Soluble OX40L and JAG1 Induce Selective Proliferation of Functional Regulatory T-Cells Independent of canonical TCR signaling

Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Treg numbers/functions has been shown to ameliorate autoimmune diseases. However, common Treg expansion approaches use T-Cell Receptor (TCR)-mediated stimulation which also causes proliferation of effector T-cells (Teff). To overcome this limitation, purified patient-specific Tregs are expanded ex vivo and transfused. Although promising, this approach is not suitable for routine clinical use. Therefore, an alternative approach to selectively expand functional Tregs in vivo is highly desired. We report a novel TCR-independent strategy for the selective proliferation of Foxp3+Tregs (without Teff proliferation), by co-culturing CD4+ T-cells with OX40 L+Jagged(JAG)-1+ bone marrow-derived DCs differentiated with GM-CSF or treating them with soluble OX40 L and JAG1 in the presence of exogenous IL-2. Tregs expanded using soluble OX40 L and JAG1 were of suppressive phenotype and delayed the onset of diabetes in NOD mice. Ligation of OX40 L and JAG1 with their cognate-receptors OX40 and Notch3, preferentially expressed on Tregs but not on Teff cells, was required for selective Treg proliferation. Soluble OX40L-JAG1-induced NF-κB activation as well as IL-2-induced STAT5 activation were essential for the proliferation of Tregs with sustained Foxp3 expression. Altogether, these findings demonstrate the utility of soluble OX40 L and JAG1 to induce TCR-independent Treg proliferation.

migrate to the periphery [13][14][15] . In the periphery they undergo proliferation upon interaction with dendritic cells (DCs) through their TCR 16,17 while receiving survival signal from IL-2 18,19 . Tregs constitutively express genes such as Ctla-4, Cd39 and Helios, which are associated with suppressive functions 20 . Furthermore, Tregs can suppress Teff function irrespective of their antigen specificity as well 21,22 . Therefore, Tregs expanded by TCR-independent methods can still retain their suppressive functions and will have significant clinical utility.
We and others have previously shown that Foxp3 + Treg proliferation, independent of TCR stimulation, can be induced by co-culturing them with a particular subset of dendritic cells (DCs) [23][24][25][26][27][28] . Bone marrow (BM) precursor cells differentiated in the presence of GM-CSF (G-BMDCs), upon co-culture with CD4 + T-cells, caused Treg proliferation mediated through OX40L-JAG1 co-signaling 23 . In the present study, we demonstrate that soluble OX40 L and JAG1 along with IL-2 were sufficient to cause Treg proliferation in the absence of TCR stimulation. Unlike TCR-stimulation approach, OX40L-JAG1 caused selective proliferation of Tregs with little or no proliferation of Teff cells. OX40L-JAG1 expanded Tregs were of a stable-suppressive phenotype and functionally competent. Furthermore, treatment of NOD mice with soluble OX40 L and JAG1 increased Tregs and delayed the onset of hyperglycemia in NOD mice. Using OX40 and Notch3 deficient mice, we showed that this TCR-independent Treg expansion was critically dependent on the signaling mediated through these cognate receptors for OX40 L and JAG1 respectively. Signaling studies revealed that NF-κ B activation induced by OX40L-OX40 and JAG1-Notch3 interactions, and STAT5 induced by IL-2, were essential for Treg proliferation with sustained Foxp3 expression. Thus, we report a novel "TCR-independent" strategy for the selective expansion of functional Tregs which could have therapeutic implications in various autoimmune diseases including T1D.

G-BMDCs-induced Treg proliferation is mediated through OX40L-JAG1 co-signaling in NOD mice and soluble OX40L-JAG1 are sufficient to cause Treg proliferation in the presence of IL-2.
