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 5

CTPS1 expression and CTPS activity in CTPS1-deficient cells from patients.

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CTPS1 expression and CTPS activity in CTPS1-deficient cells from patient...
(A) Immunoblots for CTPS1, CTPS2, and actin protein expression in lysates of control (healthy donor), mother, and patient T cell blasts stimulated with anti-CD3/CD28 beads for different periods. Data are representative of 4 independent experiments. The asterisks indicate CTPS1 signals that were not removed by stripping. Black and red arrows indicate CTPS1 and mutated CTPS1Δ18 proteins, respectively. (B) Same as A with lysates from LCLs of 4 controls and 3 patients. Data are representative of 2 independent experiments. (C) Same as A with lysates from primary fibroblasts (Prim. Fibro) or SV-40–transformed fibroblasts (SV40 Fibro) of 4 controls and 3 patients. Data were obtained from 2 independent experiments. (D) Bar graph represents the quantification of the mean of signal intensity of CTPS1 protein expression from immunoblots of 3 independent experiments. CTPS1 signal was normalized to actin protein expression in each sample. CTPS1 signal intensity was fixed to 100% in control cells. Quantification was done using ImageJ software (NIH). (E) Dot plot graphs representing the quantification of CTPS activity in lysates of resting T cells and stimulated T cells with anti-CD3/CD28 beads for 48 hours from 7 patients and 8 to 15 controls and LCLs and fibroblast cell lines from 3 patients. Patients and controls correspond to red and black circles, respectively. Data from 7 independent experiments for T cells and 2 experiments for LCLs and fibroblasts. The horizontal bars represent the median ± SEM. Groups of values were compared 2 by 2 using Mann-Whitney U tests. **P < 0.01; *** P < 0.001. The dotted line represents the threshold of CTPS activity detection.