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WO2025221808A1 - Reprogrammation épigénétique efficace de lymphocytes t effecteurs dans des lymphocytes t régulateurs stables - Google Patents

Reprogrammation épigénétique efficace de lymphocytes t effecteurs dans des lymphocytes t régulateurs stables

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Publication number
WO2025221808A1
WO2025221808A1 PCT/US2025/024796 US2025024796W WO2025221808A1 WO 2025221808 A1 WO2025221808 A1 WO 2025221808A1 US 2025024796 W US2025024796 W US 2025024796W WO 2025221808 A1 WO2025221808 A1 WO 2025221808A1
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cells
cell
treg
effector
foxp3
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Xudong Li
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Tufts University
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Tufts University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system

Definitions

  • Treg cells BACKGROUND Regulatory T cells (Treg cells) expressing the master transcription factor Foxp3 are pivotal in maintaining tolerance towards self-antigens and harmless environmental antigens. Harnessing Treg cells for antigen-specific tolerization offers a promising approach to dampen autoimmunity while preserving normal immune function.
  • endogenous naturally occurring Treg cells nTregs, or nTreg cells
  • Treg cells na ⁇ ve CD4+ T cells
  • iTregs or iTreg cells induced Treg cells
  • Reprogramming autoreactive effector T cells into functional Tregs could provide a novel reservoir of Treg cells for antigen-specific tolerization.
  • the present disclosure provides compositions and methods for the efficient reprogramming of effector T cells into Treg cells.
  • One aspect provides a method of generating an Effector T cell Reprogrammed Treg (ER-Treg) cell ex-vivo.
  • the method comprises resting an antigen specific CD4+CD44+ effector T cell in a T cell growth medium culture for at least 3 days to 5 days, and culturing the effector T cell in the presence of a primary reprogramming cocktail and a surface coated with anti-CD3 and anti-CD28 antibodies to produce an ER-Treg cell.
  • the T cell growth medium may comprise a base media, L-glutamine, buffered saline, an antibiotic, sodium pyruvate, and serum.
  • the T cell growth medium may be supplemented with IL-7, neutralizing antibodies against IL- 12, neutralizing antibodies against IFN ⁇ , and/or neutralizing antibodies against IL-4.
  • the primary reprogramming cocktail may comprise the T cell growth medium supplemented with IL-2, TGF ⁇ , retinoic acid, neutralizing antibodies against IL-12, neutralizing antibodies against IFN ⁇ , and neutralizing antibodies against IL-4.
  • the primary reprogramming cocktail may also comprise a T cell stimulus such as anti-CD3 and anti-CD28 antibodies.
  • the reprogramming cocktail additionally comprises ascorbic acid.
  • the method further comprises culturing the effector T cells in a secondary reprogramming cocktail, wherein the secondary reprogramming cocktail comprises the T cell growth medium supplemented with IL-2, TGF ⁇ and ascorbic acid.
  • the secondary reprogramming cocktail may be further supplemented with neutralizing antibodies against IL- 12, neutralizing antibodies against IFN ⁇ , and neutralizing antibodies against IL-4.
  • the ER-Treg cells express Foxp3, CD25 and/or CTLA4.
  • the ER-Treg cell has decreased methylation of Foxp3, CNS2, IL2ra, Ctla4 and/or IKzf4 as compared to the effector T cell from which it was derived.
  • Another aspect provides a composition for the generation of ER-Tregs.
  • the composition may comprise the supplemented T cell growth medium, the supplemented primary reprogramming cocktail and/or the supplemented secondary reprogramming cocktail.
  • the composition comprises a base media, L-glutamine, buffered saline, an antibiotic, Sodium Pyruvate, serum, IL-7, neutralizing antibodies to IFN ⁇ , neutralizing antibodies to IL-4, neutralizing antibodies to IL-12, IL-2, TGF ⁇ , retinoic acid, and vitamin C.
  • the composition additionally comprises a T cell stimulus.
  • a further aspect provides a method of using the composition to reprogram a T lymphocyte.
  • a further aspect provides a method of treating an autoimmune disease using the ER- Treg cells described herein.
  • the method comprises administering the ER-Treg cells described herein, wherein the effector T cells are specific for an antigen that is an autoimmunity causing autoantigen.
  • Another aspect provides a method of decreasing an antigen specific immune response in a subject in need thereof.
  • the method comprises isolating antigen- specific CD4+ T cells or CD4+CD44+ and reprograming the antigen-specific CD4+ T cells or CD4+CD44+ into ER-Treg cells ex vivo using methods described herein and administering the ER-Treg cells to a subject in need thereof.
  • the antigen-specific CD4+ T cells or CD4+CD44+ are isolated from a subject and administered to the same subject.
  • the subject is diagnosed with Graft versus host disease, allergy, infectious disease, or autoimmune disease, or the subject is an organ transplant recipient.
  • BRIEF DESCRIPTION OF THE DRAWINGS The present technology can be better understood by reference to the following drawings. The drawings are merely exemplary to illustrate certain features that may be used singularly or in combination with other features and the present technology should not be limited to the embodiments shown.
  • Figures 1A-1E Epigenetic reprogramming of CD4 + T eff cells into T regs.
  • A Flow cytometry of IFN ⁇ and GM-CSF expression in CTV lo CD4 + Teff cells, cocultured for 3 days with T-cell-depleted splenocytes serving as antigen presenting cells (APCs), in the presence of MOG and the presence or absence of Foxp3 Thy1.1 R26 Cas9 ER-Tregs transduced with the retroviral vector (RV) expressing single guide RNA targeting Foxp3 (sgFoxp3) or the non-targeting sgRNA (sgNT).
  • B Flow cytometry of IL-10 and IL-17A expression in ER-T regs as in (A).
  • C Flow cytometry of IFN ⁇ and GM-CSF expression in CTV lo CD4 + Teff cells, cocultured for 3 days with APCs, in the presence of MOG and the presence or absence of Foxp3 Thy1.1 ER-T regs transduced with MigR1 empty vector or CD4 + T eff cells forced to express Foxp3 via retroviral transduction with MigR1-Foxp3.
  • D Flow cytometry of CD25 and CTLA-4 expression in MigR1-ER-T regs and MigR1-Foxp3 CD4 + T eff cells as in (C).
  • EAE was induced via MOG/CFA immunization in CD45.2 + mice with or without adoptive transfer of CD45.1 + Foxp3 Thy1.1 MOG/CFA-primed CD4 + Teff cells reprogrammed into ER-Tregs or forced to express Foxp3 via retroviral transduction (Foxp3-RV-Teff), administered one day prior to immunization.
  • E EAE disease curve.
  • F Flow cytometry analysis of the frequencies of spinal cord CD4 + T cells.
  • FIG. 1 Flow cytometry of Foxp3 expression in adoptively transferred ER-Tregs and Foxp3-RV CD4 + T eff cells in the spinal cord.
  • H Flow cytometry of the frequencies and numbers of ER- Tregs or FRV-Tregs in the spinal cord. Mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001, one-way ANOVA and Holm- ⁇ dák test in (A, C, E, and F) and unpaired two-sided t-test in (B, D, G, and H).
  • Figures 4A-4D Inheritance of autoantigen specificity contributes to superior suppressive function of ER-T regs as compared to induced T regs.
