iTreg Generation Research Articles

Cytomegalovirus-Specific Regulatory T Cells and their Role in Repetitive CMV Infections in Solid Organ Transplanted Patients

Introduction: Cytomegalovirus (CMV) is a major cause of morbidity and mortality after transplantation. Recently, we confirmed that adoptive transfer of autologous IE-1/pp65-specific T-cell lines was able to combat severe CMV disease; however, the control of CMV infection was only temporary. We hypothesized that CMV-induced regulatory T cells (iTreg) might be related to recurring/lasting CMV infection. Methods & Results: In fact, kidney transplant patients with recurring CMV infections expressed enhanced suppression on CMV-specific cytokine and proliferative response compared to patients with non-recurring CMV infections. Analysis of in vitro expanded CMV-specific T cells revealed that CD25highCD39+ T cells functionally suppress CD25low effector T cells (Teff) upon epitope-specific reactivation. Additionally to this functional evidence, we could demonstrate that CD25highCD39+ T cells belong to the peripherally induced subset of Tregs (iTregs), for several reasons: (1) they are Helios negative, (2) they express a methylated foxp3 locus in contrast to natural thymus-derived Tregs (nTregs), and (3) they share the same dominant TCR-Vβ-CDR3 clones with functionally distinct CD25low Teff indicating that Teff and iTreg can differentiate from one clone with specificity to the same viral epitope. Moreover, the same clones were present in freshly isolated CD4+CD25high and CD4+CD25low T cells suggesting their in vivo generation. In contrast to nTregs, in vitro expanded CMV-specific iTregs produce IFNg upon antigen-specific re-stimulation. However, they secrete low amounts of IL-2 and are able to suppress IL-2 responses of Teff cells, like nTregs. Therefore, we were interested whether the effector and/or immunoregulatory cytokine expression of CMV-specific iTregs is merely a transient ability modulated by in vitro culturing or, in contrast, is a stably imprinted phenotype inherited from progenitor cells. As epigenetic modifications are an inheritable basis for memory of cytokine expression, we analyzed the DNA methylation status of the respective promoters of CMV-specific T cells compared to nTregs. Preliminary results suggest that culturing of CMV-specific T cells with rapamycin actively maintained methylation of the IL-2 promoter, thus blocked IL-2 production. Additionally, the epigenetic control of IL-2 seems to be distinct in iTregs vs Teff cells. Conclusion: Taken together, our findings demonstrate that peripheral iTreg generation seems to be related to frequent antigen appearance and that the suppressive impact of CMV-specific iTregs is of importance for solid organ transplanted patients suffering from CMV infections. If the methylation status of cytokine promoters is a tool to further differentiate natural from induced Tregs remains to be investigated.

Galectin-9 promotes TGF-β1-dependent induction of regulatory T cells via the TGF-β/Smad signaling pathway

TGF-β1 induces the conversion of CD4+CD25- Tcells into CD4+CD25+Foxp3+ regulatory T cells (Tregs). Galectin-9, one of the β-galactoside-binding animal lectins belonging to the galectin family, induces apoptosis of eosinophils, cancer cells and T cells. In this study, we report the effects of galectin-9 on the conversion of CD4+CD25- T cells into Foxp3-expressing induced Tregs (iTregs). Galectin-9 together with TGF-β1 had synergistic effects on the rate of conversion to iTregs invitro. TGF-β1 signaling appears to be essential for the effects of galectin-9 on the generation of iTregs, as galectin-9 promotes TGF-β1-induced phosphorylation of Smad2/3, ERK1/2 and formation of the Smad2/3-Smad4 complex. These data suggest a role for galectin-9 as a promoter of the TGF-β1-dependent conversion of CD4+CD25- T cells into iTregs invitro. Therefore, in addition to inducing apoptosis, galectin-9 may contribute to the regulation of inflammation via the expansion of peripheral Tregs.

