IL-2 signaling and autoimmunity Regulatory T cells (T regs ) suppress autoreactive effector T cells to prevent the occurrence of autoimmune diseases, such as type 1 diabetes (T1D). The cytokine interleukin-2 (IL-2) is critical for the development and homeostasis of T reg subsets. Polymorphisms in the genes encoding IL-2 and its receptor subunits are associated with an increased risk of developing autoimmunity. To examine the effect of decreased IL-2 signaling, Dwyer et al. expressed a signaling-defective mutant IL-2 receptor (IL-2RβY3) in T cells in NOD mice, a model of T1D. Compared to NOD mice expressing wild-type IL-2Rβ, those expressing IL-2RβY3 in their T cells had accelerated onset of T1D. This was associated with a decrease in the numbers and suppressive activity of different T reg subsets and in the infiltration of autoreactive effector T cells into the pancreas. Together, these data suggest that the use of low-dose IL-2 to therapeutically modulate different T reg subsets in the context of autoimmune disease should be evaluated.

Abstract The cytokine interleukin-2 (IL-2) is critical for the functions of regulatory T cells (T regs ). The contribution of polymorphisms in the gene encoding the IL-2 receptor α subunit (IL2RA), which are associated with type 1 diabetes, is difficult to determine because autoimmunity depends on variations in multiple genes, where the contribution of any one gene product is small. We investigated the mechanisms whereby a modest reduction in IL-2R signaling selectively in T lymphocytes influenced the development of diabetes in the NOD mouse model. The sensitivity of IL-2R signaling was reduced by about two- to threefold in T regs from mice that coexpressed wild-type IL-2Rβ and a mutant subunit (IL-2RβY3) with reduced signaling (designated NOD-Y3). Male and female NOD-Y3 mice exhibited accelerated diabetes onset due to intrinsic effects on multiple activities in T regs . Bone marrow chimera and adoptive transfer experiments demonstrated that IL-2RβY3 T regs resulted in impaired homeostasis of lymphoid-residing central T regs and inefficient development of highly activated effector T regs and that they were less suppressive. Pancreatic IL-2RβY3 T regs showed impaired development into IL-10–secreting effector T regs . The pancreatic lymph nodes and pancreases of NOD-Y3 mice had increased numbers of antigen-experienced CD4+ effector T cells, which was largely due to impaired T regs , because adoptively transferred pancreatic autoantigen–specific CD4+ Foxp3− T cells from NOD-Y3 mice did not accelerate diabetes in NOD.SCID recipients. Our study indicates that the primary defect associated with chronic, mildly reduced IL-2R signaling is due to impaired T regs that cannot effectively produce and maintain highly functional tissue-seeking effector T reg subsets.

INTRODUCTION CD4+ Foxp3+ regulatory T cells (T regs ) maintain peripheral tolerance by suppressing autoreactive T cells that escape negative selection in the thymus. Interleukin-2 (IL-2), through its interaction with the high-affinity IL-2 receptor (IL-2R), which consists of IL-2Rα (CD25), IL-2Rβ (CD122), and γc (CD132), controls multiple critical activities in T regs (1–4). IL-2R signaling is essential for thymic T reg development, promoting the maturation of CD4+ CD25lo Foxp3lo immature cells into functional T regs (5–9). IL-2 also contributes to T reg peripheral homeostasis (10), in part by providing survival signals to central or resting CD62Lhi T regs (11) and by supporting the development of terminally differentiated, short-lived Klrg1+ T regs (12). T reg identity and suppressive function are enforced by IL-2 (13) through direct regulation of the expression of Foxp3. This property also influences the development of conventional T cells into peripherally induced T regs (14–16). Because Foxp3 represses the expression of IL2, T regs constitutively express the high-affinity trimeric IL-2R to respond to IL-2 produced by effector T (Teff) cells and, in some cases, dendritic cells (17, 18). Multiple single-nucleotide