  • A Flow cytometry of Foxp3 expression in CTV lo CD4 + Tconv and nTreg cells 8 days after they were isolated from CD45.2 + Foxp3 Thy1.1 mice, labeled with CTV, and adoptively transferred into CD45.1 + Foxp3 Thy1.1 mice, which were subsequently immunized with MOG/CFA one day after adoptive transfer.
  • A-F RNA-seq analysis of the transcriptomes of ER-T regs , nT regs , and CD4 + T eff cells.
  • CD45.1 + Foxp3 Thy1.1 ER-Tregs were adoptively transferred into CD45.2 + Foxp3 Thy1.1 mice one day prior to MOG/CFA immunization.
  • the transcriptomes of transferred ER-T regs and host nTreg and CD4 + Teff cells were determined by bulk RNA-seq at 7 dpi.
  • A Principal component analysis of ER-T reg , nT reg , and CD4 + T eff transcriptomes.
  • B Volcano plot showing the differential expression of genes between ER-Tregs and Teff cells.
  • GSEA Gene Set Enrichment Analysis of the expression of T reg -specific genes in ER-T regs and nT regs .
  • D Volcano plot showing the differential expression of genes between ER-Tregs and nTregs.
  • E Normalized gene expression levels for selected lists of T helper genes in ER-T regs and nT regs .
  • F GSEA of the expression of indicated gene sets in ER-Tregs and nTregs.
  • G Flow cytometry of c-Maf and Ror ⁇ t expression in MOG/CFA-primed 2D2 nT regs and ER-T regs following in vitro activation in the presence of IL-2 for 3 days.
  • C Flow cytometry of Ror ⁇ t expression in transferred Tregs.
  • D Flow cytometric assessment of in vivo competitive fitness between 2D2 ER-T regs and nT regs retrovirally transduced with distinct fluorescent reporters, co-transferred at a 1:1 ratio into recipient mice one day prior to CFA/MOG immunization and analyzed 5 days post-immunization.
  • (F) Flow cytometry analysis of the frequencies (left) and numbers (right) of CD4 + T cells in the spinal cord.
  • (G) Total numbers of GM-CSF + CD4 + T cells in the spinal cord analyzed by flow cytometry.
  • (H) Flow cytometry of c-Maf and Ror ⁇ t expression in ER- Tregs in the draining LNs.
  • (I) Flow cytometry analysis of the frequencies and numbers of transferred ER-T regs in CD4 + T cells in the spinal cord (upper) and draining LNs (lower).
  • FIGS 8A-8E Cellularity and phenotypes of transferred ER-Tregs in mice with EAE.
  • A- B EAE was induced via MOG/CFA immunization in CD45.2 + mice with or without adoptive transfer of ER-Tregs reprogrammed from MOG/CFA-primed CD45.1 + Foxp3 Thy1.1 CD4 + Teff cells, administered one day prior to immunization.
  • (A) Flow cytometry analysis of the frequency and total number of host Tregs and transferred ER-T regs in the draining lymph nodes.
  • (B) Flow cytometry analysis of Ror ⁇ t and c- MAF expression in both host Tregs and ER-Tregs in the draining lymph nodes.
  • A T eff cells retrovirally transduced with a Foxp3 overexpression vector (FRV-T regs ) or ER-T regs were co-cultured with APCs and responder Teff in the presence of MOG for 3 days before flow cytometry analysis of IFN ⁇ , IL-17a, and GM-CSF expression.
  • B-F FRV-T regs or ER-T regs were transferred into CD45 congenically distinct mice. Recipients were immunized with MOG/CFA and cells were analyzed with flow cytometry 4 days after immunization.
  • ER-T regs Adoptive transfer of ER-T regs not only prevents autoimmune neuroinflammation in mice when administered pre-onset but also halts disease progression post-onset.
  • ER-Tregs outperform Foxp3-overexpressing T eff cells, T regs induced from na ⁇ ve precursors, and endogenous T regs in suppressing neuroinflammation. Without wishing to be bound by any theory, this superiority is attributed to their inherited autoantigen specificity and the retention of key transcriptional programs from parental Teff cells, which enhance their fitness in inflammatory niches and boost their suppressive potency.
  • This disclosure provides compositions and methods to reprogram an effector T cell. When naive T cells encounter a specific antigen, they undergo activation and differentiate into effector T cells.
  • Effector T cells are T lymphocytes that carry out the functions of an immune response. Effector T cells secrete cytokines and chemokines that recruit other immune cells, promote activation and differentiation to inflammatory phenotypes, and secrete effector molecules that eliminate pathogens. Effector T cells may be cytotoxic, helper, and regulatory and may include, but are not limited to CD4+, CD8+ Th1, Th2, Th9, Th17, Th22, Treg, Tfh, Thelper-like, ⁇ T cell, and NKT cells. Various cell surface markers known in the art can be used to identify effector T cells. The markers used will depend on the type of effector T cell. By way of example, and not limitation, markers of effector T cells may comprise CD4 and/or CD44.
  • effector T cells may be a CD4+ T cell or a CD4+CD44+ T cell.
  • effector T cells are reprogramed to have immunosuppressive functions and markers that are similar to regulatory T cells.
  • the effector T cell of the present disclosure may be an antigen specific effector T cell.
  • An antigen specific effector T cell has the ability to recognize and respond to a specific antigen presented by an antigen-presenting cell or a T cell that has been activated by a specific antigen.
  • the antigen may be derived from a pathogen such as a bacteria or virus, from a tumor or from normal tissue.
  • Regulatory T cells, or Treg cell are a type of CD4 + T cell.
  • Treg cells are a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity.
  • Forkhead box protein 3 (FoxP3) is the master transcription factor in regulating Treg cell development and function. FoxP3 activity is fine ⁇ tuned by its transcription, post ⁇ translational modifications and interaction partners.
  • Tregs-signature genes coding factors such as Il2ra (CD25), Ctla4 (CD152), Tnfrsf18 (GITR), Ikzf2 (Helios), and Ikzf4 (Eos), which are believed to play an important role in Tregs function.
  • DNA methylation is important to Treg cells, and some studies have shown more than 100 differentially methylated regions in Treg cells as compared to effector T cells. Tregs-specific hypomethylation of critical genes is a dynamic process during Tregs development.
  • Tregs-related genes such as Ctla4 exon 2, Foxp3 intron 1, Tnfrsf18 exon 5, Ikzf4 intron 1b, and Il2ra intron 1a
  • ER-Treg Effector T cell Reprogrammed Treg
  • Epigenetics describe changes in gene expression not caused by alterations in the DNA nucleotide sequence.
  • DNA methylation is a process of cytosine conversion into 5-methylocitosine catalyzed by specific enzymes called DNA methyltransferases (DNMTs), which use S- adenosylmethionine as a substrate.
  • DNMTs DNA methyltransferases
  • DNMTs are a group of enzymes—DNMT3A, DNMT3B, Atty Docket No.166118.01508 and DNMT1—that transfer the methyl group during de novo methylation or during cell replication. This reaction is presented at CpG islands, highly enriched in CG content. When CpG islands are methylated, it is believed that gene expression is silenced, and the gene is repressed. TET enzymes (Ten-Eleven Translocation family enzymes) are able to reverse the process and induce a demethylation process that leads to an open chromatin structure, and eventually to gene expression.
  • Methods One aspect of the present disclosure provides a method of generating an Effector T cell Reprogrammed regulatory T cell (ER-Treg).
  • An ER-Treg is an effector T cell that has been reprogramed through methods and compositions described herein to have properties of a Treg cell.