Cell-autonomous role of TGFβ and IL-2 receptors in CD4+ and CD8+ inducible regulatory T-cell generation during GVHD

AbstractFoxP3+ regulatory T cells (Tregs) suppress GVHD while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from the expansion of preexisting natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor Tconvs in the transplantation recipient. Using an MHC-mismatched model of acute GVHD, in the present study we found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Experiments using various combinations of T cells from wild-type and FoxP3-deficient mice suggested that both preexisting donor nTregs and the generation of iTregs in the recipient mice contribute to protection against GVHD. Surprisingly, CD8+FoxP3+ T cells represented approximately 70% of the iTreg pool. These CD8+FoxP3+ T cells shared phenotypic markers with their CD4+ counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4+ and CD8+ Tregs appeared to be protective against GVHD-induced lethality and required IL-2 and TGFβ receptor expression for their generation. These data illustrate the complex makeup of the donor-derived FoxP3+ Treg pool in allogeneic recipients and their potential role in protection against GVHD.

Metabolic control of T cell fate decision: the HIF1α-glycolysis axis in the differentiation of TH17 and iTreg cells (163.17)

Abstract Activated T cells mainly use glycolysis to suffice their bioenergetic and biosynthetic demands. However, whether glycolysis actively controls the differentiation of activated T cell is not fully understood. As a master regulator of metabolism, HIF1α regulates multiple rate-limiting glycolytic enzymes. We previously reported that HIF1α deficiency inhibited TH17 differentiation and promoted induced Treg (iTreg) generation (Shi et al, J Exp Med 2011). To further explore the roles of the glycolytic pathway in T cell differentiation, we combined genetic and pharmacological approaches to examine T cell metabolism and signaling. Inhibition of glycolysis by multiple agents diminished TH17 differentiation and promoted iTreg induction, while supplementation of the products of the glycolytic flux partially rescued these defects. Importantly, blocking glycolysis ameliorated the pathogenesis of autoimmune neuroinflammation both prophylactically and therapeutically, suggesting that the glycolytic pathway is a potential therapeutic target for autoimmune pathogenesis elicited by TH17 cells. Mechanistic studies further revealed that downregulation of IL-23R expression contributed to the diminished TH17 differentiation in HIF1α-deficient T cells, as forced expression of IL-23R restored the TH17 defects. In conclusion, our results demonstrate that the HIF1α-mediated glycolytic pathway links T cell metabolism and signaling, thereby dictating the cell fate decision between TH17 and iTreg cells.

Program death-1 regulates peripheral T cell tolerance via an anergy-independent mechanism

Program death-1 (PD-1) has been documented to negatively regulate immune responses. However, the cellular and molecular mechanisms for PD-1-mediated immune suppression have not been fully elucidated. In this study, we show that loss of PD-1 does not lead to defective induction of CD4(+) T cell anergy in vitro and in vivo. Rather, the absence of PD-1 inhibits the development of inducible CD4(+)Foxp3(+) regulatory T cells (iTregs) induced by TGF-β in vitro. In support of this finding, PD-1 deficiency impairs the generation of iTregs in vivo and leads to development of severe T cell-transfer-induced colitis. Mechanistically, defective iTreg generation in the absence of PD-1 was attributed to the heightened phosphorylation of Akt. Therefore, we first demonstrate that PD-1 controls peripheral T cell tolerance via an anergy-independent but iTreg-dependent mechanism.

Cytomegalovirus-Specific Regulatory and Effector T Cells Share TCR Clonality—Possible Relation to Repetitive CMV Infections

Cytomegalovirus (CMV) infections have a major impact on morbidity and mortality of transplant patients. Among the complex antiviral T-cell response, CMV-IE-1 antigen-specific CD8+ cells are crucial for preventing CMV disease but do not protect from recurring/lasting CMV reactivation. Recently, we confirmed that adoptive transfer of autologous IE-1/pp65-specific T-cell lines was able to combat severe CMV disease; however, the control of CMV infection was only temporary. We hypothesized that CMV-induced regulatory T cells (iTreg) might be related to recurring/lasting CMV infection. In fact, kidney transplant patients with recurring CMV infections expressed enhanced suppression on CMV response. Analysis of in vitro expanded CD4+ epitope-specific cells revealed that CMV-specific CD4+CD25(high) Treg cells functionally suppress CD25(low) effector T cells (Teff) upon epitope-specific reactivation. Their phenotype is similar to iTreg - CD39(high) /Helios-/IL-2(low) /IFNγ(high) /IL-10±/TGFß-LAP±/FOXP3+ and methylated foxp3 locus. Remarkably, in vitro expanded CD4+CD25(high) iTreg share the same dominant TCR-Vβ-CDR3 clones with functionally distinct CD4+CD25(low) Teff. Moreover, the same clones were present in freshly isolated CD4+CD25(high) and CD4+CD25(low) T cells suggesting their in vivo generation. These findings directly demonstrate that Teff and iTreg can differentiate from one "mother" clone with specificity to the same viral epitope and indicate that peripheral iTreg generation is related to frequent antigen appearance.

