ICOS-Dependent Homeostasis and Function of Foxp3⁺ Regulatory T Cells in Islets of Nonobese Diabetic Mice

ICOS expression designates a dominant Foxp3⁺ Treg subset in prediabetic islets

ICOS blockade in NOD mice results in T1D exacerbation, suggesting that ICOS may play a role in Treg-mediated self-tolerance (21). We first sought to characterize ICOS expression within the Foxp3⁺ and Foxp3⁻ T cell subsets in nondraining LN (data not shown), draining pLN, and pancreas at the early stage of insulitis in 3- to 4-wk-old BDC2.5 mice, whose TCR is specific for a β-islet–specific autoantigen. We observed a substantial increase in ICOS expression specifically on intrapancreatic Foxp3⁺ Tregs (Fig. 1A, top row). The frequency of Foxp3⁺ICOS⁺ Tregs among CD4⁺ T cells was significantly higher in the pancreas compared with that in draining pLN (8.8 ± 1.8% versus 1.5 ± 0.7%, p ≤ 0.01) (Fig. 1A, bottom row, left panel) and represents a dominant fraction of total Foxp3⁺ Treg in the pancreas compared with that in draining pLN (57 ± 10.2% and 18.9 ± 4.5%, p ≤ 0.002) (Fig. 1A, bottom row, middle panel). Similarly, the mean fluorescence intensity (MFI) of ICOS was also significantly greater on Tregs in the pancreas relative to the pLN (144.3 ± 11.9 versus 38.5 ± 15.9, p ≤ 0.001) (Fig. 1A, bottom row, right panel). Only a small proportion of ICOS⁺ Teffs in pLN and pancreas was detected among the CD4⁺ T cell pool (0.96 ± 0.4% versus 5.3 ± 1.5%) (Fig. 1A, bottom row, left panel) and particularly among Foxp3⁻ T cells (1 ± 0.4% versus 6 ± 1.6%) (Fig. 1A, bottom row, middle panel), suggesting that ICOS costimulation may not be required for the optimal function of autoreactive CD4⁺ T cells.

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FIGURE 1.

ICOS expression designates a dominant Foxp3⁺ Treg subset in prediabetic islets. Cell suspensions of draining pLN and pancreas of 3- to 4-wk old BDC2.5 mice were prepared and examined for (A) ICOS frequency and MFI, (B) CD25 expression, and (C) proportion of cycling cells (based on Ki-67 expression) within the ICOS⁺ and ICOS^– subsets of Foxp3^+/− CD4⁺ T cells and analyzed by FACS. Data are representative of at least three separate experiments (n = three mice/group). Results represent the mean ± SD. **p ≤ 0.02, ***p ≤ 0.002.

The striking difference in ICOS expression on intrapancreatic Tregs relative to their counterparts in pLN suggested that Tregs acquire distinctive functional and phenotypic properties in situ. In pLN, approximately half of the Foxp3⁺ Treg pool expressed CD25 (49.7 ± 4.3%), and only a small proportion of Foxp3⁺ Tregs coexpressed both CD25 and ICOS (7.5 ± 0.8%) (Fig. 1B). In stark contrast, a significant frequency of Foxp3⁺ Tregs coexpressed CD25 and ICOS in the pancreas of BDC2.5 mice (53.8 ± 5.2% versus 7.5 ± 0.8%, p ≤ 0.0007) (Fig. 1B). We also examined how ICOS expression is related to the proliferation of the Foxp3⁺ Treg pool within the pancreatic lesion. A significant proportion of cycling Tregs (based on Ki-67 expression) expressed ICOS in the pancreas relative to pLN (24.4 ± 4.5% versus 9.2 ± 3.3%, p ≤ 0.001) (Fig. 1C, right panel). Notably, the ICOS⁺ subset exhibited a much greater proliferative potential compared with the ICOS⁻ subset of Foxp3⁺ Tregs (53.1 ± 5.7% versus 16.9 ± 4.2%, p ≤ 0.001) (Fig. 1C, left panel). Remarkably, pancreatic ICOS⁺ and ICOS⁻ Foxp3⁺ Treg subsets did not differ in their activation status as both fractions were found within the CD62L^lowCD44^high Teff memory pool, irrespective of their proliferative potential (data not shown), suggesting that ICOS⁺Foxp3⁺ Tregs represent tissue-resident memory Tregs in islets. Thus, ICOS expression discriminates effector Foxp3⁻ Teffs from Foxp3⁺ Tregs and specifically designates a dominant subset of intra-islet Tregs, endowed with an increased potential to expand in islets of prediabetic BDC2.5 mice.

