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

Impaired Lymphocyte Function and Differentiation in CTPS1-deficient Patients Result From a Hypomorphic Homozygous Mutation

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Free PMC article

Impaired Lymphocyte Function and Differentiation in CTPS1-deficient Patients Result From a Hypomorphic Homozygous Mutation

Emmanuel Martin et al. JCI Insight. .
Free PMC article

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.

Keywords: Adaptive immunity; Immunology; Monogenic diseases; T cells.

Conflict of interest statement

Conflict of interest: Step Pharma has had a partnership agreement with Imagine Institute to support the development of CTPS1 inhibitors since 2015 and has financed the PhD program of NM since 2017.

Figures

Figure 1
Figure 1. Immunophenotyping of CTPS1-deficient patients.
(A) Percentages of αβ and γδ T cells, NK cells, B cells, monocytes, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs) in PBMCs are represented in dot plot graphs. Data were obtained from FACS analysis after cell-specific staining. Reference ranges for age-matched controls (3–18 years) correspond to 21%–98%, 0.9%–17%, 4%–22 %, 6%–24%, 2%–21%, 0.1%–7.8%, 0%–4.5%, 0.1%–3.1%, and 0.8%–0.36% for αβ T cells, γδ T cells, NK cells, B cells, classical CD14++CD16, intermediate CD14+CD16+, nonclassical CD14+CD16++ monocytes, mDCs, and pDCs, respectively. (B) Frequencies of naive (CD31+CD45RA+CCR7), central memory (CD45RACCR7+CD27), effector memory (CD45RACCR7+CD27), and exhausted effector memory/TEMRA (CD45RA+CCR7+CD27) compartments in CD4+ and CD8+ T cells are represented in the dot plot graph. Representative dot plot FACS analysis of naive and memory CD4+ cells (left) and CD8+ cells (right) are also depicted. (C) Frequencies of senescent cells (CD8+CD57+) gated on CD3+ cells are shown in the dot plot graph (left). Representative dot plot FACS analysis of senescent CD8+ cell populations are depicted at right . Data were obtained from FACS analysis of cell-specific staining. (D) Frequencies of naive (CD19+CD27), memory (CD19+CD27+) (left), circulating marginal zone (MZ) (CD19+CD27+IgM+IgD+) and switched B cells (CD19+CD27+IgMIgD) (right) are shown in the dot plot graph. Representative dot plots FACS analysis of naive/memory (left) and marginal zone/switched (right) B cell populations are depicted. Data were obtained from FACS analysis after cell-specific staining. In AD, black or red dots correspond to a healthy individual donor or a patient, respectively. Each circle represents an independent biological sample. Ten to 19 adult controls and 8 patients were analyzed. The horizontal bars represent the median. Data obtained from 6 independent experiments. Groups of values were compared 2 by 2 using Mann-Whitney U tests. *P < 0.05; **P < 0.01.
Figure 2
Figure 2. Absence of invariant T cells in blood of CTPS1-deficient patients.
(A) Percentages of CD127loCD25hi regulatory T cells (Tregs) in blood are shown in the dot plot graph (upper left). Data were obtained from FACS analysis after cell-specific staining. Representative dot plot of FACS analysis of Treg population is depicted (lower). Representative dot plot of FACS analysis of Foxp3 expression in CD127hiCD25lo mainstream T cells (black cell population) and CD127loCD25hi Tregs (red cell population) are shown (upper right). (B) Percentages of Vα7.2+CD161 and Vα24+CD161 mainstream T cells, Vα7.2+CD161+ mucosal-associated invariant T (MAIT) and Vα24+CD161+ invariant NK T (iNKT) cells in blood are shown in the dot blot graph (upper). Representative dot plot FACS analysis of MAIT cells (lower left) and iNKT cells (lower right) in control and patient blood are depicted. In AB, red and black dots represent independent biological samples of patients and controls, respectively. The horizontal bars represent the median. Data obtained from 3 independent experiments. Groups of values were compared 2 by 2 using Mann-Whitney U tests. ***P < 0.001.
Figure 3
Figure 3. Functional studies of CTPS1-deficient T cells.
(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.
Figure 4
Figure 4. Analysis of CTPS1 expression in lymphocytes subsets and activated T cells in control individuals.
