Abstract
Lymphocyte functions triggered by antigen recognition and co-stimulation signals are associated with a rapid and intense cell division, and hence with metabolism adaptation1. The nucleotide cytidine 5′ triphosphate (CTP) is a precursor required for the metabolism of DNA, RNA and phospholipids2,3,4. 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 known5,6,7. CTP synthase activity is a potentially important step for DNA synthesis in lymphocytes8,9. 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.
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Change history
25 June 2014
Nature 510, 288–292 (2014); doi:10.1038/nature13386 Owing to a production error, the vertical axis of the right panel of Fig. 3g was misaligned. The correct panel is shown below as Fig. 1 of this Erratum. In addition, the legends for Fig. 2 and Extended Data Fig. 3 should read “Induction of CTPS1 expression during T- and B-cell activation and defective proliferation of activated CTPS1-deficient T and B cells” and “Induction of CTPS1 expression in activated B cells and inhibitors of CTPS1 expression in activated T cells”, respectively.
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Acknowledgements
The authors thank the patients, their families and the healthy donors for cooperation. We thank S. Rigaud, S. Gérart, C. Synaeve and R. Rodriguez for help with experiments and P. Revy for discussion. This work was supported by grants from INSERM, ANR (ANR-08-MIEN-012-01, ANR-2010-MIDI-005-02 and ANR-10-IAHU-01), Fondation ARC (France), the European Research Council (ERC-2009-AdG_20090506 n°FP7-249816) and the Rare Diseases Fondation (France). S. L. is a senior scientist of CNRS (France). E. M. is supported by ANR (France) and Ligue contre le cancer (France). We are also grateful to the UK 1958 Birth Cohort (http://www2.le.ac.uk/projects/birthcohort) for providing DNA from 752 individuals born in the northwest of England. Access to these resources was enabled via the 58READIE Project funded by the Wellcome Trust and Medical Research Council (grant numbers WT095219MA and G1001799). A full list of the financial, institutional and personal contributions to the development of the 1958 Birth Cohort Biomedical resource is available at (http://www2.le.ac.uk/projects/birthcohort).
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E.M. performed experiments, analysed the data and participated in the writing of the manuscript. N.P., F.H., C.L., S.F., C.M. and S.S. performed experiments. A.F., S.L., S.S., J.S., C.P., P.N., J.M., N.J., C.M. and N.T. analysed the data. M.D.E., R.F.W. and P.D.A. identified the families and provided and analysed clinical information. S.L. and A.F. co-wrote the manuscript. S.L. designed and supervised the research.
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Extended data figures and tables
Extended Data Figure 1 Identification of a genetic CTPS1 defect in patients P1.1, P1.2 and P2.1.
a, Analysis of the single nucleotide variations (SNVs) detected by whole-exome sequencing in the genome of P1.1, P1.2 and P2.1. The numbers of SNVs are indicated in the triangles. SNVs were filtered by removal of non-functional intronic and synonymous mutations, heterozygous variations and those present in dbSNPs, 1000 genomes databases. The intersection of the filtered SNVs in the three patients resulted in the identification of a single common splicing site variation in the CTPS1 gene. b, Exon–intron structure and sequences of exons 17, 18 and 19 of CTPS1. The position of the variation is indicated by an arrow. The boxed nucleotide corresponds to the alternative splice site which produces a shorter transcript lacking exon 18 detected in patient cells. The alternative stop codon is indicated by an asterisk. c, Expression of a CTPS1 transcript lacking exon 18 (CTPS1Δ18) in CTPS1-deficient patients. The relative expression of full length CTPS1, CTPS1Δ18 and actin transcripts was examined by qRT–PCR in EBV-B cell lines (patient P2.1) and T-cell blasts (patient P1.2) from CTPS1-deficient patients. qRT–PCRs of actin are shown as normalization controls of the cDNA samples. Three fold-serial dilutions of cDNAs (indicated as 1, 0.3 and 0.1) were used for amplification of each transcript. Base pair markers are shown on the left. PCR products were verified by sequencing showing the expression of an abnormal CTPS1 transcript lacking exon 18 in the cells of the patients.