ICOS costimulation promotes the competitive fitness of Tregs in vivo

To determine whether ICOS expression conferred a competitive advantage to Ag-specific Foxp3⁺ Tregs in vivo, we examined Foxp3⁺ Treg responses in ICOS-deficient (ICOS^−/−) BDC2.5 mice compared to BDC2.5 (WT) mice. To this end, T cell-deficient NOD.TCRα^−/− mice were adoptively transferred with CD4⁺ T cells from congenic (Thy1.1⁺) WT or (Thy1.2⁺) ICOS^−/− BDC2.5 mice, and the relative occupancy of transferred T cells in pancreatic sites was evaluated 8 d after adoptive transfer. Our results show an increase in the proportion of WT (Thy1.1⁺) Foxp3⁺ Tregs compared with ICOS^−/− (Thy1.2⁺) Tregs in draining LN (14.0 ± 0.8 versus 7.4 ± 0.7, p ≤ 0.007) and the pancreas (25.9 ± 2.3 versus 10.7 ± 2.2, p ≤ 0.0032) (Fig. 2A, left panel). The proportion of Foxp3⁻ T cells was slightly increased from ICOS^−/− (Thy1.2⁺) donors (data not shown). We then examined whether the decreased frequency of ICOS^−/− Foxp3⁺ Tregs resulted from a reduced cycling in situ. Our analysis of Ki-67 expression showed that ICOS^−/− (Thy1.2⁺) Tregs expanded less efficiently compared with WT (Thy1.1⁺) Foxp3⁺ Tregs in draining LN (65.7 ± 2.8 versus 78.0 ± 1.2, p ≤ 0.007) (Fig. 2A, right panel) and particularly in the pancreas (77.7 ± 4.2 versus 95.9 ± 1.2, p ≤ 0.0061) (Fig. 2A, right panel). This decrease in ICOS^−/− (Thy1.2⁺) Treg expansion also coincided with a reduction in CD25 expression in ICOS^−/− Tregs compared with WT Tregs in pancreas (73.9 ± 2.4 versus 49.2 ± 4.0, p ≤ 0.0001), suggesting a possible IL-2–ICOS cross-talk in Treg homeostasis in islets (data not shown).

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FIGURE 2.

ICOS costimulation promotes Treg competitive fitness in vivo. A, NOD.TCRα^−/− mice received CD4⁺ T cells (1 × 10⁶) isolated from pooled LN and spleen of 3- to 4-wk-old donor congenic WT (Thy1.1⁺) and ICOS^−/− (Thy1.2⁺) BDC2.5 mice. Eight days posttransfer, CD4⁺ donor T cells were examined for Foxp3 expression (left panel) and proliferation of Foxp3⁺ Tregs (right panel). B, Donor WT (Thy1.1⁺) or ICOS^−/− (Thy1.2⁺) BDC2.5 CD4⁺ T cells (8 × 10⁶), isolated from pooled LN and spleen, were adoptively transferred into WT (Thy1.1⁺) or ICOS^−/− (Thy1.2⁺) BDC2.5 recipient mice. Representative FACS dot plots and bar graphs show cycling of donor or resident Foxp3⁺ Tregs in the target organ 21 d posttransfer. C, MACS-purified, CFSE-labeled CD4⁺ T cells (5 × 10⁶) isolated from pooled LN and spleen of WT and ICOS^−/− BDC2.5 mice were transferred i.v. into NOD recipient mice. The pLN of recipient mice were harvested on day 21 posttransfer, and the proliferative capacity of donor CD4⁺Foxp3^+/− T cells was determined in recipient mice (n = 3 mice/group). Similar results were obtained in three independent experiments. Results represent the mean ± SD.

We then examined the competitive fitness of Foxp3⁺ Tregs from WT or ICOS^−/− BDC2.5 mice in non-lymphopenic hosts. To this end, we performed a crisscross adoptive transfer whereby WT (Thy1.1⁺) or ICOS^−/− (Thy1.2⁺) CD4⁺ T cells were transferred in either WT (Thy1.1⁺) or ICOS^−/− (Thy1.2⁺) recipient mice, and the expansion and cellular frequency of transferred T cells in pancreatic sites were assessed. In either setting, WT (Thy1.1⁺) Tregs (of donor or host origin) expanded and accumulated in pLN (58.0 ± 0.85 versus 15.85 ± 1.95, p ≤ 0.0025) (Fig. 2B, left panel) or pancreas (66.85 ± 13.85 versus 34.4 ± 2.0, p ≤ 0.057) (Fig. 2B, left panel) more efficiently compared with ICOS^−/− (Thy1.2⁺) Tregs (Fig. 2B, left and right panels).

As an alternate approach to assess the proliferation of WT and ICOS^−/− T cell subsets, CFSE-labeled CD4⁺ T cells from WT or ICOS^−/− BDC2.5 mice were adoptively transferred into NOD recipients, and the extent of proliferation was determined in draining LN. The proportion of ICOS^−/− Foxp3⁺ Treg expansion was significantly lower relative to WT Tregs in pLN (65.1 ± 3.6 versus 81.4 ± 0.35, p ≤ 0.0126) (Fig. 2C, upper panel). No significant difference was observed between Teffs from either genotype, indicating that Teff priming and expansion is not impaired in the absence of ICOS in vivo (Fig. 2C, lower panel). Overall, our results show that ICOS⁺ Tregs have a greater proliferative capacity than Teffs and that a decreased expansion potential underlies the defective competitiveness of Ag-specific ICOS^−/− Tregs in inflamed sites.

