Efficient epigenetic reprogramming of t effector cells into stable regulatory t cells

Abstract

The present disclosure provides compositions and methods for the reprogramming of effector T cells into stable regulatory T cells and methods of using the reprogrammed regulatory T cells.

Description

[0001]Atty Docket No.166118.01508 EFFICIENT EPIGENETIC REPROGRAMMING OF T EFFECTOR CELLS INTO STABLE REGULATORY T CELLS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 634,105 filed on April 15, 2024. The contents of which is incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING An electronic sequence listing (16611801508.xml; Size: 12,413 bytes; and Date of Creation: April 15, 2025) accompanies this application. The sequence listing is incorporated by reference in its entirety. 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. However, efforts to expand endogenous naturally occurring Treg cells (nTregs, or nTreg cells) or convert naïve CD4+ T cells (Tn cells) into induced Treg cells (iTregs or iTreg cells) have shown limited efficacy in clinical studies. Reprogramming autoreactive effector T cells into functional Tregs could provide a novel reservoir of Treg cells for antigen-specific tolerization. However, uncertainties remain regarding the feasibility of inducing stable Foxp3 expression in effector T cells and their potential to be reprogrammed into functional Tregs capable of mitigating autoimmunity. Accordingly, there is a remaining need in the art for methods of reprogramming Treg cells. SUMMARY 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. In some embodiments, 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. In some embodiments Atty Docket No.166118.01508 the T cell growth medium may be supplemented with IL-7, neutralizing antibodies against IL- 12, neutralizing antibodies against IFNγ, and / or neutralizing antibodies against IL-4. The primary reprogramming cocktail may comprise the T cell growth medium supplemented with IL-2, TGFβ, retinoic acid, neutralizing antibodies against IL-12, neutralizing antibodies against IFNγ, 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. In some embodiments, the reprogramming cocktail additionally comprises ascorbic acid. In some embodiments, 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, TGFβ and ascorbic acid. In some embodiments, the secondary reprogramming cocktail may be further supplemented with neutralizing antibodies against IL- 12, neutralizing antibodies against IFNγ, and neutralizing antibodies against IL-4. In some embodiments, the ER-Treg cells express Foxp3, CD25 and / or CTLA4. In some embodiments, 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. In some embodiments, the composition comprises a base media, L-glutamine, buffered saline, an antibiotic, Sodium Pyruvate, serum, IL-7, neutralizing antibodies to IFNγ, neutralizing antibodies to IL-4, neutralizing antibodies to IL-12, IL-2, TGFβ, retinoic acid, and vitamin C. In some embodiments, 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. In embodiments, 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. In some embodiments, 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. In embodiments, the antigen-specific CD4+ T cells or CD4+CD44+ are isolated from a subject and administered to the same subject. In some Atty Docket No.166118.01508 embodiments, 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+Teffcells into Tregs.(A) Flow cytometry of Foxp3-Thy1.1 induction in CD4+Teff cells isolated from Foxp3Thy1.1mice on day 7 following immunization with MOG / CFA and subsequently activated with anti-CD3 / CD28 microbeads for 4 days under indicated conditions. Rest indicates resting Teff cells for 4 days prior to their activation. RA indicates retinoic acid. VC indicates vitamin C. (B) Schematic of epigenetic reprogramming of CD4+Teff cells into ER-Tregs. (C) Flow cytometry of Foxp3-Thy1.1 expression in 1° and 2° ER-Tregsgenerated in the presence or absence of VC. (D) Flow cytometry of Foxp3-Thy1.1 expression in 1° and 2° ER-Tregs generated in the presence or absence of VC and subsequently re-stimulated for 3 days in the presence of IL-6. (E) Heatmaps of CpG demethylation patterns at specific loci in indicated cell types, analyzed with bisulfite- sequencing. Each bar represents a CpG site. Mean ± SEM. **p < 0.01, ****p < 0.0001, one- way ANOVA and Holm-Šídák test in (A and D), and two-way ANOVA in (E). Figures 2A-2E: Adoptive transfer of ER-Tregsprevents EAE development and ameliorates established EAE. (A-C) EAE was induced via MOG / CFA immunization in CD45.2+mice with or without adoptive transfer of ER-Tregsreprogrammed from MOG / CFA-primed CD45.1+Foxp3Thy1.1CD4+Teff cells, administered one day prior to immunization. Flow cytometry analyses were conducted at 21 days post-immunization (dpi). n = 6 per group. Data are representative of two independent experiments. (A) EAE disease curve. (B) Flow cytometry analysis of the frequencies of spinal cord CD4+T cells. (C) Flow cytometry of Foxp3 expression in host CD4+T cells and transferred ER-Tregs within the draining lymph nodes (LNs) of mice that received ER-Tregs. (D and E) 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+Foxp3Thy1.1Teffcells, administered at 11 dpi. Flow cytometry analyses were conducted at 29 dpi. n = 6 per group. Data are representative of two independent experiments. EAE disease curve (D). Flow cytometry (E) of IFNγ and GM-CSF expression in CD4+T cells Atty Docket No.166118.01508 in spinal cord. Mean ± SEM. *p < 0.05, ***p < 0.001, unpaired two-sided t-test of Area under the curve (AUC) in (A and D) and unpaired two-sided t-test in (B and E). Figures 3A-3H Foxp3 expression is required but not sufficient for the suppressor function of ER-Tregs.(A) Flow cytometry of IFNγ and GM-CSF expression in CTVloCD4+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 Foxp3Thy1.1R26Cas9ER-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-Tregsas in (A). (C) Flow cytometry of IFNγ and GM-CSF expression in CTVloCD4+Teff cells, cocultured for 3 days with APCs, in the presence of MOG and the presence or absence of Foxp3Thy1.1ER-Tregstransduced with MigR1 empty vector or CD4+Teffcells forced to express Foxp3 via retroviral transduction with MigR1-Foxp3. (D) Flow cytometry of CD25 and CTLA-4 expression in MigR1-ER-Tregsand MigR1-Foxp3 CD4+Teffcells as in (C). (E- G) EAE was induced via MOG / CFA immunization in CD45.2+mice with or without adoptive transfer of CD45.1+Foxp3Thy1.1MOG / 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. Flow cytometry analyses were conducted at 16 dpi. n = 6 per group. (E) EAE disease curve. (F) Flow cytometry analysis of the frequencies of spinal cord CD4+T cells. (G) Flow cytometry of Foxp3 expression in adoptively transferred ER-Tregs and Foxp3-RV CD4+Teffcells 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-Šídák 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-Tregsas compared to induced Tregs.(A) Flow cytometry of Foxp3 expression in CTVloCD4+Tconv and nTreg cells 8 days after they were isolated from CD45.2+Foxp3Thy1.1mice, labeled with CTV, and adoptively transferred into CD45.1+Foxp3Thy1.1mice, which were subsequently immunized with MOG / CFA one day after adoptive transfer. (B-D) EAE was induced in Rag1− / −mice via MOG / CFA immunization one day after adoptive transfer of MOG / CFA-primed CD4+Tconv cells with or without co-transfer of congenically distinct ER- Tregsreprogrammed from MOG / CFA- or OVA / CFA-primed CD4+Teffcells, or co-transfer of induced Tregs (iTregs) generated with in vitro differentiation of Tn cells isolated from MOG / CFA- Atty Docket No.166118.01508 primed mice. Flow cytometry analyses were conducted at 17 dpi. n = 6 per group. (B) EAE disease curve. (C) EAE scores at 17 dpi. (D) Flow cytometry analysis of the frequencies of adoptively transferred ER-Tregs or iTregs (CD45.1−CD45.2+) in the spinal cord and draining LNs. