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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.

Cytidine 5'-triphosphate synthetase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. We have recently demonstrated that a deficiency in CTPS1 is associated with an impaired capacity of activated lymphocytes to proliferate leading to a combined immunodeficiency disease. In order to better document its role in immunomodulation, we developed a method for measuring CTPS activity in human lymphocytes. Using liquid chromatography-mass spectrometry, we quantified CTPS activity by measuring CTP in cell lysates. A stable isotope analog of CTP served as internal standard. We characterized the kinetic parameters V max and K m of CTPS and verified that an inhibition of the enzyme activity was induced after 3-deazauridine (3DAU) treatment, a known inhibitor of CTPS. We then determined CTPS activity in healthy volunteers, in a family whose child displayed a homozygous mutation in CTPS1 gene and in patients who had developed or not a chronic lung allograft dysfunction (CLAD) after lung transplantation. Linearity of the CTP determination was observed up to 451 μmol/L, with accuracy in the 15% tolerance range. Michaelis-Menten kinetics for lysates of resting cells were K m =280±310 μmol/L for UTP, V max =83±20 pmol/min and, for lysates of activated PBMCs, K m =230±280 μmol/L for UTP, V max =379±90 pmol/min. Treatment by 3DAU and homozygous mutation in CTPS1 gene abolished the induction of CTPS activity associated with cell stimulation, and CTPS activity was significantly reduced in the patients who developed CLAD. We conclude that this test is suitable to reveal the involvement of CTPS alteration in immunodeficiency., (© 2019 John Wiley & Sons, Ltd.)

Lymphocyte functions triggered by antigen recognition and co-stimulation signals are associated with a rapid and intense cell division, and hence with metabolism adaptation. The nucleotide cytidine 5' triphosphate (CTP) is a precursor required for the metabolism of DNA, RNA and phospholipids. CTP originates from two sources: a salvage pathway and a de novo synthesis pathway that depends on two enzymes, the CTP synthases (or synthetases) 1 and 2 (CTPS1 with CTPS2); the respective roles of these two enzymes are not known. CTP synthase activity is a potentially important step for DNA synthesis in lymphocytes. Here we report the identification of a loss-of-function homozygous mutation (rs145092287) in CTPS1 in humans that causes a novel and life-threatening immunodeficiency, characterized by an impaired capacity of activated T and B cells to proliferate in response to antigen receptor-mediated activation. In contrast, proximal and distal T-cell receptor (TCR) signalling events and responses were only weakly affected by the absence of CTPS1. Activated CTPS1-deficient cells had decreased levels of CTP. Normal T-cell proliferation was restored in CTPS1-deficient cells by expressing wild-type CTPS1 or by addition of exogenous CTP or its nucleoside precursor, cytidine. CTPS1 expression was found to be low in resting T cells, but rapidly upregulated following TCR activation. These results highlight a key and specific role of CTPS1 in the immune system by its capacity to sustain the proliferation of activated lymphocytes during the immune response. CTPS1 may therefore represent a therapeutic target of immunosuppressive drugs that could specifically dampen lymphocyte activation.