Fig. 6.

T_TRAP enhance Breg differentiation and function via IL-6, IL-10, and IL-21. a Flow cytometric assessment of IL-10 expression by splenic B cells after 3 d of co-culture with 3 μg/ml TRAPs or 3 μg/ml TRAPs and an equal number of CD4⁺ T cells. b T_TRAP were adoptively (i.v.) transferred into C57BL/6 mice (n = 3 per group) every other day for 3 times. The frequency and number of splenic IL-10⁺ Bregs 7 days after the last transfer of T_TRAP was determined by flow cytometry. c SN/T_TRAP was pretreated with an anti-IL-6, −IL-10 or -IL-21 neutralizing antibody and co-cultured with splenic B cells and TRAPs for 72 h. The numbers of IL-10⁺ Bregs and IL-10 secretion were determined by flow cytometry and ELISA, respectively. d The supernatants from B cells stimulated with 3 μg/ml TRAPs and SN/T_TRAP (SN/B_{TRAP + SN/TTRAP}) were untreated or pre-treated with an anti-IL-10 neutralizing antibody and then used to culture anti-CD3/28-activated CD4⁺ or CD8⁺ T cells for 3 days. The percentage of IFN-γ⁺ T cells was determined by flow cytometry. e C57BL/6 mice were i.v. injected with OT-I splenocytes and vaccinated s.c. with DC_OVA on day 1, 4 and 7, following adoptive transfer of B cells induced by TRAPs (B_TRAP) or by TRAPs and SN/T_TRAP (B_{TRAP + SN/TTRAP}) on day 2, 5 and 8. On day 15, the frequency and the number of Vβ5.1⁺CD8⁺ T cells in spleens were analyzed by flow cytometry. f C57BL/6 mice were vaccinated with DC_OVA and transferred with B_TRAP or B_{TRAP + SN/TTRAP}. At day 15, the frequencies of splenic IFN-γ⁺ CD4⁺ and CD8⁺ T cells were determined after ex vivo re-stimulation. g, h B16F10 tumor cells were mixed with B_TRAP, B_{TRAP + SN/CD4+ T}, or B_{TRAP + SN/TTRAP} and injected s.c. into C57BL/6 mice (n = 4 per group). The growth of tumor was monitored (g). B16F10 tumor cells were injected i.v. into C57BL/6 mice (n = 4 per group) to establish a lung metastasis model. Subsequently, the above-prepared B cells were i.v. transferred every other day for 3 times. Three weeks later, tumor nodules in the lungs were examined (h). Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01, ***, P < 0.001; ns, not significant