(a) Naïve T (CD4+ CD62L+ CXCR5− PD-1−), effector T (CD4+ CD62L− CXCR5− PD-1−), and TFH (CD4+ CD62L− CXCR5+ PD-1+) cells were sorted from spleens of mice immunized with NP-CGG in alum 7 days earlier and then stimulated with phorbol myristate acetate (PMA) and ionomycin for 2 hr. CD40L expression on each cell subset was analyzed by FCM (left) and represented as geometric mean fluorescence intensity (gMFI, right) (mean + s.d. of triplicates). (b) Cd40lg+/+ or Cd40lg−/− mice were transferred with splenic B cells from B1-8 ki mice, and immunized with NP-CGG in alum. Six weeks later, the frequency of donor-derived (CD45.1+) NP+ CD19+ B cells (representative data on the left) and the number of the donor-derived Bmem cells (CD19+ CD45.1+ NP+ CD38+; plotted on the right; n = 7) in each spleen were analyzed by FCM. (c, d) B6 mice were transferred with splenic B cells from B1-8 ki mice, immunized with NP-CGG in alum, and injected subcutaneously (s.c.) with an inhibitory CD40L (MR-1: 1 mg/kg) mAb (αCD40L) or an isotype-matched control (ctrl IgG) Ab every day from day −1 to day 5 after immunization. Ten days or 6 weeks after immunization, splenocytes of the recipient mice were analyzed by FCM. (c) Representative data (day 10) of the analysis showing the gating strategy. (d) The frequency (%) of CD80hi and CD80lo cells in the donor-derived, class-switched Bmem cells (CD45.1+ NP+ CD19+ CD38+ IgM−), and their absolute numbers (#) at 10 days (top, n = 9) and 6 weeks (bottom, n = 8) after immunization are plotted. (e, f) B6 mice transferred with B1-8 ki B cells and immunized as in (c, d) were injected intraperitoneally (i.p.) with PBS or a stimulatory CD40 mAb (αCD40) (FGK4.5: 250 μg) at 8 days after immunization. Ten days after immunization, splenocytes from the recipient mice were analyzed by FCM. (e) Representative data of the analysis showing the gating strategy. (f) The frequency (%) and absolute numbers (#) of CD80hi and CD80lo cells in the donor-derived, IgG1+ Bmem cells (CD45.1+ NP+ CD138– CD19+ IgG1+ CD38+), and the numbers of the donor-derived GC B cells (CD45.1+ NP+ CD19+ IgG1+ CD38−) or of plasmablasts (CD45.1+ NP+ CD138+) in splenocytes are plotted (n = 5). (g–i) B6 mice, co-transferred on day −1 with B1-8 ki B cells (1 × 105) and OT-II T cells (1 × 105) that had been transduced with control (shCtrl) or shCd40lg retroviral vectors on the previous day, were immunized with NP-OVA in alum. Ten days after immunization, spleen cells from the recipient mice were analyzed by FCM. (g) Outline of the experimental protocol. (h) Representative data showing the gating strategy. (i) The frequencies of CD80hi and CD80lo cells among the donor-derived, class-switched Bmem cells, defined as in (d) (n = 8). The mean of the values in each group is indicated by a horizontal bar (b, d, f, i). n.s., not significant (p>0.05); *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; unpaired Student’s t test (b, d, f, i). All data are representative of two independent experiments except (b) and (i), where data from two independent experiments are combined.