Haploinsufficiency in PTPN2 leads to early-onset systemic autoimmunity from Evans syndrome to lupus

Abstract

An exome sequencing strategy employed to identify pathogenic variants in patients with pediatric-onset systemic lupus or Evans syndrome resulted in the discovery of six novel monoallelic mutations in PTPN2. PTPN2 is a phosphatase that acts as an essential negative regulator of the JAK/STAT pathways. All mutations led to a loss of PTPN2 regulatory function as evidenced by in vitro assays and by hyperproliferation of patients' T cells. Furthermore, patients exhibited high serum levels of inflammatory cytokines, mimicking the profile observed in individuals with gain-of-function mutations in STAT factors. Flow cytometry analysis of patients' blood cells revealed typical alterations associated with autoimmunity and all patients presented with autoantibodies. These findings further supported the notion that a loss of function in negative regulators of cytokine pathways can lead to a broad spectrum of autoimmune manifestations and that PTPN2 along with SOCS1 haploinsufficiency constitute a new group of monogenic autoimmune diseases that can benefit from targeted therapy.

Graphical abstract

Figure 1.

Pedigrees and genetics of families carrying PTPN2 mutations. (A) Anatomopathological section of liver biopsy showing lesions of chronic hepatitis and cholangitis (Metavir A3F3) with an inflammatory infiltrate compatible with a lupus-type autoimmune etiology. Hematoxylin-eosin saffron staining: portal tract inflammatory infiltrate with lymphoid follicles (*) and piecemeal necrosis (arrows). HGVS, Human Genome Variation Society. (B) cDNA mutations, GnomAD allele frequency (v2.1), and CADD score. (C) Family pedigrees with probands indicated by A1, B1, C1, D1, E1, F1. Squares: males; circles: females; black: affected mutation carriers; grey: unaffected mutation carriers. WT: WT PTPN2 allele. Under each patient is the genotype at the specified locus. (D) Linear protein model showing the location of the described PTPN2 variants (represented in bold) together with previously published variants (in grey). Multiple alignments of PTPN2 orthologs from different species using the Clustal Omega software. The affected amino acid for Y126N is boxed in red. Residues corresponding to bipartite NLS are underlined. TC, T cell. (E) cDNA from EBV-transformed B cells of patient D1 and one HC were amplified around exon 2. Migration and sequencing of PCR products are represented along with the expected result of RNA translation with exon 2 skipping. Source data are available for this figure: SourceData F1.

Figure 2.

PTPN2 regulation and expression. (A) PTPN2 protein expression in human primary T (LT) and B lymphocytes (LB), NK cells, monocytes, Jurkat–T cell line, and EBV-derived control B cell lines (LCL). PTPN2 expression was analyzed using automated quantitative western blot. Relative expression of TC48 and TC45 isoforms in Jurkat–T cells is indicated in the lower panel (mean and SDs from five different experiments are represented). (B) PTPN2 expression in HEK293T cell transfected with different constructs. Protein expression was revealed with anti-HA (left panel) or anti-PTPN2 (right panel) antibodies and analyzed using automated quantitative western blot. Protein quantification was calculated on anti-HA expression normalized on total protein expression. Mean and SDs are shown and representative of at least three different experiments. (C) Endogenous PTPN2 protein expression in control and patient in vitro–activated T cells analyzed using automated quantitative western blot. Quantification was calculated on anti-PTPN2 expression normalized on total protein expression. Mean and SDs are shown and representative of three different experiments (ns, non-significant, *P < 0.05, **P < 0.01, one-way analysis of variance, Kruskal–Wallis test). (D) Real-time RT-QPCR assays of PTPN2 in control and patient in vitro activated T cells. Results represent the PTPN2 expression normalized to GAPDH expression, related to the expression of controls. Mean and SDs are shown and representative of at least three different experiments (ns, nonsignificant, *P < 0.05, **P < 0.01, ****P < 0.0001, one-way analysis of variance, Kruskal–Wallis test). All MW are in kDa. Source data are available for this figure: SourceData F2.

Figure S1.

