Panel A shows a schematic workflow illustrating the in vitro CD80 transendocytosis assay and lysosomal inhibition strategy. Panel B shows flow cytometry plots comparing CD80 internalization and CTLA-4-associated transendocytosis between healthy control and patient cells. Panel C shows bar graphs quantifying CD80 transendocytosis percentages and mean fluorescence intensity of internalized CD80. Panel D shows proliferation histograms comparing stimulated CD4-positive T-cell responses in healthy control and patient samples. Panel E shows bar graphs depicting percentages of proliferating CD4-positive T cells under different culture conditions. Panel F shows histograms comparing CD25 expression levels on stimulated and unstimulated CD4-positive T cells. Panel G shows bar graphs summarizing mean fluorescence intensity values of CD25 expression across experimental conditions.
CTLA4 S172P/S172P variant severely impairs CD80 transendocytosis and drives CD4 + T cell hyperproliferation. (A) Schematic representation depicting the principle of the in vitro CD80 transendocytosis assay. (B) Representative flow cytometry plots showing CD80 internalization in cells from a HC versus the patient. (C) Quantitative bar graphs showing the percentages of CD80 transendocytosis and the MFI of CD80 uptake. (D) Representative histograms showing proliferation of CD4+ T cells isolated from HC and patient after 4-day coculture with mitomycin C–treated APC-Raji cells. (E) Bar graph depicting the percentages of proliferating CD4+ T cells. (F) Representative histograms of CD25 expression on CD4+ T cells after stimulation. (G) Bar graphs showing the mean MFI of CD25 expression on CD4+ T cells. In panel C, an unpaired t test was used to analyze significance. **P < 0.01. MFI, mean fluorescence intensity; APC, antigen-presenting cells; HC, healthy control, US, unstimulated; S, stimulated; CHO, chinese hamster ovary.