TCR Stimulation of Tregs in Inflamed Islets.

To investigate TCR activation of Tregs in NOD mice, we backcrossed a strain of Nur77^GFP reporter mice (15) more than 10 generations onto the NOD background. The NOD.Nur77^GFP mice developed diabetes at comparable kinetics and overall incidence to standard NOD mice (SI Appendix, Fig. S1A) and exhibited normal cellularity and proportions of T cells in peripheral lymphoid organs (SI Appendix, Fig. S1B). We then further crossed the NOD.Nur77^GFP mice with NOD.Foxp3^RFP mice (35, 36) to aid the isolation and analysis of Tregs. Tregs in the NOD.Nur77^GFP.Foxp3^RFP mice showed constitutively high GFP expression indicative of antigen stimulation of the TCR as previously reported (14, 15). To further define the correlation between GFP expression and the strength and immediacy of TCR stimulation, we analyzed lymph node (LN) cells from NOD.Nur77^GFP.Foxp3^RFP mice that had received varying doses of TCR stimulation in vitro. CD4⁺Foxp3⁻ Tconv and CD8⁺ T cells showed dose-dependent increases in the percentages of Nur77^GFP+ cells and in GFP mean fluorescence intensity (MFI) (SI Appendix, Fig. S1 C and D). Tregs were nearly all GFP positive directly ex vivo before stimulation. Increasing TCR stimulation also led to a dose-dependent increase in GFP MFI in Tregs (SI Appendix, Fig. S1 C and D). To determine the persistence of GFP expression after the cessation of TCR stimulation, we stimulated purified Tconv cells and Tregs with plate-bound anti-CD3 and anti-CD28 in vitro for 24 h, removed the cells from stimulation, and monitored GFP levels over time. GFP MFI in both Tconv cells and Tregs decayed with a half-life of ∼3 d (SI Appendix, Fig. S1E). Unstimulated Tregs showed a rate of Nur77-GFP decay similar to that seen in in vitro stimulated Tregs and Tconv cells. Together, these data demonstrate that the intensity of GFP in T cells of Nur77^GFP reporter mice integrates both the strength of the TCR signal received and the recency of antigen exposure.

We then used the NOD.Nur77^GFP.Foxp3^RFP mice to identify Tregs with more recent islet antigen exposure. We compared GFP expression in Tregs from pancreatic LN (pancLN) and inflamed islets to those from spleen. Tregs from islets had the highest expression of Nur77^GFP compared with those from pancLN and spleen (Fig. 1A). A subset of Tregs in the inflamed islets coexpressed previously reported TCR-dependent activation markers CD103, TIGIT, and ICOS (21) (Fig. 1B and SI Appendix, Fig. S2 A and B) and had higher expression of Foxp3 and CTLA4 than CD103⁻ islet Tregs (SI Appendix, Fig. S2C). These CD103⁺ islet Tregs had the highest Nur77^GFP expression compared with CD103⁻ islet Tregs and Tregs from lymphoid organs (Fig. 1 B and C). The expression of CD5, another marker of recent TCR stimulation (37, 38), was also higher on islet Tregs, especially on the CD103⁺ subset (Fig. 1D). Moreover, islet Tregs were more proliferative than those in the pancLN by Ki67 staining (Fig. 1E), and islet CD103⁺ Tregs were the most proliferative subset by both Ki67 staining and in vivo BrdU labeling (Fig. 1 E and F). These results indicate that Tregs in islets, especially those coexpressing CD103, ICOS, and TIGIT, have been more recently and/or strongly activated by antigens.

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Fig. 1.

