Fig. 3

IFI35 increases the intratumoral number of CD8⁺ T cells. A–E 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 CD45⁺ T cells (A), CD8⁺ T cells (B), CD4⁺ T cells (C), Foxp3⁺ cells (D), and MDSC cells (E) were 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 < 0.05, **P < 0.01, ns not significant. F–J 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 CD45⁺ T cells (F), CD8⁺ T cells (G), CD4⁺ T cells (H), Foxp3⁺ cells (I), and MDSC cells (J) were 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 < 0.05, **P < 0.01, ns not significant. K–O Quantification of CD8⁺ T cells from paraffin sections of CT26 and MC38 colon cancer by IHC. K Representative IHC staining for CD8 in paraffin sections of MC38 colon cancer. L–O Average cell number per high-power field (HPF) is shown; Two tailed t-tests, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P Positive correlation of IFI35 protein levels with that of CD8 IHC levels in human colorectal cancer. Q Scatterplots showing correlation between IFI35 expression and CD8⁺ T cell expression level in The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) data collection determined by tumor immune estimation resource (Timer, http://cistrome.dfci. harvard.edu/TIMER/) website. R Violin plot showing expression of IFI35 between four CMS subtypes in the single-cell cohort. S Kaplan–Meier analysis of overall survival (OS) in CRC patients with low IFI35 and low CD8 expression (n = 11), low IFI35 and high CD8 expression (n = 37), high IFI35 and low CD8 expression (n = 26), high IFI35 and high CD8 expression (n = 103). The corresponding cutoff values was calculated by the maxstat algorithm in R package “maxstat”