polymorphisms (SNPs) in IL2, IL2RA, and IL2RB are associated with an increased risk for developing several human autoimmune diseases (19). On their own, these IL-2–related SNPs, some of which are common in the population, represent a small risk for autoimmunity and most likely act in concert with SNPs in other genes as well as environmental factors to trigger autoimmunity. This complexity has made it difficult to determine how an individual SNP promotes autoimmunity. With respect to type 1 diabetes (T1D), individuals with susceptible SNPs in IL2RA (20) have a reduced abundance of CD25 on T reg and T memory cells, which leads to reduced IL-2R signaling (21). Some data have also associated reduced IL-2R signaling in T regs with decreased fitness and suppressive function (22, 23). Nevertheless, we still poorly understand how a subtle reduction in IL-2R signaling represents a risk for autoimmunity, including T1D. The nonobese diabetic (NOD) mouse has been widely used as a model for T1D, where Il2 is a genetic risk for development of diabetes. The importance of the insulin-dependent diabetes risk locus 3 (Idd3), which contains Il2, for diabetes is illustrated by the substantial reduction in diabetes occurrence in NOD congenic mice in which the Idd3 interval is derived from C57BL/6 mice (24). NOD Idd3 results in a twofold reduction in IL-2 production by CD4+ T cells (25). Moreover, Il2 mRNA was selectively reduced in infiltrating cells in pancreatic islets of NOD mice when compared to that in peripheral immune tissues (26). Reduced IL-2 production is associated with a pancreas-specific decrease in the T reg to Teff cell ratio, which might reflect impaired T reg homeostasis. Pancreatic T regs in NOD mice have reduced amounts of CD25 and Bcl-2 but increased Ki67 abundance, the latter of which may reflect a compensatory proliferative response toward autoreactive T cells (26, 27). These studies concluded that the Il2-related genetic risk for diabetes in NOD mice is primarily due to a pancreas-specific impairment of T reg homeostasis that reduced the ability of these cells to suppress autoreactive T cells. However, these findings indirectly support these conclusions, and the effect on IL-2R signaling as a risk for diabetes remains poorly understood. An additional complication in clearly defining the Il2-related risk for diabetes in NOD mice is that the Idd3 locus also contains Il21, which is closely linked to Il2. This polymorphism in Il21 results in increased secretion of IL-21, a proinflammatory cytokine, and this increase in IL-21 abundance is linked to diabetes susceptibility in NOD mice (28). As discussed earlier, the direct contribution of the Il2-related risk for diabetes in NOD mice remains poorly understood. In addition, the reduced IL-2 production associated with the Idd3 locus does not directly test the consequences of altered IL-2R signaling, which is a risk for T1D and several other autoimmune diseases. To directly target IL-2R signaling, one must affect the activity of IL-2Rβ, because this subunit is responsible for the distinct signaling attributed to IL-2. Simply knocking out Il2rb in the germ line or selectively in T regs leads to the production of immature, nonfunctional T regs , which results in rapid lethal systemic autoimmunity (29–31); this approach is not suitable to assess how subtle changes in IL-2R signaling might promote autoimmunity. For these reasons, we developed a model in which IL-2Rβ signaling was selectively reduced in all T cells of NOD mice. We reasoned that IL-2R–dependent processes in T cells relevant to diabetes development in NOD mice would be intensified and thus distinguished from other genetic risks in this model. Diabetes was accelerated in male and female NOD mice in which IL-2Rβ signaling was modestly and selectively reduced in T cells. Furthermore, this autoimmunity was directly related to substantial changes in T regs that included, but were not limited to, altered homeostasis and function, whereas more modest alterations were noted in the Teff compartment.