ICOS-deficient Foxp3⁺ Tregs are less suppressive in vitro than WT Tregs

We then asked whether ICOS affected the functional potency of Tregs. To address this question, we first characterized the function of Tregs with differential ICOS expression levels by suppression assays in vitro. CD4⁺CD25⁺ICOS⁺ Tregs were more potent than CD4⁺CD25⁺ICOS⁻ Tregs at suppressing CD4⁺ Teffs at all ratios examined (Fig. 3A, right and left panels). To ensure that CD25⁺ICOS⁺ Tregs were not contaminated with activated Teffs expressing these markers, ICOS⁺Foxp3⁺ Tregs from BDC2.5.Foxp3^GFP reporter mice were assessed for their ability to suppress the proliferation of Foxp3⁻ (GFP⁻) Teffs in vitro. ICOS-expressing Foxp3⁺ (GFP⁺) Tregs exhibited a greater suppressive capacity relative to their ICOS⁻ counterparts (Fig. 3B), consistent with the increased ability of WT BDC2.5 Tregs, compared with ICOS^−/− Tregs, to suppress the proliferation of WT or ICOS^−/− Teffs in vitro (Fig. 3C). Blocking anti-ICOS Abs did not abolish suppression, suggesting that ICOS expression is not involved in the effector mechanism of suppression in vitro (data not shown). Moreover, whereas ICOS⁻ Tregs upregulate ICOS upon TCR stimulation in vitro, they do not suppress Teffs as efficiently as ex vivo ICOS⁺ (data not shown). This suggests that ICOS may have an important role in imprinting this potential in Tregs during their development, and possibly designates a unique Foxp3⁺ Treg subset operating in the periphery, similar to an observation made by Ito et al. (28), in human studies. Thus, ICOS-expressing Foxp3⁺ Tregs are endowed with a more suppressive phenotype compared with their ICOS⁻ subset and indicate a role for ICOS in potentiating Treg suppressive functions.

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FIGURE 3.

ICOS-deficient Foxp3⁺ Tregs are less suppressive in vitro. A, FACS-purified BDC2.5 CD4⁺CD25⁻ICOS⁻ responder T cells (5 × 10⁴) isolated from pooled LN and spleen of 3- to 4-wk-old donors were CFSE-labeled and stimulated with mimetope (RVRPLWVRME) (2 ng/ml) and irradiated spleen cells (2 × 10⁵) in the presence or absence of titrated numbers of FACS-purified ICOS⁺ (filled squares) or ICOS⁻ (open squares) CD4⁺CD25⁺ Tregs. CFSE dilution profiles (left panel) and cell division analysis (right panel) are shown at multiple Teff/Treg ratios. B, To exclude contaminating CD25⁺ICOS⁺ Teffs, FACS-purified Foxp3^GFP−ICOS⁻ responder T cells were cocultured with titrated numbers of ICOS⁺ or ICOS⁻ Foxp3^GFP+ Tregs from BDC2.5.Foxp3^GFP reporter mice. Responder T cell proliferation was measured by [³H]thymidine incorporation. C, CFSE-labeled, FACS-purified CD4⁺CD25⁻ICOS⁻ responder T cells (5 × 10⁴) from WT BDC2.5 mice were activated in the presence or absence of titrated numbers of ICOS^+/− CD4⁺CD25⁺ Tregs from WT BDC2.5 mice, or CD4⁺CD25⁺ Tregs from BDC2.5 ICOS^−/− mice. Similar results were obtained in three independent experiments. Results represent the mean ± SD.

ICOS-deficient Foxp3⁺ Tregs are unable to inhibit T1D induction in vivo

We then assessed how WT and ICOS^−/− Foxp3⁺ Tregs compared in their capacity to inhibit pathogenic T cells during the pathogenesis of T1D. WT or ICOS^−/− BDC2.5 CD4⁺ Teffs were transferred into NOD.TCRα^−/− hosts either alone or with WT or ICOS^−/− BDC2.5 Tregs (1:8 Treg/Teff ratio), and the onset of T1D was monitored. Recipient mice transferred with WT or ICOS^−/− Teffs alone developed T1D with a similar onset (11–12 d posttransfer) and similar incidence (80%) indicating that absence of ICOS costimulation is not essential for the pathogenic functions of Teffs (Fig. 4A). Expectedly, WT CD4⁺ Treg efficiently suppressed the pathogenic potential of WT Teffs, as NOD.TCRα^−/− recipient mice remained 90% T1D-free for up to 23 d posttransfer (Fig. 4A). Strikingly, recipient mice receiving WT Teffs and ICOS^−/− Tregs rapidly developed T1D by day 13–15 posttransfer, demonstrating that ICOS^−/− Tregs are unable to maintain self-tolerance compared with WT Tregs (Fig. 4A). These in vivo data are consistent with our observations that ICOS⁻ or ICOS^−/− Foxp3⁺ Tregs are unable to suppress Teffs in vitro (Fig. 3). These findings confirm that ICOS deficiency in Foxp3⁺ Tregs cripples their capacity to control autoreactive Teffs and mediate T1D protection in contrast to ICOS^−/− Teffs, which fully preserve their functions in vivo.

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FIGURE 4.

ICOS genetic deficiency or Ab blockade selectively abrogates Foxp3⁺ Treg-mediated protection from T1D. A, NOD.TCRα^−/− recipient mice were adoptively transferred with CD4⁺CD25^– Teffs (1 × 10⁶) from WT or ICOS^−/− BDC2.5 mice in the presence or absence of WT or ICOS^−/− CD4⁺CD25⁺ Tregs (0.125 × 10⁶) from 4-wk-old BDC2.5 mice. Blood glucose levels in recipient mice (n = 10/group) were monitored for T1D incidence every 48 h posttransfer. B, Twenty-one days after T cell transfer, mice from each group were sacrificed, pLN and pancreas were harvested, and the cellular frequency of cycling (as per Ki-67 expression), islet-specific Foxp3⁺ Tregs was examined relative to cycling Foxp3⁻ Teffs. C, NOD.TCRα^−/− recipient mice were adoptively transferred with CD4⁺CD25^– Teffs (1 × 10⁶) from WT in the presence or absence of WT or ICOS^−/− CD4⁺CD25⁺ Tregs (0.125 × 10⁶) from 4-wk-old BDC2.5 mice. Simultaneously, each respective group received three subsequent i.p. injections of anti-ICOS mAb (50 or 100 μg). Blood glucose levels in recipient mice (n = 10/group) were monitored for T1D incidence every 48 h posttransfer. D, Nineteen days after T cell transfer, mice from groups receiving WT or ICOS^−/− Tregs were sacrificed, pancreas was harvested, and the cellular frequency of islet-specific Foxp3⁺ Tregs was examined in each group. Data are representative of three separate experiments. Results represent the means ± SD.

