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WO2024227030A1 - Bcl11b maintenant le caractère multipotent et limitant les programmes effecteurs de lymphocytes t cd8+ à mémoire résidente intestinale - Google Patents

Bcl11b maintenant le caractère multipotent et limitant les programmes effecteurs de lymphocytes t cd8+ à mémoire résidente intestinale Download PDF

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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
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cells
bcl11b
virus
species
expression
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Dorina AVRAM
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H Lee Moffitt Cancer Center and Research Institute Inc
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H Lee Moffitt Cancer Center and Research Institute Inc
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    • 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
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • 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
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
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    • 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.

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Abstract

Sont divulguées des compositions et des méthodes de traitement, d'inhibition, de réduction, de diminution et/ou de prévention d'un cancer, d'une infection microbienne, d'un cancer récurrent ou d'une infection récurrente par inhibition de Bcl11b.
PCT/US2024/026583 2023-04-28 2024-04-26 Bcl11b maintenant le caractère multipotent et limitant les programmes effecteurs de lymphocytes t cd8+ à mémoire résidente intestinale Pending WO2024227030A1 (fr)

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WO2008006363A2 (fr) * 2006-07-13 2008-01-17 Ernst-Moritz-Arndt-Universität Greifswald Traitement de malignomes à lymphocytes t

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WO2008006363A2 (fr) * 2006-07-13 2008-01-17 Ernst-Moritz-Arndt-Universität Greifswald Traitement de malignomes à lymphocytes t

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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 *

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