Flow cytometric contour plots (a, d, g) and dot plots of T_FH (b, e, h) and T_FR (c, f, i) cells in the groups of mice described below, seven days after SRBC immunization. (a-c) Mixed bone marrow chimeras generated by sub-lethally irradiating Rag2^−/− mice and reconstituting their immune system with a 1:1 ratio of bone marrow cells from CD45.1 Cd28^+/+ and CD45.2 Cd28^−/− mice or control CD45.1 Cd28^+/+ and CD45.2 Cd28^+/+. (d-f) C57BL/6 (BL/6) and B-cell deficient μMT mice. (g-i) Sh2d1a^+/+ and Sh2d1a^−/− mice. Each symbol represents one mouse and horizontal bars represent median values. Figures represent one of 3 independent experiments with similar results. Statistical significance was determined using a Mann-Whitney Test: *P<0.05, **P<0.01.

Flow cytometric contour plots of splenic CD4⁺CXCR5^highPD-1^high cells (a) or CD4⁺ cells (b) seven days after 1×10⁵ transferred transgenic TCR^3A9 HEL-specific CD45.1 T cells were adoptively transferred into congenically distinct CD45.2 B10.Br mice and immunized with HEL in alum. Flow cytometric contour plots of splenic CD4⁺CXCR5^highPD-1^high cells (c) or CD4⁺ cells (d) seven days after adoptive transfer of 1×10⁵ OT-II OVA-specific Thy1.2 T cells into congenically distinct Thy1.1 C57BL6 mice and immunization with OVA in alum. (e) Flow cytometric contour plots of splenic CD4⁺ T cells from CD45.1 C57BL/6 mice seven days after adoptive transfer of 1×10⁶ sorted naïve CD4⁺CD44^intFoxp3⁺ T_reg (top panel) or CD4⁺CD44^lowFoxp3⁻ naïve T cells (lower panel) from unimmunized CD45.2 Foxp3^GFP mice and KLH in Ribi immunization. Transferred CD45.2 cells are shown in red, the endogenous CD45.1 cells are represented by the grey contour plots. Histograms showing Foxp3-GFP expression in transferred CD45.2⁺CD4⁺CXCR5^highPD-1^high cells. (f) Contour plots of splenic CD4⁺ T cells and quantification of T_FR cells (g) from Foxp3^DTR mice six days after SRBC immunization and administration of either 0.9% saline (top panel) or DT (lower panel). Histograms show Foxp3⁺ cells within the CD4⁺CXCR5^highPD-1^high compartment. (h) Flow cytometric contour plots of splenic CD4⁺ cells from Foxp3-cre x ROSA-Stop-flox-YFP mice immunized seven days previously with SRBC (left panel). Enumeration of the proportion of CD4⁺CXCR5^highPD-1^high cells that expressed YFP and/or Foxp3 (right panel). Each symbol represents one mouse and horizontal bars represent median values. Figures are representative of 2-4 independent experiments.

Flow cytometric contour plots (a) and graphs (b) of total GL-7⁺CD95⁺ germinal center B cells and (c) NP⁺ germinal center B cells ten days after immunization of Foxp3^WT and Foxp3^DTR mice that have been treated with DT 6 days after NP-KLH immunization. Statistical analyses performed using Mann Whitney U-test. Experimental outline (d) of immunization and DT or saline treatment scheme of Foxp3^DTR mice (n=8 per group) to examine the antigen specific immunoglobulin response over time, mice were bled prior to, and d10, d15, d20 and d28 after primary immunization. Mice were given a booster immunization 24 days after the primary immunization. (e) ELISA analysis of NP12 and NP2 antibodies in the experiment outlined in (d). Error bars represent the standard error of the mean from eight individual mice from one experiment, representative of two experiments. Statistical analyses in (e) were performed using a two-way ANOVA with Bonferroni post test to compare differences at each time point. Graphs and flow cytometric contour plots of NP⁺ germinal center B cells (f), total GL-7⁺CD95⁺ germinal center B cells (g) and NP⁺ bone marrow plasma and memory cells (h, i) 21 days after NP-CGG immunization of chimeric mice generated by reconstituting Rag2^−/− mice with a 1:1 mix of Sh2d1a^−/−:Foxp3^−/−, Sh2d1a^+/+:Foxp3^+/+, Sh2d1a^+/+:Foxp3^−/− and Sh2d1a^−/−:Foxp3^+/+ fetal liver. Statistical analyses in (f, g, h and i) were performed using a one-way ANOVA with Bonferroni post test correction. Each symbol represents one mouse and horizontal bars represent median values. *P<0.05, **P<0.01, ***P<0.001.

