CTLA-4 Y218* mutation leads to reduced protein levels in primary patient cells. (A) Representative histograms of CTLA-4 mean fluorescence intensity (MFI) levels in Treg cells in one healthy donor and P16 with p.(Tyr218*) variant after 24 h of incubation in IL-2 media alone (Unstim), with stimulation (5 μg/ml plated anti-CD3 and 1 μg/ml soluble anti-CD28; Stim) or stimulation and Baf treatment (20 ng/ml; Stim + Baf); quantified MFI on the right using multiple Mann–Whitney tests (N = 3 independent repeats). (B) Representative histograms of CTLA-4 MFI in CD4 T cells of one healthy donor and P16 after 24 h of incubation in IL-2 media alone (Unstim), with stimulation (5 μg/ml plated anti-CD3 and 1 μg/ml soluble anti-CD28; Stim) or stimulation and Baf treatment (20 ng/ml; Stim + Baf); quantified MFI on the right using multiple Mann–Whitney tests (N = 3 independent repeats). (C) Quantified fold-change data from A, normalized to the unstimulated condition. Statistical significance calculated using Kruskal–Wallis test. (D) Quantified fold-change data from B, normalized to the unstimulated condition. Statistical significance calculated using Kruskal–Wallis test. (A–D)N = 3 independent experiments with eight healthy donor (HD) samples (eight individuals) and eight patient samples (five patients: P16, P17, P12, P13, and P2). P16 and P12 contributed longitudinal samples at multiple time points (red = P16; pink = P12), with only one sample per individual used in any single experiment. Each black dot represents a distinct healthy donor or patient sample. **P < 0.001. number represents fold change. ns: not significant.