The Immune System uses iTregs to keep from giving Non-pathogenic Microorganisms a “Time-Out”

The Immune System uses iTregs to keep from giving Non-pathogenic Microorganisms a “Time-Out”

Induced Foxp3+ regulatory T cells: a potential new weapon to treat autoimmune and inflammatory diseases?

Foxp3(+) T regulatory cells (Tregs) consisting of natural and induced Treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. The actions designed to correct defects in numbers or functions of Tregs may be therapeutic in the treatment of autoimmune diseases. While recent studies demonstrated that natural Tregs are instable and dysfunctional in the inflammatory condition, induced Tregs (iTregs) may have a different feature. Here we review the progress of iTregs, particularly focus on their stability and function in the established autoimmune diseases. The advantage of iTregs as therapeutics used under inflammatory conditions is highlighted. Proper generation and manipulation of iTregs used for cellular therapy may provide a promise for the treatment of many autoimmune and inflammatory diseases.

Induction of a Novel Population of CD8+ Foxp3+ Regulatory T Cells During Graft Versus Host Disease

Abstract 821Regulatory T cells defined as CD4+ and expressing the transcription factor Foxp3 have been shown to play a pivotal role in mitigating the severity of graft versus host disease (GVHD). In the course of studies designed to define the functional role of various CD4+ Treg populations in GVHD biology, we identified a novel population of CD8+ T cells that expressed Foxp3 and were induced early during this disease. While this population has been reported in patients with autoimmune disorders, the role of CD8+ Foxp3+ T cells in GVHD is unknown. To delineate the significance of this observation, we performed studies in which lethally irradiated Balb/c [H-2d] mice were transplanted with bone marrow and spleen cells from C57BL/6J [H-2b] mice that carried an EGFP reporter gene linked to Foxp3 (Foxp3EGFP). Tissues (spleen, lung, liver and colon) were harvested 5, 7, 10, 14 and 21 days post transplantation to define the temporal kinetics and absolute numbers of CD8+ Tregs during acute GVHD. We observed that CD8+ Foxp3+ T cells were detectable as early as five days post transplantation and persisted for up to three weeks in all GVHD target tissues. This cell population was present in similar percentages and absolute numbers to CD4+ Tregs in these tissue sites which is noteworthy given that the CD4+ Treg pool is comprised of two populations (natural Tregs and induced Tregs) whereas the CD8 pool is made up almost exclusively of Tregs that are induced, since only a very small percentage of CD8+ T cells from normal mice (<1.0%) constitutively express Foxp3. To determine whether the induction of CD8+ Tregs was a function of MHC disparity, we performed similar transplant studies using murine models with varying degrees of MHC incompatibility. Notably, the relative and absolute number of CD8+ Tregs were much lower in an MHC-matched, minor antigen mismatched model of GVHD [B6→Balb.B], and were absent in a model where only three amino acids distinguish donor and recipient [B6→bm1], indicating a correlation between CD8+ iTreg generation and MHC disparity between donor and host. To confirm that in vivo-induced CD8+ Tregs were suppressive, CD8+ Foxp3+ and CD4+ Foxp3+ T cells were sorted from the spleen and liver of B6→Balb/c GVHD mice six days post transplantation and examined in standard MLC suppression assays. These studies revealed that in vivo-derived CD8+ and CD4+ Tregs equally suppressed alloreactive T cell responses. Phenotypic analysis of in vivo-differentiated CD8 iTregs revealed that these cells expressed many of the same cell surface molecules as CD4+ Tregs (e.g. GITR, CD25, CD103, CTLA-4). To determine if CD8+ Foxp3+ T cells could be induced in vitro and used as adoptive therapy for GVHD prevention, purified CD8+ Foxp3EGFP– T cells were cultured with anti-CD3/CD28 antibodies, TGF-β and IL-2 for 3 days. Under these conditions, ∼30% of cells are induced to become Foxp3+. Addition of in vitro-differentiated CD8+ iTregs to a standard MLC resulted in potent suppression which was equivalent to that observed with in vitro-differentiated CD4+ Tregs. To determine whether these cells were suppressive in vivo, in vitro-differentiated CD8+ iTregs were adoptively transferred at a 1:1 Treg: effector cell ratio into lethally irradiated Balb/c mice that also received B6.PL BM and spleen cells to induce GVHD. In vitro-derived CD8+ iTregs failed to protect mice from GVHD in comparison to animals transplanted without CD8+ iTregs. This was attributable to reduced survival and the loss of Foxp3 expression in vivo. Furthermore, approximately 30–50% of these cells reverted to a proinflammatory phenotype characterized by IFN-γ secretion, similar to what has been described for in vitro-differentiated CD4+ iTregs (Beres et al, Clin Can Res, 2011). Finally, microarray studies were performed to compare the gene signatures of in vitro versus in vivo-induced CD8+ Tregs. Ontological analysis revealed that there was a 3–16 fold increase in the transcription of cytokine (e.g. IL-10) and cytotoxic (granzyme A, perforin, granzyme B) pathway genes in in vivo versus in vitro-induced CD8+ Tregs, suggesting that the former Treg population may employ similar mechanisms of suppression as has been reported for CD4+ Tregs. In summary, these studies have identified a novel population of CD8+ Foxp3+ cells that are induced early during GVHD, are able to suppress alloreactive T cell responses, and constitute another regulatory T cell population that is operative in GVHD biology. Disclosures:No relevant conflicts of interest to declare.

