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Stable inhibitory activity of regulatory T cells requires the transcription factor Helios

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Science  16 Oct 2015:
Vol. 350, Issue 6258, pp. 334-339
DOI: 10.1126/science.aad0616

How T cells maintain their identity

Although best known for their pathogen-fighting prowess, T lymphocytes also ensure that the immune response does not run amok. A subset of T cells called regulatory T cells (Tregs) performs this function by, for example, making sure T cells only attack pathogens and not self. T cells can exhibit plasticity in their functions in the face of an inflammatory stimulus. Kim et al. sought to identify the molecules that ensure the stable maintenance of Tregs. Using genetically modified mice, they found that both CD4+ and CD8+ Tregs require the transcription factor Helios to stably maintain their identity.

Science, this issue p. 334

Abstract

The maintenance of immune homeostasis requires regulatory T cells (Tregs). Given their intrinsic self-reactivity, Tregs must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3+ CD4 and Qa-1–restricted CD8 Tregs results in defective regulatory activity and autoimmunity in mice. Helios-deficient Tregs develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 Tregs also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes Tregs in the face of inflammatory responses provides a genetic explanation for a core property of Tregs.

Regulatory T cells (CD4 and CD8 Tregs) dampen excessive immune responses and prevent or ameliorate autoimmune tissue damage, whereas immune suppression exerted by Tregs can impede anti-tumor immune responses. In contrast to effector T cells (Teff), which rely on robust activation and differentiative plasticity, Tregs depend on the preservation of a stable, anergic, and suppressive phenotype to maintain immune homeostasis (1, 2). Although FoxP3+ CD4 Tregs are remarkably stable (1, 2), the genetic mechanisms that ensure phenotypic stability after expansion during inflammation, infection, or autoimmunity (i.e., conditions that most require maintenance of an anergic and inhibitory Tregs phenotype) are poorly understood.

The Helios (Ikzf2) transcription factor (TF) is expressed by two Treg lineages: FoxP3+CD4+ and Ly49+CD8+ Tregs (fig. S1) (36). To determine the contribution of Helios to the regulatory phenotype, we analyzed mice deficient in Izkf2 (Helios–/–), the gene that encodes Helios (5). Helios–/– mice (6 to 8 weeks old) displayed reduced numbers of CD8 but not CD4 Tregs (fig. S2) and no obvious signs of autoimmune disorder. However, 5-month-old Helios-deficient mice exhibited increased numbers of activated CD4 and CD8 T cells, T follicular helper (TFH) cells, and germinal center (GC) B cells as compared to wild-type (WT) mice (Fig. 1A and fig. S3A). Autoimmune disease was apparent by 6 to 8 months of age, accompanied by the infiltration of immune cells into nonlymphoid tissues (Fig. 1B), the production of autoantibodies (Fig. 1C), and glomerular nephritis (fig. S3B). Rag2–/– mice reconstituted with bone marrow (BM) from Helios–/– donors also developed autoimmunity (fig. S4), indicating a lymphocyte-intrinsic effect.

Fig. 1 Helios–/– mice develop an autoimmune phenotype.

(A) Activated CD4 and CD8 T cells (CD44+CD62Llo), TFH (CD4+PD-1+CXCR5+) and GC B (B220+Fas+), cells in spleens from (5-month-old) Helios+/+ and Helios–/– mice were compared (n = 3 to 6 mice). Representative data from three independent experiments are shown. The mean ± SEM is indicated. *P < 0.05 and **P < 0.01 (Mann-Whitney test). (B) Microscopy (×200) of representative hematoxylin and eosin staining of salivary gland, liver, lung, pancreas, and kidney sections from (7-month-old) Helios+/+ and Helios–/– mice (n = 4 mice per group). Representative data from two independent experiments are shown. (C) The generation of autoantibodies was compared in sera from 7-month-old Helios+/+ and Helios–/– mice (n = 7 to 10 mice per group). Representative data from three independent experiments are shown. ANA, anti-nuclear Ab; SS/A, Sjögren’s syndrome–related antigen/A; SS/B, Sjögren’s syndrome–related antigen/B. The mean ± SEM is indicated. *P < 0.05, **P < 0.01, and ***P < 0.001 (Mann-Whitney test). (D) Viral infection of Helios-deficient mice. Two-month-old and 6-month-old Helios+/+ and Helios–/– mice were infected intraperitoneally (i.p.) with 2 × 105 plaque-forming units of LCMV-Armstrong. 30 days later, spleen cells were analyzed for TFH and GC B cells (n = 4 mice per group). Representative data from two independent experiments are shown. The mean ± SEM is indicated. *P < 0.05 (Mann-Whitney test). (E) Kidney sections from virus-infected mice were analyzed for IgG deposition and IgG+ areas in glomeruli (n = 4 mice per group). Representative data from two independent experiments are shown. The mean ± SEM is indicated. ***P < 0.001 (Mann-Whitney test).

