Figure 5.

Tfh Are Required to Generate Functional CD8 Memory

(A and B) (A) CD4-DTR mice received 10⁴ OT-1 cells and were then infected with 2 × 10⁴ colony-forming unit (CFU) of rLm-OVA. CD4⁺ cells were depleted with DT on days 1, 2, and 3 after infection. CD4-DTR mice were reconstituted on day 4 post-priming with total CD4⁺ T cells (w/Tfh) or with Tfh-depleted CD4⁺ T cells (w/o 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 S4A and Transparent Methods). (A) On the left, the frequency of Tfh among CD4⁺ T cells 3 days after transfer (day 7 post-infection) is shown. On the right, the percentage of Tfh cells among CD4⁺ T cells, the splenic rLm-OVA burden, the corrected geometric MFI of granzyme B in OT-1 cells (calculated as [MFI^OT-1 – MFI^non-OT-1]), and the percentage of granzyme B-positive OT-1 cells, 4 days after the challenge at the memory stage (day 46), are shown. (B) Similar experiments were conducted with tamoxifen-treated CD4-bcl6^floxed mice (Bcl-6^ko in the graph) or with tamoxifen-treated CD4-bcl6⁺ littermates (non-sensitive to tamoxifen, Bcl-6⁺ in the graph) (see Figure S4C and Transparent Methods). On the left, the frequency of Tfh in CD4⁺ T cells, the percentage of CXCR5⁺ cells among OT-1 cells, and the geometric corrected MFI of CXCR5 on OT-1 cells (as [MFI^OT-1 – MFI^non-OT-1]) were analyzed on day 4 post-infection. On the right, the splenic rLm-OVA burden after challenge, the corrected geometric MFI of granzyme B in OT-1 cells (calculated as [MFI^OT-1 – MFI^non-OT-1]), and the percentage of granzyme B-positive OT-1 cells after challenge (day 46) are shown. Graphs display median and 10th and 90th percentiles. Mann-Whitney test was used for statistical comparison. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.