Enhanced induction of CD4+CD25+FOXP3+ suppressive function in memory versus naïve CD4+CD25-FOXP3- T-cells is proportional to strength of activating stimulus. (50.2)

Abstract CD4+CD25+FOXP3+ regulatory T-cells (Tregs) play a vital role in suppressing autoimmune responses. We have previously shown that, in humans, all activated T-cells attain a state of transient FOXP3 expression that correlates with transient suppressive ability. In fact, peripheral generation of induced Tregs (iTregs) is likely the dominant source of regulatory cells in healthy adults. Through the use of a sensitive flow-based suppression assay, we observed that both memory and naïve CD4+CD25-FOXP3- T-cells developed regulatory ability following a variety of activating stimuli. This suppressive ability was not due to competition for nutrients or antigen presenting cells. Interestingly, memory T-cells displayed significantly greater FOXP3 induction compared to naïve counterparts, and this phenotype correlated to significantly enhanced suppressive function. Furthermore, acquisition of suppressive ability in both the memory and naïve iTreg compartments was proportional to the strength of activating stimulus. While blockade of activation using anti-IL-2, CTLA-4 Ig, anti-TGF-B, indomethacin or cyclosporin A did not affect iTreg generation, methotrexate drastically preempted the induction of regulatory ability. Moreover, irradiation of iTregs also abrogated regulatory function. These studies suggest that iTreg development and function may vary dependent on precursor origin and emphasize the importance of characterizing the iTreg compartment prior to therapeutic application.

Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling

AbstractCD28 costimulation is required for the generation of naturally derived regulatory T cells (nTregs) in the thymus through lymphocyte-specific protein tyrosine kinase (Lck) signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naive CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naive CD4 T cells (CD25−Foxp3−) by T-cell receptor stimulation with additional transforming growth factorβ (TGFβ) in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. Using a series of transgenic mice on a CD28-deficient background that bears wild-type or mutated CD28 in its cytosolic tail that is incapable of binding to Lck, phosphoinositide 3-kinase (PI3K), or IL-2–inducible T-cell kinase (Itk), we found that CD28-mediated Lck signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide a rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28 signaling.

Thymic Stromal Lymphopoietin Interferes with Airway Tolerance by Suppressing the Generation of Antigen-Specific Regulatory T Cells

Thymic stromal lymphopoietin (TSLP) is an essential cytokine for the initiation and development of allergic inflammation. In this study, we have investigated the role of TSLP in the breakdown of immune tolerance and generation of inducible regulatory T cells (iTregs). Our results demonstrated that TSLP diverted airway tolerance against OVA to Th2 sensitization and inhibited the generation of OVA-specific iTregs. TSLP exerted a direct inhibitory effect on both human and mouse iTreg development in vitro. Low doses of TSLP were capable of inhibiting iTreg induction without significantly promoting Th2 development, indicating that these two functions of TSLP are separable. Moreover, the TSLP-mediated inhibition of iTreg generation was only partially dependent on IL-4 and Stat6, and was effective when TSLP was present for the first 24 h of T cell activation. These results define a novel role for TSLP in regulating the balance of airway tolerance and allergic inflammation.

