Fig 2: CTLA-4-deficient B-1a cells and their IgMneg decedents induce Tfh cells and GCs in congenic recipients.a Live CD4+ T cells and CD19+ B cells from spleens of indicated mice were gated to reveal Tfh (PD-1hi ICOS+) and GC B (CD95hi CD38neg) cells, respectively. b Data summarizing the numbers of Tfh and GC B cells in spleens of indicated mouse groups is shown, n = 7–10 mice per group. Each dot represents data for an individual mouse, *p < 0.003, **p < 0.0002, ***p < 0.002, ****p < 0.0003, *****p < 0.004, ns, not significant (p < 0.2), nonparametric Wilcoxon one-way test. About 5 × 106 of peritoneal B-1a (pB-1a) and 2 × 105 of IgMneg splenic B-1a (sB-1a) cells were transferred. c TCRß CDR3 tree-map plots (upper) illustrate the CDR3 nucleotide sequences expressed by non-Tfh T and Tfh cells from indicated mice. One representative plot from four or six independent T-cell samples is shown. Each rectangle in a given tree-map represents a unique CDR3 nucleotide sequence and the size of each rectangle denotes the relative frequency of an individual CDR3 sequence. The box plot summarizes the D50 metric analysis that quantifies TCRß CDR3 nucleotide sequence diversity for each T-cell sample from indicated mouse group. Each dot represents data for a T-cell sample from an individual mouse, n = 4–6 T-cell samples per group, *p < 0.006, ns, not significant (p < 0.2), nonparametric Wilcoxon one-way test. Low D50 values are associated with less TCRß CDR3 diversity. Sequence information for each T-cell sample is summarized in supplementary Table 3. d Live myeloidneg CD3neg CD19+ splenic B cells from indicated CB.17 mice were gated to reveal GC B cells (CD19+CD95+GL7+), which were further gated to show IgG1, IgG1b and IgG1a surface expression. Statistical summary for five independent experiments is shown in Supplementary Table 5. Box plots in b, c: box draws 75% (upper), 50% (center line) and 25% (down) quartile, the maxima and minima outliers are shown as top and bottom line, respectively.

Fig 3: Analysis of PD-1, ICOS, and Bcl-6 expression in naive (A), and Th1 (B) cells.

Fig 4: Phenotype of Friend virus (FV)-specific PD-1+ SIRPa- and PD-1+ SIRPa+ CD8+ T cells in mice chronically infected with FV. Splenocytes from mice chronically infected with FV were analyzed by multiparameter flow cytometry for surface expression of (a) CD122, (b) Tim3, (c) Lag3, (d) CD95 (Fas), (e) CD43, (f) CD44, (g) CD40, (h) CD278 (inducible T cell co-stimulator), (i) KLRG1, (j) CD62L, (k) CD47, and (l) CX3CR1. A representative off-set histogram overlay is displayed for each marker as well as the average geometric mean fluorescence intensity (MFI) from one experiment is given (n = 4 mice). CD8+ dextramer- cells (non-DbgagL-specific cells from infected mice) are shown in dashed gray, PD-1+/SIRPa- CD8+ dextramer+ cells are shown in solid line gray, and PD-1+/SIRPa+ CD8+ dextramer+ are shown in black. The vertical dashed line delineates positivity relative to the FMO control). Results are from one of three independent experiments with similar results (with n = 8 additional mice). ns, p > 0.05, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001 (unpaired, two-way t tests). The flow cytometric gating strategy is shown in supplementary Fig. 6h–m

Fig 5: TFR impair TFH ICOS expression and proliferation.(a) Tonsil TFH were analysed for surface ICOS, following mock-, X4-, or R5-spinoculation (n=7). (b) CD25+ regulatory cells were depleted from each culture condition and ICOS expression on TFH was analysed (n=3). (c) Tonsil TFH and TFR were isolated, spinoculated and co-cultured for 2 days at the indicated ratios. Representative images of ICOS expression on TFH surface are shown (n=2). (d) Tonsil TFH were isolated, labelled with eFluor 670 proliferation dye, and cultured alone or with an equal number of TFR. Proliferation was measured by dye dilution (n=4). The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed using Friedman nonparametric tests (a) and significance is denoted by asterisks where *P<0.05.