Impaired lymphocyte function and differentiation in CTPS1-deficient patients result from a hypomorphic homozygous mutation
Emmanuel Martin, Norbert Minet, Anne-Claire Boschat, Sylvia Sanquer, Steicy Sobrino, Christelle Lenoir, Jean Pierre de Villartay, Maria Leite-de-Moraes, Capucine Picard, Claire Soudais, Tim Bourne, Sophie Hambleton, Stephen M. Hughes, Robert F. Wynn, Tracy A. Briggs, Genomics England Research Consortium, Smita Patel, Monica G. Lawrence, Alain Fischer, Peter D. Arkwright, Sylvain Latour
Emmanuel Martin, Norbert Minet, Anne-Claire Boschat, Sylvia Sanquer, Steicy Sobrino, Christelle Lenoir, Jean Pierre de Villartay, Maria Leite-de-Moraes, Capucine Picard, Claire Soudais, Tim Bourne, Sophie Hambleton, Stephen M. Hughes, Robert F. Wynn, Tracy A. Briggs, Genomics England Research Consortium, Smita Patel, Monica G. Lawrence, Alain Fischer, Peter D. Arkwright, Sylvain Latour
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Research Article Immunology

Impaired lymphocyte function and differentiation in CTPS1-deficient patients result from a hypomorphic homozygous mutation

Abstract

Cytidine triphosphate (CTP) synthetase 1 (CTPS1) deficiency is caused by a unique homozygous frameshift splice mutation (c.1692-1G>C, p.T566Dfs26X). CTPS1-deficient patients display severe bacterial and viral infections. CTPS1 is responsible for CTP nucleotide de novo production involved in DNA/RNA synthesis. Herein, we characterized in depth lymphocyte defects associated with CTPS1 deficiency. Immune phenotyping performed in 7 patients showed absence or low numbers of mucosal-associated T cells, invariant NKT cells, memory B cells, and NK cells, whereas other subsets were normal. Proliferation and IL-2 secretion by T cells in response to TCR activation were markedly decreased in all patients, while other T cell effector functions were preserved. The CTPS1T566Dfs26X mutant protein was found to be hypomorphic, resulting in 80%–90% reduction of protein expression and CTPS activity in cells of patients. Inactivation of CTPS1 in a T cell leukemia fully abolished cell proliferation. Expression of CTPS1T566Dfs26X failed to restore proliferation of CTPS1-deficient leukemia cells to normal, except when forcing its expression to a level comparable to that of WT CTPS1. This indicates that CTPS1T566Dfs26X retained normal CTPS activity, and thus the loss of function of CTPS1T566Dfs26X is completely attributable to protein instability. This study supports that CTPS1 represents an attractive therapeutic target to selectively inhibit pathological T cell proliferation, including lymphoma.

Authors

Emmanuel Martin, Norbert Minet, Anne-Claire Boschat, Sylvia Sanquer, Steicy Sobrino, Christelle Lenoir, Jean Pierre de Villartay, Maria Leite-de-Moraes, Capucine Picard, Claire Soudais, Tim Bourne, Sophie Hambleton, Stephen M. Hughes, Robert F. Wynn, Tracy A. Briggs, Genomics England Research Consortium, Smita Patel, Monica G. Lawrence, Alain Fischer, Peter D. Arkwright, Sylvain Latour

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Figure 3

Functional studies of CTPS1-deficient T cells.

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Functional studies of CTPS1-deficient T cells. (A) Quantification of IFN...
(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.