Although Helios–/– mice did not develop overt signs of autoimmunity until 5 to 6 months of age, upon challenge with viral infection by LCMV-Armstrong (LCMV, lymphocytic choriomeningitis virus), both young (2 months) and older (6 months) Helios–/– mice but not Helios+/+ mice developed inflammatory and autoimmune changes characterized by increased levels of TFH and GC B cells (Fig. 1D) and immunoglobulin G (IgG) deposition in the kidney (Fig. 1E), although Helios+/+ and Helios–/– mice cleared virus with equal efficiency (fig. S5).

Because autoimmunity in Helios–/– mice did not result from defective negative selection (figs. S6 to S8), we asked whether it instead reflected defective Treg activity. Analyses of BM chimeras that express a selective Helios deficiency in either CD4 or CD8 T cells revealed that mice with either Helios-deficient CD4 or CD8 T cells develop autoimmune disease with similar features (fig. S9). Moreover, tolerance was dominant, because Rag2–/– mice given Helios–/– BM + Helios+/+ BM did not develop autoimmunity (fig. S10).

Direct evidence for the contribution of Helios to CD4 Treg activity and the prevention of autoimmune disease came from analysis of Heliosfl/fl.FoxP3YFP-Cre mice, which develop autoimmune disease at >5 months of age, characterized by increased numbers of activated CD4 and CD8 T cells, TFH and GC B cells (Fig. 2, A and B), autoantibody production (Fig. 2C), and immune cell infiltration (fig. S11). Moreover, BM chimeras from Heliosfl/fl.FoxP3-Cre donors developed this disorder within 6 weeks (fig. S12).

Fig. 2 Helios-deficient CD4 and CD8 Tregs contribute to autoimmune disease.

(A) Phenotype of Heliosfl/fl.FoxP3-Cre mice at 6 months of age. The percentage and numbers of activated CD4 T cells (CD44+CD62Llo) in the spleen are shown (n = 4 or 5 mice per group). Data representative of two independent experiments are shown. The mean ± SEM is indicated. ***P < 0.001 (Mann-Whitney test). (B) Fluorescence-activated cell sorting profiles for TFH and GC B cells from spleens of FoxP3-Cre and Heliosfl/fl.FoxP3-Cre mice (n = 4 or 5 mice per group). Data are representative of two independent experiments. (C) Levels of antibody to double-stranded DNA in sera of FoxP3-Cre and Heliosfl/fl.FoxP3-Cre mice at 6 months of age (n = 4 or 5 mice per group). Data are representative of two independent experiments. The mean ± SEM is indicated. **P < 0.01 (Mann-Whitney test). (D) Lethally irradiated Rag2–/– mice were reconstituted with hematopoietic progenitors from Helios+/+, Helios–/–, Scurfy, Helios+/+/Helios–/– (1:1), Helios+/+/Scurfy (1;1), and Helios–/–/Scurfy (1;1) mice (n = 4 to 6 mice per group). The intensity of immune cell infiltration was quantified by scoring immune cell infiltration 7 weeks after reconstitution: >4 (most severe), 2 to 3 (severe), 1 (mild), and 0 (none). Data are representative of two independent experiments. (E) Defective inhibitory activity of Helios-deficient CD4 and CD8 Tregs. Rag2–/– hosts received sort-purified Teff cells (CD25CD44loCD62Lhi, CD45.1) and CD4 Tregs (CD3+CD4+YFP+) from spleens of FoxP3-Cre or Heliosfl/fl.FoxP3-Cre mice. Recipients were examined for changes in weight (n = 4 mice per group). Representative data from two independent experiments are shown. The mean ± SEM is indicated. *P < 0.05 (Kruskal-Wallis test). (F) Rag2–/– hosts received CD4 T cells (Teff: CD25CD44loCD62Lhi, CD4 Treg: CD3+CD4+CD25+) from spleens of defined donor mouse strains. Recipients were examined for changes in weight (n = 4 mice per group). Representative data from three independent experiments are shown. The mean ± SEM is indicated. *P < 0.05 (Kruskal-Wallis test). (G) WT B and CD25-depleted CD4 T cells were transferred into Rag2–/– hosts along with Ly49+ or Ly49 CD8 T cells from spleens of Helios+/+ or Helios–/– mice. Rag2–/– adoptive hosts were immunized with NP19-KLH [(4-hydroxy-3-nitrophenyl acetyl)19-keyhole limpet hemocyanin] in Freund’s complete adjuvant (FCA)at day 0 and reimmunized with NP19-KLH in Freund’s incomplete adjuvant at day 10. NP-specific IgG1 responses were measured at day 15 (n = 3 mice per group). Data are representative of three independent experiments. The mean ± SEM is indicated. *P < 0.05 (Kruskal-Wallis test).