Extended Data Figure 2 Loss of CTPS1 expression and undetectable expression of the mutant CTPS1Δ18 protein in cells from CTPS1-deficient patients.
a, Transient expression of CTPS1 and the mutant CTPS1Δ18 in 293-T cells transfected with vectors containing wild-type CTPS1 or the mutant CTPS1Δ18. Cell lysates were tested by immunoblotting for CTPS1 with different antibodies raised against CTPS1 and for actin as a control for loading. The CTPS1Δ18 mutant protein is recognized by the rabbit polyclonal antibodies raised against the 341 to 355 (anti-341-355) or the 416 to 430 (anti-416-430) residues of CTPS1 but not by the rabbit polyclonal antibody K21. b, T-cell blasts from a healthy control (Ctr.) and the CTPS1-deficient patient P1.2 (P1.2) stimulated for 48 h with anti-CD3 were analysed for CTPS1 expression with the rabbit polyclonal antibodies anti-416-430 and anti-341-355. Actin expression as control for loading. c, EBV B-cell lines from healthy controls (Ctr. 1 and Ctr.2) and CTPS1-mutated patients (P1.2 and P2.1) were analysed for CTPS1 expression with the rabbit polyclonal antibody anti-416-430. Actin expression served as control for loading.
Extended Data Figure 3 Induction of CTPS1 expression in activated B cells.
a, Immunoblots for CTPS1 expression in sorted CD19+B cells (from PBMCs of an healthy donor) stimulated with the indicated stimuli. Actin was used as a loading control. b, Kinetics of CTPS1 mRNA expression monitored by qRT–PCR in sorted B cells that have been stimulated with anti-BCR+CpG. Expression is in arbitrary units (a.u.) normalized to the expression of the GADPH gene and leukocytes were used as calibrator. c, Immunoblots for CTPS1 expression in T-cell blasts (from an healthy donor) stimulated with anti-CD3/CD28 beads in the presence of selective inhibitors of NFκB, Src kinases, Ca2+, ERK kinase and PI3Kdelta. Actin was used as a loading control. The activity of the inhibitors was controlled in parallel (see Methods and data not shown).
Extended Data Figure 4 Analysis of proximal and late TCR activation responses in CTPS1-deficient cells.
a, Immunoblots showing the phosphorylation of proximal signalling molecules in T-cell blasts from a control donor (Ctr.) and a CTPS1-deficient patient P1.2 (P1.2) stimulated with anti-CD3 antibodies for 0, 2, 5, 15, 30 and 60 min or PMA plus ionomycin (P + I). Cell lysates were immunoblotted with antibodies against tyrosine-phosphorylated residues (PY), phosphoPLCG1 (pPLCG1), PLCG1, NFAT2c, phosphoPKCtheta (pPKCtheta), IkBa, phosphoERK1/2 (pERK1/2) and actin as a loading control. Molecular weights are on the left. Data correspond to one representative experiment of 2 or 3 independent experiments. b, Flow cytometry analyses of Ca2+-flux in T cells from PBMCs or T-cell blasts of a control donor (Ctr.) and a CTPS1-deficient patient P1.2 (P1.2) loaded with the Ca2+-sensitive fluorescent dye Indo-1. Cells were then stimulated with anti-CD3 antibodies (first arrow) crosslinked with rabbit anti-mouse antibodies (second arrow) and then incubated with ionomycin (third arrow) to induce a receptor-independent Ca2+ response. Intracellular Ca2+ levels are expressed in arbitrary units (a.u.). Data with the T-cell blasts correspond to one of three representative experiments. c, Analysis of the degranulation capacity of CD8+ T-cell blasts from two control donors (Ctr.1 and Ctr.2) and a CTPS1-deficient patient P1.2 (P1.2) stimulated with the indicated concentrations of anti-CD3 antibodies for 4 h. Cells were stained with antibodies against CD107a/b (LAMP1/2), a surface-exposed marker of the secretion of lytic granules, and then analysed by FACS. Means with s.d of percentages of CD8+ CD107+ cells are presented. d, Flow cytometry analysis of intracellular IL-2 production in CD4+ and CD8+ T cells from PBMCs of a control donor (Ctr.) and two CTPS1-deficient patients P1.2 and P2.2 (P1.2 and P2.2) stimulated for 36 h with anti-CD28 and anti-CD3 antibodies. The percentages of CD4+IL-2+ and CD8+IL-2+ are shown. e, Flow cytometry analysis of intracellular IFN-γ and TNF-α production on gated CD3+ T-cell blasts of a control donor (Ctr.) and a CTPS1-deficient patient P1.2 (P1.2) stimulated for 12 h with IL-2, anti-CD3 and anti-CD28 coated beads (anti-CD3/CD28), PMA plus ionomycin or PHA. Data are representative of one of 3 independent experiments. Dot-plots in red correspond to the isotype control. f, Induction of CD25 and CD69 in CD3+ T-cell blasts from a control donor (Ctr.) and a CTPS1-deficient patient (P1.2) was assessed after 24 h of anti-CD3 stimulation for CD69 and 96 h for CD25. Expression was assessed by flow cytometry and the median fluorescence intensity (MFI) is presented. Data are means with s.d of four and eight independent experiments for CD69 and CD25, respectively. Unpaired Student’s t-test. ***P < 0.001, *P < 0.05. g, Analysis of activation-induced cell death (AICD) in CD3+ T-cell blasts from a control donor (Ctr.) and a CTPS1-deficient patient P1.2 (P1.2,) after stimulation with the indicated concentration of anti-CD3 antibodies for 12 h. Apoptotic cells were detected by annexin V and 7-AAD staining and the percentages of annexin V positive/7-AAD negative cells within the gated CD3 population are shown. Data are means with s.d. of four (P1.2, n = 4) and eight (Ctr., n = 8) independent experiments. Unpaired Student’s t-tests and *P < 0.05.
