restricts effector programs of intestinal resident memory cd8+ t cells Pending WO2024227030A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363499203P 2023-04-28 2023-04-28
US63/499,203 2023-04-28

Publications (1)

Publication Number Publication Date
WO2024227030A1 true WO2024227030A1 (en) 2024-10-31

Family

ID=93257145

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/026583 Pending WO2024227030A1 (en) 2023-04-28 2024-04-26 Bcl11b sustains multipotency and restricts effector programs of intestinal resident memory cd8+ t cells

Country Status (1)

Country Link
WO (1) WO2024227030A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008006363A2 (en) * 2006-07-13 2008-01-17 Ernst-Moritz-Arndt-Universität Greifswald Treatment of t-cell carcinomas using bcl11b inhibitors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008006363A2 (en) * 2006-07-13 2008-01-17 Ernst-Moritz-Arndt-Universität Greifswald Treatment of t-cell carcinomas using bcl11b inhibitors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FORKEL HANNES, GRABARCZYK PIOTR, DEPKE MAREN, TROSCHKE-MEURER SASCHA, SIMM STEFAN, HAMMER ELKE, MICHALIK STEPHAN, HENTSCHKER CHRIS: "BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood αβ CD8+ T cells", ONCOIMMUNOLOGY, vol. 11, no. 1, 31 December 2022 (2022-12-31), XP093232635, ISSN: 2162-402X, DOI: 10.1080/2162402X.2022.2148850 *
JIANG ZHIWU, QIN LE, TANG YUOU, LIAO RUI, SHI JINGXUAN, HE BINGJIA, LI SHANGLIN, ZHENG DIWEI, CUI YUANBIN, WU QITING, LONG YOUGUO,: "Human induced-T-to-natural killer cells have potent anti-tumour activities", BIOMARKER RESEARCH, vol. 10, no. 1, 1 December 2022 (2022-12-01), XP093058552, DOI: 10.1186/s40364-022-00358-4 *

Similar Documents

Publication Publication Date Title
JP7258802B2 (en) Treatment of cancer using anti-CD19 chimeric antigen receptor
US20230013642A1 (en) Toxicity Management for Anti-Tumor Activity of CARs
KR20220104217A (en) CD19 and CD22 chimeric antigen receptors and uses thereof
CN107567336A (en) The therapeutic alliance of cancer
KR20200044889A (en) RAR selective agonists in combination with immunomodulators for cancer immunotherapy
JP2019512271A (en) T cell exhaustion state specific gene expression regulator and use thereof
KR20200029544A (en) Immunomodulatory retinoid and rexinoid compounds combined with immunomodulators for cancer immunotherapy
US20240401044A1 (en) Exploiting il33 secretion as a therapeutic target in cancer
US20240226168A1 (en) Engineered nk cells and uses thereof
US20220241333A1 (en) Modulation of t cell cytotoxicity and related therapy
JP2022553643A (en) Compositions and methods for treating cytotoxic T-cell resistant tumors
WO2024227030A1 (en) Bcl11b sustains multipotency and restricts effector programs of intestinal resident memory cd8+ t cells
WO2023201347A1 (en) Distinct cd8+ t cell programming in the tumor microenvironment contributes to sex bias in cancer
NL2035852B1 (en) Enhancement of t cell mediated therapies
US20230338423A1 (en) Targetable immune checkpoint for immunotherapy
WO2024059757A2 (en) Methods for reprograming exhausted t cells and boosting immune checkpoint blockade therapy for cancer
WO2025207498A1 (en) Compositions of kinase inhibitors and methods of using the same for chimeric antigen receptor therapy
JP2025526679A (en) Chimeric Antigen Receptor Domain
HK40069741A (en) Toxicity management for anti-tumor activity of cars
TW202325844A (en) Systems and methods for enhanced immunotherapies
BR122021024691B1 (en) USES OF A CELL EXPRESSING A CAR MOLECULE THAT BINDS CD19 IN COMBINATION WITH ONE OR MORE KINASE INHIBITORS

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24798091

Country of ref document: EP

Kind code of ref document: A1