Given the inability of ICOS^−/− Tregs to suppress induction of T1D by Teffs, we then asked whether this was due to decreased expansion and accumulation of Foxp3⁺ Tregs in the pLN and pancreas. To examine this possibility, BDC2.5 CD4⁺ Teffs were transferred into NOD.TCRα^−/− mice either alone or with WT and ICOS^−/− BDC2.5 Tregs, and the cellular frequency of cycling Foxp3⁺ cells (Ki-67 expression) was analyzed on days 15 and 23 posttransfer. The frequency of ICOS^−/− Foxp3⁺ Tregs in the pLN and pancreas was significantly lower compared with recipients of WT Tregs (22.8 ± 4.2 versus 11.13 ± 3.02, p ≤ 0.004) (Fig. 4B), and particularly after the onset of T1D (22.8 ± 4.2 versus 5.6 ± 0.7, p ≤ 0.0001) (Fig. 4B). Moreover, T1D protection correlates with the increased proportion of WT Foxp3⁺ Tregs in the pancreas and with decline in Foxp3⁻ Teff cycling (24.8 ± 2.1 versus 33.8 ± 4.6, NS) (Fig. 4B), an observation not seen with ICOS^−/− Treg_, particularly after the onset of T1D (24.8 ± 2.1 versus 57.6 ± 4.7, p ≤ 0.0002) (Fig. 4B). This suggests that the proliferative potential of Tregs correlates directly with their functional potency in situ and strongly indicates that Tregs actively suppress autoreactive Foxp3⁻ Teffs in the pancreas. Thus, our data show that ICOS^−/− Tregs are unable to prevent T1D induction and correlate with their decreased accumulation in the pancreatic lesion.

In vivo ICOS Ab blockade selectively targets Foxp3⁺ Tregs in the target organ and exacerbates T1D

We then performed an anti-ICOS mAb blockade experiment to demonstrate the dependence and specificity of the ICOS signal in Foxp3⁺ Treg-mediated T1D protection in vivo. To this end, we administered a blocking anti-ICOS mAb in our Teff/Treg cotransfer system and then assessed the onset of T1D relative to the frequency of Foxp3⁺ Tregs in pancreatic sites. Ab-mediated ICOS blockade did not inhibit the diabetogenic potential of Teffs compared with control mice (Fig. 4C). The onset of T1D occurred similarly in both groups (by day 10–13 posttransfer), suggesting that ICOS signals are not required for induction of disease, an observation consistent with results obtained with ICOS^−/− Teffs (Fig. 4A). Expectedly, WT Treg efficiently suppressed the pathogenic potential of WT Teffs and remained T1D-free for the entire duration of the experiment (up to day 19 posttransfer, endpoint of experiment) (Fig. 4C). Notably, ICOS blockade completely prevented the protection conferred by WT Tregs and rapidly exacerbated T1D development (Fig. 4C). The rapid onset of T1D correlated with a substantial loss in Foxp3⁺ Tregs in anti-ICOS mAb-treated mice compared with the control group (9.4 ± 7.9 versus 33.5 ± 7.3, p ≤ 0.015) (Fig. 4D).

To demonstrate further that anti-ICOS mAb specifically targets Foxp3⁺ Tregs in our system, we cotransferred ICOS^−/− Tregs with WT Teffs in the presence or absence of anti-ICOS mAb treatment. Anti-ICOS mAb treatment failed to change the course of disease by 10–16 d posttransfer (Fig. 4C) or frequency of Foxp3⁺ Tregs in the pancreas (19.96 ± 7.4 versus 16.3 ± 3.3, NS) (Fig. 4D), an outcome consistent with the lack of effect of Ab treatment on disease progression in mice receiving Teffs alone (Fig. 4C). Thus, our results show that anti-ICOS mAb blockade targets Foxp3⁺ Tregs specifically and does not affect the pathogenic potential of diabetogenic Teffs in T1D. Moreover, we observe that Ab treatment specifically abolishes Foxp3⁺ Tregs in the pancreas but not in the pLN (data not shown). This correlates with our previous observations that ICOS expression on Foxp3⁺ Tregs is predominantly expressed in the pancreas and correlates with a functional potency of these cells directly in the target organ. Overall, our data point to an ICOS-dependent role in favoring the expansion and function of Foxp3⁺ Tregs within the pancreatic environment, in turn increasing the Treg/Teff ratio and tipping the balance to self-tolerance. Whether ICOS imprints such function during Treg development or specific signals received in the periphery are responsible for such a phenotype is currently unclear.

