a and b, Expression of CXCR5 and BTLA in Foxp3^-(a) and Foxp3⁺(b) CD4⁺ T cells were analyzed with lymphoid cells from the draining lymph nodes or spleen of wild-type littermates or Bcl6^-/- mice at 7 days after s.c. immunization with KLH in CFA. Data represent two independent experiments.

a, Expression of CXCR5, BTLA and Foxp3 in CD4⁺ T cells from spleen or thymus (CD4⁺CD8^-). b, Analysis of Foxp3⁺ and Foxp3^- CD4⁺ T cells in the draining lymph nodes of Tcrb^-/- recipients given mixture of CD25^-GITR^-CD44^loCD62L^hi naïve CD4⁺ T cells (CD45.1⁺) and CXCR5-negative Foxp3⁺ T cells (CD45.2⁺; CXCR5^-gfp⁺ cells from Foxp3^gfp mice) after immunization with KLH in CFA (left panel). The expression of CXCR5 and Bcl6 in Foxp3⁺ or Foxp3^- CD4⁺ T cells (middle panels). CD45.1/CD45.2 expression in CXCR5⁺Bcl6⁺ or CXCR5^-Bcl6^- population among Foxp3⁺ T_reg cells (right panels). c, Helios expression in CXCR5⁺ T_reg cells. Data shown are gated on CD4⁺ Foxp3⁺ cells. Data shown represent two independent experiments.

Expression of CD25 and Foxp3 among CD4⁺ T cells (a), GL7⁺CD95⁺ cells among B220⁺ B cells (b, c), and CXCR5⁺BTLA⁺ cells among CD45.1⁺ population (d) in the draining lymph nodes of Tcrb^-/- recipients given the mixture of naïve CD4⁺ T cells (CD45.1⁺) and regulatory T cells (CD25^hi; CD45.2⁺) from wild-type or Cxcr5^-/- mice, followed by s.c. immunization with KLH in CFA. e, The draining LNs were stained with PE-labeled PNA to visualize germinal center together with FITC-labeled anti-Foxp3. Scale bars shown are 25 μm. Data shown were obtained 9 days after immunization and represent three independent experiments. Data in c and d are pooled from two independent experiments. f-g, Mixture of naïve CD4⁺ T cells (CD45.1⁺) and T_reg (CD25^hi; CD45.2⁺) from wild-type or Bcl6^-/- mice were transferred into Tcrb^-/- mice, followed by s.c. immunization with KLH-NP₁₅ in CFA. Levels of immunoglobulin specific for NP₄ or NP₂₆ in sera obtained 10 days post immunization (f). Population of NP-specific B220⁺ cells or B220^-CD138⁺ plasma cells in the spleens of the recipients (g). Data shown are mean ± SE (n=5). *, p<0.05 in comparison with wild-type Treg recipients. p-values in ‘f’ were analyzed by two-way ANOVA. Data represent two independent experiments.

a, Foxp3⁺ cells (green) in the PNA⁺ (red) germinal center were stained in the draining LNs. White arrow heads indicate Foxp3⁺ cells in PNA⁺ area. Scale bar shown is 50 μm. b, Lymphoid cells from the indicated secondary lymphoid tissues from naïve mice were analyzed for the expression of CXCR5, BTLA and Foxp3 in CD4⁺ T cells by flow cytometry. c-d, Expression of Bcl6 and CXCR5 in Foxp3⁺ and Foxp3^- CD4⁺ T cells were analyzed with draining lymph node cells obtained 7 days after s.c. immunization with KLH in CFA (‘Immunized’) or without immunization (Nil). Values in d are mean ± SE. *, p<0.05; **, p<0.01 in comparison with ‘Nil’ group. a-d, Data represent two or three independent experiments. e, Expression of CXCR5, Foxp3 and Bcl6 in human tonsillar CD4⁺ T cells were analyzed. The dot plots represent the T cell staining from three donors.

a, Surface expression of the indicated molecules on CXCR5⁺ Foxp3⁺ T cells. Data shown are gated on CD4⁺ Foxp3⁺ cells, and represent two independent experiments. b, The relative expression levels of the indicated genes in FACS-sorted CD25^hiCD4⁺ T cells from Bcl6^-/- mice or wild-type littermates. Data were normalized with expression amounts of Actb. *, p<0.05; **, p<0.01 in comparison with wild-type T_reg. c, Suppressive activity of CD25^hiCD4⁺ T cells from Bcl6^-/- mice or wild-type littermates. Data shown are mean ± SE. **, p<0.01 in comparison with ‘naïve T cell alone’ condition. Data represent two independent experiments.

Expression of Bcl6 and CXCR5 on CD4⁺ T cells (a) or GL7 and CD95 on B cells (b) from the indicated secondary lymphoid tissues of scurfy mice and littermates (4-5 weeks old). Data are shown on CD4 gate (a) or B220 gate (b).