Upon co-culturing total CD4 + T-cells with splenic (SpDCs) and G-BMDCs from NOD mice for 5 days, we observed a significant (***p < 0.001) increase in selective Treg proliferation in G-BMDC co-cultures compared to co-cultures with SpDCs ( Fig. 1A,B). To determine whether OX40 L and JAG1 co-signaling is involved in this G-BMDCs induced Treg proliferation, we pre-treated G-BMDCs with blocking antibodies against OX40 L, JAG1, neurolpilin and ligand for glucocorticoid-induced TNFR family related protein (GITRL) and then co-cultured with total CD4 + T-cells. We observed a significant reduction in Treg proliferation in the presence of blocking antibodies to OX40 L (**p < 0.01) and JAG1 (**p < 0.01) but not GITRL or neuropilin. This underscored the specific involvement of OX40 L and JAG1 signaling in G-BMDCs-induced Treg proliferation from NOD mice (Fig. 1C,D). To confirm the involvement of Notch signaling induced by JAG1, we pre-treated CD4 + T-cells with γ -secretase inhibitor (GSI) to inhibit Notch signaling and co-cultured with G-BMDCs. As shown in Fig. S1A, we found a dose-dependent inhibition of Treg proliferation indicating the critical role of Notch signaling. Among the various Notch receptors, Notch3 is preferentially over-expressed on Tregs when compared to Teff cells 29 . Therefore, we sorted for Notch3 − OX40 − , Notch3 + OX40 L − , Notch3 − OX40 + , Notch3 + OX40 + subsets of CD4 + CD25 + Tregs from NOD mice and co-cultured them with G-BMDCs. The G-BMDC-induced proliferation was maximal in Notch3 + OX40 + Tregs compared to Notch3+ OX40-and Notch3-OX40+ Treg subsets (Fig. S1B). Next, we checked whether soluble OX40 L and JAG1 were sufficient to cause proliferation of Tregs. We treated CD4+ T-cells with soluble OX40 L and JAG1 in the presence of IL-2 without any exogenous antigenic stimulation for 3 days. Since we anticipated OX40L-JAG1 treatment not to cause Teff cell activation, exogenous IL-2 was added to maintain Treg survival in ex vivo cultures. As shown in Fig. 2C,D, among the different combinations tested OX40L-JAG1-IL-2 treatment caused maximum increase in the percentage of proliferating Tregs (**p < 0.01) followed by OX40L-IL-2 and JAG1-IL-2. Further, CD4+ T-cells treated with IL-2 alone or OX40L-JAG1-IL-2 were stained for proliferation marker Ki67 and percentage of Ki67+ Tregs were found to be more in OX40L-JAG1-IL-2 treated cells compared to IL-2-treated controls (Fig. S2). Taken together, these results showed that soluble OX40 L and JAG1 were sufficient to cause Treg proliferation independent of TCR stimulation in an IL-2 dependent manner.
Soluble OX40L-JAG1-IL-2 can cause selective proliferation of Tregs independent of TCR stimulation. To validate whether OX40L-JAG1-induced Treg proliferation differs from TCR-stimulation approach, we compared the T-cell proliferation induced by TCR-dependent anti-CD3-CD28 versus TCR-independent OX40L-JAG1 stimulation. As shown in Fig. 2A, we observed robust proliferation of Tregs upon both OX40L-JAG1 and anti-CD3/CD28 treatment. However, unlike anti-CD3/CD28 treatment which also induced very strong Teff cell proliferation, OX40L-JAG1 treatment induced selective proliferation of Tregs without significant Teff proliferation. Analyses of activation markers expression showed a significant (***p < 0.001) increase in the percentage of Teff cells expressing CD25, CD44 and CD69 upon treatment with anti-CD3/CD28 compared to control cells ( Fig. 2B-D). However, no significant difference was observed between the control and OX40L-JAG1 treated Teff cells. Moreover, Tregs from both OX40L-JAG1 and anti-CD3/CD28 treated cells had increased CD25, CD44 and CD69 expressing cells compared to control cells. These results suggested that soluble OX40L-JAG1 can cause selective proliferation of Tregs, without significantly affecting Teff cell activation and proliferation.