Intra-islet ICOS⁺ Foxp3⁺ Tregs preferentially secrete IL-10

ICOS expression on activated murine and human T cells enables them preferentially to produce IL-10 (29–31). ICOS-deficient patients show a severe reduction in IL-10 production, reinforcing the role of ICOS in IL-10 secretion (32). IL-10 is an important immunomodulatory cytokine, which has been shown to be critical for the control of T1D in BDC2.5 mice (33, 34). The link between ICOS and IL-10 led to the hypothesis that ICOS⁺Foxp3⁺ Treg-mediated T1D protection is related to this production of IL-10. To this end, ICOS⁺ and ICOS⁻ Treg subsets were FACS purified from BDC2.5.Foxp3^GFP mice, activated with NOD BMDCs and mimetope in vitro, and the capacity to produce IL-10 was assessed by flow cytometry. Our results show that only ICOS⁺ Tregs were able to secrete IL-10 after Ag-specific stimulation compared with ICOS⁻ Treg (19 ± 7.1% versus 1.91 ± 4.1%, p ≤ 0.0001) (Fig. 5A). Similarly, ICOS expression correlated with IL-10 production in recently in vitro-activated CD4⁺ T cells from pLN and pancreas of prediabetic NOD.Foxp3^GFP mice. More specifically, a substantial proportion of ICOS-expressing CD4⁺ T cells produced IL-10 (4.8 ± 1.8%) (Fig. 5B, left panel), and a striking proportion of Foxp3^gfp+ Tregs secreted IL-10 upon in vitro polyclonal stimulation (47.2 ± 6.4%) (Fig. 5B, right panel). The frequency of IL-10–secreting Foxp3⁺ Tregs is significantly greater in the pancreas than in pLN (47.2 ± 6.35 versus 3.6 ± 0.5, p ≤ 0.0001), where little IL-10 production is detected (Fig. 5B, right panel). Surprisingly, the in vitro suppressive activity of ICOS⁺ or ICOS⁻ Treg subsets was not affected by IL-10 neutralization suggesting that redundant effector mechanisms of Treg suppression operate in vitro (data not shown).

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FIGURE 5.

Intra-islet ICOS⁺ Foxp3⁺ Tregs preferentially secrete IL-10. A, FACS-purified ICOS⁺ and ICOS⁻ Foxp3^GFP+ T cells were isolated from pooled LN and spleen of 3- to 4-wk-old BDC2.5^GFP reporter mice and then activated in the presence of NOD BMDCs at a 4:1 ratio and BDC2.5 mimetope (100 ng/ml) for 72 h. The frequency of IL-10–producing Foxp3⁺ Tregs, relative to ICOS expression, was assessed by flow cytometry (left panel). The proportion of IL-10–producing Foxp3⁺ Tregs among ICOS⁺ and ICOS⁻ subsets are shown (right panel). B, Cell suspensions from pLN and pancreas of 4-wk-old NOD.Foxp3^GFP reporter mice were reactivated in vitro for 4 h with PMA/ionomycin, and the frequency of IL-10–producing Foxp3⁺ Tregs, relative to ICOS expression, was determined by FACS (left panel). The proportion of IL-10–producing ICOS⁺Foxp3⁺ Tregs in pLN and pancreas are shown (right panel). C, NOD.TCRα^−/− mice were transferred with CD4⁺ T cells (1 × 10⁶) isolated from pooled LN and spleen of 3- to 4-wk-old WT (Thy1.1⁺) or ICOS^−/− (Thy1.2⁺) BDC2.5 mice. Eight days posttransfer, the frequency of IL-10–secreting Foxp3⁺ Tregs in each donor group was determined by FACS in pancreas and pLN, as in B. Data are representative of three separate experiments. Results represent the means ± SD.

To assess directly the requirement for ICOS expression in the production of IL-10 by Foxp3⁺ Tregs, we examined the potential for IL-10 secretion in BDC2.5 ICOS^−/− mice. To this end, WT or ICOS^−/− BDC2.5 CD4⁺ T cells were transferred into NOD.TCRα^−/− recipients and the frequency of IL-10–producing Foxp3⁺ Treg assessed in pLN and pancreas 8 d posttransfer. We observed that the most significant IL-10 production by Tregs occurs directly in the pancreas compared with that in the draining LN (Fig. 5C). Furthermore, we observe that the frequency of IL-10–producing Foxp3⁺ Tregs is dramatically reduced in the pLN, and particularly in the pancreas, of ICOS^−/− mice compared with that in WT mice (16.7 ± 7.7 versus 3.0 ± 0.7, p ≤ 0.002) (Fig. 5C). Overall, our results show that ICOS expression is required for IL-10 production by Tregs and likely drives this effector differentiation in the pancreas.

Loss in ICOS expression and IL-10 production in Foxp3⁺ Tregs coincides with T1D progression

We and others have previously shown that Foxp3⁺ Treg function wanes with T1D progression, in turn enabling the diabetogenic T cells to escape regulation and promote destructive infiltration of islets (8, 9, 11–13, 35, 36). We then explored the possibility that breakdown in self-tolerance may be attributed to an age-related decline in ICOS expression on Tregs, at checkpoints known to demarcate the early (≤4 wk) from late (≥12 wk) stages of insulitis in the BDC2.5 model. We observed a remarkable decline in the cellular frequency of ICOS-expressing Foxp3⁺ Tregs within the pancreas (68.6 ± 1.4 versus 47.4 ± 1.8, p ≤ 0.0008) (Fig. 6A, right panel), in stark contrast to the pLN. Similarly, ICOS expression on intrapancreatic Foxp3⁺ Tregs is significantly reduced with age (144.3 ± 11.9 versus 50.7 ± 17.6, p ≤ 0.003) (Fig. 6A, left panel), suggesting that the suppressive potential of Tregs correlates with ICOS-mediated costimulation in Tregs. Moreover, this reduction in the cellular frequency of ICOS⁺Foxp3⁺ Tregs was directly related to the loss in expansion (Ki-67 expression) of ICOS-expressing Foxp3⁺ Tregs directly within the pancreas of prediabetic mice (50.2 ± 3.8 versus 25.0 ± 2.6, p ≤ 0.003) (Fig. 6B) with time, a trend not observed in pLN (10.4 ± 0.3 versus 7.9 ± 0.7, NS) (Fig. 6B).