DISCUSSION Past studies provided some initial information consistent with the notion that variation in the genes encoding IL-2 or the IL-2R in mice and humans reduces the activity of T regs (21, 24, 26), but little is understood regarding how this genetic variation represents a risk for autoimmunity. Here, we directly reduced IL-2Rβ signaling in T cells and showed that modestly, but chronically, reducing IL-2 responsiveness by about 2.6-fold accelerated diabetes in male and female NOD mice. Our findings demonstrate that accelerated diabetes occurs because of a multiplicity of effects on T regs that affect their homeostasis and function. Altered T reg homeostasis by IL-2RβY3 was evident because T regs from NOD-Y3 mice were broadly characterized by having increased amounts of Ki67 but decreased Bcl-2 abundance. This phenotype is consistent with T regs that undergo increased proliferation to counterbalance enhanced cell death. Reduced IL-2R signaling affected T reg homeostasis in two distinct ways. First, cT regs (as defined by high expression of CD62L) were found at a reduced proportion under several different experimental conditions when they expressed IL-2RβY3, and this effect may reflect impaired survival because IL-2 promotes the survival of this T reg subset (11). Second, the development of more highly activated eT regs [identified on the basis of the expression of CD103 and Klrg1 by CD62Llo T regs (38)] was less efficient for T regs with reduced IL-2R signaling. This was most obviously seen in experiments with mixed bone marrow chimeras and the direct development of eT regs from purified adoptively transferred cT regs . The expression of Klrg1 by eT regs represents a terminally differentiated state of activation and occurs only after more than eight or nine cell divisions as cT regs develop into eT regs (12). The observation that reduced IL-2R signaling led to fewer Klrg1+ eT regs is likely explained by eT regs that die before reaching this state of activation. In other systems, blockade of IL-2R signaling destabilizes T regs so that they become IFN-γ+ and IL-17+ Teff cells; this process is most evident for peripherally derived T regs (42, 43). Our experimental design did not enable us to directly assess this process, and some of the decrease in T reg number that we noted might be due to their development into Teff cells. However, several observations suggest that this mechanism does not fully account for the accelerated onset of diabetes in NOD-Y3 mice. First, we did not detect increases in the numbers of IFN-γ– and IL-17–producing CD4+ T cells in NOD-Y3 mice. If T reg conversion to T H 1 and T H 17 Teff cells was a main mechanism, these cells should have increased in number, particularly in the pancreas. Second, TCR repertoire studies indicate that most T regs in the peripheral immune tissues are thymus-derived cells (44) and have a stable phenotype (45). Thus, the poor persistence of adoptively transferred IL-2RβY3 T regs is unlikely to be due to the few peripherally derived T regs that may be part of the donor inoculum. This study also begins to provide a molecular underpinning for the dysregulation associated with IL-2RβY3 T regs . IRF4 is required for the cT reg -to-eT reg developmental step (40). Blimp-1 is selectively expressed by eT regs and contributes to the production of IL-10 (40). Broad immune mRNA profiling by NanoString analysis showed that all of these mRNAs were less abundant in pancreatic T regs of NOD-Y3 mice than in those of WT mice. These data are consistent with reduced IL-2R signaling interfering with the development of eT regs through an IRF4–Blimp-1–IL-10 axis. Activated STAT5 directly regulates the expression of Il2ra, Foxp3, Irf4, and Prdm1 (46–48). In addition, Il2ra, Irf4, and Prdm1 are direct targets of Foxp3 (49). Thus, subtly and chronically reducing both IL-2R signaling and the abundance of Foxp3 by IL-2RβY3 might cooperatively act to impair eT reg development. TCR signaling also importantly contributes to the development of eT regs (41). Thus, the reduced abundance of CD3ζ in pancreatic IL-2RβY3 T regs raises the possibility that IL-2RβY3 might also influence eT reg development by reducing TCR signaling, but this requires further investigation. We showed that IL-2RβY3 directly reduced functional activity by demonstrating the inability of T regs from NOD-Y3 BDC2.5 mice to suppress autoreactive BDC2.5 T cells and diabetes development in a NOD.SCID transfer model. The effect of IL-2RβY3 on T regs is predicted to reduce T reg function in several important ways. The impaired homeostasis of cT regs and eT regs likely diminishes the capacity of T regs that reside in lymph nodes and tissue sites, respectively, to properly balance their numbers with autoreactive T cells. Furthermore, Klrg1+ and CD103+ eT regs are characterized by having increased amounts of several T reg functional molecules and tissue-targeting chemokine receptors (38, 39). Thus, IL-2–dependent alterations