Soluble OX40L-JAG1 treatment selectively induces Treg proliferation in vivo. To examine whether soluble OX40L-JAG1 can cause in vivo proliferation of Tregs, we treated 10 week-old pre-diabetic NOD mice with soluble OX40 L and JAG1 for 3 weeks and analyzed Treg numbers in their spleen, pancreatic and peripheral lymph nodes (LN). We did not treat these mice with exogenous IL-2 as we expected IL-2 required for Treg survival to be available in vivo. As shown in Fig. 3A,B, we found significantly increased percentages of Tregs in the spleen (**p < 0.01), pancreatic LNs (**p < 0.01) and peripheral LNs (***p < 0.001) of OX40L-JAG1 treated mice compared to PBS treated control mice. Furthermore, to demonstrate OX40L-JAG1 induced Treg Scientific RepoRts | 7:39751 | DOI: 10.1038/srep39751 proliferation in vivo, we analyzed Ki67 expression in CD4 + Foxp3-(Teff), and CD4 + Foxp3+ (Treg) cells from PBS and OX40L-JAG1 treated mice. As shown in Fig. 3C,D, OX40L-JAG1 treated mice had significantly increased percentage of Ki67+ cells only in Tregs (**p < 0.01) but not in Teff population when compared to PBS treated mice. These results showed that OX40 L and JAG1 can induce preferential proliferation of Tregs both ex vivo and in vivo. Further, we treated MHC class-II deficient mice with soluble OX40L-JAG1 to determine whether these ligands can increase Tregs in vivo in the absence of canonical antigen presentation through MHC class-II. These mice also had reduced CD4+ T-cells and increased number of CD8+ Foxp3+ T-cells when compared to wild type mice (data not shown). OX40L-JAG1 treatment significantly increased CD4 + Foxp3 + Tregs (**p < 0.05) and CD4-Foxp3+ T-cells (**p < 0.01) in these mice compared to PBS treated control mice (Fig. 3E,F). These results indicated that soluble OX40L-JAG1 can induce selective proliferation of Tregs in vivo independent of canonical antigen presentation to TCR.
OX40L-JAG1-IL-2 expanded Tregs retain stable-suppressive phenotype and delay the onset of diabetes in NOD mice. Next we examined whether these OX40L-JAG1 expanded Tregs retained their suppressive phenotype and functions. We analyzed the expression of suppressive markers such as CTLA4, CD39, Helios and TIGIT in Tregs from control and OX40L-JAG1 treated mice. As shown in Fig. 4A,B, OX40L-JAG1 expanded Tregs had significantly increased expression of suppressive markers such as CTLA4 (***p < 0.001),  Helios (***p < 0.001) and TIGIT (**p < 0.01) when compared to control Tregs. CD39 expression was not significantly different between control and OX40L-JAG1 expanded Tregs. Furthermore, we setup an ex vivo suppression assay using control Tregs and OX40L-JAG1 expanded Tregs to confirm the functional competency of OX40L-JAG1-IL-2 expanded Tregs. In line with the phenotypic results, we found OX40L-JAG1 expanded Tregs to efficiently suppress Teff proliferation similar to control Tregs (Fig. 4C,D). Taken together, these results suggested that OX40L-JAG1 could expand functional Tregs without loss of their suppressive phenotype and function.