Evidence of antigen exposure at the site of inflammation. (A) Representative histograms of flow cytometric analysis of Nur77^GFP expression in CD4⁺Foxp3⁺ Tregs and CD4⁺Foxp3⁻ Tconv cells from prediabetic NOD mice. Results represent at least 80 mice analyzed in more than 20 independent experiments. (B) Representative contour plots of CD103 and Nur77^GFP expression by Tregs from prediabetic NOD mice. Results represent at least 80 mice analyzed in more than 20 independent experiments. (C) Quantification of Nur77^GFP in CD103⁺ or CD103⁻ Tregs in B. Results shown are a summary of three independent experiments. Statistical significance was determined using a repeated measures one-way ANOVA followed by Tukey’s multiple comparison test; ***P < 0.001; ****P < 0.0001. (D) CD5 MFI in CD103⁺ or CD103⁻ Tregs from prediabetic NOD mice. Results shown are a summary of two independent experiments. Statistical significance was determined using a repeated measures one-way ANOVA followed by Tukey’s multiple comparison test; **P < 0.01; ***P < 0.001; ns, not significant. (E) Ki67 staining in CD103⁺ or CD103⁻ Tregs from prediabetic NOD mice. Results shown are a summary of three independent experiments. Statistical significance was determined using a repeated measures one-way ANOVA followed by Tukey’s multiple comparison test; **P < 0.01; ***P < 0.001; ns, not significant. (F) BrdU staining in CD103⁺ or CD103⁻ Tregs from prediabetic NOD mice. BrdU was administered continuously for 1 wk before analysis. Results shown are a summary of two independent experiments. Statistical significance was determined using a paired t test; *P < 0.05; ns, not significant.

Islet Tregs Have a Distinct and Restricted TCR Repertoire.

The activation and proliferation of Tregs in inflamed islets may result in local enrichment of islet antigen-specific clonotypes and formation of distinct TCR repertoires. We therefore compared islet Treg TCRβ sequences with those used by Tregs from pancLN, spleen, and nondraining inguinal-popliteal-sciatic LN (iLN) using high-throughput TCRβ sequencing analysis. In each organ, we divided Tregs into CD103⁻ and CD103⁺ subsets, and the CD103⁻ islet Tregs were further divided into Nur77^GFP high and low fractions (Fig. 2A). Between 1,600 and 600,000 Tregs were analyzed from different compartments, and 1,144 to 62,604 unique TCRβ sequences were identified (SI Appendix, Table S1). We used the clonality index—the inverse of normalized Shannon’s entropy—to quantify the clonal dominance in each population, with 0 indicating that each clone appears only once and 1 representing a monoclonal population. While all Treg populations showed polyclonality, with a diversity index of less than 0.4, the CD103⁺ islet Tregs were the least diverse (Fig. 2B). Clonal dominance can also be visualized by the contribution of the top 10 clones to the repertoire they reside in, which was 30% for CD103+ islet Tregs, markedly higher than the CD103⁻ islet Treg subsets (Fig. 2 C and D). CD103⁻ Tregs in spleen, pancLN, and iLN were highly diverse, with the 10 most abundant clones representing less than 2% of the population (Fig. 2D). These results further support local clonal expansion of activated Tregs in the inflamed islets.

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Fig. 2.

Treg TCR repertoires in prediabetic NOD mice. (A) A summary of Treg populations used for TCRβ chain sequencing analysis. CD103⁺ and CD103⁻ Treg populations were sorted from spleen, pancLN, and iLN. Islet Tregs were additionally sorted on Nur77^GFP expression from islets: CD103⁺Nur^hi, CD103⁻Nur^hi, and CD103⁻Nur^lo. A total of 32 populations from four mice were analyzed individually (SI Appendix, Table S1). (B) Clonality of each Treg population’s TCR repertoire. Each triangle represents a single mouse, and the columns represent the mean and SD of the group. Statistical significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test; ***P < 0.001. (C) Frequency of the 10 most abundant clones in different islet Treg populations in a representative mouse. (D) Summary graph of the sum frequencies of the 10 most abundant clones in different Treg populations in each mouse. Each triangle represents a single mouse, and the columns represent the mean and SD of the group. Statistical significance determined as described in B; *P < 0.01.

Islet Antigen Specificity of Islet Tregs.

Our results thus far suggest that Tregs specific to islet autoantigens are locally activated, clonally expand, and accumulate in the islets. However, distinct TCRs may recognize the same antigen. To further determine the specificity of islet Tregs, we performed single-cell sequencing of paired TCRαβ chains and then further interrogated for their specificity using an in vitro reporter assay (39, 40). FACS-purified ICOS⁺TIGIT⁺ islet Tregs, which also express high levels of CD103 and Nur77^GFP (SI Appendix, Fig. S2A), were sorted into single wells for barcoding and sequencing. Among the 71 paired TCRαβ sequences identified, six TCRs were found more than once in a single mouse, indicating clonal expansion, and two other TCRs were found in multiple mice (SI Appendix, Table S2). We thus selected these eight TCRs for further analysis of their reactivity to islet antigens.

