Fig 2: Enhanced activation and proliferation of PTPN22C129S T cells.(A) Gene expression of PTPN22 in splenocytes shown as fold change over WT. (B) Immunoblot analysis of PTPN22 expression in sorted CD4 T cells either unstimulated or activated in vitro with 1 µg/ml anti-CD3/CD28 for 24 hr. Each lane shows cells from a distinct mouse, and relative expression to the right was calculated by normalizing to loading control (actin). (C, D) Analysis of T cell populations in naïve mice via flow cytometry. (C) Analysis of double negative (DN), double positive (DP), and single positive CD4 and CD8 thymic populations in 10-week-old littermate mice as well as expression of TCRb, CD5, and CD69 on various subsets. (D) Frequencies of activated (CD44+/ CD69+) and regulatory (FOXP3+) CD4+ T cells in thymus and secondary lymphoid organs. (E) Intracellular calcium measurement in CD4 T cells at baseline, after stimulation with anti-CD3, and ionomycin to achieve maximum Ca2+ influx via staining with Fluo4 and FuraRed. Shown is a representative image of the change in ratio of fluo4 to FuraRed expression over time (blue: wild-type; red: PTPN22C129S). Quantification to the right shows slope value that describes how fast the peak of Ca2+ influx is reached (Bajnok et al., 2013). (F) Proliferation of CD4+ T cells as assessed by CellTrace Violet dilution after 96 hr in vitro stimulation with 1 µg/ml anti-CD3/CD28. Proliferation Index is the total number of divisions divided by the number of cells that went into division. Representative proliferation peaks on the left (clear: wild-type; black: PTPN22C129S) and quantification on the right (ctrl refers to unstimulated samples). (G, H) Ex vivo-stimulated CD4 T cells were assessed for CD69 expression/IFNγ production after stimulation with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin, respectively. Representative gating shown. Error bars represent mean ± SEM. Figure 5—source data 1.Uncropped Western blot images showing PTPN22 expression in CD4+ T cells. Figure 5—source data 2.Raw images of PTPN22 expression in CD4+ T cells.

Fig 3: PTPN22 is essential for THY1 to inhibit SRC activation. Coomassie staining of immunoprecipitated and total lysates of cell lines as indicated. Arrows indicate (A) PDGF-Rβ and (B) PTPN22, identified by mass spectrometry and validated with co-IP. The interaction of THY1 with PDGF-Rβ and PTPN22 were verified with co-IP analysis in HK1 cells in (C), and NP460 cells in (D). (E) Chamber invasion assay for SRC and PTPN22 siRNA depletion in HONE1-VA, HONE1-THY1, HK1-VA, and HK1-THY1 cells. *: p < 0.05. (F) Western blot analysis of SRC and PTPN22 in siRNA knockdown cells. (G) Representative images of the invaded HONE1-VA and HONE1-THY1 cells with SRC or PTPN22 knockdown. (H) IF staining of adherens junctions (merged images of green E-cadherin and red β-catenin) for cell lines described in Figure 4G. (I) Chamber invasion assay using PDGF-BB as a chemoattractant for HONE1-VA and HONE1-THY1 cells, HK11.19 and THY1-knockdown HK11.19 cells. *: p < 0.05. (J) Co-IP analysis of phospho-SRC in the presence of PDGF-BB in HONE1. (K) Western blot analysis of pSRC (Y419) and total SRC in THY1-expressing cells in response to PDGF-BB. (L) Schematic illustration of how THY1 can regulate EMT and SRC activity through the interaction with PTPN22 and PDGF-Rβ.

Fig 4: HDAC10 up-regulates ERK1/2 phosphorylation by inhibiting PTPN22(A) Real-time PCR analysis shows that HDAC10 regulates the mRNA levels of several members of the PTPN family. (B) Western blot analysis showing that HDAC10 regulates PTPN22 protein expression. (C) Quantitative analysis of the Western blots shown in panel B. (D, E) Results of the chromatin immunoprecipitation assay (ChIP assay). (D) HDAC10 binds to the PTPN22 promoter. (E) HDAC10 decreases the acetylation of histones H3 and H4 at the PTPN22 promoter. Acetylated histone H3 and H4 antibodies were used to precipitate DNA in the ChIP assay. Primers that target the transcription factor binding sites in the PTPN22 promoter were used for quantitative real-time PCR. The data were obtained from three independent experiments. Reduced acetylation of H3 and H4 blocks the binding of RNA polymerase II to the transcription factor binding sites. DNA was precipitated from HUVECs in the control group and the HDAC10 overexpression group using an anti-RNA polymerase II antibody in the ChIP assay. Data are presented as the means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control or scrambled control.

Fig 5: Protein tyrosine phosphatase non-receptor 22 (PTPN22) knockout (KO) and risk edited T cells are more likely to secrete pro-inflammatory cytokine in response to low-avidity TCR engagement.Cord blood CD4 T cells from human donors were edited as described in Figure 3, then stimulated with peptide loaded APCs for 3 days. (A–B) Representative flow plots of IFNy (A) and IL-2 (B) expression in mock edited or PTPN22 KO cells transduced with L-TCR and summary data of %L-TCR+ cells that secrete IFNy (A) or IL-2 (B) grouped by gene edit. N=5–7 independent donors (shapes correspond to individual donors). All summary data analyzed by paired t test. Columns and error bars represent mean ± SEM. All data is from four independent experiments. *p<0.05, ***p<0.001. Figure 5—source data 1.Peptide stim cytokine production, protein tyrosine phosphatase non-receptor 22 (PTPN22) edited Cells.