Response to Taraban, Ferdinand, and Al-Shamkhani

We demonstrated that the TNF receptor superfamily member 25 (TNFRSF25) agonistic antibody 4C12 drives the in vivo proliferation of preexisting CD4+FoxP3+ cells (Tregs) but not that of conventional CD4 cells (Tconvs) (1). TNFRSF25-mediated Treg proliferation depends upon MHC II loaded with self-antigen and is blocked by calcineurin inhibitors. If antigen is provided to Tconvs in vitro or in vivo, TNFRSF25 signals also costimulate proliferation and effector cell activity (2–5). Thus, Treg proliferation to TNFRSF25 signals is due to the continuous presence of self-antigenic stimulation of the TCR in vivo, rather than to the level of TNFRSF25 expression. TNFRSF25-mediated Treg expansion is not unique to the agonistic 4C12 antibody, as a TL1A-Ig fusion protein mediates similar Treg expansion in vivo (unpublished observations). In support of this conclusion, the subsequent reports by Al-Shamkhani’s group and Siegel’s group demonstrated that in transgenic mouse models overexpressing TL1A, Tregs proliferate at high frequencies throughout the secondary lymphoid organs and have increased frequencies of Tconvs with an effector/memory phenotype systemically, resulting in mice that are prone to spontaneous inflammatory bowel disease (6, 7). Meylan et al. also demonstrate that TL1A inhibits the de novo induction of Tregs from Tconvs in vitro (7). Both of these recent reports agree that TNFRSF25 attenuates the suppressive capacity of Tregs. In a letter to the JCI, Al-Shamkhani and colleagues provide evidence that the level of expression of TNFRSF25 appears much more similar between Tregs and Tconvs than we originally reported. In recent studies, we have further explored the binding of additional anti-TNFRSF25 antibodies and TL1A-Ig fusion proteins generated in our laboratory to Tregs and Tconvs by flow cytometry. We have found clone-specific variations in the intensity of TNFRSF25 staining between Tconvs and Tregs, which suggests that, as indicated by Al-Shamkhani, the absolute expression of TNFRSF25 between Tconvs and Tregs is not a key determinant in the proliferative specificity of TNFRSF25 agonists or TL1A to Tregs. Instead, as both our and Siegel’s group concluded, the determining factor of TNFRSF25 action in vivo is the availability of cognate antigen, with self-antigen continuously present for Treg expansion. Together, these three reports agree that TNFRSF25 is a regulator of Treg proliferative responses, both with the natural ligand (TL1A) and with receptor agonistic reagents (antibodies and fusion proteins). In the transgenic systems used by Taraban and colleagues and Meylan et al., the continuous availability of TL1A stimulates both the proliferation of Tregs and activation of antigen-experienced effector/memory phenotype Tconvs systemically (6, 7). The finding by Meylan et al. with regard to inhibition of in vitro iTreg generation by TL1A is consistent with the possibility of a role for TL1A in inflammatory bowel disease, in which tolerance to endogenous microbial antigens may have been disrupted by transgenic overexpression of TL1A (7). The balance of TNFRSF25 triggering regulatory or effector immunity is likely dependent upon the availability of cognate antigen to Tregs or Tconvs and not on absolute differences in the expression of the receptor between each subset.

Peroxisome Proliferator-Activated Receptor α and γ Agonists Together with TGF-β Convert Human CD4+CD25− T Cells into Functional Foxp3+ Regulatory T Cells

