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Keywords:

  • Autoimmunity;
  • Diabetes;
  • IL-17;
  • iNKT;
  • NOD

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Invariant natural killer T (iNKT) cells are a distinct lineage of innate-like T lymphocytes and converging studies in mouse models have demonstrated the protective role of iNKT cells in the development of type 1 diabetes. Recently, a new subset of iNKT cells, producing high levels of the pro-inflammatory cytokine IL-17, has been identified (iNKT17 cells). Since this cytokine has been implicated in several autoimmune diseases, we have analyzed iNKT17 cell frequency, absolute number and phenotypes in the pancreas and lymphoid organs in non-obese diabetic (NOD) mice. The role of iNKT17 cells in the development of diabetes was investigated using transfer experiments. NOD mice exhibit a higher frequency and absolute number of iNKT17 cells in the lymphoid organs as compared with C57BL/6 mice. iNKT17 cells infiltrate the pancreas of NOD mice where they express IL-17 mRNA. Contrary to the protective role of CD4+ iNKT cells, the CD4 iNKT cell population, which contains iNKT17 cells, enhances the incidence of diabetes. Treatment with a blocking anti-IL-17 antibody prevents the exacerbation of the disease. This study reveals that different iNKT cell subsets play distinct roles in the regulation of type 1 diabetes and iNKT17 cells, which are abundant in NOD mice, exacerbate diabetes development.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Invariant natural killer T (iNKT) cells represent a distinct lineage of T cells that co-express a highly conserved αβ T-cell receptor TCR along with typical surface receptors for natural killer cells. The invariant TCRα chain of iNKT cells is encoded by Vα24-Jα18 gene-segments in humans and Vα14-Jα18 gene-segments in mice. The TCRβ chain is also strongly biased, encoded by Vβ11 gene-segment in humans and Vβ8.2, Vβ7 and Vβ2 gene-segments in mice. These lymphocytes recognize both self and microbial glycolipid antigens presented by the non-classical class I molecule CD1d. iNKT cells are characterized by their capacity to produce rapidly large amounts of both Th1 (IFN-γ, TNF-α) and Th2 (IL-4, IL-13) cytokines, which enables them to exert beneficial, as well as deleterious, effects in a variety of inflammatory or autoimmune diseases 1, 2.

Converging studies in mouse models suggest that iNKT cells can prevent the development of type 1 diabetes 3. iNKT cells are reduced in number in diabetes-prone NOD mice 4, 5, and increasing the number of iNKT cells by adoptive transfer 6, 7 or via the introduction of a Vα14-Jα18 transgene, reduces significantly the progression of the disease 6. A similar protection was observed after specific iNKT cell stimulation with exogenous ligands, α-galactosylceramide (α-GalCer) and its analogues 8–11. Early reports suggested that iNKT cell protection was associated with the induction of a Th2 response to islet auto-antigens 8, 10–12. However, following studies using the transfer of anti-islet T cells showed that iNKT cells inhibit the differentiation of these auto-reactive T cells into effector cells during their priming in pancreatic lymph nodes (PLNs) 13, 14. This regulatory role of iNKT cells could be explained by their ability to promote the recruitment of tolerogenic DCs 14, 15.

It is now well established that iNKT cells can be divided into several subpopulations using various cell surface markers, these subsets exhibiting diverse functions. According to the expression of the CD4 molecule, human iNKT cells have been shown to express a Th1 or Th0 cytokine profile 16, 17. In the mouse, CD4 iNKT cells are more potent to promote tumor rejection 18. Recently, a new population of CD4 NK1.1 iNKT cells producing high levels of the pro-inflammatory cytokine IL-17 together with low IL-4 and IFN-γ levels in response to several iNKT cell ligands, has been identified and named iNKT17 cells 19. Consistent with their ability to produce IL-17 rapidly and independently of IL-6, iNKT17 cells, unlike naive T cells, were found to express constitutively IL-23R and Retinoic acid receptor – related orphan receptor γt (RORγt) 20–22.

Much of the focus on IL-17-secreting cells has been on their role in promoting organ-specific autoimmunity and chronic inflammatory conditions 23. In the past few years, results have suggested that it was not IL-12 and Th1 cells that are required for the induction of experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA) but rather IL-23 and Th17. EAE can be induced by the transfer of IL-17 producing autoreactive T cells and IL-17 deficient mice had reduced susceptibility to CIA and EAE. Unregulated Th17 responses or overwhelming IL-17 production from T cells and other sources is also associated with chronic inflammation in rheumatoid arthritis patients 23.

