Figure 5
YY1lo NKT cells produce IL-10 following primary activation. (a–c) C57BL/6J (WT) mice were injected I.V. with α-GalCer (10ug) suspended in saline. 90 minutes after injection, spleens were harvested and NKT cells (MHCII−, CD3+ CD1dtet+ CD24−) were enriched for by automacs. A cell surface capture assay was used to determine IL-10 production by YY1lo PLZFlo, YY1hi PLZFlo, and YY1hi PLZFhi NKT cells. (a) Representative histogram depicting IL-10 production by each subset. (b) Cumulative data from 5 mice, depicting the % of IL-10+ cells. (c–f) NKT cells were isolated from the spleens (c,d) or thymuses (e,f) of WT mice and activated in vitro for 3 hours by culture with PMA (50 ng/mL) and Ionomycin (500n/mL). NKT cell IL-10 production was measured by a cell surface capture assay. Representative histograms (c,e) and aggregate data (d, f), compare the production of IL-10 between YY1lo PLZFlo, YY1hi PLZFlo, and YY1hi PLZFhi NKT cells following in vitro activation. (g–h) C57Bl/6 (WT) mice were IV injected with 10 ug of α-GalCer or vehicle. Mice were sacrificed 72 hours or four weeks after injection. Spleen NKT cells (MHCII−, CD3+ CD1dtet+ CD24−) YY1 and PLZF expression were measured by FACs. (g) Representative FACS plots and (h) cumulative data. (i) C57Bl/6 (WT) mice were IV injected with 10 ug of α-GalCer or vehicle. 4 weeks later, NKT cells were enriched from spleens, activated in vitro with PMA/Ion, and IL-10 production was measured using a cell surface capture assay. Graph shows YY1 MFI and whether or not the cells produced IL-10. Data in histograms are representative of at least 3 independent experiments representing 3 or more biological replicates. For (b) N = 5, (d) N = 3, (f,h,i) N = 4. The horizontal lines indicate the mean (±s.e.m.). *P < 0.05determined by One-Way Anova (b,d,f,h,i).