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-sided t-test in (B), one-way ANOVA and Holm-Šídák test in (C-D). Figures 5A-5D: ER-Tregsconfer autoantigen-specific suppression of EAE without compromising vaccine-elicited immune responses against a foreign antigen. (A-C) EAE was induced via MOG / CFA immunization in CD45.1+mice with or without adoptive transfer of CD45.2+Foxp3Thy1.1MOG-specific or MOG-non-specific ER-Tregs, administered at 11 dpi. Flow cytometry analyses were conducted at 33 dpi. n = 6 per group. (A) EAE disease curve. (B) Flow cytometry of IFNγ and GM-CSF expression in CD4+T cells in the spinal cord. (C) Flow cytometry analysis of the frequencies and total numbers of adoptively transferred ER- Tregs(CD45.1−CD45.2+) in the spinal cord. (D) Flow cytometry analysis of the frequencies of NP-specific (NP-PE+) germinal center B cells in the spleens of mice 11 days post-immunization with NP-OVA / Alum with or without adoptive transfer of MOG-specific or OVA-specific ER- Tregs administered one day prior to immunization. Mean ± SEM. *p < 0.05, **p < 0.01, one- way ANOVA and Holm-Šídák test in (A, B, and D), and unpaired two-sided t-test in (C). Figures 6A-6H: ER-Tregsexhibit elevated expression of select parental Teffgenes. (A-F) RNA-seq analysis of the transcriptomes of ER-Tregs, nTregs, and CD4+Teffcells. CD45.1+Foxp3Thy1.1ER-Tregs were adoptively transferred into CD45.2+Foxp3Thy1.1mice one day prior to MOG / CFA immunization. The transcriptomes of transferred ER-Tregsand host nTreg and CD4+Teff cells were determined by bulk RNA-seq at 7 dpi. (A) Principal component analysis of ER-Treg, nTreg, and CD4+Tefftranscriptomes. (B) Volcano plot showing the differential expression of genes between ER-Tregs and Teff cells. (C) Gene Set Enrichment Analysis (GSEA) of the expression of Treg-specific genes in ER-Tregsand nTregs. (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-Tregsand nTregs. (F) GSEA of the expression of indicated gene sets in ER-Tregs and nTregs. (G) Flow cytometry of c-Maf and Rorγt expression in MOG / CFA-primed 2D2 nTregsand ER-Tregsfollowing in vitro activation in the presence of IL-2 for 3 days. (H) Flow cytometry of Rorγt and Foxp3-Thy1.1 expression in CD4+Tncells and in vitro differentiated T helper cells following their 1° stimulation under Atty Docket No.166118.01508 the ER-Treg reprogramming condition. Mean ± SEM. ****p < 0.0001, one-way ANOVA and Holm-Šídák test in (H). Figures 7A-7J Elevated expression of specific Teffgenes contributes to ER-Tregfitness and suppressive function in EAE. (A – C) EAE was induced in Rag1− / −mice via MOG / CFA immunization one day after adoptive transfer of MOG / CFA-primed CD45.1+CD4+Tconvcells with or without co-transfer of CD45.2+ER-Tregs reprogrammed from MOG / CFA-primed CD4+Teffcells, or CD45.2+nTregsisolated from MOG / CFA-primed mice and cultured in vitro in the presence of IL-2 for 3 days. Flow cytometry analyses were conducted at 22 dpi. n = 6 per group. (A) EAE disease curve. (B) Flow cytometry analysis of the frequencies of transferred Tregs. (C) Flow cytometry of Rorγt expression in transferred Tregs. (D) Flow cytometric assessment of in vivo competitive fitness between 2D2 ER-Tregsand nTregsretrovirally 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. (E-I) EAE was induced via MOG / CFA immunization in Rag1− / −mice one day after adoptive transfer of MOG / CFA- primed CD45.1+CD4+Tconv cells with or without co-transfer of CD45.2+Foxp3Thy1.1R26Cas9ER-Tregs transduced with sgRNA-RV targeting Stat3 (sgStat3), Maf (sgMaf), or a non-targeting sgRNA-RV (sgNT). Flow cytometry analyses were conducted at 22 dpi. n = 6-7 per group. (E) EAE disease curve. (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 Rorγt expression in ER- Tregs in the draining LNs. (I) Flow cytometry analysis of the frequencies and numbers of transferred ER-Tregsin CD4+T cells in the spinal cord (upper) and draining LNs (lower). (J) Flow cytometry of IFNγ and GM-CSF expression in CTVloCD4+Teff cells cocultured for 3 days with APCs and MOG in the presence or absence of Foxp3Thy1.1R26Cas9ER-Tregstransduced with sgMaf-RV or sgNT-RV. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA and Holm-Šídák test in (A, E, F, G, H, I, and J) and unpaired two- sided t-test in (B, C, and D). Figures 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+Foxp3Thy1.1CD4+Teff cells, administered one day prior to immunization. Flow cytometry analyses were conducted at 21 days post-immunization (dpi). n = 6 per group. Data are representative of two independent Atty Docket No.166118.01508 experiments. (A) Flow cytometry analysis of the frequency and total number of host Tregs and transferred ER-Tregsin 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. (C-E) 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+Foxp3Thy1.1Teff cells, administered at 11 dpi. Flow cytometry analyses were conducted at 29 dpi. n = 6 per group. Data are representative of two independent experiments. (C) Flow cytometry analysis of the frequency of ER-Tregsand the total number of host Tregsand ER-Tregsin the spinal cords. (D) Flow cytometry analysis of Ror^t and c-MAF expression in both host Tregs and ER-Tregs in the spinal cords. (E) Flow cytometry analysis of cytokine expression in spinal cord ER-Tregs. Mean ^ SEM. *p < 0.05, **p < 0.01. Unpaired two-sided t-test in (B and D). Figures 9A-9F: Cellularity, proliferation, and phenotypes of FRV-Tregsand ER-Tregs.(A) Teffcells retrovirally transduced with a Foxp3 overexpression vector (FRV-Tregs) or ER-Tregswere 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-Tregsor ER-Tregswere transferred into CD45 congenically distinct mice. Recipients were immunized with MOG / CFA and cells were analyzed with flow cytometry 4 days after immunization. Flow cytometry analysis of the frequencies (B), numbers (C), and expression of Ki-67 (D), Helios (E), and CD25 (F) of transferred cells in the spleens of recipient mice. Mean ^ SEM in. *p < 0.05, **p < 0.01. Unpaired two-sided t-test in (A-F). Figures 10A-10C: Stability, phenotype, and cellularity of MOG-specific and MOG- nonspecific ER-Tregsin EAE. (A-C) EAE was induced via MOG / CFA immunization in CD45.1+mice with or without adoptive transfer of CD45.2+Foxp3Thy1.1MOG-specific or MOG-non-specific ER-Tregs, administered at 11 dpi. Flow cytometry analyses were conducted at 33 dpi. n = 6 per group. (A) Flow cytometry analysis of the frequency and total cell number of MOG specific and MOG non-specific ER-Tregs in the spleen. (B) Flow cytometry analysis of Thy1.1 expression in MOG specific vs MOG non-specific ER-Tregs in the spinal cord. (C) Flow cytometry analysis of Ror^t and c-MAF expression in host Tregsand ER-Tregsin the spinal cord. Mean ^ SEM. ***p < 0.001, ****p < 0.0001. Unpaired two-sided t-test in (A) and (B), one-way ANOVA and Holm-Šídák test in (C). Atty Docket No.166118.01508 Figure 11: Cellularity, stability, and phenotype of sgNT, sgStat3, and sgMaf transduced ER-Tregs.EAE was induced via MOG / CFA immunization in Rag1− / −mice one day after adoptive transfer of MOG / CFA-primed CD45.1+CD4+Tconv cells with or without co-transfer of CD45.2+Foxp3Thy1.1R26Cas9ER-Tregstransduced with sgRNA-RV targeting Stat3 (sgStat3), Maf (sgMaf), or a non-targeting sgRNA-RV (sgNT). Flow cytometry analyses were conducted at 22 dpi. n = 6-7 per group. (A) Total number of ER-Tregsin the spinal cords. (B) Flow cytometry analysis of ER-Treg Foxp3 expression in the spinal cord. Mean ^ SEM in (A-C). *p < 0.05, ***p < 0.001, one-way ANOVA and Holm-Šídák test. DETAILED DESCRIPTION The present disclosure provides compositions, methods, and kits for the efficient reprogramming of effector T cells into stable Treg cells. Reprogramming autoreactive CD4⁺ effector T (Teff) cells into immunosuppressive regulatory T (Treg) cells offers a novel therapeutic strategy. Here, the inventors demonstrate that epigenetic activation of Tregidentity genes in Teff cells generates lineage-stable Effector T cell Reprogrammed Tregs (ER-Tregs). Adoptive transfer of ER-Tregsnot only prevents autoimmune neuroinflammation in mice when administered pre-onset but also halts disease progression post-onset. ER-Tregs outperform Foxp3-overexpressing Teffcells, Tregsinduced from naïve precursors, and endogenous Tregsin 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, γδT 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. Thus, effector T cells may be a CD4+ T cell or a CD4+CD44+ T cell. In the present Atty Docket No.166118.01508 disclosure, 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. Besides Foxp3, there are many other 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. DNA hypomethylation in Tregs-related genes, such as Ctla4 exon 2, Foxp3 intron 1, Tnfrsf18 exon 5, Ikzf4 intron 1b, and Il2ra intron 1a, is limited to Tregs and persists after cell stimulation. Described herein, the inventors demonstrate methods of reprogramming effector T cells into a type of Treg cells, termed Effector T cell Reprogrammed Treg (ER-Treg) in which ER-Treg cells have epigenetic reprogramming similar to Treg cells. Epigenetics describe changes in gene expression not caused by alterations in the DNA nucleotide sequence. The most important and well-known type of epigenetics are DNA methylation, histone posttranslational modifications, and inhibition by non-coding RNA (ncRNA). Each epigenetic mark is responsible for maintenance of DNA availability and results in changes in gene expression as well as in chromatin structure. Subsequent chromatin remodeling has an impact on the production of many crucial proteins for the proper action of the immune system. 