Fig. 3
DEF6 mutations affect CTLA-4 cycling. a Flow cytometric analysis of CTLA-4 expression in stimulated memory Tregs (CD4+CD45RA−FOXP3+CD25+), compared to unstimulated naïve Tconv as described in37. CTLA-4 expression in patients P1 and P3 was compared and normalized to respective healthy donor controls. Cells were stimulated for 16 h with anti-CD3/anti-CD28 antibody-coated beads. CTLA-4 expression after stimulation was reduced (P3) or unaltered (P1, n > 3). Data are overlaid with mean ± SD. Statistics: ****p < 0.0001, ns: p = 0.067 (Welch’s t test). b Schematic representation of CTLA-4 cycling assay performed on purified CD4 T cells. c–e CD4 T cells of P1 and P3 show reduced CTLA-4 cycling, compared to HD. Representative FACS traces of memory T cells of P3 (c) and time-course quantifications of cycling traces of memory Treg (d, CD4+CD45RA−FOXP3+) and memory Tconv cells (e, CD4+CD45RA−FOXP3−), normalized to respective total CTLA-4 expression of P1, P3 or HDs. Purified CD4 T cells were stimulated with anti-CD3/anti-CD28 antibody-coated beads for 16 h. Total stain was performed with standard intracellular staining. Cycling staining was performed by adding the labeled antibody before the cell harvest and incubation at 37 °C for the indicated times. Gating as in Fig. S3d. Representative of two independent blood shipments. Data are overlaid with mean ± SD. Source data of Fig. 3 are provided as a Supplementary Source Data file
