Panel A shows flow cytometry plots comparing frequencies of CD4-positive FOXP3-positive regulatory T cells across groups. Panel B shows a bar graph quantifying percentages of CD4-positive FOXP3-positive regulatory T cells examined. Panel C shows histogram overlays comparing CD25 and FOXP3 expression levels within regulatory T cells. Panel D shows bar graphs summarizing mean fluorescence intensities of CD25 and FOXP3 markers. Panel E shows stacked bar charts depicting proportions of CD25-positive and CD25-negative regulatory cells. Panel F shows flow cytometry plots illustrating CD39 expression within CD4-positive FOXP3-positive regulatory T cells. Panel G shows bar graphs quantifying CD39-positive and Helios-positive regulatory T-cell subpopulation frequencies. Panel H shows histograms and bar graphs comparing unstimulated CTLA-4 expression among study groups. Panel I shows histograms and bar graphs evaluating CTLA-4 expression following cellular stimulation. Panel J shows histograms and bar graphs assessing CTLA-4 recycling under unstimulated and stimulated conditions. Panel K shows a bar graph comparing CTLA-4 expression in wild-type and mutant-transfected HEK293T cells.
CTLA4 S172P/S172P variant disrupts CTLA-4 expression in CD4 + FOXP3 + Tregs. (A) Representative flow cytometry plot showing the frequency of CD4+FOXP3+ Tregs. (B) Bar graph depicting the percentages of CD4+FOXP3+ Tregs. (C) Representative histograms illustrating CD25 and FOXP3 expression levels within CD4+FOXP3+ Tregs. Indicated: gray, HC; blue, patient before abatacept (abt); purple, patient after abatacept (12 mo). (D) Bar graphs showing MFI of CD25 and FOXP3 in CD4+FOXP3+ Tregs. (E) Bar graph representing the proportion of CD25+ and CD25− cells within the CD4+FOXP3+ Treg compartment. Indicated: black, CD25+; gray, CD25−. (F) Representative flow cytometry plot of CD39 expression within CD4+FOXP3+ Tregs. (G) Bar graphs showing the percentages of CD4+FOXP3+CD39+ and CD4+FOXP3+Helios+ Treg subsets. (H) Representative histograms of CTLA-4 expression in unstimulated CD4+FOXP3+ Tregs, with corresponding bar graphs of relative CTLA-4 expression normalized to HC. Black, HC; red, mother; blue, CTLA4S172P/S172P patient; purple, CTLA-4–insufficient patients. (I) Representative histograms and normalized bar graphs showing CTLA-4 expression in stimulated CD4+FOXP3+ Tregs. Black, HC; red, mother; blue, CTLA4S172P/S172P patient. (J) Representative histograms and relative bar graphs of recycling CTLA-4 expression in CD4+FOXP3+ Tregs, normalized to HC. Black, HC; red, mother; blue, CTLA4S172P/S172P patient. Open histograms indicate US conditions, whereas filled histograms indicate stimulated conditions. (K) CTLA-4 levels in HEK293T cells overexpressing WT or the S172P mutant CTLA-4. Data are from three independent experiments, normalized to WT. Statistical significance was determined by the Wilcoxon signed-rank test. ns = nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. MFI, mean fluorescence intensity; US, unstimulated; WT, wild-type; HC, healthy contol.