Fig 2: Haploinsufficiency of BCL6 impairs a polyclonal response(A) The experimental scheme.(B and C) Representative flow-cytometry profiles and frequencies of FAShi GL7hi GC B cells in total B220+ B cells (B) or CXCR5hiPD-1hi Tfh cells in CD44hi CD4 T cells of the donor origin (C) 13 days after NP-KLH immunization in CD45.1 Sap-/- recipients. All donors are on the CD4-Cre background, and genotypes of the Bcl6 allele are indicated. Each symbol represents one mouse, and lines denote the means. Data are pooled from 2 independent experiments. n.s., not significant. *p < 0.05.

Fig 3: Endothelin-1 Expression Highly Correlates with Localization of IAHCs(A) Expression overlap between EDN1, CDH5, and REN1. The arrowhead indicates a REN1+ cell below the CDH5+ endothelium. The images in (Ai) show a magnification of the boxed region in (A).(B) Expression of REN+ cells enveloping the endothelium of an Ao V branching vessel (BV) directed toward the Mn. The images in (Bi) show a magnification of the boxed region in (B).(C and D) Immunostaining highlighting V CDH5+Runx1+CD45+ IAHCs (C’ and D’) and, on the sister section, a higher EDN1 signal in a corresponding position (C’’ and D’’). Arrowheads indicate positions of IAHCs.(E) Representative binary image of EDN1 expression across the Ao with EDN1 hotspots (pixels > 300, 2,048 × 2,048 pixel image) numbered and outlined in red. A line divides the AoD (top) from the AoV (bottom). The box-and-whisker plot shows the percentage of EDN1 hotspots found in the AoV or AoD in each section (n = 14). p < 0.01, t test.(F) CL of rounded EDN1-expressing cells attached to the CDH5+ endothelial lining (arrows). Images in (Fi) show a magnification of the boxed region in (F).(G) Correlation between the position of Runx1+ IAHCs in each section with the position of EDN1 hotspots (R, correlation coefficient).For (A)–(C), (E), and (F), protein or RNA expression is indicated by non-italicized and italicized names, respectively. SMA, superior mesenteric artery. The D-V axis is indicated. Images show transverse sections of CS15–CS16 embryos. Scale bars, 50 µm.

Fig 4: Partial CD40 deletion was able to largely rescue the aberrant GC B phenotypes resulting from miR-146a deficiency. a Expression of CD40 in B-KO B cells, B-KO/CD40+/- B cells vs. WT B cells was assessed by immunoblotting. Densitometric values of CD40 were normalized to ß-actin expression values and n-fold increase on the basis of WT controls. b Quantitative PCR of AID mRNA levels and c FACS analysis and frequencies of IgG1+ cells in in vitro CD40-stimulated B cells from B-KO, B-KO/CD40+/- or WT mice are shown. d Schematic of generation of mixed BM chimeras. e FACS analyses of Ly5.1- and Ly5.1+ PNA+GL7+ GC B cells in spleen from indicated mixed BM chimeras 14 days after SRBC immunization. f Ratios of frequencies of Ly5.1- and Ly5.1+ GC B cells in spleen from indicated chimeric mice at days 5, 10 and 14 days after SRBC immunization. Data are representative of three independent experiments. Each symbol represents an individual mouse, and the bar represents the mean. Significance was determined by unpaired Student’s t-test with a 95% confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: PDK1 is required for both early differentiation and late maintenance of Tfh cells.(A) Schematic of the SMARTA cell transfer system used for characterization of early Tfh cell commitment. SMARTA CD4+ T cells from Pdk1fl/fl::Rosa26CreER::SMARTA mice were transferred into C57BL/6J (CD45.2+) host mice, followed by Tamoxifen treatment for four consecutive days, LCMV infection, and analyzed on 3 dpi. (B) Quantitative RT-PCR analysis of Pdk1 abundance in donor-derived CXCR5+ Tfh cells from recipients on 3 dpi as in (A) (n = 6). (C) Flow cytometry analysis of Bcl-6+CXCR5+ Tfh cells gated on SMARTA CD4+ T cells from recipients on 3 dpi with representative contour plots and cumulative data (n = 3). (D) Contour plots represents BrdU+ cells gated on donor-derived activated CD4+ T cells (top panel) and CXCR5+ Tfh cells (bottom panel) from WT and Pdk1fl/fl::Rosa26CreER::SMARTA mice on 3 dpi. Cumulative data on frequency of BrdU+ cells are shown on the right (n = 6). (E) Quantitative RT-PCR analysis of selected genes in donor-derived CXCR5+ Tfh cells from recipients as in (A) (n = 3). (F) Schematic of the Tamoxifen-induced deletion system used for characterization of late Tfh cell differentiation. WT and Pdk1fl/fl::Rosa26CreER mice were treated with Tamoxifen from day 4 to day 7 post-LCMV infection and analyzed on 8 dpi. (G) Quantitative RT-PCR analysis of Pdk1 abundance in Tfh cells from WT and Pdk1fl/fl::Rosa26CreER mice on 8 dpi as in (F) (n = 5). (H) Flow cytometry analysis of CD44+CXCR5+ Tfh cells (top panel) gated on CD4+ T cells and Bcl-6+CXCR5+ GC Tfh cells (bottom panel) gated on CD44+CD62L-CD4+ T cells on 8 dpi with representative contour plots and cumulative data (n = 5). (I) Contour plots represents BrdU+ cells gated on activated CD4+CD44+ T cells (top panel) and CD44+CXCR5+ Tfh cells (bottom panel) from WT and Pdk1fl/fl::Rosa26CreER mice on 8 dpi. Cumulative data on frequency of BrdU+ cells are shown on the right (n = 5). Data are representative of at least three independent experiments (B–C, E, G–H) or pooled from two independent experiments (D). Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).Figure 4—source data 1.PDK1 is essential for Tfh cell differentiation at both early and late stages.