We reconstituted the paired TCRs using retroviral transduction in a hybridoma cell line that expresses an NFAT^GFP reporter construct so that TCR triggering can be measured by GFP expression. A hen egg lysosome (HEL)-specific TCR (PA21.14H4) was used as a negative control, and a TCR for insulin (p3w17) was used as a positive control in the screening. We stimulated all TCR-transduced hybridomas with splenic CD11c⁺ dendritic cells (DCs) pulsed with lysate from NOD.Rag2^−/− islets. Five of eight clones (i.e., 510, 518, 521, 550, and 551) showed measurable reactivity to islet lysates (Fig. 3A). To determine whether these TCRs respond to islet antigen naturally processed and presented by DCs in the inflamed islets, we stimulated the hybridomas with freshly isolated islet-derived CD11c⁺ DCs. In this assay, the same five clones responded to islet DCs, but not to control DCs from spleen or colon (Fig. 3 A and B and SI Appendix, Fig. S3B). To further map antigen specificity of these TCRs, we screened the hybridomas against 19 peptides previously reported to be stimulatory to islet antigen-specific CD4⁺ T cells in NOD mice (SI Appendix, Table S3). None of the TCRs responded to BDC mimotopes, HIPs, or peptides from ZnT8, GAD, IGRP, IAPP, or IA-2β (SI Appendix, Table S3).

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Fig. 3.

Islet antigen reactivity of activated islet Tregs. (A) Hybridomas expressing NFAT^GFP reporter and TCRs derived from activated islet Tregs were stimulated for 20 to 24 h with splenic DCs or splenic DCs with islet lysates. GFP expression was determined using flow cytometry, and contour plots for all of the hybridomas analyzed are shown. (B) As described in A, except the hybridomas were stimulated with islet DCs. (C) As described in A, except the hybridomas were stimulated with splenic DCs with insulin B:9–23 peptide, plate-bound insulin tetramers p8E and p8G, human B25D-insulin, or proinsulin B:24–36 peptide. Filled histograms are the HEL-specific hybridoma; open histograms are the TCR hybridoma listed at the top of the column. In A–C, representative flow plots for at least two independent experiments are shown.

Insulin is a dominant autoantigen in type 1 diabetes in both NOD mice and human patients (41, 42). Therefore, we tested Treg TCRs against insulin. One TCR, 521, responded to splenic DCs loaded with insulin B:9–23 peptide (Fig. 3C and Table 1). Another TCR, 510, responded to immobilized insulin B:9–23 p8E and p8G tetramers (29) (Fig. 3C and Table 1). To determine reactivity to other insulin epitopes, we stimulated the hybridomas with splenic DCs pulsed with metabolically inactive human insulin, B25D-insulin (43). The use of B25D-insulin avoided the hormonal activation of the hybridomas that altered the assay background. TCR 551 responded to B25D-insulin (Fig. 3C and Table 1). Additionally, we found that TCR 518 responded to a previously described proinsulin peptide, insulin B:24–36 (Fig. 3C and Table 1) (44). The remaining islet antigen-specific TCR, 550, did not respond to any of the above insulin stimulations (Fig. 3C, Table 1, and SI Appendix, Table S3). In summary, five of eight islet Treg-derived TCRs were stimulated by islet antigens, and four of the five were specific for insulin (Table 1).

Insulin Specificity Among Islet Tregs.

