Increased CD40L levels in effector CD4⁺ T cells in CTLA4 deficiency correlate with a decrease in the frequency of circulating FO B cells. (A) Extracellular flow cytometric analysis of peripheral blood T-cell populations in patients with CTLA4 deficiency (n = 11) and healthy controls (n = 10). Representative contour plots from a healthy control (left) and 2 patients with CTLA4 deficiency (right, heterozygous CTLA4 c.410C>T, p.P137L mutation carriers) shown at 5% of events (full description of T-cell gating strategy in supplemental Figure 1B). (B) Quantification of the major CD4⁺ T-cell subsets defined by the extracellular markers CXCR5, CCR7, and CD45RA, including TFH, naïve, CM, TEMRA, and EM populations. Symbols represent unique individuals; bars represent means (± SD) of all data. ∗P < .05; ∗∗P < .01 by 2-tailed Mann-Whitney U test. (C) Correlation between FO B-cell frequency as percent IgD⁺CD27^– B cells in the peripheral blood (data from Figure 1C; x-axis) and naïve CD4⁺ T-cell frequency as percent CD4⁺CXCR5^– T cells in the peripheral blood (data from Panel B; y-axis). Simple linear regression with correlation coefficient (R²), P value, and 95% confidence interval (CI) shown. (D) Intracellular flow cytometric analysis of peripheral blood T-cell populations in patients with CTLA4 deficiency (n = 8) and healthy controls (n = 10). Representative overlaid histograms from a healthy control (blue) and a patient with CTLA4 deficiency (red, heterozygous CTLA4 c.173G>C p.C58S mutation carrier) shown. (E) Quantification of total cellular CD40L levels (MFI) by intracellular staining in the major CD4⁺ T-cell subsets described above in patients with CTLA4 deficiency and healthy controls. Symbols represent unique individuals; bars represent means (± SD) of all data. ∗P < .05; ∗∗P < .01 or as listed by 2-tailed Mann-Whitney U test. (F) Correlation between FO B-cell frequency as percent IgD⁺CD27^– B cells in the peripheral blood (data from Figure 1C; x-axis) and total cellular CD40L levels as MFI in TEMRA CD4⁺ T cells (data from panel E; y-axis). Simple linear regression with correlation coefficient (R²), P value, and 95% CI shown. CM, central memory; EM, effector memory; TFH, T follicular helper.