Treatment with an anti-IL-17 mAb protected NOD mice against diabetes only when performed at late stage of disease development 27. Although learn more 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. 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 PS-341 order 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. 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 Ribonucleotide reductase 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.