Figure 3. T‐cell proliferation in response to CD3‐TCR activation is defective in RASGRP1‐deficient T cells.

1. Representative dot plots showing cell divisions by dilution of the CellTrace violet dye and expression of CD25 of control donor (Ctr.) or RASGRP1‐deficient T‐cell blasts gated on CD3⁺ cells of patient P1.1 (Pat.) stimulated with incremental doses of anti‐CD3 antibody or with anti‐CD3/CD28‐coated beads. Lower panels represent histograms from the dot plots of patient and control that have been overlaid. Each peak of the histograms corresponds to a cell division. One representative of four independent experiments from three different blood samples.
2. Representative dot plots of cell cycle progression of control (Ctr.) and RASGRP1‐deficient T cells of patient P1.1 (Pat.) stimulated with two concentrations of anti‐CD3 antibody. The percentages of cells in each stage are indicated. Representative data from one of two independent experiments from two different blood samples.
3. Proliferation of T cells in which RASGRP1 expression was silenced with plasmids containing shRNA for RASGRP1 (Sh RASGRP#1 or Sh RASGRP1#2), CTPS1 (Sh CTPS1), or scramble shRNA (Sh scramble) with GFP gene reporter. Immunoblots for RASGRP1 and actin as a loading control of transduced cell cultures with shRNA for RASGRP1 (#1 and #2), CTPS1, or scramble RNA at day 1 after the infection.
4. Curves showing the percentage of GFP‐positive cells at different days in long‐term expansions after CD3/CD28 stimulation at day 0. One representative of four independent experiments from blood samples of different healthy donors.
5. Representative histograms of CellTrace violet dye dilution showing the cell divisions of GFP‐positive (GFP⁺) and GFP‐negative (GFP^‐) cells after anti‐CD3 restimulation with histograms in gray corresponding to unstimulated cells (unstim.). One representative of two independent experiments.

Source data are available online for this figure.