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. 2014 Dec 15;211(13):2507–2517. doi: 10.1084/jem.20140137

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© 2014 Moalli et al.

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Figure 4. — Increased numbers of T_FH in the absence of TP in OT-II CD4⁺ T cells. (A–H) WT and TP^−/− OT-II CD4⁺ T cells were adoptively transferred separately (A–D and F–H) or combined (E) into CD45.1⁺ WT mice before immunization with OVA/CFA and analyzed on day 8 by flow cytometry. (A) Flow cytometry after pregating on CD44^high CD62L^low lymphocytes. Numbers indicate the percentage of circled CD45.2⁺ cells. (B) Percentage of adoptively transferred CD45.2⁺ T cells as pregated in A. (C) PD-1 and CXCR5 expression on transferred WT and TP^−/− OT-II T cells (red), with numbers indicating percent per quadrant field. CD4⁺ T cells from a nonimmunized mouse serve as negative control (blue). (D) Percentage of PD-1^high CXCR5^high CD45.2⁺ WT or TP^−/− OT-II CD4⁺ T cells on day 8 after OVA/CFA immunization. Shown is one of three experiments, pooling T_FH from spleens and PLNs of 3–4 mice on day 8 after OVA/CFA immunization. (E) Flow cytometric analysis of intracellular bcl-6 expression (blue) in PD-1^high CXCR5^high WT or TP^−/− OT-II CD4⁺ T cells on day 8 after OVA/CFA immunization. As negative control, we plotted the endogenous CD45.1⁺ CD4⁺ cell signal (red), which gave an overlapping labeling as the isotype control (not depicted). (F) Flow cytometric analysis of intracellular IL-21 expression (blue) in CXCR5^high WT or TP^−/− OT-II CD4⁺ T cells on day 8 after OVA/CFA immunization. As negative control, we plotted the CD45.1⁺ CD4⁺ cell signal from a nonimmunized mouse (red), which gave an overlapping labeling as the isotype control (not depicted). (G) Flow cytometry plots of B220⁺ cells containing GL-7⁺ CD95⁺ GC B cells in WT or TP^−/− OT-II T cell recipient mice on day 8 after OVA/CFA immunization with numbers indicating percent per quadrant field. (H) Percentage of GC B cells in WT or TP^−/− OT-II T cell recipient mice 8 d after OVA/CFA immunization in PLNs and spleen. Each dot represents one mouse. Data are representative of three (A–F) or pooled from two (H) independent experiments with a total of 9–10 and 5–6 mice, respectively, and analyzed using Mann-Whitney test (B, D, and H). Bars indicate mean. *, P < 0.05; **, P < 0.01.