Fig 2: Core fucosylation is required for the activation of CD4+ T cells. (A) Western blots analysis of T cell receptor (TCR). Whole cell lysates were resolved by SDS-PAGE on a 8% gel, transferred to a PVDF membrane, and probed with anti-TCRß Ab. Densitometric analysis of the bands of TCRß normalized against GAPDH. (B) Core fucose of N-glycan on TCRß in Fut8-/-CD4+ T cells was detected by LCA blot. Whole cell lysates were immunoprecipitated with an anti-TCRaß antibody. The immunoprecipitates were resolved by SDS-PAGE on a 8% gel, and probed with the LCA and anti-TCRß Ab. (C) Histograms of binding capacity with LCA. Core fucosylation level on the surface proteins of Fut8+/+CD4+ T and Fut8-/-CD4+ T cells investigated by FACS analysis. (D) Downregulation of phosphorylated Zap70 in Fut8-/-CD4+ T cells. Purified CD4+ T cells were serum-starved and were stimulated with anti-CD3/CD28 Abs for 5 min at 37°C. Cells were lysated in lysis buffer for 15 min on ice. Whole cell lysates were subjected to 10% SDS-PAGE. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are reported as the mean ± SD from three independent experiments (**p < 0.01; ***p < 0.001). (E) Loss of Fut8 reduced the CD4+CD69+ cells populations in the SPL after OVA immunization. SPLs were isolated from OVA-immunized and unimmunized mice (n = 5). Cells were stained with anti-CD69 and anti-CD4 Abs, and then detected by FACS. Data are reported as the mean ± SD from three independent experiments (*p < 0.05; ns, not significant). (F) Loss of Fut8 decreased the proliferation of CD4+ T cells. Purified CD4+ T cells were stimulated with anti-CD3e Ab-coated microbeads and anti-CD28 Ab for 0, 6, 24, and 48 h at 37°C. The growth rates of CD4+ T cell were detected by MTT assay. Data are reported as the mean ± SD from three replicate cultures (*p < 0.05; **p < 0.01). The absorbance related to the formazan dye level was measured with a microplate reader at 570 nm.

Fig 3: Lack of core fucosulation reduced interaction ability between T cell receptor (TCR) and pMHC-II and impaired the signaling via TCR. (A) ZAP70 phosphorylation of Fut8+/+OT-II T–B cells. (T) B cell conjugate formation was initiated by centrifuging together CD4+ T cells with or without 1 µg/mL OVA323–339-loaded B cells. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments. Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01). (B) ZAP70 phosphorylation of Fut8+/+OT-II CD4+ T cells with or without fucosidase treatment. Purified CD4+ T cells were treated with or without 100 mU Bovine Kidney Fucosidase, and then coincubated with 1 µg/mL OVA323–339 loaded Fut8+/+OT-II B cells for 30 min. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments. Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01; ***p < 0.001). (C) Compare of activation of Fut8+/+OT-II T + B cells and Fut8-/-OT-II T + B cells. CD4+ T cells were conjugated with OVA323–339 loaded B cell. After 30 min, the T–B cells were fixed and stained with anti-TCRß and anti-CD69 Abs. Data are shown as mean values ± SD (**p < 0.01). (D,E) Distribution of MHC on the T and B cells. The T–B cells were fixed, permeabilized, and stained with Abs to MHC-II (green). Data are from three separate experiments. The ratio of MHC II intensity at the T–B cell conjugate site relative to non-conjugate areas. Data are shown as the mean values ± SD (**p < 0.01). (F) FACS analysis of the conjugates between B cells and CD4+ T cells. B cells (OVA323–339-loaded or not loaded) were labeled with anti-MHC-II (FITC) and CD4+ T cells were labeled with anti-TCRß (PE-Cy5). T and B cells were quickly mixed and conjugated by a brief centrifugation step. They were then incubated at 37°C for 30 min. A representative staining profile with anti-MHC-II and anti-TCRß mAb is shown. The conjugate cells are double positive (MHC-II+TCRß+) cells. (G) Percentage of T–B cell conjugates (both MHC-II and TCRß positive cells) was calculated. Data are reported as the mean ± SD (**p < 0.01) in four independent experiments. (H) The secrition of IL-2 was downregulated in the culture media of Fut8-/-OT-II T–B cells. OD values were measured at 492 nm using a microplate reader. Data are reported as the mean ± SD (**p < 0.01) in three independent experiments. (I) Proliferation of Fut8+/+OT-II CD4+ T cells with OVA323–339 loaded Fut8+/+OT-II B cells. Fut8+/+OT-II CD4+ T cells were purified and labeled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE). Fut8+/+OT-II CD4+ T cells were cocultivated with 1 µg/mL OVA323–339 loaded Fut8+/+OT-II B cells for 48 h, the divided cells were analyzed by FACS. One representative experiment is shown. M1–M4 indicates daughter cell populations which have subsequently lost half of their CFSE signal.