Recent studies suggest that IL-17 might also be involved in the development of type 1 diabetes. Transfer of in vitro polarized BDC2.5 Th17 cells into NOD SCID mice induced diabetes in recipient mice with similar rates of onset as transfer of Th1 cells 24–26. However, the exact role of IL-17 in the pathogenesis of type 1 diabetes remains unclear as the neutralization of IL-17 inhibited the disease transfer in one of the studies but not in two others. Treatment with an anti-IL-17 mAb protected NOD mice against diabetes only when performed at late stage of disease development 27. Although it is clear that Th17 cells play an important role in some autoimmune disease models, their precise role in diabetes remains to be elucidated. All these observations on the role of IL-17 and iNKT cells in autoimmune diseases led us to characterize iNKT17 cells in the NOD mouse and to investigate whether these cells play a pathogenic role in diabetes.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Enhanced iNKT17 cell population in NOD mice compared with C57BL/6 mice

To investigate the role of iNKT17 cells in type 1 diabetes, we have compared the frequency and absolute number of these cells in NOD and C57BL/6 mice. C57BL/6 mice were used as the control mice, since they develop neither diabetes nor other autoimmune pathologies. iNKT17 cells were analyzed in the thymus, spleen, inguinal LNs (ILNs) and PLNs. ILNs were used as control tissue since they are enriched in iNKT17 cells 28. IL-17 production by iNKT cells was detected after CD1d-αGalCer tetramer staining and stimulation with phorbolmyristyl acetate (PMA) and ionomycin (Fig. 1A). As previously shown in C57BL/6 mice, iNKT17 cells do not express the NK1.1 marker. These cells are also NK1.1 in NK1.1 congenic NOD mice used for this analysis (Fig. 1B). Interestingly, iNKT17 cell frequency was four to six-fold increased in NOD mice as compared with C57BL/6 mice (Fig. 1B and C). This difference was also observed in terms of absolute number (Fig. 1D). Of note, in PLNs of NOD mice, iNKT17 cells represent 13% of total iNKT cells compared with only 2% in C57BL/6 mice. The high frequency and absolute number in PLNs of NOD mice suggest that iNKT17 cells could play a role in the development of type 1 diabetes.

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Figure 1. The size of the iNKT17 cell population is increased in NOD, as compared with C57BL/6, mice. The indicated tissues were harvested from 6 to 10-wk-old female NOD and C57BL/6 mice and cell suspensions were prepared. (A–C) Intracellular IL-17 staining of iNKT cells was performed after stimulation with PMA/ionomycin in the presence of brefeldin A for 4 h. iNKT cells were detected using CD1d-αGalCer tetramers in combination with anti-TCRβ and their subsets using anti-IL-17 and anti-NK1.1 mAbs. (A) Representative FACS profiles of IL-17 production by iNKT cells from thymus, spleen, ILNs and PLNs. (B) Frequency of IL-17-producing cells among iNKT cells and (C) absolute number of IL-17-producing iNKT cells in the indicated lymphoid organs. (D) Enhanced expression of IL-17 lineage-associated genes by thymic iNKT subsets in NOD mice. Thymic stage 1 (CD44 NK1.1), stage 2 (CD44+ NK1.1 CD4 or CD4+) and stage 3 (CD44+ NK1.1+) iNKT cells were sorted from C57BL/6 and NOD mice. The levels of IL-22, IL-17A, RORγt and IL-23R mRNA were evaluated by quantitative PCR. Data were normalized to the gapdh housekeeping gene. All data are from four independent experiments, each performed with cells pooled from 4 to 10 mice and are either (A) representative or (B) an analysis of all experiments (mean+SD); *p≤0.05 between NOD and C57BL/6 mice.

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Enhanced expression of IL-17-associated genes by thymic iNKT cells from NOD mice

Previous studies have shown that unlike Th17 cells, iNKT17 cells are generated during thymic differentiation 19. iNKT cell maturation can be divided in three differentiation stages; stage 1 (CD44 NK1.1), stage 2 (CD44+ NK1.1 CD4 or CD4+) and stage 3 (CD44+ NK1.1+). We have analyzed the expression of genes usually associated with the iNKT17 lineage in thymic iNKT cells. Quantitative-PCR data show that il-17a gene is mainly transcribed in stage 2 CD4 iNKT cells and to a lesser extent in stage 1 and stage 2 CD4+ iNKT cells (Fig. 1D). In agreement with our results obtained by intracellular IL-17 staining, IL-17A mRNA level is increased (10-fold) in stage 2 CD4 iNKT cells from NOD as compared with C57BL/6 mice. Analysis of mRNA encoding RORγt, which is required for iNKT17 cell differentiation 21, revealed its high expression in the stage 2 CD4 iNKT cells and 3-fold increased in NOD mice. IL-23R is constitutively expressed by iNKT17 cells 20, and its expression is high in stage 2 CD4 iNKT cells, however, there is no significant difference between NOD and C57BL/6 mice. Interestingly, stage 2 CD4 iNKT cells also expressed IL-22 mRNA and this expression is 4-fold higher in cells from NOD mice. These data showing a higher transcription of il-17a, rorγt and il-22 genes in iNKT cells from NOD mice strengthen the differences in iNKT cells between this autoimmune strain and C57BL/6 mice.