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FIGURE 6.

Temporal loss in ICOS expression and IL-10 production in Foxp3⁺ Tregs coincides with T1D onset and progression. A, Shown are the levels of ICOS expression on Foxp3⁺ Tregs (left panel) and proportions of ICOS⁺Foxp3⁺ Tregs (right panel) in pLN and pancreas of 3- to 4-wk-old and 12-wk-old BDC2.5 mice as analyzed by FACS. B, The extent (left panel) and proportion (right panel) of proliferating (based on Ki-67 expression) ICOS⁺Foxp3⁺ Tregs in pancreas of 3- to 4-wk-old and 12-wk-old BDC2.5 mice are shown. C, NOD.TCRα^−/− recipient mice were transferred i.v. with CD4⁺ T cells (1 × 10⁶) isolated from pooled LN and spleen of 3- to 4-wk-old BDC2.5.Foxp3^GFP reporter mice, and T1D onset was monitored as in Fig. 3. Fourteen to twenty-one days posttransfer, Foxp3⁺ and Foxp3⁻ CD4⁺ T cells from NOD.TCRα^−/− recipient were restimulated ex vivo with PMA/ionomycin, and the frequency of IL-10–producing Foxp3⁺ Tregs, relative to ICOS expression, was assessed in pLN and pancreas of prediabetic and diabetic mice (top panel). A representative dot plot of IL-10 production in ICOS⁺CD4⁺ T cells in the pancreas of prediabetic and diabetic mice is shown (top panel). ICOS expression (MFI, left y axis: ▴, pLN; ▽, pancreas) relative to the frequency of IL-10⁺ ICOS⁺ Tregs (right y axis: ▪, pLN; □, pancreas) in prediabetic and diabetic mice (bottom panel). Results are representative of three independent experiments, (n = 7 mice/group). Results represent the means ± SD.

We and others have previously shown that T1D progression in NOD mice correlates with a drastic reduction of intrapancreatic Treg numbers (11, 13). We then wondered whether T1D onset could be attributed to a loss of ICOS expression on Foxp3⁺ Tregs. To this end, CD4⁺ T cells from BDC2.5.Foxp3^GFP mice were transferred into NOD.TCRα^−/− recipients, and the cellular frequency of ICOS⁺Foxp3⁺ Tregs in the pLN and pancreas was analyzed relative to T1D onset. Notably, the expression level of ICOS as well as the frequency of ICOS⁺Foxp3⁺ Tregs sustained a drastic reduction as recipient mice progressed from prediabetes to overt T1D (1060 ± 92.8 versus 362.3 ± 68.6, p ≤ 0.0001) (Fig. 6C). Thus, we show that T1D progression correlates with a loss of ICOS expression on Foxp3⁺ Tregs, particularly in the pancreas, indicating a loss of Treg suppression throughout T1D progression.

Herman et al. (21) showed that IL-10 and ICOS mRNA were highly expressed by pancreatic CD25⁺CD69⁻ Tregs relative to their draining LN counterparts. As pancreatic ICOS⁺ Foxp3⁺ Tregs possess the capacity to secrete high amounts of IL-10, we then assessed whether the loss in ICOS expression with T1D progression dampened the capacity of Tregs to produce IL-10 in our BDC2.5 transfer model (34). To this end, CD4⁺ T cells from pLN and pancreas of prediabetic and diabetic recipient mice were reactivated ex vivo, and the secretion of IL-10 was assessed by flow cytometry. Notably, the marked drop in the expression level of ICOS and frequency of ICOS-expressing Foxp3⁺ Tregs within the pancreas of diabetic mice correlated with a 3-fold decline in their capacity to produce IL-10 (21.5 ± 4.7 versus 5.8 ± 1.3, p ≤ 0.03) (Fig. 6C). Taken together, these data show that ICOS drives the differentiation of IL-10–producing Tregs particularly in the pancreas of prediabetic mice and that T1D progression correlates with a loss of Foxp3⁺ICOS⁺ Tregs within the pancreas.