  • the method of generating an ER-Treg comprises resting an antigen specific effector T cell in a T-cell growth medium.
  • the antigen specific T effector cell may be rested from at least 2 to 6 days, such as for at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 6 days.
  • the antigen specific effector T cell may be rested for at least 3 days, at least 4 days, or at least 5 days.
  • the T-cell growth medium may comprise components that allow the maintenance of effector T cells.
  • the growth medium may comprise a base media, a source of L-glutamine, buffered saline, an antibiotic, Sodium Pyruvate, and serum.
  • the base media may include Roswell Park Memorial Institute (RPMI) medium
  • the saline buffer may include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
  • the serum may include fetal calf serum
  • the antibiotic may include penicillin and or streptomycin.
  • the glutamine may be used at a concentration of about 0.5mM to about 6mM, about 0.5mM to about 3mM, about 0.5mM to about 2mM, about 0.5mM to about 1mM, preferably at about 0.5mM, about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, preferably at about at 2mM.
  • the buffered saline may be used in a range of about 5mM to about 20mM, preferably about 5mM, about 10mM, about 15mM, or about 20mM, preferably about 10mM.
  • the antibiotic may be used in a range of about 50 units/mL to about 200units/mL, preferably about 100 units/mL.
  • the sodium pyruvate may be used in a range of about 0.5mM to about 2.5mM, preferably about 1mM.
  • the serum may be used at about 2.5% to about 10%, preferably about 5%.
  • the T cell growth medium may be supplemented with interleukin 7 (IL-7), neutralizing antibodies against IL-12, neutralizing antibodies against IFN ⁇ , and neutralizing antibodies against IL-4.
  • IL-7 may be used in a range of about 0.5ng/mL to about 5ng/mL, preferably about 2ng/mL.
  • the neutralizing antibodies against IFN ⁇ , IL-4, and IL-12 may be used in a Atty Docket No.166118.01508 range of about 2 ⁇ g/mL to about 25 ⁇ g/mL, preferably about 10 ⁇ g/mL.
  • a neutralizing antibody neutralizes the effect of the target of the antibody.
  • a neutralizing antibody to a cytokine can, for example, block the binding of the cytokine to its receptor.
  • the method of generating an ER-Treg may further comprise culturing the effector T cell in a presence of a primary reprogramming cocktail after resting in the supplemented T cell growth medium.
  • the primary reprogramming cocktail may comprise, the T cell growth medium supplemented with interleukin 2 (IL-2), transforming growth factor beta (TGF ⁇ ), and retinoic acid, and neutralizing antibodies against interleukin 12 (IL-12), neutralizing antibodies against interferon gamma (IFN ⁇ ), and/or neutralizing antibodies against interleukin 4 (IL-4).
  • IL-2 may be used at a concentration in a range of about 10 units/mL to about 6000 units/mL, or of about 10 units/mL to about 1000 units/mL, or of about 10 units/mL to about 1000 units/mL, preferably about 1000 units/mL.
  • the TGF ⁇ may be used in a range of about 0.1 ng/mL to about 10 ng/mL or about 0.1 ng/mL to about 5ng/mL, preferably about 5 ng/mL.
  • the retinoic acid may be used in a range of about 1nM to about 0.5 ⁇ M, preferably about 10nM.
  • the primary reprogramming cocktail may additionally comprise ascorbic acid. Ascorbic acid is also known as vitamin C, L-ascorbic acid, and ascorbate, and is an essential nutrient. Ascorbic acid can also act as a cofactor for DNA demethylases and promote DNA demethylation, including in the CNS2 region of Foxp3.
  • the ascorbic acid may be used in a range of about 1 ⁇ g/mL to about 500 ⁇ g/mL, of about 1 ⁇ g/mL to about 250 ⁇ g/mL, of about 1 ⁇ g/mL to about 100 ⁇ g/mL, preferably about 100 ⁇ g/mL.
  • the effector T cell may be cultured in the presence of a primary reprogramming cocktail and a means of activating, expanding and or proliferating T cells.
  • the means of activating, expanding, or proliferating the T cells may be stimulus, such as a surface coated with anti-CD3 and anti-CD28 antibodies.
  • the coated surface may comprise a bead surface in a culture dish or a culture dish coated with anti-CD3 antibody and anti-CD28 antibody.
  • soluble anti-CD3 and anti-CD28, or feeder cells may be used as stimulus to activate the effector T cells.
  • a specific antigen may be used to stimulate cells, for example, the antigen for which the ER-Treg cells are intended to be specific for or to target.
  • the effector T cells are cultured in the primary reprogramming cocktail in a range from at least 2 to at least 5 days, such as for at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days.
  • a new primary reprogramming cocktail is added.
  • the primary reprogramming cocktail may be replaced.
  • any amount of the reprogramming cocktail may be Atty Docket No.166118.01508 replaced, including, but not limited to 1 ⁇ 4, 1/3, 3 ⁇ 4, or 2/3 of the cocktail may be removed and replaced with new primary reprogramming cocktail.
  • the method of reprogramming effector T cells into ER-Treg cells comprises resting effector T cells in T cell growth culture in a growth media for about 3, about 4 days, or about 5 days, then stimulating the effector T cells in a primary reprograming cocktail in culture for about 3 or about 4 days, which generates primary stimulated ER-Treg cells.
  • the primary ER- Treg cells can also be stimulated again in the presence of a secondary reprogramming cocktail to generate secondary stimulated ER-Treg cells.
  • ER-Treg T cells are isolated from the primary reprogramming cocktail and the isolated cells are placed in the secondary reprogramming cocktail.
  • the ER-Treg cells of the present disclosure may be primary stimulated ER-Treg cells or secondary stimulated ER-Treg cells.
  • the secondary reprogramming cocktail may comprise T cell growth medium supplemented with IL-2, TGF ⁇ , and ascorbic acid.
  • the secondary reprogramming cocktail may be further supplemented with neutralizing antibodies against IL-12, neutralizing antibodies against IFN ⁇ , and neutralizing antibodies against IL-4.
  • the secondary reprogramming cocktail may comprise components at any range disclosed herein.
  • the IL-2 is preferably about 1000 units/mL
  • the TGF ⁇ is at about 2ng/mL
  • the and ascorbic acid is used at about 10 ⁇ g/mL in the secondary programming cocktail.
  • the ER-Treg cells of the present disclosure may have molecular and or genetic markers of Treg cells.
  • the ER-Treg cells may express Foxp3.
  • the ER-Treg cells may also express CD25 and or CTLA4.
  • the markers on the ER-Tregs may depend on the original cell type they were reprogramed from.
  • the ER-Tregs express makers that are different from cells with induced Foxp3 express and from naturally occurring Treg cells.
  • the ER-Treg may have reduced expression of quiescent T cells, for example, ER-Tregs cells may have decreased expression of Lef1, Ccr7, Bach2 and/or Sell as compared to the effector T cell from which they were derived or from naturally occurring Treg cells.
  • ER-Treg cells may express genes associated with Th17 differentiation, effector Treg cells, the IL-23 pathway, the SMAD2/3 pathway, or any combination thereof.
  • the ER-Tregs cells may have reduced expression of genes associated with IL-2 Stat5 signaling as compared to the effector T cells from which they were derived.
  • the ER-Treg cells have increased expression of Ctla4, IL-10, RORC, Maf, IL-23r, CCr6, and/or IL-1r as compared to naturally occurring Treg cells.
