Fig. 2

TRAPs induce IL-6/IL-10/IL-21 production of CD4⁺ T cells via TLR2-MyD88 pathway. a Confocal microscopy analysis of CFSE-labeled TRAPs (3 μg/ml) and mouse splenic CD4⁺ T cells (stained with anti-CD4-PE) after 24 h of co-culture. Scale bar: 5 μm. b Flow cytometric determination of the proportion of CFSE⁺ CD4⁺ T cells after incubated with CFSE-labeled TRAPs (0, 1, 3, or 10 μg/ml) in the presence of anti-CD3 and anti-CD28 for 24 h. c ELISA of IL-6, IL-10, and IL-21 secretion by WT, Tlr2^−/−, Tlr4^−/− or Myd88^−/− CD4⁺ T cells treated with TRAPs (3 μg/ml) or control media (CM) in the presence of anti-CD3 and anti-CD28 for 72 h. d Purified CD4⁺ T cells were co-cultured with CFSE-labeled TRAPs (3 μg/ml) for 24 h, and then stained for TLR2 and analyzed by confocal microscopy. e, f Flow cytometric and statistical analyses of the percentage of IL-10⁺ CD4⁺ T cells (e) or IL-21⁺ CD4⁺ T cells (f) in the tumor tissues of WT or Tlr2^−/− C57BL/6 mice (n = 6) 21 days after s.c. inoculation of B16F10 cells. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, by 2-tailed unpaired t-test or Mann-Whitney U test