IL-2 preferentially supports the fitness and survival of ICOS-expressing Foxp3⁺ Tregs

IL-2 is essential for the survival and function of Foxp3⁺ Tregs within islets, and a local deficiency in IL-2 was shown to render Tregs unfit and functionally defective, in turn provoking a Treg/Teff imbalance in situ (13). This is similar to earlier studies showing that T cells from prediabetic NOD mice become hypoproliferative and poor IL-2 producers at the onset of insulitis (3). Therefore, we examined whether the decline in the ICOS⁺ Treg pool, coincident with a lack of suppression of these cells and T1D progression, is associated with altered IL-2 levels. We first assessed IL-2Rα (CD25) expression in ICOS⁺ and ICOS⁻ Treg subsets in response to IL-2 in vitro. To this end, ICOS⁺ and ICOS⁻ Tregs from BDC2.5.Foxp3^GFP reporter mice were activated in vitro with APCs and mimetope in the presence or absence of IL-2. We observe that ICOS⁺ Tregs are more responsive to IL-2 compared with ICOS⁻ Tregs, as this resulted in a 28-fold increase in CD25 expression in ICOS⁺ Tregs and only a 10-fold increase in ICOS⁻ Tregs in response to IL-2 compared with nontreated conditions (Fig. 7A, left panel). To confirm that ICOS⁺ Tregs preferentially respond to IL-2, STAT5 phosphorylation (p-STAT5) was assayed as an indicator of activation of the IL-2 signaling pathway. Our results show that administration of IL-2 leads to a significantly higher proportion (83.3 ± 2.6% versus 44.0 ± 1.8%, p ≤ 0.002) and MFI (1443 ± 19.1 versus 779 ± 4.2, p ≤ 0.008) of p-STAT5–positive ICOS⁺ Tregs compared with ICOS⁻ Tregs (Fig. 7A, middle and right panels).

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FIGURE 7.

IL-2 is essential for ICOS expression in Foxp3⁺ Tregs and drives their fitness and survival. A, FACS-purified ICOS⁺ or ICOS⁻ Foxp3⁺ Tregs (1 × 10⁵) from pooled LN and spleen of 3- to 4-wk-old BDC2.5.Foxp3^GFP reporter mice were stimulated with BDC2.5 mimetope (40 ng/ml) and irradiated spleen cells (4 × 10⁵) in the presence or absence of IL-2 (5 μg/ml) for 36–72 h. The frequency of IL-2–responsive ICOS⁺ and ICOS⁻ Foxp3⁺ Tregs was assessed by measuring CD25 expression in ICOS⁺ and ICOS⁻ Treg subsets in unstimulated and IL-2–treated conditions demonstrated in histogram plots (left panel). The frequency of IL-2–responsive ICOS⁺ and ICOS⁻ Foxp3⁺ Tregs was assessed by measuring p-STAT5 by flow cytometry. A representative histogram plot (middle panel) and MFI values (right panel) for p-STAT5 are shown. MFI values are expressed as δ MFI of p-STAT5 in presence or absence of IL-2. B, The frequency of apoptotic cells (annexin V⁺/PI⁺) in ICOS⁺ and ICOS⁻ Tregs in the presence or absence of IL-2 was assessed by flow cytometry (left panel). The frequency of surviving ICOS⁺ and ICOS⁻ Tregs in the presence or absence of IL-2 (indicated by Bcl-2 expression) was assessed by flow cytometry. Representative histogram plots of Bcl-2 expression in ICOS⁺ and ICOS⁻ Tregs (middle panel) and MFI values (right panel) are shown. MFI values are expressed as δ MFI of Bcl-2 expression in the presence or absence of IL-2. Each experiment was repeated three times with 10–15 mice per experiment. Results represent the mean ± SD. C, The level of ICOS expression (MFI) (left and middle panels) on Foxp3⁺ Tregs and the frequency (right panel) of ICOS⁺Foxp3⁺ Tregs in pancreas of 3- to 4-wk-old and 12-wk-old BDC2.5 and BDC.Idd3 mice was analyzed by FACS. D, Three- to four-week-old female BDC2.5 mice were injected daily with IL-2 (25,000 IU) for 5 consecutive days. On day 7 after the initial injection, the proportion of Foxp3⁺ Tregs within CD4⁺ T cells (left panel), the proportion of CD25-expressing Foxp3⁺ Tregs (middle panel), and the proportion of ICOS⁺ within Foxp3⁺ Tregs (right panel) in pLN and pancreas was compared between PBS-treated and IL-2–treated BDC2.5 mice, as well as to BDC2.5Idd3 mice. Data are representative of at least three separate experiments (n = 3 mice/group). Results represent the mean ± SD.

IL-2–mediated STAT5 phosphorylation is essential for survival of Foxp3⁺ Tregs (37–39). As ICOS⁺ Tregs are more sensitive to differential IL-2 levels, we then examined whether they also differed in their survival potential. We observe that ICOS⁺ Tregs, compared with ICOS⁻ Tregs, are more susceptible to death by IL-2 withdrawal as indicated by the increased frequency of apoptotic cells in the absence of IL-2 compared with untreated cells (% annexin V⁺ PI⁺ cells: 90.3 ± 4.6 versus 62.3 ± 1.9, p ≤ 0.0003) (Fig. 7B, left panel). IL-2 treatment rescues ICOS⁺ Tregs from apoptosis, whereas ICOS⁻ Tregs remain insensitive to IL-2 (45.4 ± 1.8 versus 39.0 ± 0.9, p ≤ 0.0002) (Fig. 7B, left panel). The IL-2–driven survival of ICOS⁺ Tregs was also confirmed by the upregulation of Bcl-2 expression in ICOS⁺ Tregs (1447 ± 7.5 versus 657 ± 4.6, p ≤ 0.008) in response to IL-2 in contrast to ICOS⁻ Tregs whose Bcl-2 expression remained unchanged irrespective of IL-2 treatment (605 ± 13.2 versus 702 ± 6.8, NS) (Fig. 7B, middle and right panels). Overall, ICOS⁺ Tregs, in contrast to ICOS⁻ Tregs, are more dependent on IL-2 signals, which bolster CD25 expression and prevent apoptosis in ICOS⁺ Tregs. Notably, WT and ICOS^−/− BDC2.5 Tregs do not differ in their intrinsic IL-2 response after activation as measured by p-STAT5 levels (data not shown); rather, the ICOS⁺ Tregs, compared with ICOS⁻ Tregs, have different IL-2 requirements to maintain their functional fitness. Therefore, ICOS expression is not required for mediating IL-2 responses in ICOS⁺ Tregs, but rather delineates an IL-2–responsive Foxp3⁺ Treg subset in the pancreatic microenvironment.

