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. 2023 Dec 4;221(1):e20221927. doi: 10.1084/jem.20221927

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© 2023 Feng et al.

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Figure 3. — Ontogeny of CD62L-expressing Tfh cells in immune responses. (A and B) Naïve OT-II labeled with CFSE were adoptively transferred into CD45.1 recipient mice, which were subsequently immunized subcutaneously with OVA. Mouse draining lymph nodes were isolated for analysis at day 4 after immunization. (A) Representative flow cytometry staining of PD-1 or CD62L in each cell division of CXCR5⁺Bcl6⁺CD45.2⁺ OT-II cells. (B) Statistical analysis of (left) CD62L⁺CXCR5⁺Bcl6⁺ and (right) PD1⁺ CXCR5⁺Bcl6⁺ cell percentages in transferred CD45.2⁺ OT-II cells. (C–E) IL21-VFP and Il21^creR26^YFP mice were subcutaneously immunized with KLH. 5 or 21 days later, mice were sacrificed for analysis. (C) Representative flow cytometry analysis of PD-1 and CD62L versus IL-21-VFP or YFP expression in CD4⁺CD44^hiCXCR5⁺ cells at day 5 after KLH immunization of IL21-VFP mice or day 21 after KLH immunization of Il21^creR26^YFP mice, respectively. (D and E) Statistical analysis of IL-21-VFP⁺ or YFP⁺ percentages in PD1⁺ or CD62L⁺ Tfh cells, respectively. (F) RNA velocity analysis of cells in C1, C3, and C6 projected onto the UMAP plot. Arrows indicated the direction and speed of each cell. (G–I) Bcl6^RFPFoxp3^GFP mice were immunized with KLH, 8 days later, PD-1⁺ Tfh, CD62L⁺ Tfh, and naïve T cells were isolated into 96-well plates, and the TCRβ chain mRNA sequences were amplified and sequenced. (G) TCRβ clonotypes in PD-1⁺ Tfh, CD62L⁺ Tfh, and naïve T cells. (H) The distribution of shared TCRβ sequences in CD62L⁺ and PD1⁺ Tfh cells. Blue and red boxes indicate the uneven presence of indicated TCRβ clonotypes in PD1⁺ and CD62L⁺ Tfh cells, respectively. (I) The proportions of shared TCR in each TCR Vb subtype between CD62L⁺ and PD1⁺ Tfh cells. Blue and red lines indicate higher percentages of indicated TCR Vb in PD1⁺ and CD62L⁺ Tfh cells, respectively. Data above are representative of two independent experiments. Data of A–E represent two independent experiments. Data are shown as mean or mean ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.