Fig 1: NKT17 trans-differentiates to NKT10. (A, B) 2x106 splenocytes from IL-10GFP C57BL/6 mice was stimulated with αGalCer (100 nM) and murine IL-2 (20 ng/ml), and the production of IL-10GFP from CD4+ or CD4- iNKT cells were assessed daily. Upon antigenic stimulation, iNKT cells were not detectable for the first 3 days, likely due to the downregulation of iNK-TCR and resurged from day 4. On average, 23.4% of iNKT cells expressed IL-10 on day 4, and expanded to 29.1% on day 5 and 31.3% on day 6. The percentage of IL-10+ iNKT cells was similar between CD4+ and CD4- iNKT cells, especially on days 4 and 5, but IL-10+ CD4+ iNKT cells were significantly higher than IL-10+ CD4- iNKT cells on day 6. (C, D) IL-17A Fate+ mice (L-17ACre.Rosa26STOPf/fYFPxIL-17AKatushkaIL-10eGFPFoxp3RFP) were used to track NKT17, exNKT17 with a prior IL17 production, NKT10/NKT17 hybrid, and NKT10. Splenocytes from IL17A Fate+ reporter mice were stimulated with αGalCer and IL-2 under Treg-promoting conditions with additional TGFβ1 (5 ng/ml) or Tr1-promoting conditions with additional IL-6 (25 ng/ml), IL-23 (25 ng/ml), and TGFβ (5 ng/ml) for 4 days. The presence of IL-17AKatushka (NKT17), IL-10eGFP (NKT10), and IL-17YFP (exNKT17) in iNKT cells was assessed. In vitro antigenic stimulation of iNKT cells from IL-17A Fate+ reporter mice showed a significant expansion of IL-10eGFP iNKT cells under both Treg and Tr1-promoting conditions. However, the expansion of IL-17AKatushka iNK T cells, IL-10eGFP IL-17AKatushka iNKT cells, and IL-10eGFP IL17AYFP iNKT cells were observed only under Tr1-promoting condition. Results were one of two independent experiments. Student’s t-test was used to compare the differences between the two groups. The error bars represent mean ± standard deviation. * notes p<0.05.