iNKT17 cells infiltrate the pancreas of NOD mice

To determine whether iNKT17 cells infiltrate the pancreas of NOD mice, we have analyzed pancreatic infiltrates from NOD and Vα14 NOD transgenic mice that express iNKT cell characteristic TCRα chain and exhibit a10 fold increased frequency and number of iNKT cells in lymphoid tissues 6 as well as in the pancreas 29. iNKT17 cells represent 6% of all iNKT cells infiltrating the pancreas in NOD and Vα14 NOD mice (Fig. 2A). We next assessed whether this frequency varies at different stages of insulitis. At 6 wk of age NOD mice have a small infiltrate of hematopoietic cells, at 12-wk peri-insulitis is more abundant and at 20 wk many pancreatic islets are characterized by a destructive insulitis leading to diabetes onset 30. Indeed, we observed an increased frequency of pancreatic infiltrating hematopoietic (CD45+) cells with aging (Fig. 2B). Even though, iNKT17 cell frequency among iNKT cells as well as iNKT cell frequency among CD45+ cells infiltrating pancreas remained stable (Fig. 2B), the number of iNKT17 cells increased with the enhanced infiltration of pancreas, meaning that they could participate in the destruction of islet cells. CCR6 and CD103 integrin expression has been described on iNKT17 cells 28 and CCR6 has been involved in the recruitment of pathogenic Th17 cells in CIA 23. All iNKT17 cells from ILNs are CD103+ and the level of CD103 expression is higher in iNKT17 cells of NOD mice as compared with C57BL/6 mice (Supporting Information Fig. 1). iNKT17 cells from ILNs are mainly CCR6+, whereas in PLNs and spleen only a fraction of iNKT17 cells express CCR6 and CD103 (Supporting Information Fig. 1). The analysis of CCR6 and CD103 expression on pancreatic iNKT17 cells showed that, while 60% of iNKT17 cells expressed CD103 integrin, most of them were negative for CCR6 (Fig. 3C). These data suggest that iNKT17 cell recruitment in the pancreas is independent of CCR6, whereas CD103 could play a role in the retention of these cells.

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Figure 2. iNKT17 cells infiltrate the pancreas of NOD mice and express IL-17 mRNA in the absence of exogenous stimulation. (A–C) Cell suspensions from pancreatic islets of female NOD and Vα14 NOD mice were prepared and intracellular IL-17 staining of iNKT cells was performed as described in Fig. 1. iNKT cells were detected using CD1d-αGalCer tetramers in combination with anti-CD45, anti-TCRβ and their subsets using anti-IL-17, anti-CD4, anti-CD103 and anti-CCR6 mAbs. (A) Representative FACS profiles of IL-17 and CD4 expression by pancreatic iNKT cells from 12-wk-old NOD and Vα14 NOD mice. (B) Pancreatic islet cells of NOD and Vα14 NOD female mice were prepared. The frequencies of pancreatic infiltrating CD45+ cells (top panel), iNKT cells (middle panel) and iNKT17 cells (bottom panel) at the indicated ages and at diabetes onset (n.d=not done) are shown. (C) Representative FACS profiles of IL-17 and CD103 or CCR6 expression by pancreatic iNKT cells from three pooled Vα14 NOD mice at 12 wk of age. All data are from four independent experiments and each experiment was performed with cells pooled from 3 to 8 mice. The data are either (A, C) representative or (B) an analysis of all experiments (mean+SD). (D, E) The levels of IL-17A, IL-17F, IL-21, IL-22, RORγt, IL-23R and IFN-γmRNA in iNKT cells purified from the (D) pancreas, PLNs and ILNs from 12-wk-old Vα14 NOD mice and from (E) the pancreas of 12 wk-old Vα14 NOD and CD1dpLck Vα14 NOD mice were evaluated by quantitative PCR and the data were normalized to gapdh housekeeping gene. (D, E) Data are mean+SD of four independent experiments are shown, each performed with cells pooled from 3 to 6 mice; *p≤0.05 between the (D) different tissues or between (E) different mice.