Extended Data Figure 5 Decreased proliferation of TCR-stimulated T cells from patients P1.1, P1.2 and P2.2. and IL-2-expanded natural killer cells from patient P1.2.
a, Proliferation of CD3+ T cells from PBMCs of control donors (Ctr.) and CTPS1-deficient patients (P1.1, P1.2; left panels) or (P1.2, P2.2; right panels). Right panels and left panels correspond to 2 independent experiments. Cells were stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies during the course of 6 days. The proliferation was determined by dilution of CFSE staining analysed by flow cytometry. Histograms correspond to CFSE staining dilutions for which the number of cell divisions was indicated at the top of each peak. b, Proliferation of CD3+ T and CD16+CD56+ natural killer cells from PBMCs of a control donor (Ctr.) and a CTPS1-deficient patient (P1.2). Cells were stimulated with anti-CD3/CD28 coated beads for 3 days or IL-2 for 7 days. Representative dot plots showing cell divisions by dilution of the violet dye and expression of the activation marker CD69. Inserts with histograms showing the violet dye dilution with the number cell divisions indicated at the top of each peak.
Extended Data Figure 6 Decreased incorporation of thymidine, uridine, cytidine, leucine and asparte in CTPS1-deficient cells.
a, Incorporation of [3H]thymidine, [3H]uridine, [3H]cytidine and [3H]leucine as tracers of DNA, RNA and protein synthesis in PBMCs from a control healthy donor (Ctr.) and a CTPS1-deficient patient (P1.2) stimulated or not (no stim.) for 3 days with anti-CD3 or PHA and for 6 days with tetanus toxoid, candidin or tuberculin. The concentration of [3H]cytidine used in these experiments is under the value allowing the restoration of normal proliferation in CTPS1-deficient cells (also see Methods). Data are means with s.d of two independent experiments with triplicates. Unpaired Student’s t-tests and ***P < 0.001, **P < 0.01, *P < 0.05. b, c, Incorporation of [14C]aspartate and [3H]thymidine in PBMCs (b) or T-cell blasts (c) from a control healthy donor (Ctr.) and a CTPS1-deficient patient (P1.2) stimulated or not (no stim.) for 3 days with anti-CD3 or PHA. Data means with s.d of three independent samples.
Extended Data Figure 7 Decreased proliferation of Jurkat cells following shRNA-mediated CTPS1 downregulation.
a, Expression of CTPS1 in Jurkat cells transduced with lentiviral vectors containing two distinct CTPS1 shRNAs (Sh CTPS1 #1 or Sh CTPS1 #2) or a scrambled shRNA (Sh scramble). Cell lysates were analysed by immunoblotting for CTPS1, CTPS2 and actin protein expression. b, Proliferation of Jurkat cells, in which CTPS1 expression was silenced, was monitored as a function of the loss of GFP expression and compared with cells transduced with the scrambled shRNA. The percentages of GFP-positive cells were determined by flow cytometry at the indicated time points with ‘time 0’ corresponding to 48 h post-transduction.
Extended Data Figure 8 Measurements of nucleotide pools in CTPS1-deficient T cells and in B/EBV cell lines from patients.
a, Concentration of ATP, GTP, UTP and CTP in cell extracts of T-cell blasts stimulated with anti-CD3/CD28 coated beads or not (no stim.) from control healthy donors (Ctr.) or from patient P1.2. Control cells were treated or not with deazauridine for 24 h before and during stimulation or not. Representative data from 3 independent experiments. b, Same as in a with EBV B-cell lines from control healthy donors (Ctr.) and patients P1.1 (squares), P1.2 (circles) and P2.1 (triangles). For controls, each symbol corresponds to a different control cell line (from a different healthy donor). Representative data from two independent experiments with blinding during the measurements. Bars correspond to averages. Unpaired Student’s t-tests and *P < 0.05, **P < 0.01, ***P < 0.001. CTP data are also shown in Fig. 3g, h.
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Martin, E., Palmic, N., Sanquer, S. et al. CTP synthase 1 deficiency in humans reveals its central role in lymphocyte proliferation. Nature 510, 288–292 (2014). https://doi.org/10.1038/nature13386
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DOI: https://doi.org/10.1038/nature13386
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