Additional file 1 of Tumor-secreted IFI35 promotes proliferation and cytotoxic activity of CD8+ T cells through PI3K/AKT/mTOR signaling pathway in colorectal cancer
Creators: name: Li, Peisi
name: Zhou, Dawang
name: Chen, Dongwen
name: Cheng, Yikan
name: Chen, Yuan
name: Lin, Zhensen
name: Zhang, Xi
name: Huang, Zhihong
name: Cai, Jiawei
name: Huang, Wenfeng
name: Lin, Yanyun
name: Ke, Haoxian
name: Long, Jiahui
name: Zou, Yifeng
name: Ye, Shubiao
name: Lan, Ping
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. Additional data on the association of tumor IFI35 with CD8+ T cells expression and patient outcome. A, B Scatterplots of correlation between IFI35 and activated CD8+ T cells expression in CRC and solid cancersin published database. C–H Positive correlation of IFI35 mRNA levels with CD8+ T cells expression in human Adrenocortical carcinoma, Bladder Urothelial Carcinoma, Cervical squamous cell carcinoma and endocervical adenocarcinoma, Esophageal carcinoma, Head and Neck squamous cell carcinoma, Kidney Chromophobe, Liver hepatocellular carcinoma, Stomach adenocarcinoma. Data from the Timer database. I–K Higher levels of IFI35 expression correlated to better survival in 3 cancer patient cohorts including hepatocellular carcinoma, head and neck squamous cell carcinoma, and stomach adenocarcinoma. P values calculated by log-rank test. Data from public proteomics datasets. Figure S2. Gating strategy for immune cells population and additional data on intratumoral number of immune cells. A Live cells were selected by the live/dead dye. CD45+NKp64+ cells were defined as NK cells. CD45+CD19+ cells were defined as B cells. CD45+γδ T+ cells were defined as γδ T cells. CD8+ and CD4+ T lymphocytes were from the CD45+CD8+ and CD45+CD4+ subpopulation respectively. Tregs were subdivided from CD4+ T lymphocytes and were defined as CD45+ CD4+Foxp3+ population. DC were defined as the CD45+CD11b+CD11c+ subset. MDSC were defined as the Ly6G+Ly6Clow subpopulation of the CD45+CD11b+ subset. Total macrophages were defined as the F480+ subpopulation of the CD45+CD11b+ subset. B–F Effect of IFI35 overexpression on CT26 tumor-infiltrating various immune cells. IFI35 transfected CT26 cells were inoculated into BALB/c mice. The numbers of tumor-infiltrating γδ T cells, B cells, DC cells, NK cells, and MΦ cellswere analyzed by flow cytometry. Cell numbers are given for 1000,000 total cells from each tumor. Values are represented as mean ± SEM. P values were determined by two tailed t-tests. **P &lt; 0.01, ns: not significant. G–K Effect of IFI35 overexpression on MC38 tumor-infiltrating various immune cells. IFI35 transfected MC38 cells were inoculated into C57/BL6 mice. The numbers of tumor-infiltrating γδ T cells, B cells, DC cells, NK cells, and MΦ cellswere analyzed by flow cytometry. Cell numbers are given for 1000,000 total cells from each tumor. Values are represented as mean ± SEM. P values were determined by two tailed t-tests. **P &lt; 0.01, ns: not significant. Figure S3. The impact of IFI35 on the function of TAMs and Treg cells. A Gating strategy for detection of TAMs and exhaustion markers on the Treg cells by flow cytometry. Live cells were selected by the live/dead dye. CD45+CD11b+F480+CD86+ cells were defined as M1 macrophages. CD45+CD11b+F480+CD206+ cells were defined as M2 macrophages. B, C Proportions of M1and M2macrophages in the TME of BALB/c mice injected subcutaneously with vector control and IFI35-overexpressing CT26 cell line. Values are represented as mean ± SEM. P values were determined by two tailed t-tests. ns: not significant. D–F Proportions of exhaustion markers on the Treg cells in the TME of BALB/c mice injected subcutaneously with vector control and IFI35-overexpressing CT26 cell line. quantification of LAG3, CD39and CTLA4expression among Treg cells. Values are represented as mean ± SEM. P values were determined by two tailed t-tests. ns: not significant. Figure S4. Impact of pharmacological inhibition of STAT1 and genetic inhibition of IRF7 on the expression of STAT1, IFI35, and IRF7 in murine colon cancer cells. A–D the expression of STAT1 and IFI35 mRNA by RT-qPCR in MC38and CT26colorectal cancer cells treated with or without STAT1 inhibitor or/and IFNγ. n = 3. Error bars represent the mean ± SEM. Two tailed t-tests, *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001. E, F the expression of IRFs mRNA was measured after CT26and MC38were treated with 10 ng/mL IFNγ for up to 16 h. n = 3. Error bars represent the mean ± SEM. Two tailed t-tests, *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001. G–J Expression of IRF1, IRF7 and IFI35 mRNA by RT-qPCR in the CT26 and MC38 CRC cells transfected with IRF1 or IRF7 plasmid. n = 3. Error bars represent the mean ± SEM. Two tailed t-tests, *P &lt; 0.05, **P &lt; 0.01, ****P &lt; 0.0001. K, L the expression of IRF7 mRNA by RT-qPCR in controlor Irf7-targetedMC38and CT26colorectal cancer cells. n = 3. Error bars represent the mean ± SEM. Two tailed t-tests, *P &lt; 0.05, ***P &lt; 0.001. M, N the expression of IRF7 mRNA by RT-qPCR in MC38and CT26colorectal cancer cells treated with or without STAT1 inhibitor or/and IFNγ. n = 3. Error bars represent the mean ± SEM. Two tailed t-tests, **P &lt; 0.01, ****P &lt; 0.0001.