(A and B) Sorted tonsil CD4⁺CD25^–ICOS⁺CXCR5⁺ Tfh cells (10⁴ cells) were either stimulated (CD3 and CD28 mAb) or not and were cultured for 3 days alone or with sorted tonsil CD4⁺CD25⁺ICOS⁺CXCR5⁺ Tfr cells (10⁴ cells) or CD4⁺CD25⁺ICOS⁺CXCR5^– Tregs (10⁴ cells) in the presence or absence of sOX40L (100 ng/ml). Proliferation of Tfh cells was analyzed at 3 days of coculture. (A) Histograms of a representative experiment showing proliferation (CFSE^dim) of unstimulated Tfh cells (gray filled), stimulated Tfh cells (black line), and Tfh cells cultured with either Tfr cells or Tregs (red line) at 3 days of culture. (B) Percentage inhibition of Tfh cell proliferation. The percentage of inhibition was calculated in reference to proliferation observed with stimulated Tfh cells cultured alone. Error bars indicate the mean ± SEM, n = 3. Statistical analysis was conducted using the 1-way ANOVA test. (C and D) Soluble OX40L impairs the ability of Tfr cells and Tregs to suppress Tfh cell function. After incubation with sOX40L (100 ng/ml), Tregs or Tfr cells (4 × 10⁴ cells) were cultured for 7 days along with Tfh cells (2 × 10⁴) and memory B cells (2 × 10⁴) in the presence of SEB (0.25 ng/ml). CD27⁺CD38⁺ plasmablast percentage in cell culture (C) and concentration of IgG in cell culture supernatant (D) were determined by flow cytometry and ELISA, respectively, after 7 days of coculture. Individual values are shown with mean and SEM and were compared using nonparametric Kruskal-Wallis test with Dunn’s comparison for multiple comparisons. *P < 0.05; **P < 0.01.