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Figure 3. The CD4 iNKT cell population, which contains iNKT17 cells, enhances the incidence of diabetes. (A) Cytokine analysis by intracytoplasmic staining of the indicated iNKT cell subsets. Pooled splenocytes and mesenteric LN cells from individual Vα14 Cα−/− NOD donor mice were stimulated and stained as described in Fig. 1. The data correspond to the mean+SD of four independent mice. *p≤0.05 between CD4 NK1.1 and CD4 NK1.1+iNKT cells for IL-17 production. The iNKT cells were detected using CD1d-αGalCer tetramers in combination with anti-TCRβ, anti-CD4 and anti-NK1.1 mAbs. (B, C) iNKT cell subset purification and cell transfer into recipient mice. (B) Intracellular staining of iNKT cells from pooled splenocytes and mesenteric LN (MLN) cells of Vα14 Cα−/− NOD donor mice (left panel). CD4 or CD4+ iNKT cells were purified by cell-sorting and 1.5×106 cells were injected into 2-wk-old Cα−/− NOD mice (middle panel). Four wks later, the frequency of CD90.2+ CD4 or CD4+ iNKT cells were analyzed in the pancreas of Cα−/− NOD recipient mice (right panel). (C) The incidence of diabetes in recipient mice reconstituted with CD4 iNKT cells (triangles, n=15), CD4+ iNKT cells (asterisks, n=22) or PBS (circles, n=25), and then injected with 104 BDC2.5 T cells four wks later (day 0). (D) Similar transfer experiments to those described in (C) were performed with purified NK1.1 CD4 (inverted triangles, n=9), NK1.1+ CD4 (stars, n=6) iNKT cell subsets or PBS controls (n=11).

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Pancreatic iNKT17 cells express IL-17A mRNA in the absence of exogenous stimulation

To determine whether iNKT17 cells express IL-17A mRNA in the absence of exogenous stimulation such as PMA and ionomycin, iNKT cells were purified from the pancreas, PLNs and ILNs from Vα14 NOD mice. Expression of other genes usually associated with iNKT17 cells were also assessed by quantitative-PCR (Fig. 3D). IL-21 and IL-22 mRNA were barely detectable in the three organs analyzed. Interestingly, il-17a gene was expressed at much higher level in pancreatic iNKT cells than in iNKT cells from PLNs and ILNs (6- and 13-fold increased respectively). A similar trend was observed for il-17f gene. In contrast, rorγt and il-23r gene expression was not significantly different in iNKT cells from pancreas and ILNs. These data show that although iNKT17 cells are present in these three tissues, they are expressing IL-17A mRNA only in the pancreas.

Since previous studies have shown that iNKT17 cells can secrete IL-17 through TCR engagement 20, we investigated whether CD1d was required for IL-17A mRNA expression by iNKT17 cells in the pancreas (Fig. 3E). To address this question, we used Vα14 NOD mice expressing CD1d solely in the thymus (CD1dpLck Vα14 NOD mice) 31. RORγt, IL-23R and IFN-γmRNA expression was similar in pancreatic iNKT cells from both types of mice. However, IL-17A mRNA expression was significantly decreased (3-fold) in iNKT cells from mice lacking peripheral CD1d expression. Altogether, our data suggest that iNKT17 cells are activated locally in the pancreas in a CD1d-dependent manner.

CD4equation image iNKT cells containing iNKT17 cells enhance the incidence of diabetes

To evaluate the role of iNKT17 cells in type 1 diabetes, we reconstituted immunodeficient NOD mice with different iNKT cell subsets and analyzed the induction of diabetes after transfer of anti-islet BDC2.5 T cells 32. Since there is no specific antibody available to purify iNKT17 cells, we first determined the frequency of iNKT17 cells in different iNKT cell subpopulations divided according to CD4 and NK1.1 expression of donor cells. As shown in Fig. 3A and Supporting Information Fig. 2, iNKT17 cells are mainly present in the CD4 iNKT cell population and at a higher frequency among NK1.1 CD4 iNKT cells. Therefore, we enriched iNKT17 cells based on their lack of CD4 expression and they were found to represent around 23% of the injected CD4 iNKT cell population (Fig. 3B). Recipient NOD mice were reconstituted with CD4 or CD4+ iNKT cells, which were detected in pancreas before BDC2.5 T-cell transfer (Fig. 3B). In order to detect an eventual pathogenic role of iNKT17 cells, all recipient mice were injected with a low number of BDC2.5 T cells, which induces around 30% of diabetes in control mice devoid of iNKT cells (Fig. 3C). Interestingly, in the group of mice reconstituted with CD4 iNKT cells, the incidence of diabetes was significantly (p=0.036) increased and reached 70%. In contrast, reconstitution with CD4+ iNKT cells significantly (p=0.033) prevented the development of diabetes. Moreover, when CD4 iNKT cells were further divided according to NK1.1 expression, only NK1.1 CD4 iNKT cells containing the higher frequency of iNKT17 cells exacerbated diabetes (Fig. 3D).