PTPN2 expression following PBMC stimulation and intracellular localization. (A) PTPN2 protein in PBMCs stimulated 6 h with indicated cytokines or ligands was quantified using automated quantitative western blot. Protein quantification was calculated on anti-PTPN2 expression normalized on total protein expression. Mean and SDs are shown and representative of three different experiments. All MW are in kDa. (B) PTPN2 mRNA was measured in control PBMCs stimulated 6 h with indicated cytokines or ligands. Mean and SDs are shown and representative of three different experiments. (C) Confocal microscopy analysis of PTPN2 subcellular localization. Constructs were overexpressed in HEK293T cells and detected with anti-HA antibody together with endoplasmic reticulum and nucleus staining. Source data are available for this figure: SourceData FS1.

Figure 3.

Effects of mutations on PTPN2 phosphatase activity and JAK-STAT signaling. (A) Tyrosine PTPN2 phosphatase activity of immunoprecipitated WT or mutant PTPN2 toward fluorescent tyrosine-phosphorylated STAT1 or STAT3 peptide by RP-UFLC. Mean and SDs are shown and represent three different experiments (ns, nonsignificant, *P < 0.05, ***P < 0.001, Paired t test). (B) STAT1 transcriptional activity following 24-h activation with IFN-α of HEK293T cells expressing IFN-sensitive responsive element (ISRE) and transfected with the indicated constructs (left panel). Mean and SDs are shown and representative of at least five different experiments in triplicate STAT3 transcriptional activity following 24-h activation with IL-6 of HEK293T cells expressing STAT3 responsive element and transfected with the indicated constructs (right panel). Results were normalized on Renilla. Mean and SDs are shown and representative of at least three different experiments in triplicate (ns, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test). (C) PTPN2 protein expression in Jurkat–T cells and PTPN2 Crispr KO Jurkat–T cells. PTPN2 expression was quantified using anti-PTPN2 antibody and automated quantitative western blot (left panels). Mean and SDs are shown and representative of three different experiments. PTPN2 constructs were re-expressed in Jurkat–T cells following lentiviral transduction and protein expression was verified using anti-HA antibody and automated quantitative western blot (right panels). Mean and SDs are shown and representative of five different experiments. (D) Modified Jurkat–T cells were stimulated with IFN-α or IFN-γ as indicated and phosphorylation of STAT1 was measured by intracellular staining and analyzed by spectral cytometry. pSTAT1 phosphorylation in Jurkat transduced with PTPN2 WT was used as reference. Histograms representative of one experiment out of four are illustrated on the right of each quantification (ns, nonsignificant, *P < 0.05, **P < 0.01, one-way ANOVA, Kruskal–Wallis test). All MW are in kDa. Source data are available for this figure: SourceData F3.

Figure 4.

Study of JAK/STAT signaling pathway response in in vitro–activated T cell from patients. (A) Phospho-STAT5 analysis at steady state (NS) in CD4⁺ T cells and CD8⁺ T cells from HCs (blue bars) or patients (grey bars) after ex vivo stimulation of whole blood with IL-2 (10⁴ U/ml) for 15 min. Phospho-STAT1 analysis at steady state (NS) in CD14⁺ monocytes from HC (blue bars) or patients (grey bars) after ex vivo stimulation of whole blood with IFN-γ (10⁴ U/ml) for 15 min. Mean and SDs are shown and representative of four different experiments, with a total height of HCs. P values were obtained with two-way ANOVA: <0.0001 (****), 0.0002 (***), 0.0021 (**), 0.1234 (ns). (B) Phosphorylation of STAT5 was assessed by intracellular staining in in vitro–activated T cells from HC and patients without stimulation (left histograms), upon 15 min IL-2 stimulation (250 U/ml) (middle histograms) or 2 h following IL-2 removal after IL-2 stimulation (250 U/ml) (right histograms). Representative flow cytometry histograms are shown on the left. Normalized pSTAT5 mean fluorescence intensity (MFI) to the mean of controls from the day of the experiment for each condition, n = 7 patients, n = 13 controls. The results were obtained from five separate experiments. P values were obtained with two-way ANOVA: <0.0332 (*), 0.1234 (ns). NS, nonstimulated. (C) Proliferation of in vitro–activated T cells stimulated or not with different concentrations of IL-2 for 4 days. T cell proliferation was determined from the level of CellTrace Violet (CTV) dye dilution. Representative histograms (left panel) show cell divisions of activated CD8 T cells from HCs or PTPN2 patients as indicated. Percentage of CD8⁺ dividing cells from HCs and patients (right panel). Dividing cells represent cells having undergone at least one division (data pooled from n = 5 independent experiments including a total of 18 HCs and 7 patients). P values were determined in a two-way ANOVA: <0.0002 (***), 0.0021(**), and 0.1234 (ns). (D) Proliferation of patients’ IL2-stimulated activated T cells with or without incubation with JAK1/JAK3 inhibitor tofacitinib for 4 days (data pooled from n = 2 independent experiments including a total of seven HCs and seven patients). P values were determined in a two-way ANOVA: <0.0332 (*).