Next, we treated NOD mice with soluble OX40 L and JAG1 once a week at 10-12 weeks of age and monitored their blood glucose levels. As shown in Fig. 5A, by 27 th week 100% of control mice became hyperglycemic, while 40% of OX40 L and JAG1 treated mice were still normoglycemic (*p < 0.05). Additionally, we found significantly higher percentages of Tregs in the spleen of OX40 L and JAG1 treated mice (15.87 ± 0.80) relative to controls (10.67 ± 1.83; *p < 0.05, n = 10) (Fig. 5B). Examination of the pancreatic sections showed that OX40 L and JAG1 treated mice had more number of intact islets and reduced incidence of peri-insulitis (Fig. 5C). Nearly 70% of the islets from control mice showed severe insulitis with only 7.14% exhibiting normal architecture. In contrast, only 30% of the islets from OX40 L and JAG1 treated mice showed heavy infiltration and over 30% of the islets exhibited normal architecture (Fig. 5D). OX40 L and JAG1 treated mice also had higher proportion of insulin secreting islets relative to control mice (Fig. 5E). Further, we stimulated splenocytes from control and OX40L-JAG1 treated mice with PMA-Ionomycin, and analyzed their cytokine expression profile by RT-qPCR. We found reduced expression of Th1 cytokines such as IFN-γ , IL-12α (*p < 0.05), IL-12β (**p < 0.01) and TNF-α (*p < 0.05), and increased expression of Th2 cytokines such as IL-4 (**p < 0.01) and IL-13 (*p < 0.05) in the splenocytes from OX40L-JAG1 treated mice relative to controls (Fig. 5F) upon stimulation. We also noted increased expression of anti-inflammatory cytokine IL-10 (**p < 0.01) and pro-inflammatory cytokine IL-6 (**p < 0.01) in splenocytes from OX40 L and JAG1 treated mice. A recent study has shown that transgenic expression of Notch1 intracellular domain in Treg cells can cause lymphoproliferation, exacerbated Th1 responses and autoimmunity 30 . To see if a similar phenomenon was occurring, we stimulated splenocytes from control and OX40L-JAG1 treated mice with PMA-Ionomycin and stained both Treg and Teff cells for IFN-γ expression. Our results clearly showed that there was no change in the percentage of IFN-γ expressing Teff cells between control and OX40L-JAG1 treated mice, and there was barely any IFN-γ expressing Tregs in both control and OX40L-JAG1 treated mice (Fig. S3).
OX40 L has been shown to bind to its only known cognate receptor OX40, constitutively expressed on Tregs 31 . However, JAG1 can bind to multiple receptors such as Notch1, Notch2 32 and Notch3 33 of which Notch3 is preferentially up-regulated in Tregs 29,34 . Additionally, JAG1 has been characterized as the most abundant and specific ligand for Notch3 33 .Therefore, we hypothesized that loss of either OX40 or Notch3 might negatively affect Treg proliferation induced by OX40 L, JAG1 and IL-2. We treated CD4 + T-cells isolated from OX40 −/− , Notch3 −/− and respective wild type C57BL6 and B6129SF1 control mice with soluble OX40 L, JAG1 and IL-2 for 3 days. As shown in Fig. 7A,B, we noted a significantly lower percentage of proliferating Tregs from OX40 −/− (***p < 0.001) and Notch3 −/− (*p < 0.05) mice compared to their corresponding wild type controls. Further, we treated wild type, OX40 −/− and Notch3 −/− mice with soluble OX40 L and JAG1 for 3 weeks and analyzed Treg numbers in the spleen. As shown in Fig. 7C-E, we did not observe any significant difference in the total number of splenic Tregs among untreated OX40 −/− , Notch3 −/− and the corresponding wild type control mice. Similarly, basal Foxp3 expression was also not significantly different among OX40 −/− , Notch3 −/− and corresponding wild type control mice (Fig. S4). However, treatment with soluble OX40L-JAG1 caused a significant (***p < 0.001) increase in Treg numbers in both C57BL6/J and B6129SF1/J wild type mice, but not in OX40 −/− mice. In OX40L-JAG1 treated Notch3 −/− mice there was a significant increase of Tregs compared to PBS-treated Notch3 −/− mice (*p < 0.05), but the level of increase was still significantly less than wild type mice treated with OX40L-JAG1 (*p < 0.05 Vs OX40L-JAG1). These results suggested that although expression of OX40 or Notch3 is not required for the development of Tregs or Foxp3 expression in steady state, they are indispensable for optimal Treg proliferation induced by OX40 L and JAG1.
