Figure 3. CTPS1 is required for proliferation of T-cells in response to TCR-CD3 activation.
a, Proliferation of T-cells in which CTPS1 expression was silenced with vectors containing shRNA for CTPS1 (Sh CTPS1#1 or Sh CTPS1#2) or containing a scramble shRNA (Sh scramble) with GFP gene reporter. Representative dot plots of GFP+ cells corresponding to transduced cells (left upper panels). Representative histograms of violet dye dilution showing the cell divisions after stimulation (left lower panels). Curves showing the percentage of GFP+ transduced cells in long-term expansions after repeated stimulation (middle panel). Immunoblots for CTPS1 and CTPS2 expression in transduced cells (right panels) and ACTIN as loading control. One representative of two experiments. b,c, Proliferation of control (Ctr.) and CTPS1-deficient T-cells (patient P1.2) transduced by empty or wild-type CTPS1-containing vector. Representative histograms of violet dye dilution (b, left panels). Means of indexes of cell division after stimulation (b, right panels) from triplicate of one representative of two experiments. Curves showing the percentage of GFP+ tranduced cells same as in (a) (c, left panel). Representative data from one of 2 independent experiments. Immunoblots same as in (a) (c, right panels). d, Representative histograms of violet dye dilution showing cell divisions of control (Ctr.) and CTPS-1-deficient T cells (P1.2) incubated with the indicated nucleotides or nucleosides before stimulation. Data from one of 3 independent experiments. e, Incorporation of 14C-aspartate, a tracer of the de novo pyrimidine nucleotide synthesis and 3H-thymidine as a control of proliferation/DNA synthesis. T cells were labelled during stimulation. Means of incorporated radioactivity (cpm) (n=6) f, Same as (d) excepted that control T-cells were incubated with deazauridine. Data from one representative of 3 independent experiments. g, Concentration of CTP in control T-cells incubated with deazauridine (Ctr.+Deaza., n=4) or not (Ctr., n=6) and CTPS1-deficient cells (P1.2, n=3) after stimulation with anti-CD3/CD28 coated beads. Data from 3 independent experiments. h, Concentration of CTP in cell extracts of EBV B-cell lines from healthy controls (Ctr.; n=6), and CTPS1-deficient patients transduced (Pat.+CTPS1, n=4) or not (Pat., n=6) with wild-type CTPS1-containing vector. P1.1 (squares), P1.2 (circles) and P2.1 (triangles). For controls, each symbol corresponds to cells of a different donor. Data from 2 independent experiments. i, Proliferation of CTPS1-deficient EBV B-cell lines (P1.2 and P2.1) transduced by empty or wild-type CTPS1-containing vector. Curves showing the percentage of GFP+-tranduced cells in culture. Unpaired t-tests and ***P<0.001, **P<0.01, *P<0.05 (b, e, g, h).