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. 2019 Mar 29;10:606. doi: 10.3389/fimmu.2019.00606

Figure 5.

Figure 5

T-bet promotes TFH cell maintenance. (A,B) WT or Tbx21−/− SMARTA cells (CD45.1+) were transferred into naïve WT mice (CD45.2+), which would be infected with LCMV. Splenocytes were isolated and analyzed for TFH response at day2, 5 and 8 post infection. Flow cytometry of TFH cells derived from transferred SMARTA cells (A), and the summary of the percentages and numbers of those cells (B). (C,D) WT and iT-bet−/− mice were treated with Tamoxifen for 3 days before or after LCMV infection. Spleens were harvested at day 9 post infection. (C) Flow cytometry of TFH cells (left) with its number (right) in mice treat with Tamoxifen before infection (day−3 to−1). (D) Flow cytometry of TFH cells (left) with its number (right) in mice treat with Tamoxifen after infection (day 5–7). (E) Setup of CD4+ T cell transfer experiment. CD4+ T cells were purified form spleens of iT-bet−/− mice (CD45.2+) without Tamoxifen treatment and adoptively transferred into infection-matched recipient mice (CD45.1+). At day 8–10 post infection, recipient mice were treated with Tamoxifen or vehicle. Spleens were harvested at day 14 post infection. (F) Representative flow cytometry of TFH cells (left) and summary of TFH cell number (right) in Tamoxifen treated mice (iTbx21−/−) and vehicle treated mice (WT) was showed here. (G) Setup of TFH co-transfer experiments. WT SMARTA cells (CD45.1+CD45.2+) and Tbx21−/− SMARTA cells (CD45.1−CD45.2+) were transferred into naïve B6 mice (CD45.1+CD45.2−) separately before infecting recipients with LCMV. At day 6 post infection, WT SMARTA TFH cells and Tbx21−/− SMARTA TFH cells were FACS sorted and mixed (about 1:1), then co-transferred into infection-matched B6 mice (CD45.1+CD45.2−). Spleens were harvested and analyzed for transferred TFH cells at day 9 post infection. (H) Flow cytometry of sorted SMARTA TFH cells before (day 6 p.i.) and after (day 9 p.i.) co-transfer (left). The percentage of Tbx21−/− SMARTA TFH cells in total donor TFH cells and the number of WT and Tbx21−/− SMARTA TFH cells at day 9 post infection were summarized (right). Numbers adjacent to outlined areas in (A,C,D,F,H) indicate percent of each cell subset in parent subset. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired (B–D,F) or paired (H) two-tailed t-test). Data in (C,D,F) are representative of two independent experiments with 3–5 mice per group (error bars, SEM).