Since our micro array results suggested upregulation of genes associated with NF-kB and STAT5 signaling in proliferating Tregs compared to resting Tregs, we examined their role in TCR independent Treg proliferation. As shown in Fig. 8A, OX40 L, JAG1 and IL-2-induced Treg proliferation was significantly blocked by NF-κ B and STAT5 inhibitors, but not by MEK inhibitor. Next, we investigated whether NF-κ B and STAT5 signaling pathways were involved in the regulation of Foxp3 expression using RT-qPCR (Fig. 8B) and Western blot (Fig. 8C). While  Next, we carried out a time course analysis to determine the effect of OX40 L, JAG1 and IL-2 on Foxp3 expression, and NF-κ Bp65 and STAT5 activation. We observed a significant increase in Foxp3 expression at 24 h (*p < 0.05) which was sustained up to 120 h (Fig. 8D). While phospo-NF-κ Bp65 levels were maximal at 24 h (**p < 0.01) (Fig. 8E), STAT5 phosphorylation was maximum at 72 h (***p < 0.001, **p < 0.01) (Fig. 8F).

Discussion
Growing body of evidence demonstrates the protective role for Foxp3+ Tregs in various autoimmune diseases 6,7,35 . However, translation of Treg cell therapy to clinical settings is impeded by several limitations. One of these limitations is the inability of TCR-dependent approaches to cause selective in vivo expansion of Tregs 10 . Unlike stimulation with anti-CD3/CD28 which activated both Tregs and Teff cells, stimulation with OX40L-JAG1 caused selective proliferation of Tregs without activating Teffs as evidenced by no significant change in the expression of activation markers such as CD25, CD44 and CD69 on Teff cells. Differential gene expression analysis between resting vs proliferating Tregs showed up-regulation of expression of Foxp3 and its functional partners Gata3, Runx1, Cnot3, Cbfb, Cebpz and Bcl11b in proliferating Tregs. These molecules are known to increase the functional fitness of Tregs. For example, expression of Gata3 by Tregs is essential for their migration towards the site of inflammation and to sustain Foxp3 expression under inflammatory conditions 36 . Runx and CBF-β complex have been shown to directly bind to Foxp3 promoter and increase its transcription 37 . Similarly, transcription factor Bcl11b can bind to both Foxp3 and IL-10 promoters, and regulate their expression and help confer Treg mediated protection against IBD 38 . Besides, we observed up-regulation of Ctla-4, Helios, Tigit, Icos, Cd39, Pdcd1 and Tgf-β1, all of which are suppressive and stable phenotypic markers of Tregs. Further, in vivo treatment of NOD mice with soluble OX40 L and JAG1 resulted in a significant increase of Tregs. We also found increased expression of suppressive markers such as CTLA-4, Helios and TIGIT in Tregs expanded by in vivo treatment with OX40L-JAG1. CD39 expression was comparable between control and OX40L-JAG1 expanded Tregs. CTLA-4 is one of the most widely accepted mediators of Treg suppressive functions 35 . CD39, an ectonucleotidase that can hydrolyze ATP, is considered as a stability marker for Tregs and CD39 + Foxp3 + Tregs have been shown to suppress both Th1 and Th17 cells 39 . TIGIT is another Treg cell specific co-inhibitory molecule. TIGIT + subset of Tregs have been shown to predominantly inhibit Th1 and Th17 cells without affecting Th2 cells 40 . Helios, an Ikoras transcription factor family member, has also been reported to be associated with Treg functions 41 and suppression of autoimmune diabetes 42 . Increased expression of these suppressive markers in OX40L-JAG1 expanded Tregs could help sustain their suppressive functions. Besides, we also confirmed the functional competency of these expanded Tregs in ex vivo suppressive assays.