  • Atty Docket No.166118.01508 The inventors have further found that ER-Treg cells generated via the methods described herein express Lrrc32 encoding GARP (Glycoprotein A Repetitions Predominant), Itgae encoding ITGAE (Integrin Subunit Alpha E), Gpr15 encoding GPR15 (G Protein- Coupled Receptor 15), Il2ra encoding CD25, and Ctla4 encoding CTLA-4 higher than effector T cells.
  • GARP Glycoprotein A Repetitions Predominant
  • ITGAE Integrin Subunit Alpha E
  • Gpr15 encoding GPR15 G Protein- Coupled Receptor 15
  • Il2ra encoding CD25 and Ctla4 encoding CTLA
  • one or more of these markers may be used to distinguish ER- Treg cells from effector T cells.
  • the ER-Treg have changes in epigenetic patterns that are similar to Treg cells.
  • ER-Tregs may have decreased methylation of Foxp3, Foxp3 CNS2, IL2ra, Ctla4 and/or IKzf4 as compared to the effector T cell from which it was derived.
  • the methods of generating an ER-Treg cells described herein differ from methods used to differentiate induced Treg cells in vitro. In vitro differentiated iTregs cells are antigen- na ⁇ ve CD4+ T cells, which are CD4+ Foxp3- CD44lo CD62hi cells.
  • the cells used to generate ER-Treg cells of the present disclosure are effector and or memory CD4+ T cells which are CD4+ Foxp3- CD44hi cells.
  • Induced Treg differentiation conditions include stimulation na ⁇ ve CD4+ cells with plate or bead, coated anti-CD3/CD28 presence of IL-2, TGF-beta, and neutralizing antibodies against IL-12, IL-4 and IFN ⁇ . These conditions convert almost all Na ⁇ ve CD4+ T cells into Foxp3+ iTregs. However, these conditions only convert ⁇ 13.6% of Teff cells into Foxp3 positive cells, as shown in Figure 1A of the present disclosure.
  • the ER-Treg cell reprogramming method of the present disclosure converts greater than 50% of effector T cells into ER-Treg cells with stable Foxp3 expression.
  • the Foxp3 expression levels in ER-Treg cells, nTreg cells, and iTreg cells are similar. Further, ER-Treg cells exhibit similar stability of Foxp3 expression as nTreg cells. However, differentiated iTreg cells exhibit unstable Foxp3 expression.
  • ER-Treg cells generated by the methods described herein exhibit intermediate expression levels of Helios and Nrp1, which is lower than levels in nTreg cells but higher than levels found in iTreg cells.
  • the ER-Treg cells may retain some lineage markers from the effector T cell from which they were derived.
  • an effector T cell may be a Th17 cell.
  • the Th17 cell may express ROR ⁇ T, IL-1r1, Maf, TGF ⁇ , IL-6r, IL-21R, IL-23R, CCR6, and/or STAT3.
  • the ER-Treg cell may express one or more Th17 markers and may also express one or more Treg cell marker.
  • the ER-Treg cells generated by the methods described herein are distinct from the effector T cells from which they were derived and from naturally occurring Treg cells and induced Treg cells.
  • Atty Docket No.166118.01508 The methods described herein may be used to stably activate Foxp3 expression and confer regulatory functions to other types of cells in addition to CD4+ effector T cells.
  • gamma-delta ( ⁇ ) T cells or CD8+ T cells can express Foxp3 and exhibit regulatory functions.
  • genomic editing for example CRISPR-Cas based methods, may be used to further enhance the activation and efficiency of Foxp3 in some cell types.
  • antigen specific effector T cells from an immunized subject may be used, steady-state effector T cells, or in-vitro differentiated Th1, Th2 or Th17 cells may be used in the methods described herein to generate ER-Treg cells.
  • the antigen specific effector T cell is specific for an autoantigen.
  • An autoantigen may also be called a self-antigen.
  • Autoantigens are any molecule or chemical group of an organism which acts as an antigen in inducing antibody formation or T cell activation in another organism but to which the healthy immune system of the parent organism is tolerant.
  • An immune response to autoantigens can result in autoimmunity or the autoimmune disease.
  • the autoantigen may be any antigen that causes autoimmunity.
  • the autoantigen may be a myelin antigen, a neuron-derived antigen, or an astrocyte-derived antigen.
  • antigens include, but are not limited to myelin basic protein (MBP), proteolipid protein (PLP), and the glial cell adhesion molecule GlialCAM.
  • Another aspect of the present disclosure comprises a method of treating an autoimmune disease.
  • the method comprises administering ER-Treg cells.
  • the ER-Treg cells have been reprogramed from effector T cells which are specific for an antigen that is specific for an autoimmunity causing autoantigen.
  • Effector T cells may be isolated from blood or diseased tissue.
  • effector T cells may be isolated from cerebrospinal fluid for the treatment of multiple sclerosis, or from inflamed joint tissue for the treatment of rheumatoid arthritis.
  • ER-Treg cells of the present disclosure may be administered by any means known in the art. For example, using the methods to administer any cell based therapy. Routes of administration may include, without limitation, systemic administration, for example intravenous administration or local administration such as injection into the effected tissue.
  • An autoimmune disease is a condition in which the body’s immune system mistakes its own healthy tissues as foreign and reacts to it.
  • the autoimmune disease may be one associated with myelin degeneration or demyelination, for example, multiple sclerosis.
  • autoimmune diseases including, but are not limited to, myasthenia gravis, ankylosing spondylitis, neuromyelitis optica, rheumatoid arthritis, bullous pemphigoid, narcolepsy, Hashimoto Atty Docket No.166118.01508 thyroiditis, Graves disease dermatitis herpetiformis, type I diabetes, hemolytic anemia, thrombocytopenic purpura, Goodpasture’s syndrome, pernphigus vulgaris, acute rheumatic fever, systemic lupus erythematosus, celiac disease or vitiligo.
  • “treat”, “treating”, “treatment”, “therapy” and/or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible.
  • the aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.
  • the term "effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • Another aspect of the present disclosure provides a method of decreasing an antigen- specific immune response in a subject. In some embodiments, the method comprises administering the ER-Treg cells described herein to a subject in need.
  • the ER-Treg cells control the antigen specific immune response.
  • the ER-Teg cells have been reprogramed from antigen specific effector T cells, wherein the effector T cells are specific for the antigen causing the immune response in the subject.
  • the method comprises isolating an antigen-specific CD4+ T cell or CD4+CD44+ T cell, reprograming the antigen-specific CD4+ T cell or CD4+CD44+ T cell into ER-Treg cells ex vivo using the methods the methods as described herein, and administering the ER- Treg cells to a subject in need thereof.
  • the antigen-specific T cell may be isolated from the subject and administered to the same subject.
  • the subject may be diagnosed with Graft vs. host disease, allergy, infectious disease, or autoimmune disease, or the subject is an organ transplant recipient.
  • the effector T cells are specific for an antigen causing Graft vs. host disease, an allergy, an autoimmune reaction, or organ transplant rejection.
  • a “subject in need thereof” as utilized herein may refer to a subject in need of treatment for an antigen-specific immune response.
  • a subject in need thereof may include a subject having or suspected of having an autoimmune disease or reaction.
  • the term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non- human mammalian subjects.
  • subject refers to both mammals and non-mammals.