Since diabetes induced by diabetogenic BDC2.5 T cells is associated with their production of IFN-γ 13, we have analyzed whether the presence of iNKT cell subsets have influenced their production of IFN-γ and IL-17. As previously described 13, in diabetic control mice devoid of iNKT cells, BDC2.5 T cells produced large amount of IFN-γ in both PLNs and pancreas (Fig. 4A). In diabetic mice reconstituted with CD4 iNKT cells, production of IFN-γ by BDC2.5 T cells was similar as in diabetic control mice and production of IL-17 remained low, less than 1%. While cytokine production by BDC2.5 T cells was similar in both groups of mice, the frequency of BDC2.5 T cells in the pancreas was increased in mice reconstituted with CD4 iNKT cells (44±3.1%) compared with control mice (32±1.4%). These data suggest that the enhanced incidence of diabetes in mice reconstituted with CD4 iNKT cells is due to the increased frequency of diabetogenic BDC2.5 T cells. Indeed, the frequency of pathogenic BDC2.5 T cells is probably a key parameter controlling the development of diabetes, since non-diabetic mice reconstituted with CD4+ iNKT cells contained only 0.9±0.2% and 12±6.4% of BDC2.5 T cells in their PLNs and pancreas, respectively. Our results highlight the pathogenic role of CD4 iNKT cells. To demonstrate the key role of IL-17, produced by iNKT17 cells, we treated mice with an anti-IL-17 antibody. Importantly, this treatment abolished the deleterious role of CD4 iNKT cells whereas it does not alter the incidence of diabetes induced by BDC2.5 T cells alone (Fig. 4B). Altogether, our results show that CD4 iNKT cells containing iNKT17 cells exacerbate the development of diabetes in an IL-17-dependent manner.

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Figure 4. Analysis of diabetogenic T cells and the role of IL-17 in the exacerbation of diabetes by CD4 iNKT cells. (A) 1.5×106 CD4 or CD4+ iNKT cells or PBS were transferred into Cα−/− NOD recipient mice, followed four wks later by injection of 104 BDC2.5 T cells. The frequency of CD45.2+ CD4+ BDC2.5 T cells (left panel) and their production of IFN-γ and IL-17 (right panel) in the PLNs and pancreas of each group of recipient mice were determined twelve days after BDC2.5 T-cell transfer after stimulation and staining as described in Fig. 1. Representative plots are shown and the values given in the plots represent the mean±SD of two independent experiments with two pooled mice. (B) The incidence of diabetes in the Cα−/− NOD recipient mice reconstituted with CD4 iNKT cells or PBS as a control and injected with 104 BDC2.5 T cells four wks later. In addition, the mice were treated with anti-IL-17 antibodies (filled triangle) or isotype control (empty triangle) on days 0, 2, 4 and 6 after BDC2.5 T-cell transfer.

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αGalCer treatment abolishes IL-17 production by iNKT cells

It has been well established that activation of iNKT cells by repeated αGalCer injections prevents the development of diabetes in NOD mice 8, 10, 15. Autoimmunity prevention correlated with the ability of αGalCer to induce iNKT cell anergy and to strongly suppress their IFN-γ production while IL-4 production was less inhibited 33. Interestingly, we have observed that αGalCer treatment suppressed not only IFN-γ by iNKT cells but also their IL-17 production whereas it does not inhibit IL-10 production (Fig. 5). This inhibition of IL-17 production could be critical in the protective role of αGalCer treatment.

