Reduced frequency of circulating FO B cells correlates with degree of congenital Treg dysfunction in CTLA4 deficiency. CHO-CD80-GFP cells were cocultured with activated T cells from the peripheral blood of patients with CTLA4 deficiency (n = 10) and healthy controls (n = 7). Gated CD4⁺FOXP3⁺ Tregs that had acquired GFP from CHO expressing CD80-GFP cells were scored positive for intact CTLA4-mediated transendocytosis. (A) Representative contour plots from a healthy control (left) and a patient with CTLA4 deficiency (right, heterozygous CTLA4 c.410C>T, p.P137L mutation carrier), shown at 2% of events with gating strategy outlined. Quantification of (B) total cellular CTLA4 levels by intracellular staining or (C) CTLA4 function by CD80-GFP transendocytosis as percent GFP⁺CTLA4⁺ in stimulated peripheral blood CD4⁺FOXP3⁺ Tregs from patients with CTLA4 deficiency compared with healthy controls (left) and compared by CTLA4 mutation type (right). Symbols represent unique individuals and bars represent means (± SD) of all data. ∗∗P < .01 by 2-tailed Mann-Whitney U test. (D-E) Correlations between FO B-cell frequency in the peripheral blood and severity of the underlying CTLA4 mutation type. Y-axes are FO B-cell frequency as percent IgD⁺CD27^– B cells in the peripheral blood (data from Figure 1C, here shown as means ± standard error of the mean for all analyzed patients with the indicated CTLA4 mutation type or healthy control state, respectively). (D) X-axis is total cellular CTLA4 levels as MFI by intracellular staining in stimulated CD4⁺FOXP3⁺ Tregs (data from panel B, here consolidated as geometric mean per CTLA4 mutation type or healthy control state, respectively). (E) X-axis is CTLA4 function as CD80-GFP transendocytosis (percent GFP⁺CTLA4⁺) in stimulated CD4⁺FOXP3⁺ Tregs (data from panel C, here consolidated as geometric mean per CTLA4 mutation type or healthy control state, respectively). Simple linear regression with correlation coefficient (R²) and P value shown. Homo, homozygous; het, heterozygous.