  • “Mammals” include any member of the class Mammalia, such as humans, non- human primates (e.g., chimpanzees, other apes and monkey species), farm animals (e.g., cattle, horses, sheep, goats, and swine), domestic animals (e.g., rabbits, dogs, and cats), and laboratory Atty Docket No.166118.01508 animals (e.g., rats, mice, and guinea pigs).
  • the term “subject” does not denote a particular age or sex. In one embodiment, the subject is a human.
  • the subject is diagnosed with hyperinflammation, a hypersensitivity reaction, cytokine storm, or a dysregulated immune response.
  • administering and “administration” refer to any method of providing a pharmaceutical preparation or composition to a subject comprising the one or more ER-Treg cells described herein. Such methods are well known to those skilled in the art and include, but are not limited to, transdermal administration, administration by inhalation, nasal administration, and parenteral administration, including injectable such as intramuscular administration, intradermal administration, and subcutaneous administration.
  • the term “decrease” or the related terms “decreased,” “reduce” or “reduced” refers to a statistically significant decrease.
  • compositions for the generation of ER-Treg cells.
  • the compositions may comprise the supplemented T-cell growth medium, and/or a supplemented primary or supplemented secondary reprogramming cocktail, as described herein.
  • the composition comprises a base media, L-glutamine, buffered saline, an antibiotic, Sodium Pyruvate, serum, IL-7, neutralizing antibodies to IFN ⁇ , neutralizing antibodies to IL-4, neutralizing antibodies to IL-12, IL-2, TGF ⁇ , retinoic acid, and vitamin C.
  • alternative means to activate Foxp3, such as CRISPR-based tools may be used, and may replace the use of retinoic acid and/or TGF ⁇ .
  • the composition may additionally comprise a T cell stimulus.
  • the T cell stimulus may be any stimulus that activates, and/or induces proliferation of a T cell.
  • the T cell stimulus may comprise anti-CD3 and/or CD28 antibodies.
  • Various T cell stimuli are commercially available.
  • the T cell stimulus may be comprised as part of a bead added in culture, as a culture plate bound stimulus, a feeder cell, or may be a soluble stimulus, as described herein.
  • the T cell stimulus may also be a specific, target antigen.
  • the composition may be used to reprogram an effector T cell, for example a CD4+CD44+ T cell into an ER-Treg cell.
  • the CD4+CD44+ effector T cell may be cultured in the primary reprogramming cocktail composition for a range of at least 2 days to at least 5 Atty Docket No.166118.01508 days, such as for at least 2 days, at least 3 days, at least 4 days, or at least 5 days.
  • the composition may also include a T cell stimulus as described herein.
  • the effector T cell may be rested prior to culturing in the composition, for example in the T cell growth medium culture composition.
  • the effector T cell may be placed in the composition more than one time.
  • the effector T cell may be placed in the primary reprogramming cocktail composition along with a stimulus for at least 2-5 days, for example at least 3 days or at least 4 days, to generate a primary stimulated ER-Treg cell, and then a secondary reprogramming composition and stimulus may be added to generate a secondary ER-Treg cell.
  • Kits In another aspect, the present disclosure provides kits for the reprogramming of effector T cells into ER-Treg cells.
  • the kit may comprise compositions described herein.
  • the kit may comprise an antigen specific CD4+CD44+ effector T cell, a T-cell growth medium as described herein, and a primary and secondary reprogramming cocktail as described herein.
  • the kit may also comprise supplements for the growth medium and/or the reprogramming cocktails, including neutralizing antibodies against IL-12, neutralizing antibodies against IFN ⁇ , and/or neutralizing antibodies against IL-4 and IL-7.
  • the kit may also include a T cell stimulus, for example an anti-CD3 and/or CD28 antibody. Additional definitions The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein.
  • compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows.
  • the phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims.
  • Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
  • the terms “a”, “an”, and “the” mean “one or more.”
  • a molecule should be interpreted to mean “one or more molecules.”
  • “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ⁇ 10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
  • the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
  • the terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims.
  • the term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
  • Example 1 In the following example, the inventors describe compositions and methods of use to reprogram effector T cells into immunosuppressive ER-Treg cells. These ER-Treg cell can prevent disease when administered pre-onset, and halt disease progression when administered Atty Docket No.166118.01508 post-onset. These ER-Tregs are more immunosuppressive than FoxP3 overexpressing T cells or induced Tregs from naive precursors.
  • Treg Regulatory T cells expressing the transcription factor Foxp3 play an essential role in immune homeostasis by preventing autoimmunity against self-antigens and curtailing deleterious immune responses towards environmental antigens (1, 2).
  • endogenous T regs nT regs
  • Treg functional deficiency can arise from either inadequate Treg suppressor function on a per-cell basis or a paucity of autoantigen specific T regs .
  • Tregs cellular identity and suppressive function depends on the stable expression of the transcription factor Foxp3, along with Foxp3-independent core identity genes (15-19).
  • pre-exist Teff gene expression may antagonize Treg identity establishment (20), raising concerns that residual effector signatures could destabilize lineage commitment or impair suppressor activity in reprogrammed Treg populations.
  • Teff gene expression when combined with the reprogramming process—could actually enhance suppressor function by imparting an effector Treg-like gene expression profile, where heightened expression of specific T eff genes correlates with superior suppressive capabilities (21-34).
  • Treg cytosine- guanine
  • CpG cytosine- guanine
  • CpG cytosine- guanine
  • pharmacologically enhancing DNA demethylation in pre-existing T regs augments their fitness and function and accelerates repair of experimental lung injury (43).
  • CNS2 non- coding sequence 2
  • ER-Tregs The autoantigen specificity inherited by ER-Tregs from autoreactive CD4 + Teff cells enables antigen-specific suppression of autoimmune inflammation without compromising normal immune function. Additionally, the selective inheritance of parental Teff gene expression confers ER-Tregs superior fitness and suppressor functionality under inflammatory conditions. Thus, epigenetic activation of Treg identity genes in autoreactive CD4 + Teff cells can establish a bona fide Treg gene expression program, giving rise to T regs capable of quelling established autoimmune inflammation. Atty Docket No.166118.01508 RESULTS Epigenetic reprogramming enables stable induction of Foxp3 and other core T reg identity genes Foxp3 expression is essential for establishing T reg cellular identity (17-19).
  • CD4 + Teff cells from mice with experimental autoimmune encephalomyelitis (EAE)
  • EAE experimental autoimmune encephalomyelitis
  • CD4 + Foxp3-Thy1.1 ⁇ CD44 hi Teff cells from Foxp3 Thy1.1 reporter mice (50) immunized with myelin oligodendrocyte glycoprotein peptide (MOG) emulsified in complete Freund’s adjuvant (CFA).
  • MOG myelin oligodendrocyte glycoprotein peptide
  • ER-T regs comprised only about 2% of all T regs in the draining lymph nodes (Figure 8A), they exhibited a significantly higher frequency of ROR ⁇ t+ and ROR ⁇ t+c-MAF+ populations compared to endogenous T regs ( Figure 8B).
  • This distinct phenotypic profile combined with their scarcity, collectively suggests that ER-Tregs possess enhanced per-cell suppressive potency compared to endogenous T regs under inflammatory conditions.
  • To assess the suppressive capacity of ER-T regs in established EAE we adoptively transferred ER-Tregs derived from MOG/CFA-primed CD4 + Teff cells into CD45 congenically distinct mice at disease onset (clinical score ⁇ 1).