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Figure 5. Repeated αGalCer injections inhibit IL-17 production by iNKT cells in NOD mice. Five wk-old NOD female mice were treated twice a wk for three wks with αGalCer (5 μg, i.p.). One wk after the last injection, the mice were sacrificed, the spleens harvested and the splenocytes from individual mice were cultured with or without 100 ng/mL of αGalCer for three days. IL-4, IL-10, IL-17 and IFN-γ levels were measured in the supernatants by ELISA as previously described 6, 19. Data correspond to the mean+SD of four individual mice; similar data were obtained in four independent experiments. *p≤0.05 between mice pretreated, or not, by αGalCer.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Our study reveals that NOD mice exhibit a high frequency of iNKT17 cells, which produce IL-17 in the pancreas and can exacerbate diabetes development upon cell transfer. This study suggests that IL-17 can participate in the pathology of type 1 diabetes. The role of IL-17 in autoimmune diabetes was first suggested by the low IL-17 production observed in NOD mice protected against the disease after treatment with a modified self-peptide 25. More recent studies showed that IL-17 neutralization with specific antibodies prevents the development of diabetes in NOD mice 27. Different immune cell populations can secrete IL-17 34. The role of Th17 cells in diabetes remains unclear. Indeed the induction of the disease in NOD SCID mice after transfer of in vitro polarized Th17 anti-islet T cells was abolished by anti-IL-17 treatment in one study but not in two others 25, 26. It has been reported that IL-17-producing γδT cells do not exacerbate diabetes upon co-transfer into NOD/SCID mice 35.

iNKT17 cells represent a new subset of IL-17-producing cells 19 and we observed an increased frequency of this cell population in NOD mice as compared with non-autoimmune C57BL/6 mice. iNKT17 cells from NOD and C57BL/6 mice exhibit a similar phenotype, mainly CD4 and NK1.1. iNKT17 cells are generated in the thymus where they constitutively express IL-17 mRNA 21, 22. The analysis of thymic iNKT cells showed higher frequency and absolute number of iNKT17 cells in NOD mice compared with C57BL/6 mice. Furthermore the analysis of the thymic stage 2 CD4 iNKT cell subset (containing iNKT17 cells) showed an enhanced expression of RORγt and IL-23R mRNA, two key molecules controlling IL-17 lineage 21. Thus, our data suggest that the high frequency of iNKT17 cells in the peripheral tissues is subsequent to an elevated frequency of iNKT17 cells in the thymus of NOD mice, which could be due to an elevated expression of RORγt in thymic iNKT cells upon their IL-17 lineage commitment.

Not only are iNKT17 cells present at high frequency in NOD mice but more importantly, they infiltrate pancreatic islets of NOD mice. NOD pancreatic islets express the adhesion molecule E-cadherin, which interacts with the integrin CD103 36. Interestingly, 60% of pancreatic iNKT17 cells expressed CD103 integrin and retention of iNKT17 cells in the pancreas could be due to CD103/E-cadherin interactions as previously described for diabetogenic CD8 T cells in the context of islet allografts 37. Moreover, CD103 can act as a co-activation molecule in human T lymphocytes 38 and could play a similar role in the activation of iNKT17 cells in the pancreas. While CCR6 is involved in the recruitment of Th17 cells in the target tissue in autoimmune CIA 39, the recruitment of iNKT17 cells in the pancreas is probably independent of CCR6 since most of them do not express this molecule. Alternatively, lack of expression of CCR6 might be due to downregulation upon entry into inflamed pancreas. Even though it has been suggested that iNKT17 cells are characterized by CCR6 and CD103 expression, the expression of these molecules by iNKT17 cells varies depending on tissues.

Since IL-17 protein is not detectable in absence of exogenous activation 19, 20, we analyzed IL-17 mRNA and other mRNAs associated with the IL-17 response. Importantly, IL-17 mRNA level was much higher in iNKT cells from the pancreatic islets than from PLNs and ILNs. No such difference in the mRNA level was observed for RORγt and IL-23R between these three tissues. Flow cytometry data showed that iNKT17 cells represent respectively 40% of iNKT cells in ILNs, 12% in PLNs and 6% in pancreas. The discrepancy between the frequency of iNKT17 cells in these three tissues and the spontaneous level of IL-17 mRNA suggests that pancreatic iNKT17 cells are locally activated in this tissue. Interestingly, IL-17, but not IFN-γ, mRNA expression by pancreatic iNKT cells was strongly decreased in mice lacking peripheral CD1d expression, demonstrating that local iNKT17 cell activation involves CD1d recognition. The residual expression of IL-17 mRNA in the absence of peripheral CD1d expression suggests that other local factors, such as IL-23 or IL-1β, could participate in the activation of iNKT17 cells 40. IL-1β is an interesting candidate since it is present in inflamed pancreatic islets 41.

