Identification of CD62L-expressing Tfh cells through single-cell transcriptomic analysis. (A) Unsupervised clustering of KLH immunization–derived Tfh single-cell RNA-seq data was performed on a UMAP. Selected DEGs of the indicated cluster were highlighted in the UMAP. (B) UMAP visualization showing gene expressions of Pdcd1, Il21, Hif1a, Lag3, Sell, Klf2, S1pr1, Il7r, Bcl6, Icos, Tcf7, Lef1. (C) DEGs of each cluster were shown by heatmaps. Selected genes of marker genes in C1 and C3 were highlighted. (D) Cluster distributions of each single-cell sample were shown by stacked bars. (E) Flow cytometry analysis of PD-1 and CD62L expression in Tfr cells derived from draining lymph nodes on day 8 after KLH immunization and GC-Tfh cells derived from draining lymph nodes at day 8 after KLH immunization (KLH), day 9 after influenza infection (Flu), as well as PP at steady state. (F) Statistical analysis of PD1⁺ and CD62L⁺ percentages in indicated cell types. Data of E and F represent two independent experiments. Data are shown as mean ± SD; two-tailed t test; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.