(a,b) After SRBC immunization Foxp3⁺ cells were identified in the CXCR5^highPD-1^highCD4⁺ ‘T_FH’ compartment, these cells follow the same kinetics as classic T_FH cells. (c) Foxp3⁺ cells (red) are present within the Bcl6⁺ germinal center area (green) following SRBC immunization. Scale bar represents 100μm. (d) Heat map comparing the gene expression profiles of different CD4⁺ T cell subsets from Foxp3^GFP mice seven days after immunization. Red: high gene expression; blue: low gene expression. The cells were sorted using the following markers and for simplicity will be referred by the abbreviations in parentheses throughout: CD4⁺CD44^lowFoxp3⁻ naïve (T_N) cells, CD4⁺CD44^highCXCR5^int/lowPD-1^int/lowFoxp3⁻ effector/memory (T_EM) cells, CD4⁺CD44^intCXCR5^int/lowPD-1^int/lowFoxp3⁺ regulatory T cells (T_reg), CD4⁺CXCR5^highPD-1^highFoxp3⁻ T follicular helper (T_FH) cells and CD4⁺CXCR5^highPD-1^highFoxp3⁺ follicular regulatory (T_FR) cells. (e) Il21 and Il4 mRNA measured by quantitative PCR from sorted cells using the strategy described in (d) normalized to Gapdh. Heights of the bars represent the mean and error bars represent the range of expression from 3 biological replicates. nd: gene expression not detected. (f) Left: Intracellular expression of CD40L as determined by flow cytometry in T_reg (blue), T_FH (green) and T_FR (red) cell populations; the grey histogram represents a staining control from an immunized CD40L-deficient mouse. Right: Cxcl13 mRNA measured by quantitative RT-PCR as described in (e). (g) Cell surface expression of GITR, CD25 and intracellular CTLA4 in T_reg (blue), T_FH (green) and T_FR (red) cell populations; grey histograms represent the isotype control. (h) Relative Gzmb and Gzma mRNA determined by quantitative RT-PCR as described in (e). (i) Percentage of CD103⁺ cells within the T_reg, T_FH & T_FR populations, each symbol represents one mouse. (j) Left: Cell surface expression of ICOS as determined by flow cytometry in T_reg (blue), T_FH (green) and T_FR (red) cell populations; the grey histogram represents staining level of an isotype control. Right: Il10 mRNA detected by quantitative RT-PCR of as described in (e). Flow cytometric and RT-PCR data are representative of at least three independent experiments. In (e)-(i): Statistical significance was determined using a one-way ANOVA analysis with Bonferroni’s multiple testing correction; * P<0.05; **P<0.01; ***P<0.001.

(a) Bcl6 and Prdm1 mRNA normalized to Gapdh determined by quantitative RT-PCR from sorted cells using the strategy described in Fig. 1d and Supplementary Fig. 1. Heights of the bars represent the mean and error bars represent the range of expression from 3 biological replicates. Statistical significance was determined using a one-way ANOVA analysis with Bonferroni’s multiple testing correction; * P<0.05; **P<0.01; ***P<0.001. Bar graphs are representative of 3 experiments. (b) Immunofluorescence of frozen spleen sections from mice immunized seven days previously with SRBC. The germinal center is demarcated by the white dotted line in the three consecutive sections. Upper panel: AID (red) and CD3 (green); middle panel: Foxp3 (red) and Bcl-6 (green); lower panel: Foxp3 (red) and Blimp1 (green). Scale bar represents 100μm. (c) Flow cytometric contour plots of T_FH (upper panels) & T_FR (lower panels) formation in the draining (mediastinal) lymph node ten days after intranasal influenza infection of mixed fetal liver chimeras reconstituted with a 1:1 ratio of fetal liver cells from E14.5 CD45.2 Prdm1^gfp/gfp : CD45.1 Prdm1^+/+ embryos, E14.5 CD45.2 Bcl6^−/− : CD45.1 Bcl6^+/+ embryos or control E14.5 CD45.2 Prdm1^gfp/+ : CD45.1 Prdm1^+/+ embryos.

Flow cytometric contour plots and graphs of T_FH cells (a) and germinal center B cells (b) from the spleens of Foxp3^DTR mice immunized eight days previously (d0) with SRBC. Five days after immunization the mice were treated with either DT or saline. (c-f) Analysis of mixed bone marrow chimeras generated by sub-lethally irradiating Rag2^−/− mice and reconstituting their immune system with either a 1:1 ratio of Sh2d1a^−/− CD45.2 : Foxp3^DTR CD45.1 bone marrow or control Sh2d1a^+/+ CD45.2 : Foxp3^DTR CD45.1 bone marrow. Eight weeks after reconstitution chimeric mice were immunized with SRBC and treated with 50μg/Kg of DT on one day prior to immunization and d2 and d5 thereafter. Splenocytes were analyzed on d8 for the proportion and total number of CD4⁺CXCR5^highPD-1^highFoxp3⁺ T_FR cells (c), CD4⁺Foxp3⁺ T_reg (d), CD4⁺CXCR5^highPD-1^high T_FH cells (e) and B220⁺ GL-7^highCD95^high germinal center B cells (f). Each symbol represents one mouse and horizontal bars represent median values. Statistical significance was determined using a Mann-Whitney Test: *P<0.05, **P<0.01.