Helios-sufficient but not Helios-deficient FoxP3+ CD4 Tregs exerted dominant, lymphocyte-intrinsic inhibition that prevented autoimmune disease in the presence of highly activated self-reactive T cells from scurfy mice, which have no FoxP3 forkhead domain. BM chimeras reconstituted with Helios–/–/Scurfy BM but not Helios+/+/Scurfy BM cells rapidly developed autoimmunity (Fig. 2D and fig. S13, A and B).

Impaired suppressive activity of Helios-deficient FoxP3+ CD4 Tregs was observed when FoxP3+ CD4 cells (YFP+) from Heliosfl/fl.FoxP3YFP-Cre were cotransferred into Rag2–/– hosts with naïve CD4+ T cells, resulting in wasting disease (Fig. 2E and fig. S14A). Analysis of CD4 Tregs from Helios–/– mice with a global Helios deletion (5) showed that mice given naïve CD4 cells developed colitis that could be prevented by Helios+/+ but not Helios–/– FoxP3+ CD4 Tregs (Fig. 2F and fig. S14B).

Helios deficiency also resulted in defective CD8 Treg function. CD8 Tregs (CD44+CD122+Ly49+) recognize target TH cells through a Qa-1/peptide–T cell receptor (TCR) interaction that prevents autoantibody-mediated autoimmune disease (3). Helios-deficient CD8 Tregs failed to inhibit the adoptive helper response of TFH cells (Fig. 2G). Defective suppressive activity of Helios–/– CD8 Tregs was lymphocyte-intrinsic, because Ly49+ CD8 cells isolated from Rag2–/– recipients reconstituted with Helios+/+ but not Helios–/– BM cells mediated inhibitory activity (fig. S14C), which is consistent with impaired suppressive activity by CD8 Tregs from Heliosfl/fl.CD4-Cre mice (fig. S14D). These data indicate that Helios-dependent suppressive activity exerted by both CD4 and CD8 Tregs is required to maintain self-tolerance.

These findings suggested that Helios may control common genetic pathway(s) in CD4 and CD8 Tregs. To address this issue, we defined the genome-wide distribution of Helios binding sites in these cells by chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) (7). The chromatin state of Helios-bound regions was determined according to acetylation of histone H3 at Lys27 (H3K27ac) for active regulatory regions and trimethylation of histone H3 at Lys27 for polycomb-repressed regions (H3K27me3) (8).

This analysis revealed that Helios bound mainly to promoter regions of (~85%) target genes in both CD4 and CD8 Tregs (Fig. 3A); 1602 and 828 genes, respectively, with 649 shared target genes (Fig. 3B). Analysis of DNA regions bound by Helios showed significant enrichment of NRF1, Sp1/Sp4, and IKAROS binding motifs (Fig. 3C), as well as genes that regulate cell cycle progression and apoptosis/cell survival (table S1A), including STAT5b, Jak2, NFAT5, and Birc2, and autophagy genes, whose loci showed activated chromatin marks, as evidenced by expression of H3K27ac (Fig. 3D and table S1B). Pathway analysis of Helios target genes in CD4 and CD8 Tregs revealed STAT5b/interleukin-2 receptor α (IL-2Rα) as a central node within a Helios target gene network, suggesting that Helios might regulate genes involved in IL-2 signaling and sustained survival (Fig. 3E).

