Figure 6.

Tfh Provide Help through IL-21 and CD40 Ligation

(A) IL-21R expression by the secondary effectors (4 days after challenge, at day 46) generated in the two experimental models of Tfh depletion shown in Figure 5 (see also Figures S4A and S4C). The results are expressed as corrected geometric MFI, i.e., [MFI^OT-1 – MFI^non-OT-1]. Representative plot profiles are also shown.

(B and C) (B) IL-21 expression by Tfh and non-Tfh CD4⁺ T cells, at day 4 post-infection (p.i.). (C) CXCR5⁺ OT-1 cells sorted at day 3.5 p.i. were treated with anti-IL-21R (+αIL-21R, in blue) or with anti-IL-21R isotype control (in red) and were adoptively transferred into recipient mice (see Figure S5 and Transparent Methods). Bcl6 and CD127 expression, at day 10 p.i., are shown in both conditions. Isotype control staining (open histograms) is also shown.

(D) OT-1 memory response was analyzed 4 days after the challenge at the memory stage (day 46). Percentages of granzyme B, Blimp-1, eomesodermin- and T-bet-positive cells, and corrected geometric MFI of IL-21R (i.e., [MFI^OT-1 – MFI^non-OT-1]) in secondary effectors derived from CXCR5^- OT-1 cells, anti-IL21R-treated CXCR5⁺ OT-1 cells, or isotype control-treated CXCR5⁺ OT-1 cells are shown.

(E and F) (E) At day 4 p.i., geometric MFI of CD40 ligand at the cell surface of Tfh and CXCR5⁺ or CXCR5^- OT-1 primary effectors. Representative plot profiles are also shown. (F) CD4-DTR mice received 10⁴ OT-1 cells and were then infected with 2 × 10⁴ colony-forming unit (CFU) of rLm-OVA. CD4⁺ T cells were depleted with DT on days 1, 2, and 3 after infection. CD4-DTR mice were reconstituted on day 4 post-priming with Tfh-depleted CD4⁺ T cells and either Tfh treated with neutralizing anti-CD40L (w/αCD40L Tfh) or Tfh treated with the corresponding isotype control (w/Tfh). On day 42, mice were challenged with 2 × 10⁵ CFU of rLm-OVA, and secondary responses and bacterial burden were evaluated 4 days later (day 46) (see Figure S6 and Transparent Methods). Data shown are, from left to right, corrected geometric MFI of IL-21R at the cell surface of OT-1 cells (calculated as [MFI^OT-1 – MFI^non-OT-1]), splenic rLm-OVA burden, granzyme B-corrected geometric MFI in OT-1 cells (calculated as [MFI^OT-1 – MFI^non-OT-1]), and the percentage of granzyme B-positive OT-1 cells. The data shown in Figure 6 are the median and 10^th and 90^th percentiles of values obtained in three independent experiments with at least three mice per condition. Data from (A, C, and F) were analyzed with the Mann-Whitney test, data from (B) with the Wilcoxon test, data from (D) with the Kruskal-Wallis test, and data from (E) with the Friedman test. *p < 0.05; **p < 0.01; ***p < 0.001.