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments the growth medium may comprise a base media, a source of L-glutamine, buffered saline, an antibiotic, Sodium Pyruvate, and serum. By way of example and not limitation 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, and 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 IFNγ, and neutralizing antibodies against IL-4. The 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 IFNγ, IL-4, and IL-12 may be used in a Atty Docket No.166118.01508 range of about 2µg / mL to about 25µg / mL, preferably about 10µg / 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 (TGFβ), and retinoic acid, and neutralizing antibodies against interleukin 12 (IL-12), neutralizing antibodies against interferon gamma (IFNγ), and / or neutralizing antibodies against interleukin 4 (IL-4). The 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 TGFβ 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 0.5µM, 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 1µg / mL to about 500µg / mL, of about 1µg / mL to about 250µg / mL, of about 1µg / mL to about 100µg / mL, preferably about 100µg / 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. Alternatively soluble anti-CD3 and anti-CD28, or feeder cells may be used as stimulus to activate the effector T cells. In some embodiments, 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. In some embodiments, 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. In some embodiments, after at least 2-5 days in the primary reprogramming cocktail, a new primary reprogramming cocktail is added. In some embodiments, 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 ¼, 1 / 3, ¾, 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. In some embodiments, 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, TGFβ, and ascorbic acid. The secondary reprogramming cocktail may be further supplemented with neutralizing antibodies against IL-12, neutralizing antibodies against IFNγ, and neutralizing antibodies against IL-4. The secondary reprogramming cocktail may comprise components at any range disclosed herein. In some embodiments, the IL-2 is preferably about 1000 units / mL, the TGFβ is at about 2ng / mL, and the and ascorbic acid is used at about 10µg / 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. In some embodiments, 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. For example, 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. In some embodiments, 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. In some embodiments 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. In some embodiments, 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. In some embodiments, one or more of these markers may be used to distinguish ER- Treg cells from effector T cells. In some embodiments the ER-Treg have changes in epigenetic patterns that are similar to Treg cells. In some embodiments 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- naïve 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 naïve 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 IFNγ. These conditions convert almost all Naïve 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. By way of example and not limitation, an effector T cell may be a Th17 cell. The Th17 cell may express RORγT, IL-1r1, Maf, TGFβ, IL-6r, IL-21R, IL-23R, CCR6, and / or STAT3. After reprogramming the Th17 cell into an ER-Treg cell, 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. For example, gamma-delta (γδ) T cells or CD8+ T cells can express Foxp3 and exhibit regulatory functions. In some embodiments, genomic editing, for example CRISPR-Cas based methods, may be used to further enhance the activation and efficiency of Foxp3 in some cell types. In some embodiments, 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. In some embodiments, 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. By way of example, and not limitation, the autoantigen may be a myelin antigen, a neuron-derived antigen, or an astrocyte-derived antigen. Examples of 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. In some embodiments, the method comprises administering ER-Treg cells. In some embodiments, 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. For example, 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. Suitably, the autoimmune disease may be one associated with myelin degeneration or demyelination, for example, multiple sclerosis. The methods and compositions provided herein may also be used with additional 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. As used herein, “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. In some embodiments, the ER-Treg cells control the antigen specific immune response. In some embodiments, 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. In some embodiments, 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. In the methods, the antigen-specific T cell may be isolated from the subject and administered to the same subject. In the described methods of decreasing an antigen-specific immune response, the subject may be diagnosed with Graft vs. host disease, allergy, infectious disease, or autoimmune disease, or the subject is an organ transplant recipient. In some embodiments, 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. As used herein, “subject” or "patient" 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. In some embodiments, the subject is diagnosed with hyperinflammation, a hypersensitivity reaction, cytokine storm, or a dysregulated immune response. As used herein, the terms “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. As used herein, the term “decrease” or the related terms “decreased,” “reduce” or “reduced” refers to a statistically significant decrease. For the avoidance of doubt, the terms generally refer to at least a 10% decrease in a given parameter, and can encompass at least a 20% decrease, 30% decrease, 40% decrease, 50% decrease, 60% decrease, 70% decrease, 80% decrease, 90% decrease, 95% decrease, 97% decrease, 99% or even a 100% decrease (i.e., the measured parameter is at zero). Compositions Another aspect of the present disclosure provides 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. In some embodiments, the composition comprises a base media, L-glutamine, buffered saline, an antibiotic, Sodium Pyruvate, serum, IL-7, neutralizing antibodies to IFNγ, neutralizing antibodies to IL-4, neutralizing antibodies to IL-12, IL-2, TGFβ, retinoic acid, and vitamin C. In some embodiments, alternative means to activate Foxp3, such as CRISPR-based tools may be used, and may replace the use of retinoic acid and / or TGFβ. 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. In some embodiments, the effector T cell may be rested prior to culturing in the composition, for example in the T cell growth medium culture composition. In some embodiments, the effector T cell may be placed in the composition more than one time. For example, 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. In an embodiment, 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 IFNγ, 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. The 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 use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be Atty Docket No.166118.01508 construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “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. As used herein, 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. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise. In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the Atty Docket No.166118.01508 art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.” No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. EXAMPLES 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. Regulatory T (Treg) 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). However, naturally occurring endogenous Tregs(nTregs) are often inadequate at suppressing ongoing inflammation in established autoimmune diseases (3, 4), limiting their therapeutic efficacy (5). This highlights the urgent need to better understand the mechanisms underlying disease-associated Treg functional deficiencies so that novel approaches can be developed to reinvigorate their function for the treatment of autoimmune diseases. Treg functional deficiency can arise from either inadequate Treg suppressor function on a per-cell basis or a paucity of autoantigen specific Tregs. The latter may result from impaired differentiation and lineage stability of autoantigen specific Tregs, leading to their diversion into effector CD4+T (Teff) cell fates (6-11). In this regard, reprogramming autoreactive CD4+Teffcells into Tregs for adoptive cell therapy presents an attractive option for restoring Treg function. This approach could generate Tregs that share the autoantigen specificities of their target Teff cells, potentially enhancing antigen specific suppression (12). However, the development of this approach is hindered by the lack of an effective method to convert CD4+Teff cells into bona fide Tregsthat exhibit Treg-specific gene expression and function (12-14). The maintenance of 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). However, whether stable induction of Treg identity genes in committed CD4+Teffcells can establish bona fide Tregidentity and suppressive capacity remains unresolved. Pre- existing 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. Alternatively, the inherent 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 Teffgenes correlates with superior suppressive capabilities (21-34). Clarifying these possibilities is crucial not only for advancing this therapeutic strategy but also for providing novel insights into how the core Tregidentity gene program interacts with effector gene expression to shape Treg function. Nevertheless, the fundamental question of whether the core Tregidentity genes can be stably activated in CD4+Teffcells remains unresolved, hindering deeper exploration of this approach. Atty Docket No.166118.01508 Treg gene expression is controlled by intricate epigenetic mechanisms, including DNA methylation and demethylation (11, 35-39). During Treg development, the stable activation of Foxp3 and other core Treg identity genes requires the demethylation of DNA at cytosine- guanine (CpG) motifs in conserved cis-regulatory regions (16, 40-42), presenting therapeutic targets for boosting Treg function. Indeed, pharmacologically enhancing DNA demethylation in pre-existing Tregsaugments their fitness and function and accelerates repair of experimental lung injury (43). Importantly, we and others have shown that demethylation at conserved non- coding sequence 2 (CNS2) within the Foxp3 gene stabilizes its expression in effector Tregsby counteracting the transcriptional inhibitory effects of TCR and pro-inflammatory cytokine signaling (44, 45). These Foxp3-inhibitory signals are also highly active in Teffcells (46-48), suggesting that induction of stable Foxp3 expression in CD4+Teff cells likely requires CNS2 demethylation. Furthermore, we recently demonstrated that CNS2 demethylation requires sustained Foxp3 transcriptional activation (49), indicating a positive feedback loop where Foxp3 transcriptional activation and DNA demethylation mutually reinforce each other, facilitating Treg lineage commitment during differentiation. These findings suggest that fostering sustained transcriptional activation of Foxp3 and other core Treg identity genes in an environment conducive to DNA demethylation may enable epigenetic reprogramming of CD4+Teff cells into Tregs. To determine whether autoreactive CD4+Teffcells can be reprogrammed into bona fide Tregs for mitigating established autoimmunity, we developed an approach to achieve stable epigenetic activation of Tregidentity genes in CD4+Teffcells. Our findings show that this approach generates bona fide Tregs, which we term Effector T cell Reprogrammed Tregs (ER- Tregs). These ER-Tregsexhibit a superior ability to ameliorate established autoimmune neuroinflammation compared to CD4+Teff cells forced to express Foxp3 exogenously, induced Tregs(iTregs) derived from naïve CD4+T (Tn) cells, and endogenous nTregs. 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 Tregscapable of quelling established autoimmune inflammation. Atty Docket No.166118.01508 RESULTS Epigenetic reprogramming enables stable induction of Foxp3 and other core Tregidentity genes Foxp3 expression is essential for establishing Tregcellular identity (17-19). To induce this identity in pro-inflammatory CD4+Teff cells from mice with experimental autoimmune encephalomyelitis (EAE), we first optimized conditions for efficient Foxp3 induction. We purified CD4+Foxp3-Thy1.1−CD44hiTeff cells from Foxp3Thy1.1reporter mice (50) immunized with myelin oligodendrocyte glycoprotein peptide (MOG) emulsified in complete Freund’s adjuvant (CFA). When activated in vitro under an iTreg differentiation condition consisting of IL-2, TGFβ, and neutralizing antibodies against pro-inflammatory cytokines IL-12, IFNγ, and IL-4 (51, 52), approximately 14% of the Teff cells began expressing Thy1.1 (Figure 1A). Pre- resting Teffcells before activation, along with the addition of retinoic acid (RA) to promote Foxp3 induction (53-56) and vitamin C (VC) to facilitate DNA demethylation (38, 39), progressively enhanced Foxp3 induction, resulting in nearly 60% of cells expressing Foxp3 (Figure 1A). Sustained transcriptional activation of Foxp3 enhances its epigenetic stabilization by promoting CNS2 demethylation (49). To test whether maintaining Foxp3 activation in ER-Tregs through re-stimulation with the ER-Treg reprogramming cocktail improves Foxp3 stability, we re-stimulated the cells in this context (Figure 1B). Both re-stimulation and the inclusion of VC during reprogramming increased Foxp3 expression (Figure 1C), and more importantly, improved its stability when ER-Tregswere subsequently exposed to the pro-inflammatory cytokine IL-6 (Figure 1D). Notably, restimulated ER-Tregs displayed significant DNA demethylation at Treg-specific demethylation regions, including Foxp3 CNS2, Il2ra, Ctla4, and Ikzf4 (16), compared to parental Teff cells (Figure 1E). Collectively, these findings suggest that both features defining Tregcellular identity – stable Foxp3 expression and epigenetic activation of core Treg identity genes – can be successfully established in committed CD4+Teff cells by epigenetic reprogramming. Adoptive transfer of ER-Tregsprevents EAE development and ameliorates established EAE To assess the therapeutic potential of ER-Tregs in curbing autoimmune inflammation, we adoptively transferred ER-Tregsderived from MOG / CFA-primed CD4+Teffcells into CD45 congenically distinct mice one day before inducing EAE via MOG / CFA immunization and pertussis toxin (PT) injections. Remarkably, ER-Tregtransfer nearly abolished clinical manifestations of EAE, in stark contrast to the rapid disease progression observed in control Atty Docket No.166118.01508 animals (Figure 2A). Consistent with this protection, CD4⁺ T cell infiltration into the spinal cord was significantly reduced in ER-Tregs-treated mice (Figure 2B). Approximately 90% of transferred ER-Tregs maintained Foxp3 expression, demonstrating robust in vivo stability under inflammatory conditions (Figure 2C). Although ER-Tregscomprised only about 2% of all Tregsin the draining lymph nodes (Figure 8A), they exhibited a significantly higher frequency of RORγt⁺ and RORγt⁺c-MAF⁺ populations compared to endogenous Tregs(Figure 8B). This distinct phenotypic profile, combined with their scarcity, collectively suggests that ER-Tregs possess enhanced per-cell suppressive potency compared to endogenous Tregsunder inflammatory conditions. To assess the suppressive capacity of ER-Tregsin 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-Tregtransfer attenuated disease progression (Figure 2D) and significantly reduced spinal cord infiltration by GM-CSF- producing CD4⁺ Teffcells (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 RORγt⁺ and RORγt⁺c-MAF⁺ subsets compared to endogenous Tregs(Figure 8D), further underscoring their distinct phenotype. Notably, ER-Tregslacked expression of inflammatory cytokines IFNγ or GM-CSF, although about 20% produced IL-17A (Figure 8E). Collectively, these findings demonstrate that ER-Tregsretain robust lineage stability and suppressive potency in vivo, even within an established inflammatory niche, with a single transfer sufficient to ameliorate