  • ER-T reg transfer attenuated disease progression (Figure 2D) and significantly reduced spinal cord infiltration by GM-CSF- producing CD4+ T eff cells (Figure 2E), a key driver of neuroinflammation (57-59).
  • Detectable ER-Tregs were observed in only two recipients, likely reflecting the contraction of the transferred population as inflammation resolved. In these mice, ER-Tregs accounted for approximately 20% of spinal cord Foxp3+ cells (Figure 8C). Strikingly, spinal cord ER-Tregs exhibited a significantly higher frequency of ROR ⁇ t+ and ROR ⁇ t+c-MAF+ subsets compared to endogenous T regs ( Figure 8D), further underscoring their distinct phenotype.
  • ER-T regs lacked expression of inflammatory cytokines IFN ⁇ or GM-CSF, although about 20% produced IL-17A (Figure 8E).
  • Foxp3-expressing Teff cells failed to suppress GM-CSF production in responder CD4+ Teff cells upon MOG stimulation. Instead, they amplified inflammatory responses, likely due to their inherently elevated expression of pro-inflammatory cytokines (Figure 3C; Figure 9A). Furthermore, these cells exhibited markedly reduced expression of critical Treg effector molecules, including CD25 and CTLA-4, which are encoded by TSDR- containing Il2ra and Ctla4, respectively (61, 62) ( Figure 3D). To evaluate the in vivo relevance of these findings, we compared the therapeutic efficacy of Foxp3-expressing Teff cells with that of ER-Tregs in EAE.
  • Foxp3-expressing Teff cells displayed reduced splenic engraftment, lower proliferation (as indicated by Ki-67 staining), and diminished expression of Helios and CD25 compared to ER-Tregs (Figure 9B- F).
  • Helios promotes T reg stability and survival (63, 64) and CD25 enhances IL-2– dependent survival and function (62, 65, 66)
  • the diminished suppressive capacity of Foxp3- expressing T eff cells thus likely results from inadequate epigenetic priming of critical genes (e.g., Helios, Il2ra), thereby compromising their resilience in inflammatory environments.
  • the diminished suppressive capacity of autologous iT regs may reflect diminished autoantigen specificity, as inflammatory conditions preferentially drive the differentiation of autoreactive Tn cells into Teff cells.
  • ER-T regs suppress EAE in an autoantigen-specific manner without inhibiting immune response against a non-myelin foreign antigen
  • MOG-specific ER-T regs Transfer of MOG-specific ER-T regs , but not their nonspecific counterparts, significantly attenuated EAE severity and reduced spinal cord infiltration by GM-CSF+ CD4+ T eff cells (Figure 5, A and B). Moreover, MOG-specific ER-Tregs exhibited enhanced tissue fitness, as evidenced by substantially higher frequencies in the spinal cord (Figure 5C), despite comparable splenic engraftment ( Figure 10A).
  • ER-T regs selectively inherit parental T eff gene expression
  • RNA-seq on ER-Tregs, nTregs, and CD4+ Teff cells—all isolated from MOG/CFA-immunized mice to ensure uniform in vivo exposure.
  • Principal component analysis revealed that the ER-Treg transcriptome more closely resembles that of nTregs than CD4+ Teff cells ( Figure 6A).
  • ER-Tregs expressed significantly higher levels of core T reg identity genes, including Foxp3, Itgae, Il2ra, Ctla4, and Ikzf4 (22), compared to CD4 + Teff cells (Figure 6B).
  • GSEA Gene set enrichment analysis
  • ER-Tregs Comparative transcriptomic analysis of ER-Tregs and nTregs reveals that ER-Tregs exhibit reduced expression of genes linked to T cell quiescence, such as Lef1, Ccr7, Bach2, and Sell, and increased expression of Treg effector genes, including Ctla4, and Il10 (69), as well as Th17- associated genes like Rorc, Maf, Il23r, Ccr6, and Il1r1 (70, 71) ( Figure 6D). Moreover, ER- Tregs express high levels of Th17 markers but not those typical of Th1 or Th2 cells (Figure 6E).
  • GSEA further indicates that ER-T regs upregulate genes involved in effector T reg function, Th17 differentiation, and the IL-23 pathway (Figure 6F), supporting the notion of enhanced Th17 polarization in ER-T regs derived from MOG/CFA-primed Teff cells. Additionally, the observed amplification of SMAD2/3 signaling in ER-Tregs suggests that TGF- ⁇ in the reprogramming cocktail significantly contributes to their unique gene expression program.
  • ER-Tregs bearing the MOG specific 2D2 TCR express higher levels of c- Maf and Ror ⁇ t compared to nT regs with the same 2D2 TCR (72) (Figure 6G), indicating enhanced Th17 polarization in myelin autoantigen-specific ER-Tregs.
  • Figure 6G 2D2 TCR
  • ER-T regs reprogrammed from Th17 cells exhibited significantly higher Ror ⁇ t expression than those reprogrammed from Th1 cells, Th2 cells, or iT regs (Figure 6H), suggesting that the inheritance of parental Th17 characteristics drives the elevated expression of selective Th17 genes in ER-Tregs. Elevated expression of Th17 genes contributes to ER-T reg fitness and function in EAE Adoptive transfer of a limited number of ER-Tregs (2 ⁇ 106 per mouse) into lymphoreplete mice harboring endogenous nT regs significantly ameliorated EAE (Figure 2), demonstrating the superior suppressive capacity of ER-Tregs over endogenous nTregs.
  • ER-Tregs exhibited significantly greater accumulation and elevated Ror ⁇ t expression in the spinal cord compared to nTregs ( Figure 7B–C), suggesting that retained Th17-associated transcriptional programming enhances their tissue fitness.
  • ER-Tregs exhibited significantly greater accumulation and elevated Ror ⁇ t expression in the spinal cord compared to nTregs ( Figure 7B–C), suggesting that retained Th17-associated transcriptional programming enhances their tissue fitness.
  • Th17-related gene expression in ER-Treg fitness we performed a competitive fitness assay using ER-T regs and nT regs from 2D2 MOG-specific mice. Both cell types were cultured under identical reprogramming conditions, transduced with distinct fluorescent reporters, and co-transferred at a 1:1 ratio into MOG/CFA-immunized mice.
  • T regs acquire their identity through tolerogenic signals that imprint T reg - specific transcriptional and epigenetic programs onto na ⁇ ve precursors.
  • T eff cells which retain inflammatory epigenetic memory
  • TGF- ⁇ drives Foxp3 expression via Smad3 binding to the conserved noncoding sequence 1 (CNS1) enhancer of the Foxp3 locus (73).
  • RA amplifies this process by enhancing TGF- ⁇ /Smad3 signaling while concurrently suppressing inflammatory pathways: it downregulates IL-6 and IL-23 receptor expression, neutralizes cytokine-mediated inhibition of Foxp3, and reduces pro-inflammatory cytokine secretion by T eff cells (74-77).
  • VC stabilizes Foxp3 expression by promoting TET enzyme- dependent DNA demethylation at TSDRs, including the Foxp3 locus itself (38, 39, 78).
  • Stable Foxp3 induction further requires re-stimulation, which sustains transcriptional activation of Foxp3 and facilitates demethylation of the CNS2 enhancer (49), establishing a self-reinforcing Atty Docket No.166118.01508 loop to stabilize Treg identity.
