Supplementary Figure 2: Functional characterization of T cells.
From: RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics
(a) Invariant natural killer T (iNKT) cells. A prominent TCRvα24-expressing cell population was detectable in patient peripheral blood, however these cells expressed CD8 and CD45RA surface markers, suggesting that they belong to oligoclonally expanded exhausted memory CD8 T cells. HD – healthy donor; SC – Shipment control. (b) Proliferative response of T cells determined by [3H]-thymidine incorporation assay after stimulation with various stimuli after 3 days (OKT3, anti-CD3 antibody (clone OKT3)); PMA, phorbol 12-myristate 13-acetate; SEA: Staphylococcal enterotoxin A; SEB: Staphylococcal enterotoxin B; PHA: phytohaemagglutinin). (c) Proliferation of Jurkat T cells upon shRNA-mediated knockdown of RASGRP1. shRNA against Renilla luciferase was used as negative control. Cells were labeled with violet proliferation dye and analyzed by flow cytometry over the period of 5 days. (d) TCRVb spectratyping of patient and control T cells indicating oligoclonality of the TCR repertoire of the patient. (e) Calcium flux of Jurkat T cells upon inducible shRNA-mediated knockdown of RASGRP-1. shRNA against Renilla luciferase was used as negative control. (f) Fluorescence microscopy of Ca2+-flux of one representative cell is displayed below the graph. Scale bar represents 10 μm. HD–healthy donor; DIC–differential interference contrast. (g) ICAM Ring formation and cSMAC exclusion of hTERT immortalized patient and control T cell lines on a lipid bilayer following OKT3 stimulation. (h) Cropped immunoblot showing downstream TCR signaling in expanded patient and shipment T cells upon CD3/CD28 stimulation. Cells were starved and restimulated with anti-CD3/anti-CD28 antibodies and subsequently analyzed for ERK1/2 phosphorylation. Data is representative of three (h), two (a,c,d,g) or one (b,f,e) independent experiment.
