Fig. 4. Bob1 supports the memory stage of Tfh cells.

a Experimental protocol for the immunization of FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice and control FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice. NP₄₉-CGG was injected intraperitoneally with CFA on day 0 and with PBS on day 42. b, c Representative flow cytometric profiles and graphs of Tfh cell subsets in spleens. Specimens were analyzed on days 0, 7, 42, and 49 in (b), and on day 42 in (c). b Profiles of PD-1^lo Tfh cells (GFP^-B220^-CD4⁺CD44⁺CXCR5⁺PD-1^lo) and graphs showing the % of PD-1^lo Tfh cells among the total CD4⁺ T cells. *P = 0.0334. c Profiles of Ki67⁺PD-1^lo Tfh cells and graphs showing the % of Ki67⁺PD-1^lo Tfh cells among the total PD-1^lo Tfh cells. *P = 0.0472. d–g Adoptive cell transfer experiments of PD-1^lo Tfh cells using TCRβ^−/− mice. d Experimental protocol for immunized cell transfer. OVA was administered intraperitoneally with CFA on day 0 to CD45.2⁺CD4^Cre/+Bob1^fl/fl mice and control CD45.2⁺CD4^+/+Bob1^fl/fl mice. PD-1^lo Tfh cells (B220^-CD4⁺CD44⁺CXCR5⁺PD-1^lo) from the spleen were sorted on day 42 and intravenously transferred into CD45.1⁺TCRβ^−/− hosts. The next day, mice were intraperitoneally immunized with OVA in incomplete Freund’s adjuvant (IFA). Seven days later, spleen cells were analyzed. e, f Flow cytometric analysis of PD-1^lo Tfh cells, prior to being injected into CD45.1⁺TCRβ^−/− hosts. e Representative profiles of OVA-specific tetramer-positive (Tet⁺) Tfh cells and Tet⁺ PD-1^lo Tfh cells. Graphs showing the % of Tet⁺ Tfh cells and Tet⁺ PD-1^lo Tfh cells among the total CD4⁺ T cells. *P = 0.0286. f Representative profiles of FR4⁺PD-1^lo Tfh cells and graphs showing the % of FR4⁺PD-1^lo Tfh cells among the total PD-1^lo Tfh cells. *P = 0.0286. g Representative profiles and graphs of CD45.2⁺ Tfh cells in CD45.1⁺TCRβ^−/− hosts. The % of CD45.2⁺ Tfh cells among the total CD45.2⁺CD4⁺ T cells are shown. *P = 0.0411. Data represent the mean ± SD of 4–10 mice per group. Statistical significance was analyzed by the Mann-Whitney U-test (b-c, e-g); *P < 0.05. Data shown in (b, c) are from FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice (open triangle) and FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice (closed triangle), and data shown in (e–g) are from CD4^+/+Bob1^fl/fl mice (open circle) and CD4^Cre/+Bob1^fl/fl mice (closed square). Similar results were obtained across two to three independent experiments.