Transfer experiments of iNKT cell subsets reveal the pathogenic role of CD4 iNKT cells containing the iNKT17 cell population in the development of diabetes. Reconstitution of immunodeficient NOD mice with CD4 iNKT cells enhanced the incidence of diabetes after injection of a low dose of BDC2.5 T cells. Similar exacerbation of diabetes incidence was observed after reconstitution with the NK1.1 CD4 iNKT cell population, which exhibits a high frequency of iNKT17 cells. However, due to cell number limitations most of our experiments were performed with the whole CD4 iNKT cell population. Treatment with anti-IL-17 antibodies abolished the pathogenic role of CD4 iNKT cells suggesting that iNKT17 cells are the critical players in the exacerbation of diabetes, however, we cannot rule out that other cell types producing IL-17 are also participating. Unfortunately, we could not directly demonstrate that only iNKT17 cells were involved in the deleterious effect of CD4 iNKT cells since there is presently no specific surface marker to purify this cell population. IFN-γ is also produced by CD4 iNKT cells and this cytokine could also participate in the exacerbation of diabetes; however, no exacerbation was observed after reconstitution with NK1.1+ CD4 iNKT cells producing high amounts of IFN-γ but low levels of IL-17. Of note, CD4 iNKT cells alone do not induce diabetes after transfer into immunodeficient NOD mice (data not shown). Therefore, we can propose that iNKT17 cells enhanced diabetes incidence through different mechanisms. In vitro data have shown that IL-17 synergizes with other cytokines such as IFN-γ and IL-1α/β to induce iNOS expression and subsequent NO production in insulinoma cells or in pancreatic islets of NOD mice 42. Similarly in the pancreas, IL-17 produced by iNKT cells could synergize with IFN-γ secreted by BDC2.5 T cells to induce high expression of NO in β-cells resulting in their destruction. A deleterious loop could take place since β-cell death induced by NO would promote self-antigen presentation by DCs to BDC2.5 T cells. This mechanism could explain the higher frequency of BDC2.5 T cells observed in the PLNs and the pancreas of mice transferred with CD4 iNKT cells as compared with mice devoid of iNKT cells. Furthermore, it has been shown that IL-17A and IL-17F can induce CXCL10 chemokine expression in lung epithelial cells 43, 44. Production of CXCL10 by pancreatic β-cells could contribute to the recruitment of auto reactive T cells expressing the CXCR3 chemokine receptor as previously shown in several mouse models of type 1 diabetes (T10) 45, 46. Thus, iNKT17 cells might not be involved in the initiation of the insulitis but rather could participate in the exacerbation of -β-cell death and diabetes onset.

Our data reveal a functional dichotomy between CD4+ and CD4 iNKT cell subsets in the control of diabetes development. While CD4 iNKT cells exacerbate the incidence of diabetes, CD4+ iNKT cells strongly protect mice against diabetes induced by BDC2.5 T cells. Our transfer experiments demonstrate the protective role of CD4+ iNKT cells as it was previously suggested in NOD mice deficient for CD38 47. iNKT cells represent a heterogeneous population, each subset of iNKT cells exhibiting different functions, either deleterious or beneficial toward diabetes development. Protection by iNKT cells is probably not only due to their total frequency but also to the ratio between the different iNKT cell subsets. This hypothesis is a possible explanation for the controversial role of iNKT cells in diabetic patients. In contrast to studies in NOD mice, some authors failed to detect differences in iNKT cell frequencies and IL-4 production between diabetic patients and healthy subjects 48. Autoimmune diabetes is generally considered a Th1-type pathology, but recent reports have suggested that IL-17-producing cells are enhanced in diabetic patients and allegedly contribute to disease severity 49. We have recently reported that human iNKT cells produce IL-17 under pro-inflammatory conditions 50. IL-17-producing cells in T1D patients 49 express CCR6 similarly to IL-17-producing human iNKT cells 50. Therefore, our data prompt further analysis of iNKT cell subpopulations in patients with a peculiar emphasis on determining the cytokine profile not only of circulating iNKT cells, but more relevantly of iNKT cells from tissues such as PLNs and pancreas.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Mice

C57BL/6J, NOD, Cα−/− NK1.1 NOD, BDC2.5 Cα−/− NOD, Vα14 NOD, CD1dpLck Vα14 NOD, Vα14 Cα−/− NOD mice have already been described 6, 13, 31. NK1.1 Vα14 Cα−/− NOD were generated for iNKT cell subset transfer experiments. NK1.1 NOD females were used for flow cytometry analysis of Fig. 151. Females were used between 6 and 20 wk of age. All experimental protocols were approved by the local ethic committee on animal experimentation.

