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. 2020 Mar 12;5(5):e133880. doi: 10.1172/jci.insight.133880

Figure 3. Functional studies of CTPS1-deficient T cells.

Figure 3

(A) Quantification of IFN-γ, TNF-α, IL-17A, granzyme B, IL-4, and IL-2 in supernatants of activated T cell blasts from 10 healthy donors and patients using coated beads array technology. T cell blasts were stimulated or not with 1 μg/mL of anti-CD3 (OKT3) antibody, anti-CD3/CD28 beads, or PMA plus ionomycin for 48 hours. Data were obtained from 4 independent experiments. (B) Flow cytometry analysis of IFN-γ and IL-2 intracellular detections gated on CD3+ T cell blasts from healthy donors and CTPS1-deficient patients. T cell blasts were stimulated or not with 1 μg/mL of anti-CD3 (OKT3) antibody, anti-CD3/CD28 beads or PMA plus ionomycin for 12 hours. Representative dot plots of FACS analyses corresponding to IFN-γ and IL-2 stainings after PMA stimulation of control and patient T cell blasts are depicted (lower left and right panels, respectively) with isotype staining (upper panels). Frequencies of IFN-γ– and IL-2–secreting T cell blasts are shown in the dot plot graphs (lower left and right panels, respectively). Data were normalized on isotype staining and obtained from 4 independent experiments. (C) Expression of CD25-based mean fluorescence intensity (MFI) calculated from FACS histograms following anti-CD3 antibody or anti-CD3/CD28 bead stimulation of control and patient T cell blasts for 96 hours. Graph from 5 independent experiments. (D) Index values of proliferation of CD3+ T cell blasts from controls and CTPS1-deficient patients after anti-CD3/CD28 beads (left) or coated anti-CD3 antibody (right) stimulation for 4 days. The index value was obtained from histogram analyses of cell trace violet staining of healthy donors and patient T cells using FlowJo software. Data were obtained from 1 experiment. (E) Same as D, except that stimulated CD3+ T cell blasts were cultured in medium supplemented or not with cytidine (200 μM). (F) Analysis of control (black histogram) and patient (red histogram) T cell blast proliferations using cell trace violet staining. Cells were stimulated with anti-CD3 antibody for 4 days and supplemented or not with IL-2 (100 U/mL) or cytidine (200 μM). Histogram analysis was performed using FlowJo software. Data shown are representative of 3 independent experiments. In A–E, red and black dots represent independent biological samples of patients and controls, respectively. The horizontal bars represent the median. Group of values were compared 2 by 2 using Mann-Whitney U tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.