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 (*).

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 (*).

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