Flow cytometry

CD1d-αGalCer tetramer staining was performed as previously described 52. Then cells were stained at 4°C in PBS containing 5% FCS and 0.1% NaN3. FcγR were blocked with 2.4G2 mAb. Surface staining was performed with anti-CD44 (clone IM7), anti-NK1.1 (clone PK136), anti-TCRβ (clone H57-597), anti-CD4 (clone RM4-5), anti-CD45 (clone 30F11), anti-CD90.2 (clone 30H12), anti-CD45.2 (clone 104), anti-CD103 (clone 2E7) (BD Pharmingen) and anti-CCR6 (clone 140706 – R&D). For intracellular staining, cells were stimulated for 4 h at 37°C with 10 ng/mL of PMA, 1 μg/mL of ionomycin in the presence of 10 μg/mL of brefeldin A (all from Sigma). Then cells were surface stained, fixed, permeabilized using a commercial kit (BD Pharmingen) and stained with anti-IL-17 (clone TC11-10H10), anti-IFNγ (clone XMG1.2), anti-IL-4 (clone 11B11) and anti-IL-10 (clone JES5-16E3) (BD Pharmingen). Cells were analyzed on a FACSAria (BD).

Preparation of iNKT cells and quantitative PCR

Thymic cells were expanded 5 days in the presence of 20 ng/mL of IL-7 (R&D). iNKT cells were sorted as TCRβ+ CD1d-αGalCer tetramer+ cells and according to various markers CD44, NK1.1 and CD4 expression, using FACSAria. Ten thousand iNKT cells were collected in RLT buffer with 1% of β-mercaptoethanol. mRNA was isolated using RNeasy Mini Kit (Qiagen) and reverse transcripted with Superscript III (Invitrogen). Quantitative-PCR was realized with SYBR Green (Roche) and analyzed with LightCycler 480 (Roche).

Preparation of pancreatic islet cells

Pancreatic islet cells were prepared as previously described 53. Pancreata were perfused with a solution containing collagenase P (Roche), dissected free from surrounding tissues and digested at 37°C for 10 min. Islets were then purified on a Ficoll gradient and disrupted by adding cell dissociation buffer (GIBCO) for 10 min at 37°C.

Cell purification for transfer experiments

iNKT cells from spleen and mesenteric LNs of CD45.1+/+ CD90.1+/+ Vα14 Cα−/− NOD mice were enriched by negative selection and then sorted as CD4 or CD4+ CD1d-αGalCer tetramer+ cells. Sorted cell purity was >96%. CD62L+ BDC2.5 T cells were isolated from CD45.2+/+ CD90.1+/+ BDC2.5 Cα−/− NOD mice. Splenocytes were enriched in T cells by negative selection and CD62L+ cells were positively selected using biotinylated anti-CD62L mAb and Streptavidin microbeads (Miltenyi Biotec). CD62L+ BDC2.5 T-cell purity was >92%. Similar procedures were used for the reconstitution with NK1.1 or NK1.1+ CD4 iNKT cells. Donor cells were obtained from NK1.1 Vα14 Cα−/− NOD mice.

Adoptive transfer experiments and diabetes diagnosis

At 2 wks of age, CD45.1+/+ CD90.1+/+−/− NOD mice were reconstituted i.v with 1.5×106 CD4 or CD4+ iNKT cells from CD45.1+/+ CD90.2+/+ Vα14 Cα−/− mice. Mice were injected i.p with PK136 mAb (50 μg/mouse of on days 15, 17, 26 and with 100 μg/mouse on day 32). At 6 wks of age, recipient mice were injected i.v with 104 naïve CD62L+ BDC2.5 T cells from CD45.2+/+ CD90.1+/+ BDC2.5 Cα−/− mice. Diabetes analysis was also performed in mice reconstituted with NK1.1 or NK1.1+ CD4 iNKT cells. In some experiments mice were injected i.p with 200 μg of blocking anti-mouse IL-17 Ab (CA028_00511) or isotype control (101.4) on days 0, 2, 4, 6 and 8 after BDC2.5 T cell transfer (day 0). Reagents were provided by UCB Celltech. Overt diabetes was defined by two consecutive positive glucosuria tests and glycemia >200 mg/dL.

Statistical analysis

Statistical analyses were performed with the nonparametric Mann–Whitney U test. The log-rank test was used for the comparison of diabetes incidence.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank UCB Celltech for the generous gift of anti-IL-17 and isotype control reagents, L. Breton and the staff of the mouse facility for help in animal care and L. Ghazarian and J. Diana for critical reading of the manuscript. This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale and the Centre National pour la Recherche Scientifique, grant from ANR-09-GENO-023 to A. L.. Anne-Sophie Gautron and Yannick Simoni were supported by doctoral fellowships from the Ministère de l'Education Nationale et de la Recherche et Technique and from Région Île-de-France.

Conflict of interest: The authors declare no commercial or financial conflict of interest.

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  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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