in eT reg development affect T reg function indirectly by reducing the proportions of T regs with enhanced suppressive function, as well as directly by decreasing the amount of IL-10. Previous studies designed to ascertain the contribution of IL-2R signaling to diabetes in NOD mice have been indirect in that they assessed the potential effects of reduced IL-2 production, especially by pancreatic T cells, on T regs ’due to the Idd3 polymorphism (26). These studies implied that NOD T reg homeostasis was impaired as reflected by their altered amounts of Ki67 and Bcl-2, which we confirmed. A complication of the past results is that Idd3 not only reduces IL-2 but also increases the production of the proinflammatory cytokine IL-21 (28), which might also contribute to the development of diabetes. Thus, an advantage of IL-2RβY3 is that IL-2R signaling is directly targeted. The caveat with IL-2RβY3 is that IL-15R signaling is also reduced. This was detected in T regs and CD8+ T cells. However, the T reg compartment is largely normal in IL-15–deficient mice (6), and we showed that in situ pSTAT5 activation by T regs was inhibited by anti–IL-2, indicating that impaired IL-2R signaling largely accounted for the intrinsic effects of IL-2RβY3 on T regs . In contrast, the lower proportion of CD8+ T cells associated with NOD-Y3 mice was unaffected by anti–IL-2. These results, in conjunction with the known role of IL-15 in CD8+ T cell homeostasis (50, 51), suggest that the decrease in the number of CD8+ T cells is accounted for by reduced IL-15R signaling. CD8+ T cells and increased IL-15 signaling have been implicated in promoting diabetes in NOD mice (52). Thus, a reduction in these two parameters by IL-2RβY3 is not expected to be a contributing factor to the accelerated disease in NOD-Y3 mice. Upon reducing IL-2R signaling in T cells, T regs were intrinsically affected in a manner that would reduce peripheral tolerance, as discussed earlier. Reduced IL-2R signaling is also expected to reduce T H 1 and T H 2 responses (53–56), which should not accelerate diabetes. However, decreased IL-2R signaling could intrinsically increase the concentration of IL-2, T H 17s, and T follicular helper (Tfh) cells (57, 58), whereas increased IL-17 has been linked to diabetes in NOD mice (59). We observed an increase in antigen-experienced CD4+ T cells in NOD-Y3 mice, which included increased numbers of IL-2–producing, but not IL-17–producing, T cells in the PLN. Increased anti-insulin autoantibodies were also observed, which may reflect an increase in the number of Tfh cells due to reduced STAT5 and Blimp-1–mediated repression (57). However, transfer studies of CD4+ Foxp3− T cells into NOD.SCID mice did not provide clear evidence that IL-2RβY3 enhanced the activity of autoreactive T cells. A more difficult question to resolve is whether accelerated diabetes in NOD-Y3 mice is due to the reduced intrinsic IL-2Rβ signaling by autoreactive T cells that enhances their activity or is simply the result of reduced T reg activity. Although male NOD mice are relatively resistant to diabetes, a particularly marked result was that NOD-Y3 males developed diabetes at a similar rate to that of NOD females. Sex-biased development of T1D in NOD mice has been attributed to differences in microbiota and sex hormones (60–62). Differences in sex hormones have been suggested to influence the type of microbiota in male and female NOD mice (62), raising the possibility that these two factors are interrelated. Although our study did not address the factors responsible for the lack of sex bias in NOD-Y3 mice, we speculate that altering the number of eT regs (which dominate the gut mucosa) through reduced IL-2R signaling distinctively shapes immune homeostasis in the gut mucosa to influence the microbiota in a way that promotes diabetes in both male and female mice. Alternatively, decreased T reg function as a result of reduced IL-2R signaling in NOD-Y3 mice may be sufficient to counteract the protective effects in male NOD mice that are attributed to microbiota and sex hormones. Experimentation is required to assess these points. The IL-2–IL-2R axis has been implicated as a risk factor not only for T1D but also for other autoimmune diseases, including multiple sclerosis, celiac disease, and rheumatoid arthritis. IL-2RβY3 reduced IL-2R signaling in all T cells, and this situation is predicted to mimic genetic variation due to SNPs within the noncoding region of IL2RA that may alter gene expression. Thus, the effects that we noted in NOD-Y3 mice might also be detected in humans that carry some IL2RA risk alleles. In addition, low-dose IL-2 represents a promising strategy to boost T regs in patients with autoimmunity (63). Our study raises the possibility that along with evaluating the capacity of low-dose IL-2 to increase the total number of T regs , one may also wish to assess how this treatment affects particular subsets of T regs to maximize therapeutic benefits.