ongoing autoimmune pathology. Foxp3 expression is required but not sufficient for ER-Tregsuppressor function While Foxp3 expression is essential for the development and function of most endogenous Tregs (17-19), a recent study demonstrated that Foxp3 is dispensable for the fitness of microbiota-dependent peripherally induced Tregs(pTregs) and their ability to suppress colonic T cell expansion (60). To determine whether Foxp3 activation is necessary for ER-Treg suppressor function, we used CRISPR / Cas9 to ablate Foxp3 in Foxp3Thy1.1R26Cas9ER-Tregs. We transduced these cells with a retroviral vector expressing a single guide RNA targeting Foxp3 (sgFoxp3) and compared their capacity to suppress GM-CSF expression in MOG / CFA-primed responder CD4⁺ Teff cells to that of ER-Tregs transduced with a non-targeting sgRNA (sgNT). Foxp3 ablation completely abolished ER-Treg-mediated suppression of GM-CSF in CD4⁺ Teff cells following MOG stimulation (Figure 3A). Moreover, Foxp3-deficient ER-Tregs exhibited Atty Docket No.166118.01508 increased IL-17A expression (Figure 3B), consistent with studies showing that Foxp3 is critical for repressing IL-17A in pTregs(60). In addition, Foxp3 ablation led to reduced IL-10 expression in ER-Tregs (Figure 3B). Collectively, these results indicate that activation of Foxp3 is indispensable for the suppressive function of ER-Tregs. To investigate whether epigenetic activation of Foxp3-independent Treg identity genes (Figure 1E) is essential for ER-Tregsuppressive function, we forced Foxp3 expression in CD4⁺ Teff cells via retroviral transduction and assessed their regulatory capacity. In contrast to ER- Tregs, 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. Adoptive transfer of Foxp3-expressing Teff cells neither attenuated disease progression nor reduced CD4⁺ T cell infiltration in the spinal cord (Figure 3E-F). Although these cells expressed Foxp3 at levels comparable to ER-Tregs (Figure 3G), their relative abundance in the spinal cord was significantly lower (Figure 3H). These results underscore the necessity of Foxp3-independent epigenetic reprogramming for effective ER-Treg functionality. To evaluate differences between Foxp3-expressing Teffcells and ER-Tregs, we directly compared their fitness and phenotype in immunocompetent EAE hosts. 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). Given that Helios promotes Tregstability and survival (63, 64) and CD25 enhances IL-2– dependent survival and function (62, 65, 66), the diminished suppressive capacity of Foxp3- expressing Teffcells thus likely results from inadequate epigenetic priming of critical genes (e.g., Helios, Il2ra), thereby compromising their resilience in inflammatory environments. Inherited autoantigen specificity confers superior functionality to ER-TregsAntigen specificity is critical for Treg suppressor function, suggesting that the inheritance of parental Teffautoantigen specificity may enhance ER-Tregactivity. To investigate this possibility, we examined whether the pro-inflammatory environment during autoimmune inflammation hinders the de novo differentiation of MOG-specific Tregsin EAE. We adoptively transferred CellTrace Violet (CTV)-labeled Foxp3-Thy1.1−conventional CD4+T (Tconv) cells Atty Docket No.166118.01508 or Foxp3-Thy1.1+nTregs from unimmunized donor mice into CD45 congenically distinct mice, followed by immunization with MOG / CFA. Fewer than 1% of CTVlodonor Tconvcells expressed Foxp3-Thy1.1, whereas the majority of CTVlodonor nTregs maintained Foxp3 expression (Figure 4A), indicating that de novo differentiation of Tconvcells into Tregsdoes not occur in EAE. Together, these findings suggest that the pro-inflammatory environment in EAE drives MOG-specific CD4⁺ naïve T cells to differentiate into Teffcells rather than Tregs. To assess whether inherited myelin autoantigen specificity contributes to ER- Tregsuppressor function, we performed adoptive transfers into Rag1⁻ / ⁻ mice. Specifically, we transferred MOG / CFA-primed CD4+Tconv cells alone or co-transferred them with CD45 congenically distinct ER-Tregsreprogrammed from CD4+Teffcells primed in vivo with either MOG / CFA or Ovalbumin peptide (OVA) emulsified in CFA. We also included iTregs differentiated from Tncells from MOG / CFA-immunized mice. Following recipient immunization with MOG / CFA and PT administration, mice receiving only Tconv cells developed severe EAE (Figure 4, C and D). In contrast, co-transfer of ER-Tregsderived from MOG / CFA-primed CD4+Teff cells substantially mitigated EAE, whereas co-transfer of ER- Tregs derived from OVA / CFA-primed CD4+Teff cells or iTregs did not. Notably, the suppressive efficacy of the transferred Tregs correlated positively with their frequencies in the spinal cord (Figure 4E). These findings suggest that inherited myelin autoantigen specificity contributes to the ability of ER-Tregsto curtail EAE development, at least in part by enhancing their ability to accrue in the inflamed tissue. Conversely, the diminished suppressive capacity of autologous iTregsmay reflect diminished autoantigen specificity, as inflammatory conditions preferentially drive the differentiation of autoreactive Tn cells into Teff cells. ER-Tregssuppress EAE in an autoantigen-specific manner without inhibiting immune response against a non-myelin foreign antigen Initial adoptive transfer experiments revealed that both iTregsand OVA-specific ER- Tregs transiently delayed EAE progression at early stages (Figure 4C), suggesting the possibility of antigen-nonspecific immunosuppressive effects. To clarify this, we developed a refined reprogramming protocol leveraging MOG-induced CTV dilution to isolate MOG- specific from MOG-nonspecific ER-Tregpopulations. Transfer of MOG-specific ER-Tregs, but not their nonspecific counterparts, significantly attenuated EAE severity and reduced spinal cord infiltration by GM-CSF⁺ CD4⁺ Teffcells (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). They also demonstrated superior lineage stability, with elevated frequencies of Foxp3-Thy1.1⁺ cells and Atty Docket No.166118.01508 higher Thy1.1 MFI, as well as enrichment for c-MAF⁺RORγt⁺ subsets (Figure 10B–C), a phenotype linked to enhanced Th17 suppression (31-34). These findings align with previous studies showing that TCR activation enhances Foxp3 expression, functional specialization, and tissue homing in Tregs(67, 68), and underscore the necessity of myelin antigen specificity for ER-Tregs to durably suppress CNS inflammation. To evaluate antigen-nonspecific immunosuppression, we transferred ER-Tregsgenerated from either CFA / MOG- or CFA / Ova-primed Teff cells into mice immunized with nitrophenol-conjugated ovalbumin (NP-Ova) in Alum. Neither MOG-specific nor OVA- specific ER-Tregs significantly inhibited NP-specific germinal center B cell responses (Figure 5D), although OVA-specific ER-Tregsexhibited a modest, non-significant trend toward suppression. These results suggest that ER-Treg-mediated suppression is tightly restricted to their cognate antigen and relies on the inflammatory context. ER-Tregsselectively inherit parental Teffgene expression To determine whether parental Teffgene expression confers a distinct transcriptional profile to ER-Tregs, we performed bulk 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). Moreover, ER-Tregs expressed significantly higher levels of core Tregidentity genes, including Foxp3, Itgae, Il2ra, Ctla4, and Ikzf4 (22), compared to CD4+Teff cells (Figure 6B). Gene set enrichment analysis (GSEA) further demonstrated that genes typically upregulated (or downregulated) in Tregsrelative to CD4+Tconvcells are similarly upregulated (or downregulated) in ER-Tregs relative to CD4+Teff cells (Figure 6C), confirming the successful establishment of a Treggene expression program in 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-Tregsupregulate genes involved in effector Tregfunction, Th17 differentiation, and the IL-23 pathway (Figure 6F), supporting the notion of enhanced Th17 polarization in ER-Tregsderived from MOG / CFA-primed Teff cells. Additionally, the observed amplification of SMAD2 / 3 signaling in ER-Tregs suggests that TGF-β in the reprogramming cocktail significantly contributes to their unique gene expression program. Atty Docket No.166118.01508 Additionally, ER-Tregs bearing the MOG specific 2D2 TCR express higher levels of c- Maf and Rorγt compared to nTregswith the same 2D2 TCR (72) (Figure 6G), indicating enhanced Th17 polarization in myelin autoantigen-specific ER-Tregs. To assess