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. 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 click here 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 find more 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 these 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. 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.