Fig. 5: Bob1 regulates the reactivation of Tfh cells from the Tcm pool.

a–d Adoptive cell transfer experiments of CD4⁺ Tcm and Tem cells. a Experimental protocol for immunized cell transfer. OVA was administered intraperitoneally with CFA on day 0 to CD45.2⁺FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice and control CD45.2⁺FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice. Tcm cells (GFP^-B220^-CD4⁺CD44⁺CD62L⁺) or Tem cells (GFP^-B220^-CD4⁺CD44⁺CD62L^-) from the spleens of immunized mice were sorted on day 42 and transferred to CD45.1⁺TCRβ^−/− mice via tail veins using the sorting strategy shown in Supplementary Fig. 9. The next day, mice were immunized intraperitoneally with OVA in IFA, and then the sera and spleen cells were analyzed 7 days later. b Representative flow cytometric profiles and graphs of CD45.2⁺ Tfh cells (CD4⁺ CD45.2⁺CXCR5⁺PD-1⁺) in CD45.1⁺TCRβ^−/− hosts transferred with CD45.2⁺ Tcm or Tem cells. The % of CD45.2⁺ Tfh cells among the total CD4⁺CD45.2⁺ T cells are shown. *P = 0.0159 (CD4^+/+Bob1^fl/fl Tcm cells vs. CD4^Cre/+Bob1^fl/fl Tcm cells) and ^†P = 0.0159 (CD4^+/+Bob1^fl/fl Tcm cells vs. CD4^+/+Bob1^fl/fl Tem cells). c Representative flow cytometric profiles and graphs of CD45.2⁺ICOS⁺ Tfh cells in CD45.1⁺TCRβ^−/− hosts transferred with CD45.2⁺ Tcm cells. The % of CD45.2⁺ICOS⁺ Tfh cells among the total CD45.2⁺ Tfh cells are shown. d Serum levels of OVA-specific IgG1 antibodies in CD45.1⁺TCRβ^−/− hosts transferred with CD45.2⁺ Tcm cells as assessed by ELISA. *P = 0.0159. e–g Histological distribution of Tfh cells originated from CD4⁺ Tcm cells. e Experimental protocol for immunized cell transfer. OVA was administered intraperitoneally with CFA on day 0 to CD45.1⁺OT-II⁺FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice and control CD45.1⁺OT-II⁺FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice. CD45.1⁺OT-II⁺CD4⁺ Tcm cells were sorted on day 42 and then intravenously transferred into CD45.2⁺ wild-type mice. The next day, mice were immunized intraperitoneally with OVA emulsified in IFA. Seven days later, spleens were examined by immunostaining with a laser confocal microscope. f Representative confocal images of spleen sections stained for CD45.1 (green), PNA (red), and DAPI (blue). Scale bar: 50 μm. g Graphs show the fluorescence intensity of transferred cells (CD45.1⁺OT-II⁺) localized within the PNA⁺ GC areas of CD45.2⁺ wild-type hosts. Imaging data were analyzed by ZEN software. **P = 0.0020. h, i Dual DNA labeling experiment. h Experimental protocol for immunization in FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice and control FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice. NP₁₆-OVA was injected intraperitoneally with CFA on day 0 and with PBS on day 30. BrdU was administered intraperitoneally on days −1, 1, 3, 5, and 7. On day 37, EdU was administered through the tail vein 3 h before analysis. i Representative flow cytometric profiles and graphs of BrdU⁺EdU⁺ Tfh cells (FoxP3^-CD3⁺CD4⁺CXCR5⁺PD-1⁺) derived from spleens. The % of BrdU⁺EdU⁺ Tfh cells among the total Tfh cells are shown. **P = 0.0015. Data represent the mean ± SD in (b, c, d, g, i) of 4–11 mice per group. Statistical significance was analyzed by the Mann-Whitney U-test for all groups: *P < 0.05, **P < 0.01. Data in (b–d) are shown from FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice (open triangle) and FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice (closed triangle), and data in (g) are shown from CD45.1⁺OT-II⁺FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice (open triangle) and CD45.1⁺OT-II⁺FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice (closed triangle). Data in (i) are shown from FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice (open triangle) and FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice (closed triangle). Similar results were obtained across two to three independent experiments.