Additional file 1 of IL-39 promotes chronic graft-versus-host disease by increasing T and B Cell pathogenicity
Creators: name: Lv, Kangkang
name: Hu, Bo
name: Xu, Mingzhu
name: Wan, Li
name: Jin, Ziqi
name: Xu, Mimi
name: Du, Yuanyuan
name: Ma, Kunpeng
name: Lv, Quansheng
name: Xu, Yang
name: Lei, Lei
name: Gong, Huanle
name: Liu, Haiyan
name: Wu, Depei
name: Liu, Yuejun
Publisher: name: figshare
Year: 2022
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Table S1. Sequences of primers used. Table S2. Clinical characteristics of the patients. Table S3. Antibodies used in flow cytometry. Table S4. Primary data of CXCL13 and IL-39 concentrations in patients. Figure S1. Relative expression of IL-23p19 and EBi3 in spleen and target tissues of cGVHD mice. Irradiated BALB/c recipients were infused with 1×107 bone marrow cells and 1×106 splenocytes from the C57BL/6 mice. Mice infused with bone marrow cells were used as the controls. The expression of IL-39 in the lungs, liver, small intestine, and spleen of recipients was quantified by qPCR on days 30, 40, and 50 after transplantation (n=3, each group and time point). Data are representative of at least three independent experiments. Values are presented as mean ± SEM. **P&lt; 0.01, ***P&lt; 0.001. Figure S2: Survival of transduced flag-tagged IL-39 mice in scleroderma and lupus-like cGVHD models. Scleroderma-like (A) and lupus-like (B) cGVHD models were established. The survival of mice was observed for 56 days. Survival was assessed using the Kaplan-Meier method and compared using the log-rank test. Figure S3: IL-39 promotes the activation of T cells in the lupus-like cGVHD mice model. Irradiated BALB/c recipients were infused with 5×106 bone marrow cells and 4×107 CD25- splenocytes from the DBA/2 mice. Splenocytes (n=6 per group) were collected and stained for FACS analysis 8 weeks post transplantation. The percentages and numbers of CD4+T, CD69+CD4+T, CD8+T, and CD69+CD8+T cells in lymphocytes from the spleens of the recipients are shown (A). Lymphocytes were isolated from the spleens of recipients and treated with PMA, brefeldin A, and ionomycin for 4-6h. The percentage and number of TNF-α-(B), IL-4-(C) positive T cells and Tregs (D) in the spleens of recipients are shown. Values are presented as mean ± SD. *P&lt; 0.05. Figure S4. Anti-IL-39 antibody suppressed secretion of the pro-inflammatory cytokines in vitro. CD3+T cells were isolated from the spleens of C57BL/6 mice using magnetic bead sorting. T cells were stimulated with anti-CD3 and anti-CD28 and then treated with PBS, rIL-39, or anti-IL-39 antibody for 72h. The percentage of cytokines in the CD3+T cells is shown. Data are representative of at least three independent experiments. Values are presented as mean ± SD. *P&lt; 0.05. Figure S5: Effect of IL-39 blockade on scleroderma-like cGVHD development. Irradiated BALB/c recipients (n=4 each group) were infused with 1×107 bone marrow cells and 1×106 splenocytes from the C57BL/6 mice. Fourteen days after transplantation, each mouse in the antibody group received 100μl (100μg) of IL-39 antibody, while each mouse in the control group received 100μl (100μg) of isotype control antibody via intraperitoneal injection twice a week for 6 weeks. The overall survival (A), body weight (B) and GVHD scores (C) are shown. Values are presented as mean ± SEM. *P&lt; 0.05; ***P&lt; 0.001. Figure S6: Effect of IL-39 blockade on cGVHD development and immune cells in the target tissues. Irradiated BALB/c recipients (n=4 each group) were infused with 1×107 bone marrow cells and 1×106 splenocytes from C57BL/6 mice. Fourteen days after transplantation, each mouse in the antibody group received 100μl (100μg) of IL-39 antibody, while each mouse in the control group received 100μl (100μg) of isotype control antibody via intraperitoneal injection twice a week for 6 weeks. The percentage and number of CD8+TNF-α+cells and CD4+IL-4+ T cells in the intestine (A), the numbers of CD69+CD4+T cells and CD69+CD8+T cells in the liver (B) and the percentage and number and of CD8+T cells in the lungs (C) are shown. Values are presented as mean ± SEM. Figure S7: Phosphorylation of STAT1 and total STAT1 was detected by western blotting in sorted primary T cells from the mice. Plates were coated with 2 mg/ml anti-CD3 and 0.4 mg/ml anti-CD28 Abs overnight. T cells (2×105 T cells were cultured with various concentrations of recombinant mouse IL-39 proteins for 72h. The phosphorylation of STAT1 and total STAT1 detected by western blotting in sorted primary T cells from mice is shown. The data are representative of three independent experiments. Figure S8: Effects of IL-39 blockade on IFN-γ and IL-17A expression in CD4+ and CD8+ donor T cells in the spleens of cGVHD mice. Irradiated BALB/c recipients (n=4 in each group) were infused with 1×107 bone marrow cells and 1×106 splenocytes from the C57BL/6 mice. Fourteen days after transplantation, each mouse in the antibody group received 100μl (100μg) of IL-39 antibody, whereas each mouse in the control group received 100μl (100μg) of isotype control antibody via intraperitoneal injection twice a week for 6 weeks. The percentages and numbers of donor Th1(A), Tc1 (B), Th17 (C), and Tc17 (D) cells in the spleen on day 56 are shown. Values are presented as mean ± SD. Figure S9: Effects of IL-39 overexpression on donor CD4+ and CD8+T cells in the spleens of cGVHD mice four weeks post-transplantation. Irradiated BALB/c recipients were infused with 1×107 bone marrow cells and 1×106 splenocytes from the C57BL/6 mice. Splenocytes (n=4 in each group) were collected and stained for FACS analysis four weeks after transplantation. The percentages and numbers of CD4+T, CD69+CD4+T, CD8+T, and CD69+CD8+T cells in CD3+ cells from the spleens of the recipients are shown (A). Lymphocytes were isolated from the spleens of recipients and treated with PMA, brefeldin A, and ionomycin for 4-6h. The percentage and number of TNF-α (B) and IL-4 (C) positive T cells in the spleens of recipients are shown.