Fig. 3 Helios-dependent STAT5 activation and stabilization of CD4 Tregs.

(A) Distribution of genome-wide Helios binding sites in activated FoxP3+ CD4 and Ly49+ CD8 Tregs. (B) Number of Helios target genes and overlapping Helios binding sites in CD4 and CD8 Tregs. (C) DNA motif analysis of Helios-bound regions. (D) ChIP-Seq analysis of Helios binding and modified histones at STAT5b, Jak2, NFAT5, and Birc2 in CD4 and CD8 Tregs. Start sites of each gene locus are indicated. Vertical lines in gene diagrams (bottom) indicate exons. (E) Pathway analysis of genes targeted by Helios in CD4 and CD8 Tregs. Solid lines represent genes known to have a direct connection. Dotted lines represent a presumptive interaction based on reported studies. Major nodes of genes are marked by red circles. Solid symbols indicate Helios target genes, and white symbols indicate neighboring genes that are functionally associated but not included in the Helios target genes.

The IL-2Rα–STAT5 pathway is essential for CD4 Treg survival, whereas the maintenance of FoxP3 expression reflects binding of activated STAT5 to the Foxp3 promoter and CNS2 regions (9). Identification of the IL-2Rα–STAT5 pathway as a major Helios gene network opened the possibility that Helios contributed to Treg survival and/or FoxP3 stability through increased STAT5 activation and enhanced IL-2 responsiveness (10, 11). We noted reduced survival of Helios–/– CD4 Tregs after transfer into lymphopenic hosts (Fig. 4A, left and middle), which is consistent with an essential contribution of the IL-2Rα–STAT5 signaling pathway to Treg viability. Moreover, Helios-deficient CD4 Tregs displayed reduced coexpression of FR4 and CD73 (Fig. 4A, right), markers that indicate anergy by self-reactive CD4 cells (12). Indeed, a significant portion of Helios-deficient CD4 Tregs developed into non-anergic cells under inflammatory conditions, including autoimmunity and infection (figs. S12C and S15). Because cytokine signaling can induce binding of activated STAT6 to the Foxp3 locus and competition with p-STAT5 (10), diminished STAT5 activation in Helios-deficient CD4 Tregs may allow increased STAT6 binding to the Foxp3 locus and diminished FoxP3 expression. Reduced STAT5 activation by Helios-deficient Tregs was accompanied by reduced expression of FoxP3 under conditions including progressive autoimmune disease (6-month-old Heliosfl/fl.FoxP3-Cre mice) and colitis (Fig. 4B), consistent with the contribution of STAT5 to stable FoxP3 expression (10).

Fig. 4 Helios-dependent STAT5 activation and stabilization of CD4 Tregs.