the contribution of parental Teffgene expression to this Th17 polarization, we compared Rorγt levels in ER- Tregs reprogrammed from in vitro differentiated Th1, Th2, and Th17 cells, as well as in iTregs derived from naïve T cells. Notably, ER-Tregsreprogrammed from Th17 cells exhibited significantly higher Rorγt expression than those reprogrammed from Th1 cells, Th2 cells, or iTregs(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-Tregfitness and function in EAE Adoptive transfer of a limited number of ER-Tregs (2 × 10⁶ per mouse) into lymphoreplete mice harboring endogenous nTregssignificantly ameliorated EAE (Figure 2), demonstrating the superior suppressive capacity of ER-Tregs over endogenous nTregs. To directly compare their therapeutic efficacy, we transferred MOG / CFA-primed CD4⁺ Tconvcells into Rag1⁻ / ⁻mice either alone or alongside ER-Tregs (derived from MOG / CFA-primed Teff cells) or nTregs (isolated from MOG / CFA-immunized mice). Recipients were immunized with MOG / CFA and treated with PT to induce EAE. ER-Treg co-transfer, but not nTreg co-transfer, effectively suppressed disease progression (Figure 7A). Moreover, ER-Tregs exhibited significantly greater accumulation and elevated Rorγt expression in the spinal cord compared to nTregs (Figure 7B–C), suggesting that retained Th17-associated transcriptional programming enhances their tissue fitness. To directly evaluate the role of Th17-related gene expression in ER-Treg fitness, we performed a competitive fitness assay using ER-Tregsand nTregsfrom 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. ER-Tregs outcompeted nTregs in vivo (Figure 7D), confirming that Th17-associated gene signatures enhance their survival and expansion. Collectively, these findings indicate that inherited Th17-related transcriptional program underpins the enhanced fitness and suppressive efficacy of ER-Tregsrelative to nTregsin EAE. To determine whether heightened Th17 polarization underpins ER-Treg functionality in EAE, we used CRISPR / Cas9 to delete the Th17-associated transcription factors STAT3 or c- Maf in ER-Tregs and assessed their suppressive capacity. Co-transfer of control sgNT- transduced Foxp3Thy1.1R26Cas9ER-Tregsrobustly attenuated EAE progression, whereas ER-Tregslacking STAT3 (sgStat3) or c-Maf (sgMaf) failed to suppress disease (Figure 7E). Genetic Atty Docket No.166118.01508 ablation of Stat3 or Maf impaired the ability of ER-Tregs to reduce spinal cord infiltration by total CD4⁺ T cells and GM-CSF⁺CD4⁺ Teffcells (Figure 7F–G), which was accompanied by diminished expression of RORγt and c-Maf in ER-Tregs (Figure 7H). Although deletion of Stat3 or Maf reduced the frequency of ER-Tregsin the spinal cord (Figure 7I), their absolute numbers remained unchanged or even elevated, respectively (Figure 11A), likely due to increased CD4⁺ T cell accumulation in mice receiving knockout ER-Tregs(Figure 7E). Notably, Foxp3 expression levels remained consistent across all groups (Figure 11B), ruling out gross instability. These findings establish STAT3 and c-Maf as critical drivers of ER-Tregfitness and function, enabling their suppression of neuroinflammation via Th17-associated transcriptional programs. To determine whether heightened Th17 polarization enhances ER-Treg suppressor function on a per-cell basis, we performed in vitro suppression assays comparing Maf-deficient ER-Tregs to control ER-Tregs transduced with sgNT. Genetic ablation of Maf significantly impaired the ability of ER-Tregsto suppress GM-CSF⁺CD4⁺ Teffcell responses (Figure 7J), indicating that elevated Maf expression is essential for their per-cell suppressive potency. Together, these results demonstrate that Th17-skewed transcriptional program in ER-Tregs is critical for their superior tissue fitness and functional efficacy in EAE compared to nTregs. DISCUSSION Endogenous Tregsacquire their identity through tolerogenic signals that imprint Treg- specific transcriptional and epigenetic programs onto naïve precursors. However, whether such programs can be stably established in committed CD4⁺ Teffcells, which retain inflammatory epigenetic memory, remains unknown. To address this, we developed an epigenetic reprogramming strategy to activate core Tregtranscriptional circuitry in Teffcells, creating a model system to probe Treg plasticity and therapeutic potential. Foxp3 induction in Teffcells is achieved through the synergistic actions of TGF-β, RA, and VC, each contributing distinct mechanistic pathways. TGF-β 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-β / 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 Teffcells (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. Notably, our reprogramming approach recapitulates the epigenetic remodeling observed in endogenous Tregs, inducing robust DNA demethylation at TSDRs of key Treg identity genes such as Ctla4, Il2ra, and Ikzf4. Crucially, this demethylation occurs independently of Foxp3 (16), indicating that TGF-β, RA, and VC cooperatively remodel the epigenome of Teff cells to activate Treg transcriptional programs through both Foxp3- dependent and Foxp3-independent mechanisms. These findings highlight the ability of tolerogenic signals to override inflammatory epigenetic memory in Teff cells, enabling their conversion into functionally stable Tregs. Previous studies have established that ectopic Foxp3 expression in conventional CD4⁺ T cells—composed of both naïve and effector T cells—can confer suppressive activity (17-19). However, whether Foxp3 expression alone suffices to induce authentic Treg phenotypes and suppressive function in Teffcells, which retain inflammatory epigenetic memory, has remained unresolved. Our findings reveal that both stable Foxp3 induction and epigenetic activation of Foxp3-independent Tregidentity genes are indispensable for establishing functional Tregprograms in Teff cells. Foxp3 critically suppresses IL-17A expression in reprogrammed cells, mirroring its role in pTregs (60), while also enhancing IL-10 production, potentially amplifying their suppressive capacity. Notably, forced Foxp3 expression in Teff cells, without concurrent epigenetic reprogramming, failed to confer suppressor function, underscoring the necessity of Foxp3-independent epigenetic remodeling at Tregidentity loci. This aligns with prior work demonstrating that Treg-specific DNA demethylation, unattainable through Foxp3 overexpression alone, is essential for Tregfunctionality (16). Consistent with this, Foxp3- expressing Teff cells exhibited diminished expression of CD25, CTLA-4, and Helios—proteins encoded by genes harboring TSDRs (16)—suggesting that these epigenetic modifications regulate key aspects of Treg gene expression. Collectively, our work highlights a dual requirement for Foxp3 and TSDR-driven epigenetic activation to override Teffcell transcriptional programs and enforce stable Treg identity. Future studies are warranted to dissect the precise contributions of individual TSDRs to Tregtranscriptional programs and their therapeutic potential in reprogramming autoimmune inflammation. While MOG-nonspecific ER-Tregsalone failed to ameliorate established EAE, bystander suppression mechanisms—potentially mediated by nonspecific ER-Tregs in the presence of MOG-specific counterparts—may contribute to disease control (79, 80). Notably, molecular mimicry between myelin antigens and foreign antigens (e.g., Epstein-Barr virus, gut microbiota) has emerged as a key driver of MS / EAE pathogenesis (81-83). Although MOG- specific ER-Tregs did not impair OVA-specific vaccine responses, they may suppress immunity Atty Docket No.166118.01508 against microbial antigens sharing epitopes with autoantigens. These findings highlight the need to rigorously assess the long-term effects of autoantigen-specific ER-Tregtherapy on immune homeostasis and infection resilience. In contrast to ER-Tregs, adoptive transfer of MOG / CFA-primed nTregsfailed to suppress EAE induced by co-transferred MOG / CFA-primed Tconv cells in Rag1⁻ / ⁻ mice. While prior studies reported EAE mitigation using nTregsisolated from naïve mice or recovery-phase mice (84-86), these protocols transferred nTregs into naïve hosts at disease induction—a context lacking pre-existing Teffcell differentiation and inflammation. By contrast, our co-transfer model in Rag1⁻ / ⁻ mice recapitulates the challenge of suppressing primed Teff cells in an inflammatory milieu. Further, MOG / CFA immunization destabilizes MOG-specific nTreglineage commitment (7), likely impairing their suppressive capacity upon isolation and transfer. This instability, coupled with intrinsic limitations in effector Tregdifferentiation, may explain nTreg inefficacy in our model and prior studies (87). Future studies reprogramming destabilized nTregscould clarify their functional potential in EAE suppression. We observed heightened expression of Rorγt and c-Maf in ER-Tregs compared to nTregs, even when both cell types expressed the same MOG specific 2D2 TCR. This suggests that the upregulation of Rorγt expression in endogenous nTregs, which has been recently shown to be associated with the ability of nTregs to suppress Th17 inflammation in EAE (33), is hindered by the pre-existing gene expression and / or epigenetic landscape in nTregs. This phenomenon may serve as a regulatory mechanism preventing endogenous Tregs from impeding beneficial anti- pathogen immune responses, while potentially contributing to the development of pathogenic autoimmune inflammation under specific conditions. Further investigations into the molecular mechanisms underlying the constrained or delayed Th17 polarization of nTregsin EAE and its impact on disease progression are warranted and may yield insights into the dysfunction of endogenous Tregsin Th17-cell-driven autoimmune diseases. 