Fig 4: C24D binding to CD45 triggers the CD45 signaling pathway: PBMCs from ten hospitalized patients, ± C24D treatment, were lysed, separated on 10% SDS and blotted with antibodies for the determination of phosphorylation of the tyrosine 505 and 394 in Lck, the tyrosine 493 in ZAP-70 and the tyrosine 174 in VAV-1. PBMCs from healthy volunteers served as controls. The percentage of phosphorylation was calculated individually vs. GAPDH as protein control. (A) Percentage change of protein phosphorylation in PBMCs from 10 patients, compared to 10 healthy donors, after addition of C24D for 5–60 min to patient PBMCs, or 24 h to PBMCs from healthy donor (values represent mean± SE, *p < 0.01, **p < 0.005, ***p < 0.0001). (B) Western blot results obtained in three representative patients for each phosphorylated protein, ± C24D.

Fig 5: Enhanced phosphorylation of PTPN22C129S targets and increased PKC? expression.(A, B) Lymph node cells were stimulated for indicated time points with anti-CD3 plus anti-CD28, and phosphorylation was detected via immunoblotting of total cell lysates with antibodies against activated Zap-70 (A) and Src kinases (B). Antibody against p-Src (Y416) binds to Lck Y394 and Fyn Y417 with the top band representing pFyn and the bottom band pLCK, see Davidson et al., 2016. Each lane reflects distinct wild-type/PTPN22C129S mice and unlabeled lanes show molecular weight markers. Quantification to the right was calculated as phosphorylation signal/total signal normalized to loading control. (C) Lymph node cells were cultured 24 hr with/without buthionine sulphoximine (BSO), a glutathione depletion agent, before in vitro activation with anti-CD3 plus anti-CD28 for indicated time points. Total lysate was analyzed for PKC? expression via immunoblotting. Representative image shown. Quantification to the right was calculated as phosphorylation signal/total signal normalized to loading control (WT = 6, PTPN22C129S = 8). (D) Volcano plots comparing the proteomic profile of sorted CD4+ T cells from wild-type and PTPN22C129S mice, which were stimulated with anti-CD3 plus anti-CD28 for 5 hr either with or without prior treatment (24 hr) with BSO. Four mice per group were used for proteomic analysis, and p-values were calculated by Welch’s t-test. Proteins that were significantly up/downregulated in PTPN22C129S mutants are highlighted in red. (E) A proposed model for redox regulation of PTPN22 in vivo: upon oxidative pressure within the cell, C227 of PTPN22 forms a disulfide bond with the back-door cysteine C129 as a means of protection from irreversible oxidation and inactivation. When the C227-C129 bond cannot be formed, as is the case in PTPN22C129S mutant, PTPN22 is less sensitive to reactivation by the thioredoxin system, which leads to increased T cell receptor (TCR) signaling and inflammation. Error bars represent mean ± SEM. Figure 6—source data 1.Uncropped Western blot images showing phosphorylation of ZAP70, Src, and PKC upon CD3/CD28 stimulation. Figure 6—source data 2.Raw images of ZAP70, Fyn, and LCK expression. Figure 6—source data 3.Raw images of pZap70 and pSrc expression. Figure 6—source data 4.Raw images of pPKC expression. Figure 6—source data 5.Raw data from proteomics analysis.