(A) CD4 Tregs from spleens of Helios+/+ and Helios–/– mice were transferred into Rag2–/–γc–/– mice and numbers, apoptosis, and the anergic phenotype of recovered CD4 Tregs from spleens were analyzed 5 days after transfer (n = 4 mice per group). Representative data from three independent experiments are shown. The mean ± SEM is indicated. *P < 0.05 and **P < 0.01 (Mann-Whitney test). (B) Levels of FoxP3 expression were compared between splenic CD4 Tregs from 6-month-old FoxP3-Cre and Heliosfl/fl.FoxP3-Cre mice (left panel) and from indicated hosts in which colitis had been induced (n = 4 mice per group) (middle, right panels). Representative plots of more than three different experiments are shown. The mean ± SEM is indicated. *P < 0.05 and **P < 0.01 (Mann-Whitney test). (C) BM chimeras were generated by reconstituting Rag2–/– mice with hematopoietic progenitors from Helios+/+, Helios–/–, or Heliosfl/fl.FoxP3-Cre mice. After 6 to 8 weeks, the IL-2 responsiveness of FoxP3+ CD4 cells from spleens was tested as described in the methods. Representative histograms for the expression of p-STAT5 from two independent experiments are shown. (D) CD4 Tregs from Helios+/+ and Helios–/– mice were transduced with retrovirus expressing green fluorescent protein (GFP) or STAT5-CA/GFP, before stimulation with antibodies to CD3/CD28 in the presence of IL-2 (0 to 50 ng/ml) and IL-4 (20 ng/ml). Levels of FoxP3 expression after 5 days at 5 ng/ml IL-2 are shown (left panel). IFN-γ production by FoxP3+ cells is shown (right panel). Representative data from two independent experiments are shown. The mean ± SEM is indicated. *P < 0.05 and **P < 0.01 (Mann-Whitney test). (E) Two-month-old Heliosfl/fl and Heliosfl/fl.FoxP3-Cre mice were immunized i.p. with 8 × 108 sheep red blood cells. After 7 days, FoxP3+ cells from spleens were analyzed for IFN-γ and IL-17A expression (n = 4 or 5 mice per group). Representative data from two independent experiments are shown. The mean ± SEM is indicated. *P < 0.05 and ***P < 0.001 (Mann-Whitney test). (F) OT-II cells (1 × 106) were transferred into Rag2–/– hosts along with CD25+CD4+ T cells (2 × 105) from spleens of CD45.1+ Helios+/+ or CD45.2+ Helios–/– mice followed by immunization with OT-II peptide (10 μg) in FCA. After 5 days, spleen cells from Rag2–/– hosts were analyzed for FoxP3 expression and effector cytokine expression by CD4 Tregs (n = 4 mice per group). Representative data from three independent experiments are shown.

Examination of STAT5-dependent IL-2 responsiveness revealed decreased STAT5 activation in FoxP3+ CD4 cells from Helios–/– and Heliosfl/fl.FoxP3-Cre mice as compared to Helios+/+ mice (Fig. 4C). Enforced expression of constitutively active STAT5 (STAT5-CA) (13) in Helios-deficient CD4 Tregs restored FoxP3 expression to levels similar to WT CD4 Tregs and prevented the expression of interferon-γ, an effector cytokine (Fig. 4D and fig. S16).

Decreased FoxP3 expression may result in phenotypic instability, including derepression of Teff programs (10, 11). Analysis of the CD4 Treg phenotype after immunization of mice with sheep red blood cells revealed that Helios-deficient (Heliosfl/fl.FoxP3-Cre) but not Helios sufficient (Heliosfl/fl) CD4 Tregs express effector cytokines, including interferon-γ (IFN-γ) and IL-17. (Fig. 4E). Acquisition of a Teff phenotype under inflammatory conditions by Helios-deficient CD4 Tregs was a cell-intrinsic phenotype. Rag2–/– hosts were injected with CD4 T cells that transgenically express the OT-II T cell receptor (Helios WT), Helios+/+ (CD45.1+), and Helios–/– (CD45.2+) Tregs and were immunized with OT-II peptide. Helios-deficient but not Helios-sufficient CD4 Tregs displayed reduced FoxP3 expression and produced effector cytokines, including IFN-γ, IL-17, and tumor necrosis factor–α (Fig. 4F). Decreased FoxP3 expression by Helios-deficient CD4 Tregs during colitis progression was also accompanied by the expression of effector cytokines, including IFN-γ and IL-17 and the FR4loCD73lo phenotype (fig. S17).

Defective suppressive activity by Helios-deficient CD8 Tregs was associated with a similar phenotypic defect under inflammatory conditions. In adoptive Rag2–/–Prf1–/– hosts, Helios-deficient CD8 Tregs exhibited increased apoptosis and reduced cell recovery as compared to Helios WT CD8 Tregs, as observed for Helios-deficient FoxP3+ CD4 Tregs (fig. S18A).

In CD8 T cells, cytokines IL-2 and IL-15 induce STAT5 activation, whereas sustained activation depends on IL-2 (14, 15). Helios-deficient CD8 Tregs displayed reduced IL-2 responsiveness and diminished STAT5 activation (figs. S18B), suggesting that Helios may serve an overlapping function in both regulatory cell types through promotion of STAT5-dependent IL-2 responsiveness and increased survival under inflammatory conditions. Indeed, in vitro stimulation of Ly49+ CD8 Tregs versus Ly49 CD8 Tcon in the presence of STAT5 inhibitor (AG490) revealed impaired survival of CD8 Tregs but not of conventional memory CD8 cells (fig. S18C).