Human peripheral CD4(+)CD25(-) T cells can be induced to express Foxp3 when activated in vitro by TCR stimulation with TGF-β and IL-2. However, these TGF-β-induced Foxp3(+) regulatory T cells (iTregs) lack a regulatory phenotype. From libraries of nuclear receptor ligands and bioactive lipids, we screened three peroxisome proliferator-activated receptor (PPAR)α (bezafibrate, GW7647, and 5,8,11,14-eicosatetraynoic acid) and two PPARγ agonists (ciglitazone and 15-deoxy-Δ-(12,14)-PG J(2)) as molecules that increased Foxp3 expression in human iTregs significantly compared with that in DMSO-treated iTregs (control). These PPARα and PPARγ agonist-treated iTregs maintained a high level of Foxp3 expression and had suppressive properties. There were no significant differences in the suppressive properties of iTregs treated with the three PPARα and two PPARγ agonists, and all of the treated iTregs increased demethylation levels of the Foxp3 promoter and intronic conserved noncoding sequence 3 regions. Furthermore, PPARα and PPARγ agonists, together with TGF-β, more strongly inhibited the expression of all three DNA methyltransferases (DNMTs) (DNMT1, DNMT3a, and DNMT3b) in activated CD4(+) T cells. These results demonstrate that PPARα and PPARγ agonists together with TGF-β elicit Foxp3 DNA demethylation through potent downregulation of DNMTs and induce potent and stable Foxp3 expression, resulting in the generation of functional iTregs. Moreover, trichostatin A and retinoic acid enhanced the generation of iTregs synergistically with PPARα and PPARγ agonists.

In Vitro expanded STAT1−/− Regulatory T Cells Prevent Acute Graft Versus Host Disease (GVHD) and Promote Donor Cell Engraftment In Allogeneic Fully MHC-Mismatched Bone Marrow Transplant

AbstractAbstract 732Treg cells have been recognized as critical regulators of the immune response and shown to prevent the development of GVHD. However, little is known about of the role of STAT1 signaling in Treg cells during the development of GVHD. In this study, we tried to investigate how STAT1 signaling controls donor Treg development and function in the setting of GVHD. For this purpose we studied the role of STAT1 in natural and inducible Treg (nTreg and iTreg, respectively). To better understand the influence of STAT1-deficiency on the proliferation of nTreg cells, purified splenic STAT1−/− or STAT1+/+ CD4+CD25+ cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and cultured on anti-CD3 coated plates in the presence of anti-CD28 and IL-2 for 3 days and analyzed for proliferation and viability. After 72h of in vitro culture 50% of the STAT1+/+ starting population were no longer viable compared to only 10% of STAT1−/− cells. Furthermore, we noted a significantly increased expansion of STAT1-deficient CD4+CD25+Foxp3+ Treg cells compared to STAT1+/+ Treg cells (p<0.001). In line with these findings, STAT1-deficiency resulted in a significantly higher proportion of CFSElo cells indicating vigorous proliferation (85% Foxp3+CFSElo in STAT1−/− compared to only 65% Foxp3+CFSElo in STAT1+/+ Treg cells. Furthermore, at the end of the culture 30% of the STAT1+/+ CD4+CD25+ population were Foxp3-negative compared to only 10% of the STAT1−/− cells. We next determined the impact of STAT1 on the generation of iTreg cells in vitro. For this purpose CD4+CD25− cells from STAT1−/− or STAT1+/+ mice were cultured for 3 days on anti-CD3 coated plates in the presence of anti-CD28 antibodies, hTGF-β, mIL-2, anti-IFN-γ and anti-IL-4 for 3 days. Compared to STAT1+/+, we observed significantly enhanced generation of iTregs from STAT1−/− splenocytes (19.9%±3.0% vs. 10.6%±1.3%, p=0.008). We then performed studies to assess the in vivo generation of iTreg. For that purpose BALB/c mice were reconstituted with T Cell Depleted (TCD) 129.STAT1+/+Bone Marrow Cells (BMC) following lethal irradiation and recipients were co-injected with CD4+CD25− cells purified from either 129.STAT1+/+ or 129.STAT1−/− splenocytes. We again noted a significantly higher proportion of CD4+CD25+ Foxp3+ cells in recipients of CD4+CD25−STAT1−/− cells compared to recipients of STAT1+/+ T cells indicating a significantly increased conversion of CD4+CD25- cells into Treg cells. To confirm the in vitro results we tested the functional ability of in vitro expanded (using anti-CD3, anti-CD28, IL-2 and TGF-β) STAT1+/+ or STAT1−/− Treg cells to block induction of GVHD. GVHD was induced in BALB/c mice following lethal irradiation (800rad) and fully MHC-mismatched BMT using 129.STAT1+/+ bone marrow cells plus 129.STAT+/+ conventional T cells (Tcon). Animals were co-injected with expanded Treg cells from either 129.STAT1+/+ or 129.STAT1−/− donors at a ratio of 1:1 or 1:4 (Treg:Tcon). STAT1−/− or STAT1+/+ Treg cells were equipotent in completely preventing GVHD mortality. However, compared to recipients of STAT1+/+ Treg recipients of STAT1−/− Treg showed reduced signs of GVHD morbidity as determined by a significantly improved weight development. Furthermore, recipients of STAT1−/− Treg showed significantly increased donor cell engraftment compared to recipients of STAT1+/+Treg (donor CD4+ [87% vs. 60%, p=0.03], CD8+[99% vs. 96%, p=0.04], Mac1+[96% vs. 77%, p=0.02] and B220+[100% vs. 96%, p=0.007]) cells in the recipient spleen. These observations clearly demonstrate that STAT1 is a critical regulator of Treg cell development and expansion and that targeting STAT1 in CD4+ T cells may facilitate in vitro and in vivo generation/expansion of Treg cells for therapeutic use in GVHD while also promoting donor cell engraftment. Disclosures:Lentzsch:Celgene Corp: Research Funding. Mapara:Resolvyx: Research Funding; Gentium: stocks.