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. CRISPR-mediated deletion of c-Maf or Stat3—key Th17 transcription factors—significantly impaired ER-Tregsurvival and suppressive function in EAE, highlighting the critical role of Th17-like polarization in their therapeutic efficacy. While prior studies suggest Tregsexpressing lineage-specific transcription factors (e.g., Th1, Th17) display enhanced suppression of corresponding Teff subsets (23-30), the mechanistic basis remains unclear. Our data suggest that improved cellular fitness, mediated by transcription factor-driven adaptation to inflammatory niches, may underpin this phenomenon. Atty Docket No.166118.01508 The heightened Th17 signature in ER-Tregs may arise from epigenetic inheritance of parental Th17 cell programs or de novo activation during reprogramming. ER-Tregsalso displayed amplified SMAD2 / 3 signaling, indicative of enhanced TGF-β activity, which likely drives Maf and Rorc expression—both established TGF-β targets in Tregs(31, 34, 88, 89). Given the inclusion of TGF-β in the reprogramming cocktail, these findings underscore how lineage-specific epigenetic memory synergizes with extrinsic signals to shape ER-Tregfunctionality. Future studies should delineate how epigenetic landscapes of distinct T helper subsets influence ER-Tregdifferentiation and function, particularly in inflammatory contexts. Our study highlights the superior in vivo fitness of ER-Tregs over other Treg subsets, driven by intrinsic properties like their polarization state. CRISPR-mediated ablation of c- Maf impaired ER-Treg suppression of GM-CSF⁺ Teff cells in vitro, suggesting that Th17 polarization enhances their per-cell suppressive potency. While APC interactions were not directly assessed, bulk RNA-seq revealed elevated Ctla4 expression in ER-Tregs (Figure 6D). CTLA-4 is a key mediator of Tregsuppression 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 Tregswith bona fide transcriptional and suppressive programs. The resulting ER-Tregsoutperform endogenous nTregs, iTregs, and Foxp3-overexpressing Teff cells in suppressing established autoimmune inflammation, underscoring their therapeutic potential. This enhanced efficacy arises from dual mechanisms: (1) inherited autoantigen specificity, which promotes antigen specific and tissue-localized suppression, and (2) retention of parental Tefftranscriptional programs, which bolsters fitness in inflammatory niches. These insights advance our understanding of Tregepigenetic regulation while offering a blueprint for engineering antigen-specific Treg therapies with tailored functionality to treat autoimmune diseases. METHODS Sex as a biological variable. Though women are more susceptible to developing MS than men by a ratio of approximately 3:1, men that develop MS exhibit greater cognitive impairment and more rapid disability progression than women. Thus, our study examined both male and female mice and similar findings are reported for both sexes. Mice Atty Docket No.166118.01508 Animals were housed at the Tufts University School of Medicine (TUSM) animal facility under specific pathogen-free conditions according to institutional guidelines. All studies were performed under protocol B2022-85 and approved by Tufts Institutional Animal Care and Use Committee. All mouse strains used were on the C57BL / 6 genetic background. CD45.1 (#002014), Rosa26Cas9-eGFP(#026179), 2D2 (#006912), and Rag1- / -(#002216) mice were purchased from The Jackson Laboratory. Foxp3Thy1.1mice were a gift from Dr. Y. Zheng. The above mouse strains were bred in-house at TUSM to produce the Foxp3Thy1.1Rosa26Cas9, Foxp3Thy1.1CD45.1+ / +, and 2D2+ / -Foxp3Thy1.1CD45.1+ / -mouse strains. Male and female 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-Treggeneration CD4+CD44hiTeff 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. Cells 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). For experiments using transduced ER-Tregs, CD4+Thy1.1+Reporter+cells were sort purified and restimulated. 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). Production of retrovirus Retrovirus was produced by transfecting HEK293T cells 2 days prior to transduction using the pCL-Eco packaging vector and Fugene HD transfection reagent. Medium was replaced with half the transfection volume 1 day before transduction. Retroviral transduction Rested CD4+Teffcells 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 Reverse (5’- ATACACACCAAACAAACACTACACC-3’) (SEQ ID NO: 13). EAE induction EAE was induced by subcutaneous injection of 50 uL of an emulsion containing 50 ug MOG35-55and 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. Cell transfer For all preventive EAE experiments in Rag- / -mice, cells were transferred intravenously one day prior to disease initiation. Mice received 50,000 Tregsand 100,000 CD4+Tconvfrom CD45 congenically distinct Foxp3Thy1.1donor 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). For preventive EAE experiments in lymphoreplete mice, 2 x 106Tregsfrom CD45 congenically distinct Foxp3Thy1.1donor mice were transferred intravenously 1 day prior to disease initiation. For therapeutic EAE experiments in lymphoreplete mice, 2 x 106polyclonal or 0.5 x 106MOG specific CD45 congenically distinct Tregs were transferred intravenously at first evidence of disease (tail paralysis, typically day 11 post disease initiation). For in vivo fitness experiments in lymphoreplete mice, 0.5 x 1062D2 TCR transgenic CD45 congenically distinct Tregs were transferred intravenously one day prior to immunization with MOG / CFA. Cell isolation For lymph node and spleen, tissues were mechanically dissociated using the back of a syringe plunger and filtered through a 70 um nylon mesh. For analysis of immune cell infiltrates in the spinal cord, 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. 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. For 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. In vitro suppression assays CD45 congenically distinct CD4+CD44hiTeffcells 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 Tregsin the presence of 10 ug / mL MOG35-55. 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. Differential expression analysis was performed with DESeq2. 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). For comparisons of a single variable between 2 groups, Atty Docket No.166118.01508 significance was determined by 2-tailed Student’s t tests. For comparisons of multiple groups where variance did not significantly differ across groups, 1- or 2-way ANOVA with Šidák’s (for comparisons between preselected pairs) multiple-comparison corrections was used. EAE disease scores were analyzed with 1-way ANOVA of area under the curve of clinical scores. P values below 0.05 were considered statistically significant and are shown by the exact number or by asterisks in the figures. References 1. Josefowicz SZ, Lu LF, and Rudensky AY. Regulatory T cells: mechanisms of differentiation and function. Annual review of immunology.2012;30:531-64. 2. Sakaguchi S, Yamaguchi T, Nomura T, and Ono M. Regulatory T cells and immune tolerance. Cell.2008;133(5):775-87. 3. Viglietta V, Baecher-Allan C, Weiner HL, and Hafler DA. 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Claims