MATERIALS AND METHODS Mice NOD CD45.2 congenic (NOD.B6-Ptprcb/6908MrkJacJ) and NOD.SCID (NOD.CB17-PrkdcSCID/J) mice were purchased from Jackson Laboratories. NOD-Y3 IL-2Rβ+/+, NOD-Y3 IL-2Rβ+/−, and NOD-Y3 IL-2Rβ−/− mice, where Y3 refers to the mutant IL-2Rβ transgene, were developed by backcrossing C57BL/6-Y3 IL-2Rβ−/− mice (32) to NOD/ShiLtJ mice for 12 generations. All Idd loci were confirmed to be of NOD origin. NOD/Foxp3-RFP reporter (FIR) mice were previously described (64) and crossed to NOD BDC2.5 and NOD-Y3 IL-2Rβ+/+ mice to yield NOD-Y3-FIR, NOD BDC2.5-FIR, and NOD-Y3 BDC2.5-FIR mice. All mice were housed in a specific pathogen–free animal facility at the University of Miami. The Institutional Animal Care and Use Committee at the University of Miami reviewed and approved all animal studies conducted in this study. Assessment of diabetes, insulin autoantibodies, and islet infiltration scoring Diabetes was determined by following urine glucose two to three times per week using Diastix strips (Bayer). After two positive urine tests (>250 mg/dl), blood glucose was assessed using OneTouch Ultra strips. Mice were considered diabetic when blood glucose readings were >250 mg/dl. Serum was analyzed for insulin autoantibodies by radioimmunoassay as previously described (65). To assess islet inflammation, the pancreas was fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 5-μm sections, and stained with hematoxylin and eosin. Lymphocytic infiltration was scored for each islet and assessed blindly by light microscopy. Insulitis scoring was conducted per the following criteria: 0, no insulitis (free of infiltration); 1, peri/polar insulitis (infiltration confined to the periphery of islets); 2, mild insulitis (<50% of the islet area infiltrated); 3, severe insulitis (≥50% of the islet area infiltrated); and 4, massive insulitis (≥90% of the islet area infiltrated) (66). Fluorescence-activated cell sorting analysis Cell staining for fluorescence-activated cell sorter (FACS) analysis was performed as previously described (67). Monoclonal antibodies (mAbs) specific for the following targets were used for flow cytometry: CD25 (PC61), pSTAT5 (pY641), Ki67 (B56), and CD11c (HL3) were purchased from BD Biosciences; and Foxp3 (FJK-16s), CD62L (MEL-14), IL-2 (JES6-5H4), CD103 (2E7), Klrg1 (2F1), ICOS (15F9), and streptavidin were purchased from eBioscience. Antibodies against CD122 (Tm-b1), CD4 (RM4-5), CD8 (53-6.7), IFN-γ (XMG1.2), TNF-α (MPG-XT22), IL-17 (TC11-18H10.1), CD69 (H1.2F3), Bcl-2 (BCL/10C4), CD45.1 (A20), CD45.2 (104), CD19 (GD5), CD49b (DX5), Ly6G (1A8), CD11b (M1/70), and streptavidin were purchased from BioLegend. Antibodies against CD4 (GK1.5) and CD44 (PGP-1) were purified and conjugated in-house. Intracellular staining for Foxp3 (eBioscience) was performed as recommended by the manufacturer. For intracellular staining of cytokines, the BD Cytofix/Cytoperm kit was used according to the manufacturer’s protocol. Samples were analyzed on a BD LSRFortessa-HTS or a BD LSR II flow cytometer. Typically, 200,000 total events were collected per sample. Flow cytometry data were analyzed with BD FACSDiva software (version 8.0.1). Cell isolation and sorting To isolate lymphoid cells from the mouse pancreas, pancreases were minced into about 2-mm2 pieces in Hanks’ balanced salt solution (HBSS) containing 50% fetal calf serum (FCS) and incubated at 37°C for 3 hours in a 7% CO 2 incubator. The total pancreas pieces were then passed through a 70-μm nylon strainer (Falcon). The resulting single-cell suspension was centrifuged at 480g for 5 min, pelleted, and then washed once with HBSS. To prepare T cell–depleted bone marrow cells, mAbs against CD4 (0.2 ml of culture supernatant; RL1724), CD8 (0.2 ml of culture supernatant; HO22), Thy-1.2 (10 μg of purified antibody; 30-H12, Sigma-Aldrich), deoxyribonuclease (10 μg; Sigma-Aldrich), and rabbit complement (0.12 ml; Cedarlane Laboratories) were