Strong CD28 Costimulation Suppresses Induction of Regulatory T Cells From Naïve Precursors through Lck Signaling.

AbstractAbstract 3728CD28 costimulation is required for the generation of naturally-derived regulatory T cells (nTregs) in the thymus through Lck-signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naïve CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naïve CD4 T cells (CD25−Foxp3−) by TCR-stimulation with additional TGFβ in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. By using a series of transgenic mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk, we found that CD28-mediated Lck-signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide the rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28-signaling. Disclosures:No relevant conflicts of interest to declare.

Defects of Regulatory T Cell function In The Wiskott-Aldrich Syndrome. (49.25)

Abstract Natural Tregs from both WAS patients and WASp-deficient (WKO) mice were previously reported as unable to suppress the proliferation of conventional T-cells in vitro. Because WAS T-cells have a known defect of IL-2 production and this cytokine is important for the generation of Treg cells induced from naïve T-cells, we assessed the efficiency with which WKO naïve T-cells can differentiate in Tregs and observed a significant defect compared to WT. Such defect was not corrected by increasing concentration of anti-CD3 and TGF-β. Preliminary experiments indicated a role for endogenous IL-2 production in the generation of iTregs and pointed again to the IL-2 pathway as a major determinant of the Treg cells defects in WAS. Furthermore, we previously observed that the impaired suppression of T-cell proliferation could be partially rescued by pre-activation of nTregs. Because activated nTregs are known to be able to induce B-cell apoptosis, we set out to evaluate the function of WKO nTregs on B-cells. We found that pre-activated WKO Treg cells fail to effectively suppress B-cell proliferation. The reduced granzyme-B secretion by WKO nTregs compared to WT, suggest that the defective suppressive activity on B-cells may be due to a degranulation defect of WKO cells. In summary, our data suggest that both natural and induced arms of T regulatory cells are defective in WAS and that defective peripheral Treg induction may be involved in the pathogenesis of autoimmunity in this disease.

Smad3 Differentially Regulates the Induction of Regulatory and Inflammatory T Cell Differentiation

Transforming growth factor beta (TGF-beta) is a crucial cytokine with pleiotropic functions on immune cells. In CD4(+) T cells, TGF-beta is required for induction of both regulatory T and Th17 cells. However, the molecular mechanism underlying this differential T cell fate decision remains unclear. In this study, we have evaluated the role of Smad3 in the development of Th17 and regulatory T cells. Smad3 was found to be dispensable for natural regulatory T cell function. However, induction of Foxp3 expression by TGF-beta in naive T cells was significantly reduced in the absence of this molecule. On the contrary, Smad3 deficiency led to enhanced Th17 differentiation in vitro and in vivo. Moreover, Smad3 was found to interact with retinoid acid receptor-related orphan receptor gammat (RORgammat) and decrease its transcriptional activity. These results demonstrate that Smad3 is differentially involved in the reciprocal regulatory and inflammatory T cell generation.

Hyperactivable NFAT1 Ameliorates Autoimmune Encephalitis In Vivo.

