Developmental features of PD-1 ⁺ and CD62L ⁺ Tfh cells. (A and B) Naïve OT-II labeled with CFSE were adoptively transferred into CD45.1 recipient mice, which were subsequently immunized subcutaneously with OVA. Draining lymph nodes were isolated for analysis on day 4 after immunization. (A) Immunofluorescence intensity of CFSE in each cell division of indicated cell populations. (B) CD62L⁺ cell percentages in transferred CD45.2⁺ OT-II cells. (C and D) μMT and control mice were subcutaneously immunized with KLH. 7 days later, draining lymph nodes were isolated for analysis. (C) Representative flow cytometry analysis of CXCR5 and Bcl6 expression in CD4⁺CD44^hi cells of μMT and control mice. (D) Representative flow cytometry analysis of PD-1 and CD62L expression in Tfh (CXCR5^hiBcl6^hi) and Tcm (CXCR5^midBcl6^mid) cells of μMT and control mice. (E) C57BL/6 mice were subcutaneously immunized with KLH, and the draining lymph nodes were isolated, embedded in OCT, and subjected to cryosectioning for subsequent immunofluorescence staining. Representative immunofluorescence staining of PNA (blue), PD-1 (green), CD62L (red), and CD4 (violet) and four-color overlay image of the frozen sections. The length of scale bars represents 100 µm, unless labeled in the image. Data of A–E represent two independent experiments. Data are shown as mean; two-tailed t test; **, P < 0.01; ****, P < 0.0001 ns, no significance.