Figure 2. RASGRP1deficiency causes defective TCR signaling and an aberrant immunophenotype.

(a–d) T cell immunophenotyping displaying relative proportions of CD4⁺ cells and CD8⁺ cells amongst CD3⁺ cells in lymphocyte gate (a), proportions of naive (CD45RA⁺CCR7⁺), central memory (CD45RA⁻CCR7⁺)T cells, effector memory (CD45RA⁻CCR7⁻) T cells and exhausted effector memory (CD45RA⁺CCR7⁻) T cells among CD8⁺ T cells (b) and CD4⁺ T cells (c), and the relative abundance of CXCR5⁺CD45RA⁻ cells and naive (CXCR5⁻CD45RA⁺) cells among CD4⁺ cells (d). Full gating strategy, Supplementary Figure 3. (e) Proliferation of CD4⁺ and CD8⁺ T cells at day 4 after stimulation with anti-CD3 and anti-CD28. (f) Expression of CD25 and CD69 24 h after stimulation with anti-CD3 and anti-CD28. (g) TCR V_β spectra-typing of the patient’s peripheral blood T cells. (h) Expression of perforin and granzyme B on the patient’s CD8⁺ T cells. (i) Killing of target cells by CD8⁺ T cells with (right) or without (left) OKT3. CTL, cytotoxic T lymphocytes. (j) Microscopy of intracellular Ca²⁺ flux in immortalized T cell lines following stimulation with anti-CD3, assessed by detection of the Fura-2 excitation ratio. (k) Cropped immunoblot analysis of total and phosphorylated (p-) ERK in primary T cells after stimulation with anti-CD3 and anti-CD28. (l) Quantification of the results in k. (m) Geometric mean (GM) of phosphorylated ERK in the patient’s CD8⁺ T cells and shipment control CD8⁺ T cells transfected to express a vector encoding wild-type RASGRP1 or a vector encoding green fluorescent protein (GFP) as control. Data are representative of eight (a), four (b,c), three (e,f,h,i,m) or two (d,g,j,k) independent experiments.