Intriguingly, treatment of NOD mice with either OX40 L or JAG1 alone failed to significantly alter the course of diabetes (data not shown). We and others have observed that treatment of 6-week old NOD mice with either OX40 L or an anti-OX40 agonistic antibody (OX86) can increase CD4 + CD25 + Foxp3 + Treg cells and protect NOD mice from developing diabetes 43,44 . However, treating 12-week old NOD mice with OX40 L accelerated diabetes development likely due to an increased pro-inflammatory environment associated with aging in these mice 44 . Thus, the outcome of treatment with OX40 L alone appears to be age-dependent in NOD mice. In contrast, co-treatment of 10-12-week old NOD mice with OX40 L and JAG1 significantly increased functional Treg numbers and delayed the onset of diabetes. These results indicated a critical requirement for co-signaling induced by OX40 L and JAG1 to increase and sustain functionally competent Tregs. It should be noted that we treated mice with OX40L-JAG1 once a week for only three weeks near the time of diabetes onset. However, repeated follow-up treatments might yield better results. Additionally, IL-2 deficiency in older NOD mice might have also led to poor survival of expanded Tregs 45 and thus supplementation with IL-2 might have also increased the longevity of expanded Tregs. Interestingly, splenocytes from OX40 L and JAG1 treated mice upon PMA-Ionomycin stimulation showed reduced expression of inflammatory cytokines i.e. IFN-γ , IL-12α , IL-12β and TNF-α and increased expression of anti-inflammatory cytokines such as IL-10, IL-4 and IL-13 relative to controls. Previous reports have demonstrated the protective effects of anti-inflammatory cytokines such as IL-4, IL-10 and IL-13 in autoimmune diabetes [46][47][48] . Together, these results suggested that OX40 L and JAG1 co-treatment might have restored the balance between anti-and pro-inflammatory cytokines, and created a favorable cytokine milieu in which Tregs could proliferate and retain their suppressive functions. This notion is further evident from the reduced incidence of severe insulitis and preservation of more numbers of intact insulin secreting islets in OX40 L and JAG1 treated mice. However, further studies are required to optimize the therapeutic efficacy of this treatment.
In general, signaling required for Treg proliferation is primarily understood in the context of TCR stimulation. Previously, it has been reported that engagement of TCR with anti-CD3/CD28 can induce ZAP70, PI3K, Akt and MAPK signaling and activation of transcription factors NFAT, c-Jun, NF-κ B and AP1 49 . However, the signaling that drives TCR-independent Treg proliferation is not well defined. Interestingly, while OX40L-JAG1-IL-2 induced Treg proliferation was significantly abrogated in OX40 −/− and Notch3 −/− mice, Treg proliferation induced by TCR stimulation remained unaffected in these mice (data not shown). Additionally, differential gene expression analysis revealed selective activation of genes associated with OX40, Notch and IL-2R receptor signaling in proliferating Tregs. Collectively, these results showed that the ligation of OX40 L and JAG1 with their cognate receptors OX40 and Notch3 are the major upstream events regulating this TCR-independent Treg proliferation.
The relevance of OX40 L induced signaling in Treg expansion and function has remained elusive. OX40 expression has been shown to be essential for Treg migration to inflamed sites [50][51][52] . While OX40L-OX40 stimulation can cause Treg proliferation, it could also adversely affect Foxp3 expression and Treg suppressive functions depending upon the local cytokine milieu 53,54 . During TCR stimulation OX40L-OX40 interaction has been shown to activate PI3K (PI-3-kinase)/PKB (protein kinase B/Akt) and NF-κ B1 pathways 55,56 . Two members of the TRAF family of proteins such as TRAF2 and TRAF5 have been identified as key adaptor proteins recruited by OX40 to drive NF-kB1 activation 57 . In the absence of TCR stimulation, OX40 has been shown to form a signalosome containing TRAF2, CARMA1, MALT1, BCL10, PKCθ , RIP and IKKα /β /γ to cause NF-kB activation required for T-cell survival 58 . Our results showed that activation of NF-kB by OX40L-OX40 is indispensable for Treg proliferation in the absence of TCR-stimulation. OX40 deficient Tregs showed impaired NF-kB activation as well as proliferation induced by OX40L-JAG1. Several lines of evidence suggest that Notch signaling plays a positive role in Treg homeostasis by increasing Treg numbers in thymus and periphery, and by maintaining Scientific RepoRts | 7:39751 | DOI: 10.1038/srep39751 Foxp3 expression 34,59,60 . In particular, Notch3 has been shown to positively regulate nTreg development and Foxp3 expression 34 . It has been shown that Notch3 and canonical NF-κ B signaling pathways could co-operatively regulate Foxp3 expression 61 . Hence, JAG1 induced Notch3 signaling, along with transactivation of NF-κ B-p65 by OX40 L, could co-operatively regulate Treg proliferation and Foxp3 expression.