Figure S2.

Proliferation of patients’ T cells following TCR activation. Proliferation of activated T cells stimulated or not with different concentrations of anti-CD3–coated beads (OKT3) for 4 days. T cell proliferation was determined from the level of CellTrace Violet dye dilution. Percentage of CD8⁺ dividing cells from HCs and patients. Dividing cells represent cells having undergone at least one division (data pooled from n = 5 independent experiments including a total of 13 HCs and 7 patients). P values were determined in a two-way ANOVA (ns: not significant). NS, nonstimulated.

Figure 5.

Global cytokine responses in in vitro–activated T cells from patients. (A) RNA-seq analysis of in vitro–activated T cells from patients (n = 5) compared with HCs (n = 3) in unstimulated condition or upon IL-2 or IFN-α stimulation for 3 h. Significant pathways enrichment in patients (A1, A2, B1, C1, and D1) on a list of 36 pathways from MSigDB database are represented. NS, nonstimulated. (B) Significantly upregulated pathways from NCI Nature database in in vitro–activated T cells from patients (n = 5) (A1, A2, B1, C1, and D1) as compared with HCs (n = 3) after IL-2 stimulation (10⁴ U/ml for 3 h). *P < 0.05, **P < 0.01. (C) Heatmap of the normalized gene expressions found upregulated in the signaling events mediated by PTPN2 for patients (A1, A2, B1, C1, and D1) and HC cells after IL-2 stimulation. (D) Heatmap of cytokine dosage in supernatant of resting or activated T cells stimulated by IFN-α or IL-2 (10⁴ U/ml) for 24 h. Z-score of log10 cytokine levels normalized to HC means in indicated stimulation conditions. Data from n = 2 independent experiments including a total of 13 HCs and 6 patients.

Figure 6.

Immunophenotyping of circulating patients’ cells and cytokine signatures. (A) Representative UMAP plots were generated from immunophenotyping CyTOF data of whole blood from adult controls (n = 4), pediatric controls (n = 5), and patients’ cells (B1, C1, D1 and F1). At the day of prelevement, the B1 patient is considered as pediatric (9 years old) while patients C1, D1, and F1 were adults (22, 26, and 20 years old, respectively). pDC: plasmacytoid dendritic cell; mDC: myeloid dendritic cell; CM: central memory; EM: effector memory; TEM: terminal effector memory; TEMRA: terminally differentiated effector memory; MAIT: mucosal-associated invariant T cell; GD: gamma delta. (B) Quantification of immune cell populations in adult (n = 4) or pediatric (n = 5) control or patient (B1, C1, D1 and F1) groups. Patient’s age group is indicated on the graph’s legend (A: adult; P: pediatric). P values were obtained using Kristal–Wallis test: <0.0332 (*), 0.1234 (ns). (C) Heatmap of cytokine dosage in plasma of HCs (n = 7) and patients with STAT1 GOF (n = 3), STAT3 GOF (n = 2), SOCS1 LOF (n = 3), and PTPN2 mutation (n = 5). Z-score of log10 cytokine levels normalized to HC mean. D1 received corticoids and B1 Imurel before sampling.

Figure S3.

Quantification of immune subsets defined following CyTOF analysis. Barplot showing the proportion of each identified immune subset across all groups (x-axis) with dots shaped and colored by patient identification and bar colored by groups. Indicated proportion is the frequency among all singlet viable PBMCs. A, adult; P, pediatric. P values were obtained using Kruskal–Wallis test: <0.0332(*), and 0.1234 (ns). DN: double negative; cDC: conventional dendritic cell; CM: central memory; EM: effector memory; TEM: terminal effector memory; TEMRA: terminally differentiated effector memory; pDC: plasmacytoid dendritic cell; GD: gamma delta.