added to 1 ml of bone marrow cells (25 × 106/ml in RPMI 1640 containing 5% FCS). This mixture was incubated on ice for 15 min and then incubated at 37°C for 30 min. After this time, the cells were washed once in RPMI 1640. In most experiments, CD4+ T regs and T conventional cells from the spleen or pancreas were surface-stained and sorted on the basis of the presence or absence, respectively, of the Foxp3-RFP reporter with FACSAria II or Beckman Coulter MoFlo Astrios EQ cell sorters. Cell purity was usually greater than 95%. For the adoptive transfer of T regs into NOD-Y3 IL-2Rβ−/− mice, where some donor mice were not on the Foxp3-RFP reporter background, T regs were sorted on the basis of CD4+ CD62Lhi CD25hi expression. This population was greater than 93% Foxp3+ cells. Functional and molecular analyses To measure intracellular cytokines by flow cytometry, cells (1 × 106 to 2 × 106) were cultured in 24-well flat-bottom plates with RPMI 1640 complete medium (68) containing PMA (50 ng/ml), ionomycin (1 μM), and brefeldin A (5 μg; BioLegend) for 4 hours. To assess mRNA abundance, cell lysates from FACS-purified cells were prepared with RLT lysis buffer (Qiagen). These lysates (10,000 cell equivalents) were subjected to NanoString analysis using the nCounter Mouse Immunology Gene Expression Code set (NanoString). Expression data were analyzed by normalizing counts to housekeeping genes and positive and negative controls using nSolver version 2.5 (38). Normalized counts were log 2 −transformed, and the data were analyzed by Student’s two-tailed t test. IL-2–dependent (eBioscience) and IL-15–dependent (PeproTech) pSTAT5 activation was assessed by flow cytometry as previously described (7), either directly ex vivo or after stimulation in vitro with the appropriate cytokine for 15 min. The resulting dose-response curve data were subjected to nonlinear regression analysis using GraphPad Prism software to determine the EC 50 . In vivo studies For adoptive transfer studies, the appropriate cell populations were transferred intravenously through the tail vein into NOD.SCID or NOD-Y3 IL-2Rβ−/− recipient mice. Glucose concentrations were monitored in the NOD.SCID recipients for up to 24 days after transfer, as required. For bone marrow chimeras, 1 day before cell transfer, NOD.SCID recipients were subjected to 3.5 Gy of γ irradiation. Donor T cell–depleted bone marrow was mixed, as indicated in Results, and injected intravenously into the tail vein. Blood was collected to monitor donor cell reconstitution before the recipient mice were sacrificed 8 weeks after transfer. Statistical analysis All data are represented as means ± SEM. Statistical tests used are listed in the figure legends. Data were analyzed using Wilcoxon signed-rank tests for FACS expression based on the MFI, where the MFI of cells from WT NOD mice were normalized to 1. The log-rank test was used to assess diabetes instances, the Kruskal-Wallis test was used to assess multigroup comparisons, and the Mann-Whitney test was used to assess autoantibody titers and two group comparisons. Two-tailed unpaired t tests were used to analyze pancreatic T reg NanoString data. Statistical comparisons were performed using GraphPad Prism (version 7.01). Statistical significance is indicated in the figure legends as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

SUPPLEMENTARY MATERIALS www.sciencesignaling.org/cgi/content/full/10/510/eaam9563/DC1 Fig. S1. Analysis of STAT5 activation in conventional T cells from male NOD-Y3 mice. Fig. S2. Flow cytometry analysis of T lymphocytes from female NOD-Y3 mice. Fig. S3. Analysis of IL-2RβY3 expression on T lymphocytes from mice on the NOD BDC2.5 background. Fig. S4. Analysis of cell distribution in the non–T cell compartment in male NOD-Y3 mice. Table S1. NanoString analysis of mRNA expression in pancreatic T regs from male NOD and NOD-Y3 mice.

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