Il2 protective allelic variants or prophylactic IL-2 therapy restores ICOS expression in Tregs in BDC2.5 mice

A local IL-2 deficiency in islets is one of the underlying mechanisms in the age-related waning of Treg functions and loss of self-tolerance in NOD mice (13, 14, 24). Consistently, we have previously shown that Tregs within the pancreatic lesion of WT BDC2.5 mice exhibited reduced expansion, survival, and function, defects readily corrected by prophylactic IL-2 therapy or Il2 allelic variation in NOD mice (13). Thus, we hypothesized that IL-2 promotes ICOS expression in vivo and sought to determine whether Il2 allelic variants of T1D-protected congenic BDC2.5Idd3 mice could restore ICOS expression on Tregs and the enhanced frequency of ICOS⁺Foxp3⁺ Tregs in situ. We observed an increase in the proportion of ICOS⁺Foxp3⁺ Tregs (86.4 ± 1.7 versus 68.6 ± 1.4, p ≤ 0.001) (Fig. 7C, right panel) and extent of ICOS expression (MFI) (282.3 ± 54.0 versus 144.3 ± 11.9, p ≤ 0.04) (Fig. 7C, left and middle panels) on Foxp3⁺ Tregs in the pancreas of 4-wk-old BDC2.5Idd3 mice relative to age-matched WT mice. Despite the substantial drop in the frequency of ICOS-expressing Foxp3⁺ Tregs by 12 wk of age in prediabetic BDC2.5Idd3 mice, the frequency was nonetheless significantly greater than that in WT mice (86.37 ± 1.7 versus 72.3 ± 0.4, p ≤ 0.005) (Fig. 7C, right panel). Moreover, the enhanced proportion of ICOS⁺Foxp3⁺ Tregs coincided with a 2-fold enhancement in the fraction of actively proliferating ICOS-expressing Foxp3⁺ Tregs in 4-wk-old BDC2.5Idd3 mice relative to WT mice (39.1 ± 5.7% versus 24.4 ± 4.5%, p ≤ 0.01) (data not shown). This significant difference is maintained at later time points with disease progression (22.3 ± 2.8% versus 14.7 ± 3.4%, p ≤ 0.04) (data not shown). Overall, Il2 allelic variation, known to increase IL-2 production in Teffs, also increases ICOS expression on Foxp3⁺ Tregs within the target organ, in turn, promoting their functions and T1D protection.

To confirm whether IL-2 deficiency is an underlying cause of the temporal loss of ICOS expression observed in Tregs within the pancreas (Fig. 5), a prophylactic IL-2 therapeutic regimen, known to promote T1D protection, was initiated in prediabetic BDC.2.5 mice. In stark contrast to draining pLN, we observed a significant increase in the proportion of Foxp3⁺ Tregs within the pancreatic infiltrate of mice receiving IL-2 relative to PBS controls (13.3 ± 3.1% versus 9.4 ± 1.9%, p ≤ 0.03) (Fig. 7D, left panel). The proportion of ICOS⁺Foxp3⁺ Tregs within the CD4⁺ T cell infiltrate of the pancreas was also enhanced in IL-2–treated BDC2.5 mice relative to PBS-treated controls (87.2 ± 4.3 versus 66.4 ± 3.8, p ≤ 0.0095), comparable with levels observed in BDC2.5Idd3 mice (87.2 ± 4.3 versus 84.25 ± 1.2) (Fig. 7D, right panel). IL-2 therapy also drives the expression of CD25 on Foxp3⁺ Tregs as the proportion of Foxp3⁺CD25⁺ICOS⁺ was enhanced to levels observed in BDC2.5Idd3 mice and in IL-2–treated mice versus PBS controls (8.8 ± 2.6% versus 5 ± 0.7%, p ≤ 0.05) (Fig. 7D, middle panel). In addition, our results show that the inability of ICOS^−/− Tregs to suppress T1D in our T cell transfer model could not be reversed by prophylactic administration of IL-2, a condition that only partially bolsters Foxp3⁺ Tregs of ICOS^−/− origin in contrast to WT Tregs (data not shown). Our data show that protective Il2 allelic variants or low-dose IL-2 treatment restored ICOS and CD25 expression in intrapancreatic Foxp3⁺ Tregs, both conditions promoting T1D protection (13, 14). Overall, this suggests that ICOS expression by Foxp3⁺ Tregs plays an important role in IL-2–mediated rescue of Foxp3⁺ Tregs and T1D protection. Moreover, our results are reminiscent of those of Grinberg-Bleyer et al. (24), who demonstrated that IL-2 therapy can even reverse established T1D in NOD mice via a local effect on pancreatic Tregs by upregulating the expression of genes involved in Treg function and survival such as Bcl-2, CD25, Foxp3, and ICOS.