  • our reprogramming approach recapitulates the epigenetic remodeling observed in endogenous T regs , inducing robust DNA demethylation at TSDRs of key Treg identity genes such as Ctla4, Il2ra, and Ikzf4.
  • MOG/CFA immunization destabilizes MOG-specific nT reg lineage commitment (7), likely impairing their suppressive capacity upon isolation and transfer.
  • This instability coupled with intrinsic limitations in effector T reg differentiation, may explain nTreg inefficacy in our model and prior studies (87).
  • Future studies reprogramming destabilized nT regs could clarify their functional potential in EAE suppression.
  • ER-Tregs reprogrammed from MOG/CFA-primed CD4+ Teff cells exhibit a unique transcriptional profile, characterized by elevated expression of Th17-associated genes (Rorc, Maf) compared to nTregs.
  • T regs expressing lineage-specific transcription factors e.g., Th1, Th17
  • T regs expressing lineage-specific transcription factors display enhanced suppression of corresponding Teff subsets (23-30)
  • CTLA-4 is a key mediator of T reg suppression via CD80/CD86 downregulation on APCs through transendocytosis (90-93).
  • Future studies will delineate how ER-Tregs extrinsically modulate APCs and other immune populations in EAE.
  • Our study establishes that coordinated epigenetic activation of Foxp3 and Foxp3- independent Treg identity genes enables the conversion of committed CD4+ Teff cells into functional T regs with bona fide transcriptional and suppressive programs.
  • the resulting ER-T regs outperform endogenous nTregs, iTregs, and Foxp3-overexpressing Teff cells in suppressing established autoimmune inflammation, underscoring their therapeutic potential.
  • mice used were at least 6 weeks old and had no prior exposure to drugs or experimentation.
  • Antibodies and reagents Flow cytometry antibodies anti-CD3 (2C11), anti-CD4 + (RM4-5), anti-Thy1.1 (HIS51), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), anti-IFNg (XMG1.2), anti-GMCSF (MP1-22E9), anti-B220 (RA3-6B2), anti-GL7 (GL7), anti- CD138 (281-2), anti-CXCR5 (L138D7), anti-NGFR (ME20.4) were purchased from BioLegend.
  • Anti-IL-17 (eBio17B7), anti-Foxp3 (FJK-16s), anti-Rorgt (B2D), anti-c-MAF (sym0F1) were purchased from eBioscience.
  • Anti-CD95 (Jo2) was purchased from BD Biosciences.
  • Neutralizing antibodies toward IFNg (XMG1.2), IL-4 (11B11), and IL-12 (C17.8) were purchased from BioXcell.
  • Human IL-2 and IL-7 were purchased from PeproTech.
  • Mouse TGFb, IL-6, IL-23, and IL-1b were purchased from R&D systems.
  • Retinoic acid was purchased from Sigma-Aldrich. Vitamin C was purchased from Fisher Scientific.
  • CD3/CD28 Dynabeads were purchased from Thermo Fisher Scientific. Incomplete Fruend’s Adjuvant was purchased from Thermo Fisher Scientific.
  • Myelin oligodendrocyte glycoprotein (amino acids 35-55) was purchased from GeneMed Synthesis.
  • Heat killed Mycobacterium tuberculosis strain H37 Ra was purchased from BD Biosciences.
  • ER-T reg generation CD4 + CD44 hi Teff were sort purified from donor mice that were immunized with CFA/MOG 7 days prior.
  • Sorted cells were rested in T cell growth medium (RPMI 1640 supplemented with 2mM GlutaMAX, 10mM HEPES, 100 U/mL penicillin/streptomycin, 1 mM sodium pyruvate, 5% fetal calf serum) for 4 days in the presence of 2 ng/mL IL-7 and 10 ug/mL neutralizing antibodies against IFNg, IL-4, and IL-12.
  • T cell growth medium RPMI 1640 supplemented with 2mM GlutaMAX, 10mM HEPES, 100 U/mL penicillin/streptomycin, 1 mM sodium pyruvate, 5% fetal calf serum
  • CD3/CD28 Dynabeads were then stimulated with CD3/CD28 Dynabeads for 4 days in T cell growth medium supplemented with 1000 U/mL IL- 2, 5 ng/mL TGFb, 100 ug/mL vitamin C, 10 nM retinoic acid, and 10 ug/mL cytokine neutralizing antibodies (primary reprogramming cocktail).
  • CD4 + Thy1.1 + cells were sort purified and restimulated with Dynabeads in T cell growth medium supplemented with 1000 Atty Docket No.166118.01508 U/mL IL-2, 2 ng/mL TGFb, 10 ug/mL vitamin C, and 10 ug/mL cytokine neutralizing antibodies (secondary reprogramming cocktail).
  • Retroviral vectors MG2A and MG2N were generated by modifying MSCV-P2GM-FF (plasmid no. 19750, Addgene).
  • Mouse Foxp3, Maf, Rorc, and Stat3 single guide RNAs (sgRNA) were cloned into BbsI-digested MG2N or MG2A.
  • MIGR-mFoxp3 was a gift from D. Littman (plasmid no. 24067, Addgene).
  • the guide sequences are nontargeting (NT) (5’- GCACTACCAGAGCTAACTCA-3’) (SEQ ID NO: 1), Foxp3 (5’- GTTCCTGGGTGTACCCGAGCG-3’) (SEQ ID NO: 2), Maf (5’- GCCCGCAGCAGCTCAACCCGG-3’) (SEQ ID NO: 3), Rorc (5’- GTCATCTGGGATCCACTACG-3’) (SEQ ID NO:4), and Stat3 (5’- GAGATTATGAAACACCAACG-3’) (SEQ ID NO: 5).
  • NT nontargeting
  • NT GCACTACCAGAGCTAACTCA-3’
  • Foxp3 5’- GTTCCTGGGTGTACCCGAGCG-3’
  • Maf 5’- GCCCGCAGCAGCTCAACCCGG-3’
  • Rorc 5’- GTCATCTGGGATCCACTACG-3’
  • Stat3 5’- GAGATTATGAAACACCAACG-3’
  • Retroviral transduction Rested CD4 + T eff cells were stimulated for 1 day with CD3/CD28 Dynabeads in the presence of the primary reprogramming cocktail. Cells were then transduced by spin infection with viral supernatant supplemented with the primary reprogramming cocktail and 4 ug/mL polybrene. Spin infection was performed in a Sorvall Legend X1R centrifuge for 90 minutes at 2,800 RPM and 37C.
  • Bisulfite sequencing Genomic DNA was isolated using the GeneJet Genomic DNA Purification Kit (Thermo Fisher Scientific) and then bisulfute converted using EpiTect Bisufite Conversion Kit (Qiagen).
  • Converted DNA was amplified with Q5U polymerase (New England Biolabs) and gel purified after agarose gel electrophoresis. Purified PCR product was cloned into pJET1.2 (Thermo Fisher Scientific) for Sanger sequencing.
