Figure 5.

IL-4 was the downstream, not the inducer, of STAT6 phosphorylation in Treg-of-B (P) cells and responsible for Treg-of-B (P) cell viability maintenance. To determine whether IL-4 played the role in the generation of Treg-of-B (P) cells, CD4+CD25- T cells were cultured with anti-CD3, anti-CD28 antibodies and Peyer’s patch B cells in presence of DMSO, AS (50nM, STAT6 inhibitor), isotype IgG1 and anti-IL-4 (10 µg/ml) antibody. After B-T three-day cocultured, phosphorylated STAT6, LAG3, cell apoptosis and anti-apoptotic protein of Treg-of-B (P) were analyzed by flow cytometer. Supernatant of B-T coculture was collected at days 1 to 3 for IL-4 detection by ELISA. Gene expressions of anti-apoptotic and apoptotic protein were determined by real-time PCR. (A) phospho-STAT6 (left) expression and the suppressive ability of different treated groups of Treg-of-B (P) cell (right). (B) LAG3 expression of Treg-of-B (P) cells. (C) IL-4 production in B-T cell cocultured medium. Black bar: wild type group or DMSO group. White bar: STAT6KO group or AS group. (D) The apoptosis of wild type, IL-4 knockout (IL4KO) and STAT6 knockout (STAT6KO) Treg-of-B (P) cells. Apoptosis was evaluated by PI and Annexin V expression (early apoptosis: Annexin V+ PI-; late apoptosis: Annexin V+ PI+). Total apoptotic cells (Annexin V positive cells) were calculated for the bar graph. (E) The expressions of anti-apoptotic protein, Bcl2 and Bcl_XL, and apoptotic protein, Bax, were quantified by real-time PCR (upper) and FACS analysis (lower) in IL4KO and STAT6KO Treg-of-B cells, respectively. RQ stands for the relative quantification of the PCR signal of IL4KO group compared to WT group. Results are expressed as the mean ± SEM. Data are representative of three to four different experiments. *p < 0.05, **p < 0.001, compared with WT group. ***p < 0.005, compared with the responder T cell group. ^##p < 0.01, compared with STAT6KO group. ^###p < 0.005 compared with DMSO group.