(A) Dot plot graph representing the MFI of CTPS1 expression in αβ T cells (black circle), γδ T cells (blue circle), NK cells (green circle), B cells (red circle), and monocytes (gray circle). PBMCs were stimulated (right) or not (No stim) (left) with PMA plus ionomycin for 48 hours. (B) MFI of CTPS1 expression in T cell subpopulations: MAIT (red circle), iNKT (green circle), naive (circle), memory (square), CD45RA (up triangle) and CD45RA+ (down triangle), effector memory CD4 (black symbols), or CD8 (blue symbols). T cells were stimulated (middle) or not (upper) with 1 μg/mL of coated anti-CD3 antibody for 48 hours. CTPS1 MFI ratios of unstimulated and anti-CD3–stimulated T cells are represented in the lower panel. (C) Immunoblots for CTPS1, CTPS2, and actin protein expression in Jurkat cells and control T cell blasts stimulated or not with anti-CD3/CD28–coated beads for 48 hours, control (Ctrl), LCLs, primary fibroblast cell lines, SV-40 T antigen–immortalized fibroblast cell lines (SV40 Fibro), and telomerized SV40-transformed fibroblast cell lines (Tel. SV40 Fibro). (D) Immunoblots (upper) and quantitative PCRs (qPCRs) (lower) for CTPS1, CTPS2, and actin protein expression in T cell blasts stimulated with anti-CD3/CD28 beads for different periods. In lower panels, the bar graphs depict relative CTPS1 (left) and CTPS2 (right) mRNA expression normalized on GAPDH mRNA expression. Data were obtained from 3 independent experiments. In A–B, all MFIs were calculated from FACS histogram analysis (data not shown). Each symbol corresponds to an independent biological sample of a healthy individual. The horizontal bars represent the median ± SEM.
Figure 5
Figure 5. CTPS1 expression and CTPS activity in CTPS1-deficient cells from patients.
(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.
Figure 6
Figure 6. Expression of CTPS1D18 mutant partially restores the proliferation of CTPS1-deficient Jurkat cells.
(A) Histograms representing flow cytometry analysis of CTPS1 expression in WT (gray) or CTPS1-KO (red) Jurkat cells. Isotype staining is represented by dotted lines. (B) Immunoblots for CTPS1 and actin protein expression in lysates of WT, CTPS1-KO, or complemented CTPS1-KO Jurkat cell lines. CTPS1-KO Jurkat cells were complemented with lentivirus expressing GFP, CTPS1 plus GFP, CTPS1Δ18 plus GFP, or the empty lentivirus (shown with a slash). Black and red arrows indicate CTPS1 and CTPS1Δ18 proteins, respectively. (C) Analysis of WT (gray histogram) and CTPS1-KO (red histogram) Jurkat cell proliferation using cell trace violet staining. Cells were treated or not with 200 μM of cytidine for 4 days. Data shown are representative of 2 independent experiments. (D) Immunoblots for CTPS1, actin, and GFP protein expressions in lysates of CTPS1 and CTPS1Δ18 complemented CTPS1-KO Jurkat cell lines at different times of culture in the presence or not of cytidine. (E) Same as D except that GFP protein expression was quantified by flow cytometry and represented in histograms. (F) Percentage of GFP+ cells in cocultures of CTPS1-KO Jurkat cells complemented with GFP alone (green symbols), CTPS1Δ18 plus GFP (red symbols), or WT CTPS1 plus GFP (gray symbols) with noncomplemented CTPS1-KO Jurkat cells at a 1:10 ratio for 16 days. Cells were cultured with cytidine (left) or not (right). Percentage values were obtained from FACS analysis. Data shown are representative of 2 independent experiments.
Figure 7
Figure 7. Preserved enzymatic activity of CTPS1D18 protein.
(A) Immunoblots for CTPS1 and actin protein expression in CTPS1-KO Jurkat cell lines complemented with GFP, CTPS1Δ18 plus GFP, or CTPS1 plus GFP and maintained in culture with or without 200 μM of cytidine. (B) Percentages of GFP+ cells of a coculture of CTPS1-KO Jurkat cells complemented with GFP (green symbols), CTPS1Δ18 plus GFP (red symbols), or CTPS1 plus GFP (gray symbols) with noncomplemented CTPS1-KO Jurkat cells at a 1:10 ratio for 16 days. Coculture was supplemented with 200 μM cytidine (triangle) or not (circle). Complemented cells were preselected for high CTPS1 expression by cytidine starvation before the coculture. Percentage values were obtained from FACS analysis. The values represent the mean ± SEM of 2–3 independent experiments. (C) Dot plot graph representing the quantification of CTPS activity in lysates of WT Jurkat cells (black circle), CTPS1-KO Jurkat cells (white circle), CTPS1-KO Jurkat cells complemented with GFP (green circle), CTPS1Δ18 plus GFP (red circle), or CTPS1 plus GFP (gray circle). Complemented cells were preselected for high CTPS1 expression by cytidine starvation. Data from 3–5 independent experiments. Each symbol corresponds to the CTPS activity of an independent biological replicate. The horizontal bars represent the median ± SEM. Values were compared 2 by 2 using Mann-Whitney U tests. **P < 0.01; ***P < 0.001.

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