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. 2019 May 23;216(7):1664–1681. doi: 10.1084/jem.20190337

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© 2019 Zhu et al.

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Figure 3. — VHL is indispensable for the initiation of Tfh cell development. (A) Representative flow-cytometric plots of CD44⁺CD4⁺ T cells in the mLN from WT and VHL cKO mice under steady state. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5⁺ICOS⁺ or CXCR5⁺SLAM^lo) cells or GC-Tfh (CXCR5⁺PD-1^hi) cells. (B) Quantification of frequency (among CD44⁺CD4⁺ T cells) and number of Tfh or GC-Tfh cells of mice as in A (n = 3–5 per group). (C) Immunofluorescence of GCs in the mLNs as in A. Sections of mLNs were stained with IgD (red), CD4 (blue), and PNA (green). Bars, 50 µm. (D) Representative flow-cytometric plots of CD44⁺CD4⁺ T cells in the PP from WT and VHL cKO mice under steady state. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5⁺ICOS⁺ or CXCR5⁺SLAM^lo) cells or GC-Tfh (CXCR5⁺PD-1^hi) cells. (E) Quantification of frequency (among CD44⁺CD4⁺ T cells) of Tfh or GC-Tfh cells of mice as in D (n = 3 per group). (F) Representative flow-cytometric plots of CD44⁺CD4⁺ T cells in the spleen from WT and VHL cKO mice 3 d after infection with LCMV. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5⁺ICOS⁺ or CXCR5⁺SLAM^lo) cells or GC-Tfh (CXCR5⁺PD-1^hi) cells. (G) Quantification of frequency (among CD44⁺CD4⁺ T cells) and number of Tfh or GC-Tfh cells of mice as in F (n = 3–4 per group). (H) RT-PCR analysis of mRNA of Tfh cell–related genes in CD44⁺CD4⁺ T cells from WT and VHL cKO mice 3 d after LCMV infection; results were normalized to those of Actb mRNA (encoding β-actin). (I) Representative flow-cytometric plots of donor CD45.2⁺CD4⁺ T cells obtained from WT CD45.1⁺ host mice receiving naive CD4⁺ T cells from WT and VHL cKO SMARTA mice, followed by infection with LCMV and analysis 3 d after infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5⁺ICOS⁺ or CXCR5⁺SLAM^lo) cells or GC-Tfh (CXCR5⁺PD-1^hi) cells. (J) Quantification of frequency (among CD45.2⁺CD4⁺ T cells) and number (CD45.2⁺CD4⁺) of Tfh cells and GC-Tfh cells of WT CD45.1⁺ host mice as in I (n = 4 per group). Each symbol (B, E, G, and J) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant (Student’s t test). Data are representative of three independent experiments.