Our results suggest that insulin specificity may be common among islet-infiltrating Tregs. To further test this notion, we analyzed Tregs and Tconv cells in islets and pancLN of NOD mice (8 to 28 wk old) using insulin I-A^g7 tetramer containing modified insulin B:9–23 peptides p8E and p8G (29). Insulin B:9–23-specific cells were readily detectable among islet Treg and Tconv cells but were not consistently found in the pancLN (SI Appendix, Fig. S4). Insulin B:9–23-specific islet Tregs were often present at higher frequencies than insulin B:9–23-specific Tconv cells in prediabetic mice (Fig. 4B). Overall, the total numbers of insulin-specific Tconv cells per islet were slightly higher than the total number of insulin-specific Tregs (Fig. 4C). Moreover, insulin tetramer-positive Tregs had higher Nur77^GFP expression than insulin tetramer-negative Tregs, indicating stronger TCR activation (Fig. 4D). Thus, the insulin tetramer stain result is consistent with single-cell TCR sequencing analysis and shows that a demonstrable proportion of islet Tregs is insulin specific.

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Fig. 4.

Insulin-specific Tregs in NOD mice. (A) Representative contour plots of pooled insulin B:9–23 tetramers p8G and p8E staining versus control HEL tetramer staining in islet Tconv cells (Upper) and Tregs (Lower). Cells were gated as live CD45⁺Thy1.1⁺CD8⁻ and Foxp3⁺ (Tregs) or Foxp3⁻ (Tconv cells). Results are representative of at least eight mice analyzed in three independent experiments. (B) Quantification of insulin tetramer staining in islet Tconv cells and Tregs. Each pair of connected circles represents one mouse, and the mice (n = 24) are grouped according to age. Results shown are a summary of six independent experiments. Statistical significance was determined using a paired Student’s t test, and P values are listed at the top of the groups. (C) Total number of insulin tetramer-positive Tregs and Tconv cells from islets. Results shown are a summary of three independent experiments. Statistical significance was determined using a paired Student’s t test; *P < 0.05. (D) Nur77^GFP MFI of insulin tetramer-positive and -negative Tregs in islets. Results shown are a summary of three independent experiments. Statistical significance was determined using a paired Student’s t test; **P < 0.001.

Function of Islet Tregs.

Islet antigen-specific Tregs are more effective at protecting against diabetes (17, 18). Given their islet antigen specificity and propensity to be activated in the islets, we hypothesized that islet Tregs in the NOD mice would be effective at suppressing diabetes. We assessed the in vivo capacity of islet Tregs to prevent diabetes in an adoptive transfer model using NOD.CD28^−/− mice as recipients. These mice develop diabetes at a younger age and at higher penetrance due to Treg defects (12, 13). More than 80% of NOD.CD28^−/− mice become diabetic between 8 and 12 wk of age, which can be largely prevented by adoptive transfer of 50,000 islet antigen-specific Tregs from BDC-2.5 TCR transgenic mice (Fig. 5A). The same dose of Tregs isolated from spleen, pancLN, or nonpancreas draining LNs (mesenteric LN and inguinal LN) of prediabetic NOD mice were unable to prevent diabetes (Fig. 5B). Transfer of 50,000 islet Tregs prevented diabetes as efficiently as BDC-2.5 Tregs (Fig. 5C). Taken together, this study demonstrates that inflamed islets contain an enrichment of islet antigen-specific Tregs that exhibit potent antidiabetes activity.

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Fig. 5.

In vivo function of islet Tregs. (A) CD28^−/− mice between 2 and 3 wk of age were treated with 50,000 BDC-2.5 TCR transgenic Tregs sorted from nonpancreas draining LN (dashed line, n = 17) or untreated (bold solid line, n = 30). Diabetes development was monitored until the mice were 25 wk of age or until diabetes development. Statistical significance was determined using the log-rank (Mantel–Cox) test; ****P < 0.0001. (B) Same as in A, except polyclonal Tregs sorted from nonpancreas draining LN (ndLN, n = 7), pancLN (n = 9), and spleen (n = 14) of NOD mice were used to treat CD28^−/− mice and compared with nontreated controls (bold solid line, n = 30). Statistical significance was determined using the log-rank (Mantel–Cox) test; ns, not significant. (C) Same as in A, except polyclonal Tregs sorted from inflamed islets of NOD mice (dashed line, n = 23) were used to treat CD28^−/− mice and compared with nontreated controls (bold solid line, n = 30). Statistical significance was determined using the log-rank (Mantel–Cox) test; ****P < 0.0001. Results in A–C are summaries of more than 10 independent experiments performed over a 24-mo period.