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. Author manuscript; available in PMC: 2017 Sep 1.
Published in final edited form as: Biochim Biophys Acta. 2017 Jun 3;1863(9):2149–2157. doi: 10.1016/j.bbadis.2017.06.001

Fig. 5.

Fig. 5

Effects of combination of IL-21, anti-Tim1 and Cd40L treatment on CD1dhighCD5+ B cells frequency, IL-10 protein expression, and IL-10, RANKL and other cytokines mRNA levels. Splenocyte B cells were separated from C57/BL6J mice and cultured under multiple conditions including untreated control, CD40L (1 μg/ml) alone, CD40L (1 μg/ml) + IL-21 (100 ng/ml) +anti-Tim1 (5 μg/ml) and CD40L (1 μg/ml) +IL-21 (1 μg/ml)+ anti-Tim1 (5 μg/ml) for 48 h, and CD40L (1 μg/ml) 48 h with IL-21 (100 ng/ml) + anti-Tim1 (5 μg/ml) or IL-21 (1 μg/ml) + anti-Tim1 (5 μg/ml) for 24 h. CD1dhighCD5+ B cells were detected using flow cytometry in control and CD40L + IL-21+ anti-Tim1 combination treatment groups (A) (Xaxis: CD5 PE staining; Y-axis: CD1d APC staining). The percentage (B) and quantity (C) of CD1dhighCD5+ B cells were quantified and analyzed by FlowJo software (mean ± SD, n =4 mice per group, compared with control group no significant differences). IL-10 mRNA levels in total cell lysis were determined by real-time PCR in control and CD40L +IL-21 +anti-Tim1 combination treatment groups (D) (mean ± SD, n = 4 mice per group, compared with control group, *p < 0.05, **p < 0.01). Medium supernatants were collected and secreted IL-10 protein levels were measured by ELISA in control and CD40L + IL-21 +anti-Tim1 combination treatment groups (E) (mean ± SD, n = 4 mice per group, compared with control group, *p < 0.05, **p < 0.01). Splenocyte B cells were separated from C57/BL6J mice and cultured in two groups including untreated control and CD40L (1 μg/ml) +IL-21 (1 μg/ml) + anti-Tim1 (5 μg/ml) for 48 h. RANKL (F), TNFα (G) and ICAM-1 (H) mRNA levels in total cell lysis were determined by real-time PCR in control and CD40L + IL-21+ anti-Tim1 combination treatment group (mean ± SD, n = 4 mice per group, compared with control group, *p < 0.05). CD1dlowCD5−, CD1dhighCD5−, CD1dhighCD5+, CD1dlowCD5+ B cell subsets were sorted from control and CD40L +IL-21 +anti-Tim1 combination treatment groups respectively. IL-10 mRNA levels of each subsets were determined by real-time PCR in control and CD40L + IL-21+ anti-Tim1 combination treatment group (I) (mean ± SD, n =4 mice per group, compared with control group, *p < 0.05).