  • the bisulfite amplification primers are CNS2 Forward (5’ -TGGGTTTTTTTGGTATTTAAGAAAG-3’) (SEQ ID NO: 6), CNS2 Reverse (5’-AACCAACCAACTTCCTACACTATCTAT-3’) (SEQ ID NO: 7), CTLA4 Forward (5’- TGGTGTTGGTTAGTAGTTATGGTGT-3’) (SEQ ID NO: 8), CTLA4 Reverse (5’- AAATTCCACCTTACAAAAATACAATC-3’) (SEQ ID NO: 9), IL2ra Forward (5’- TTTTAGAGTTAGAAGATAGAAGGTATGGAA-3’) (SEQ ID NO: 10), IL2ra Reverse (5’- Atty Docket No.166118.01508 TCCCAATACTTAACAAAACCACATAT-3’) (SEQ ID NO: 11), Ikzf4 Forward (5’- AGGATGGTTTTTATTGAAGGTGAT-3’) (SEQ ID NO: 12), Ikzf4
  • EAE induction was induced by subcutaneous injection of 50 uL of an emulsion containing 50 ug MOG 35-55 and 250 ug M. tuberculosis strain H37 Ra in Incomplete Fruend’s Adjuvant into each hind flank. Mice also received an intraperitoneal injection of 200 ng pertussis toxin in 200 uL PBS on days 0 and 2 after immunization. Clinical signs of EAE were assessed by the following criteria: 0, no signs of disease; 1, loss of tail tone; 2, hind limb paresis; 3, hind limb paralysis; 4, tetraplegia; 5, moribund or dead. Mice with a score greater than 4 were euthanized and carried as 5 for the duration of the experiment.
  • mice were transferred intravenously one day prior to disease initiation.
  • Mice received 50,000 T regs and 100,000 CD4 + T conv from CD45 congenically distinct Foxp3 Thy1.1 donor mice.
  • CD4 + Tconv were procured from mice which were immunized with CFA/MOG 7 days prior to transfer using the mouse CD4 + T Cell Isolation Kit (Miltenyi Biotec) followed by Treg depletion using anti-Thy1.1 PE (HIS51) and anti-PE nanobeads (BioLegend).
  • mice were perfused with PBS. Tissues were removed from the spinal column, cut into small pieces, and enzymatically digested with 1 mg/mL collagenase D and 0.1 mg/mL DNase (Sigma Aldrich) in T cell growth medium for 30 minutes at 37C shaking at 1,500 RPM. After digesting, cells were filtered through a 40 um nylon mesh and remaining tissue was mechanically dissociated with the back of a syringe plunger.
  • 1 mg/mL collagenase D and 0.1 mg/mL DNase Sigma Aldrich
  • Dissociated spinal cord cells were Atty Docket No.166118.01508 then placed into a 30%-37%-70% isotonic Percoll (Cytiva) gradient and centrifuged for 30 minutes at room temperature at 800xg to enrich the infiltrating mononuclear cells.
  • Flow cytometry For surface staining, cells were stained in FACS buffer (PBS, 0.5% BSA, 1mM EDTA) for 15 minutes at 4C.
  • FACS buffer PBS, 0.5% BSA, 1mM EDTA
  • intracellular cytokine staining cells were incubated at 37C in the presence of 50 ng/mL PMA and 500 ng/mL ionomycin for one hour. GolgiStop (BD Biosciences) was added, and the cells were incubated at 37C for an additional 3 hours.
  • Stimulated cells were surface stained, fixed and permeabilized with Foxp3/Transcription Factor Staining Kit (Tonbo Biosciences) according to manufacturer instructions, and stained for cytokines in permeabilization buffer.
  • CD45 congenically distinct CD4 + CD44 hi T eff cells were sorted from donor mice immunized 7-10 days prior and labeled with CellTrace Violet (Invitrogen). Labeled cells were co-cultured with T cell depleted APCs and T regs in the presence of 10 ug/mL MOG 35-55 . Dilution of CellTrace Violet and cytokine expression were measured 4 days later.
  • RNA sequencing CD45 congenically distinct ER-Tregs were transferred to lymphoreplete mice and the mice were immunized with CFA/MOG. After 7 days, ER-Tregs, nTregs, and CD4 + Teff cells were sorted from the draining lymph nodes directly into TRIZOL. RNA was isolated using phenol- chloroform extraction. Uniquely indexed libraries were pooled in equimolar ratios and sequenced on a single Illumina NextSeq500 run with single-end 75-bp reads by the Tufts University Genomics Core Facilities. RNA-seq analysis Sequence reads were aligned with the mm39 reference genome assembly and gene counts were quantified with FeatureCounts.
  • GSEAPreranked Gene-set enrichment analyses were performed with GSEAPreranked, in which genes ranked according to their fold changes were compared with the following MSigDB signature collections: GSE7852_Treg_VS_Tconv_DN gene set, GSE7852_Treg_VS_Tconv_UP gene set, WP_TH17_CELL_DIFFERENTIATION_PATHWAY, PID_IL23_PATHWAY, PID_SMAD2_3NUCLEAR_PATHWAY, as well as a CNS2_Dependent_Effector_Treg gene set generated from GSE57272. Statistics Except for RNA-Seq analysis, statistical significance was determined using GraphPad Prism 10.0 (GraphPad Software).
  • T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity.2012;37(5):785-99. 17. Fontenot JD, Gavin MA, and Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol.2003;4(4):330-6. 18. Hori S, Nomura T, and Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science.2003;299(5609):1057-61. 19. Khattri R, Cox T, Yasayko SA, and Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells.
  • Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses. Nature. 2009;458(7236):351-6. 24. Cretney E, Xin A, Shi W, Minnich M, Masson F, Miasari M, et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nat Immunol.2011;12(4):304-11. Atty Docket No.166118.01508 25. Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, and Campbell DJ. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation.
  • GM-CSF production by autoreactive T cells is required for the activation of microglial cells and the onset of experimental autoimmune encephalomyelitis.
  • IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells.
  • Int Immunol.2004;16(2):249-56. 86. McGeachy MJ, Stephens LA, and Anderton SM. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. J Immunol.2005;175(5):3025-32. 87. Korn T, Reddy J, Gao W, Bettelli E, Awasthi A, Petersen TR, et al. Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nature medicine.2007;13(4):423-31. 88.

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Abstract

La présente invention concerne des compositions et des procédés pour la reprogrammation de lymphocytes T effecteurs en lymphocytes T régulateurs stables et des procédés d'utilisation des lymphocytes T régulateurs reprogrammés.
PCT/US2025/024796 2024-04-15 2025-04-15 Reprogrammation épigénétique efficace de lymphocytes t effecteurs dans des lymphocytes t régulateurs stables Pending WO2025221808A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090136470A1 (en) * 2007-06-13 2009-05-28 Hilde Cheroutre Regulatory t cells and methods of making and using same
US20120114675A1 (en) * 2008-11-13 2012-05-10 Graca Luis Ricardo Simoes Da Silva Foxp3+ natural killer t-cells and the treatment of immune related diseases
US20150368611A1 (en) * 2014-06-24 2015-12-24 Seoul National University R&Db Foundation Induced regulatory t cell and use thereof
US20210238551A1 (en) * 2018-08-22 2021-08-05 Osaka University Method for Generating Regulatory T Cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090136470A1 (en) * 2007-06-13 2009-05-28 Hilde Cheroutre Regulatory t cells and methods of making and using same
US20120114675A1 (en) * 2008-11-13 2012-05-10 Graca Luis Ricardo Simoes Da Silva Foxp3+ natural killer t-cells and the treatment of immune related diseases
US20150368611A1 (en) * 2014-06-24 2015-12-24 Seoul National University R&Db Foundation Induced regulatory t cell and use thereof
US20210238551A1 (en) * 2018-08-22 2021-08-05 Osaka University Method for Generating Regulatory T Cells

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