Analysis of CD8 Treg responses under inflammatory conditions revealed that Helios–/– CD8 Tregs expressed high levels of PD-1, TIM-3, and Lag3 and low levels of CD127 (figs. S18D and S19A). Transfer of Helios+/+ and Helios–/– CD8 Tregs (>99% Ly49+ from 2-month-old mice) into Rag2–/– hosts along with OT-II cells and antigen also revealed that Helios–/– Ly49+ CD8 cells expressed high levels of PD-1 and TIM3 (fig. S19B) and reduced survival (fig. S19C). These findings suggest that a Helios-dependent genetic program that enhances the STAT5-dependent IL-2 responsiveness of Tregs may promote survival and maintenance of an inhibitory CD8 Treg phenotype.

Earlier studies of Helios and FoxP3+ CD4 Tregs suggesting no impact on Tregs (4, 5) relied mainly on analysis in the steady state of non-immune mice. Although young Helios–/– mice do not develop signs of disease, they develop a dysregulated immune response at ~5 months of age that is markedly accelerated by immune stimulation, including viral infection. Although Helios belongs to a set of TFs that regulate FoxP3+ Tregs (1618), Helios does not form protein complexes with FoxP3 (19) nor bind to the Foxp3 locus (fig. S20). Down-regulation of FoxP3 expression and expression of effector cytokines by Helios-deficient Tregs may reflect reduced activation of the IL-2Rα–STAT5 pathway and diminished binding of STAT5b to CNS-2, resulting in Treg conversion into Teff cells (fig. S21) (10). The ability of Tregs to sense IL-2 may also be particularly critical under inflammatory conditions, where small changes in proliferation rate or apoptosis can exert major changes in the niche-filling response by Tregs and Treg insufficiency (20). Current views that thymic-derived Tregs are phenotypically and functionally more stable than induced Tregs may reflect, in part, Helios-dependent activation of the Treg IL-2Rα–STAT5 pathway described here. Recently developed surrogate surface markers may allow the isolation of stable Helios+ CD4 Tregs for the treatment of autoimmune disease or after organ transplantation (21, 22).

Helios-dependent maintenance of CD8 Treg integrity also includes the inhibition of terminal differentiation and maintenance of suppressive/cytolytic activity by activating the STAT5 signaling pathway (fig. S21). Although Helios-dependent regulation of overlapping genetic pathways, including cell survival, may stabilize the suppressive phenotype of both FoxP3+ CD4 Tregs and CD8+ Tregs, much of the Helios+ phenotype may reflect subset-specific disparities in lineage commitment and development and distinct mechanisms of suppression.

Identification of bona fide signaling pathways that induce and maintain Helios expression in FoxP3+ CD4 Tregs may allow Helios inhibition and conversion of memory Tregs into Teff cells that express self-reactive TCR with specificity for tumor antigens (23). Because Treg→Teff conversion may be confined to inflammatory intratumoral microenvironments, antibody- or small-molecule–based approaches that target Helios may lead to improved Treg-dependent cancer immunotherapy.

Supplementary Materials

www.sciencemag.org/content/350/6258/334/suppl/DC1

Materials and Methods

Figs. S1 to S22

Table S1

References (24, 25)

References and Notes

  1. Acknowledgments: We thank A. Thornton and E. Shevach (NIH) for provision of Heliosfl/fl.CD4-Cre mice, S. Crotty (La Jolla Institute for Allergy and Immunology) for provision of retroviral vector STAT5-CA, R. Bronson (DF/HCC Rodent Histopathology Core) for histology analysis, and A. Angel for manuscript and figure preparation. The data reported in this manuscript are tabulated in the main paper and in the supplementary materials. Raw data are archived in the Gene Expression Omnibus under accession numbers GSE72997 (ChIP-Seq) and GSE73015 (microarray. These studies were supported in part by research grants NIH R01AI37562 and the LeRoy Schecter Research Foundation to H.C. and the Arthritis National Research Foundation to H.-J.K. A provisional patent (U.S. patent application 62/170,379) has been filed pertaining to biological applications relating to the conversion of regulatory T cells into effector T cells for immunotherapy.
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