The role of IL-2 induced STAT5 signaling in Treg survival and stable Foxp3 expression is well established 62 . Foxp3 promoter has a STAT5 binding site 63 through which Foxp3 expression is regulated in both human and mouse Tregs 64 . In addition, Foxp3 gene has a T-Cell Specific Demethylated Region (TSDR) in its promoter which can be demethylated through IL-2/STAT5 signaling to sustain Foxp3 expression 65,66 . Consistent with these earlier findings, we noted enhanced Foxp3 expression upon addition of IL-2. Thus, enhanced activation of NF-κ Bp65 by OX40 L and JAG1, and STAT5 signaling by IL-2 likely promoted Treg proliferation with sustained Foxp3 expression in the absence of TCR signaling. However, further studies are needed to fully understand the intricate signaling mechanisms involved in OX40L-JAG1-IL-2 induced Treg proliferation. In summary, our findings demonstrate that OX40L-JAG1 co-signaling can cause selective proliferation of Tregs in a TCR-independent mechanism which will have potential utility in treating autoimmune diseases. This TCR-independent Treg proliferation and Foxp3 expression is critically dependent on NF-kB signaling induced by OX40L-JAG1 and STAT5 signaling induced by IL-2.

Materials and Methods
Animals. NOD, C57BL/6 J, B6129SF1/J, OX40 and Notch3 deficient mice were purchased from Jackson Laboratories. MHC class-II deficient mice (ABBN12 (H2-Ab1)) were from Taconic biosciences. Breeding colonies were established and maintained in a pathogen-free facility of the biological resources laboratory (BRL) of the University of Illinois at Chicago (Chicago, IL). All animal experiments were approved and performed in accordance with the guidelines set forth by the Animal Care and Use Committee at University of Illinois at Chicago.

RNA Isolation, Micro-array and RT-qPCR analyses.
Resting and proliferating Tregs were sorted based on cell trace violet dilution. Total RNA was isolated from these cells by using RNAeasy columns (Qiagen). The cDNA synthesized from total RNA was used for RT-qPCR analysis with Fast SYBR green master mix (Applied Biosystems) and gene specific primers (listed in supplementary table-1) by using AB ViiA7 RT-qPCR instrument (Applied Biosystems). Gene expression values were calculated by comparative Δ Ct method after normalization to GAPDH internal control and expressed as fold change over respective controls.
Micro array analysis was performed in duplicate using the Affymetrix GeneChip Mouse Genome 430 2.0 microarray at Center for genomics core facility, University of Illinois at Chicago. Briefly, biotinylated cDNA was synthesized from total RNA using biotinylated dNTPs and allowed to hybridize with microarrays and scanned. Arrays which passed quality control tests were further subjected to gene expression analysis after normalization with housekeeping gene controls. Data were analyzed using the R-package software. Student's t-test was used to filter differentially expressed genes Micro array has been submitted to NCBI-Gene Expression Omnibus database and publicly available (Accession No. GSE81051).
Animal experiments. Six week old female NOD mice were divided into two groups each containing13 mice. Mice were injected (i.p) with recombinant OX40 L (200 μ g) and JAG1 (200 μ g) on weeks 10, 11 and 12. Age and sex matched control mice received PBS. All the reagents used for animal experiments were endotoxin free (< 0.1 EU/ml) when tested by using Pierce endotoxin quantification kit (Thermo scientific). Blood glucose levels were monitored weekly from week 9 to 28. On week15, three mice from each group were sacrificed and analyzed for Treg cell numbers. At the end of week 28, all animals were sacrificed and tissue sections of pancreas were subjected to histopathological examination to determine lymphocyte infiltration and β -cell destruction.

Statistical analysis.
Statistical analyses were performed using Prism GraphPad (V6.0). Data were expressed as Mean ± SEM of multiple experiments. Paired Student's t-test was used to compare two groups, whereas ANOVA with multiple comparisons was used to compare more than two groups. Differences in the frequency of hyperglycemia were determined by Kaplan-Meier survival analysis using the log-rank test. A p value < 0.05 was considered as significant.