Abstract 711Naïve CD4 T cells differentiate into diverse effector and regulatory subsets to coordinate the adaptive immune response. TH1 and TH2 effector subsets produce IFN-γ and IL-4, respectively, whereas proinflammatory TH17 cells are key regulators of autoimmune inflammation, characteristically produce IL-17 and IL-22 and differentiate in the presence of inflammatory cytokines like IL-6 and IL-21 together with TGF-β. Naive T cells can also differentiate into tissue-protective induced T regulatory (iTreg) cells.NFAT proteins are highly phosphorylated and reside in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the role of the Ca/NFAT signaling pathway in regulating T cell differentiation and the development of autoimmune diseases.We generated transgenic mice conditionally expressing a hyperactivable version of NFAT1 (AV-NFAT1) from the ROSA26 locus. To restrict AV-NFAT1 expression to the T cell compartment, ROSA26-AV-NFAT1 transgenic mice were bred to CD4-Cre transgenic mice. Naïve CD4 T cells freshly isolated from AV mice produced significantly less IL-2 but increased amounts of the inhibitory cytokine IL-10. To investigate the role of NFAT1 in the generation of TH1, TH2, Tregand TH17 cells, the respective cell types were generated from CD4 T cells of AV mice by in vitro differentiation. T cells from AV-NFAT1 mice exhibited a dysregulation of cytokine expression, producing more IFN-γ and less IL-4. While the numbers of CD4+CD25+ “natural” Treg cells in peripheral lymphoid organs and their in vitro suppressive functions were slightly decreased in AV mice, iTreg generation from CD4+CD25- T cells of AV mice as compared to wild type cells was markedly enhanced. Moreover, TH17 cells generated in vitro from CD4 T cells of AV mice in the presence of IL-6, IL-21 and TGF-β exhibited dramatically increased expression of both IL-10 and IL-17 as compared to wild type controls.To investigate putative NFAT binding sites in the IL-10 and IL-17 gene loci, we performed chromatin immunoprecipitation experiments. We show that NFAT1 can bind at the IL-17 locus at 3 out of 9 CNS regions which are accessible specifically during TH17 but not during TH1 and TH2 differentiation. Furthermore, we provide evidence that NFAT1 binds one CNS region in the IL10-locus in TH17 cells.To verify our observations in vivo, we induced experimental autoimmune encephalitis (EAE) in AV mice and wild type controls with the immunodominant myelin antigen MOG33-55 emulsified in complete Freund‘s adjuvant. While wild type animals showed a normal course of disease with development of tail and hind limb paralysis after approximately 10 days, AV mice showed a markedly weaker disease phenotype with less severe degrees of paralysis and accelerated kinetics of remission. Moreover at the peak of the response, there were fewer CD4+CD25- but more CD4+CD25+ T cells in the CNS of AV animals compared to wild type controls. Surprisingly, these cells produced significantly more IL-2, IL-17 and IFN-γ upon restimulation, even though they displayed decreased disease.In summary, our data provide strong evidence that NFAT1 contributes to the regulation of IL-10 and IL-17 expression in TH17 cells and show that increasing NFAT1 activity can ameliorate autoimmune encephalitis. This could occur in part through upregulation of IL-10 expression as observed in vitro, but is also likely to reflect increased infiltration of regulatory T cells into the CNS as well as increased conversion of conventional T cells into Foxp3+ regulatory T cells within the CNS. Disclosures:No relevant conflicts of interest to declare.

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4(+) T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN-gamma-deficient-mice had more forkhead box P3 (FOXP3(+)) cells than WT mice in all secondary lymphoid organs except the thymus. However, T-bet- or IL-4Ralpha-deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3(+) cells (neo-generated inducible Treg (iTreg)) by TGF-beta was significantly inhibited by IFN-gamma in a STAT-1-dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3(+) iTreg generation by IFN-gamma was a T-cell autocrine effect. This inhibitory effect of IFN-gamma on iTreg generation was significantly abrogated after N-acetyl-L-cysteine treatment both in vitro and in vivo, indicating that IFN-gamma regulation of iTreg generation is dependent on ROS-mediated apoptosis. Therefore, our results suggest that autocrine IFN-gamma can negatively regulate the neo-generation of FOXP3(+) iTreg through ROS-mediated apoptosis in the periphery.