Fig. 3. Factors required for B cell–elicited polarization of inflammatory TH subsets.
(A) Neutralizing indicated cytokines separately or together hardly affected B cell–mediated TH subset differentiation on day 7. (B) Effects of total B cells or conditioned medium from the culture of total B cells (B-CM) or of total B cells plus T cells (BT-CM) on TH subset differentiation on day 7. (C) Analysis of TH subsets after cultured for 7 days in medium or with total B cells directly or in a transwell chamber. (D and E) FACS analysis of ICOSL in indicated cells from blood of healthy donors (D) or in total B cells left untreated or stimulated with αIgM, Pam3CysSK4, LPS, or CpG for 18 hours (E). (F) FACS analysis of ICOS in indicated cells left untreated or stimulated with CD3–cross-linking antibodies for 24 hours. (G and H) Blocking (G) or silencing (H) ICOS in T cells abrogated B cell–mediated TH subset differentiation on day 7. (I and J) FACS analysis of CD40 in indicated cells from blood of healthy donors (I) or in total B cells left untreated or stimulated with αIgM, Pam3CysSK4, LPS, or CpG for 18 hours (J). (K) FACS analysis of ICOSL in indicated cells cultured for 18 hours in medium or with TCR-primed TH cells in the presence or absence of CD40-neutralizing antibody. (L) Blocking CD40 abrogated B cell–mediated TH subset differentiation on day 7. (M) Analysis of STAT activation in TH cells cultured alone or stimulated with total B cells, ICOS agonist, or conditioned medium from LPS-stimulated monocytes for 2 days (n = 3). Data are presented as means ± SEM (A to L). *P < 0.05, **P < 0.01, ***P < 0.001 [one-way ANOVA test for (A) to (G) and (I) to (L); two-way ANOVA test for (H)].