Atty Docket No.166118.01508 CLAIMS What is claimed:

1. A method of generating an Effector T cell Reprogrammed Treg (ER-Treg) cell ex- vivo, the method comprising: a) resting an antigen specific CD4+CD44+ effector T cell in a T-cell growth medium supplemented with IL-7, neutralizing antibodies against IL-12, neutralizing antibodies against IFNγ, and neutralizing antibodies against IL-4 for at least 3 days to 5 days; and b) culturing the antigen specific CD4+CD44+ 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; wherein the T cell growth medium comprises a cell culture medium, a base media, L- glutamine, buffered saline, an antibiotic, sodium pyruvate, and serum, and wherein the primary reprogramming cocktail comprises T cell growth medium supplemented with IL-2, TGFβ, retinoic acid, neutralizing antibodies against IL-12, neutralizing antibodies against IFNγ, and neutralizing antibodies against IL-4.

2. The method of claim 1, wherein the primary reprogramming cocktail additionally comprises ascorbic acid.

3. The method of claim 1 or 2, wherein the method further comprising c) isolating an antigen specific CD4+CD44+ effector T cell from step b) and culturing the antigen specific CD4+CD44+ effector T cell in a secondary reprogramming cocktail and a surface coated with anti-CD3 and anti-CD28 antibodies, wherein the secondary reprogramming cocktail comprises T cell growth medium, supplemented with IL-2, TGFβ and ascorbic acid.

4. The method of claim 3, wherein the secondary reprogramming cocktail further comprises neutralizing antibodies against IL-12, neutralizing antibodies against IFNγ, and neutralizing antibodies against IL-4.

5. The method of any one of the previous claims, wherein the effector T cell is cultured in the primary reprogramming cocktail or the secondary reprogramming cocktail for at least 3 to at least 4 days.Atty Docket No.166118.01508 6. The method of any one of the previous claims, wherein the ER-Treg cell expresses Foxp3.

7. The method of any one of the previous claims, wherein 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.

8. The method of any one of the previous claims, wherein the ER-Treg cell expresses CD25 and / or CTLA4.

9. The method of any one of the previous claims, wherein the ER-Treg cell has reduced expression of Lef1, Ccr7, Bach2 and / or Sell as compared to the effector T cell from which they were derived or naturally derived Treg cells.

10. The method of any one of the previous claims, wherein the ER-Treg cell has increased expression of Ctla4, IL-10, RORC, Maf, IL-23r, CCr6, and / or IL-1r as compared to a naturally occurring Treg cell.

11. The method of any one of the previous claims, wherein the antigen specific CD4+ CD44+ effector T cell is a Th1, Th2, Th9, Th17, Th22, Treg, or Tfh cell.

12. The method of any one of the previous claims, wherein the ER- Treg cell expresses one or more lineage markers of the effector T cell from which it was derived.

13. The method of any one of the previous claims, wherein the antigen specific CD4+CD44+ effector T cell is a Th17 cell and the ER-Treg cell expresses one or more Th17 lineage marker, wherein the lineage markers comprise RORγT, IL-1r1, Maf, TGFβ, IL-6r, IL- 21R, IL-23R, CCR6, and / or STAT3.

14. The method of any one of the previous claims, wherein the antigen specific CD4+CD44+ effector T cell is specific for an autoantigen.

15. The method of claim 14, wherein the autoantigen is a myelin antigen, neuron-derived antigen, or astrocyte-derived antigen.Atty Docket No.166118.01508 16. The method of any one of the preceding claims, wherein the surface comprising CD3 and CD28 is a bead and the bead is added to the reprogramming cocktail during the culturing step.

17. A composition for the generation of ER-Treg cells, the composition comprising: a base media, L-glutamine, buffered saline, an antibiotic, Sodium Pyruvate, serum, IL-7, neutralizing antibodies to IFNγ, neutralizing antibodies to IL-4, neutralizing antibodies to IL- 12, IL-2, TGFβ, retinoic acid, and vitamin C.

18. The composition of claim 16, additionally comprising retinoic acid.

19. The composition of claim 16 or 17, additionally comprising vitamin C.

20. The composition of any one of claims 17-19, additionally comprising a T cell stimulus.

21. A method of using the composition of any one of claims 17-20 to reprogram a T lymphocyte comprising culturing an antigen specific CD4+CD44+ effector T cell in the composition for at least 3 days in the presence of a surface coated with CD3 and CD28, and selecting for ER Treg cells after the culturing step.

22. A method of treating an autoimmune disease, the method comprising administering the ER-Treg cell of any one of claims 1-20, wherein the CD4+CD44+ effector T cell is specific for an antigen that is an autoimmunity causing autoantigen.

23. The method of claim 22, wherein the autoimmune disease comprises Multiple sclerosis, Rheumatoid arthritis, Lupus, Celiac disease, Sjögren's syndrome, Polymyalgia rheumatica, Ankylosing spondylitis, Type 1 diabetes, Alopecia areata, Vasculitis, and Temporal arteritis.

24. A method of decreasing an antigen-specific immune response in a subject comprising administering the ER-Treg cell of any one of claims 1-20 to a subject in need of a decreased immune response, wherein administration of the ER-Treg cell controls an antigenic specific immune response.Atty Docket No.166118.01508 25. The method of claim 24, wherein the antigen specific CD4+CD44+ effector T cell is isolated from the subject in need.

26. The method of claim 24 or 25, wherein the subject is diagnosed with Graft vs. host disease, allergy, infectious disease, autoimmune disease or the subject is an organ transplant recipient.

27. A method of decreasing an antigen-specific immune response in a subject in need thereof, the method comprising, a) isolating antigen-specific CD4+ T cells; and b) reprograming the antigen-specific CD4+ T cells into ER-Treg cells ex vivo using the method of any one of claims 1-15; and c) administering the ER-Treg cells to a subject in need thereof or wherein the antigen-specific T cells are isolated and administered to the same subject.

28. The method of claim 27, wherein the subject is diagnosed with Graft vs. host disease, allergy, infectious disease, autoimmune disease or the subject is an organ transplant recipient.

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