Scale bar 100 m. PI3K-AKT-mTOR pathway was highly activated in CD8+ effector T cells of colitic lesions. Moreover, we developed a mouse melanoma model to recapitulate the gastrointestinal toxicity of anti-PD-1 treatment in clinical settings. Anti-PD-1 treatment significantly contributed to the infiltration of CD8+ T cells, and correspondingly induced severe enteritis. Immunohistochemistry experiments showed that the PI3K-AKT-mTOR pathway of T cells was activated by anti-PD-1 treatment. Blockade of the pathway with mTOR inhibitor sirolimus not only inhibits tumor growth but also suppresses the T cell infiltration in colitic lesions. More importantly, combination with sirolimus and anti-PD-1 synergistically inhibits tumor growth Lycorine chloride inducing the immunogenic cell death of tumor cells 0.05; ** 0.01, analyzed by two-sided Wilcoxon test comparing control and colitis groups, average and SEM are shown for each patient group. (D) Effector score of CD8+ T cells among patient groups. **** 0.0001, two-sided Wilcoxon test. (E) Violin plots showing the expression of selected effector marker genes among patient groups. **** 0.0001, two-sided Wilcoxon test. (F) Effector scores computed for each CD8+ T cell sub-cluster (top) and effector T cell cluster (down). **** 0.0001, two-sided Wilcoxon test. (G) Gene set enrichment analysis Lycorine chloride of effector T cells and other CD8+ T cells. Method used to adjust the p-values is B-H. Anti-PD-1 Treatment Induced irAEs in Mouse Melanoma Model To illuminate the correlation between irAEs and immunotherapy, we recapitulated the clinical therapeutic process in a mouse model. SMM103, an anti-PD-1 antibody responsive cell line, was subcutaneously inoculated in immune competent C57BL/6 mice, the mice were randomly divided into two groups when the tumor grew up to 80 mm3, and were treated with saline and Lycorine chloride anti-PD-1 antibody (5mg/kg), respectively ( Figure?2A ). Tumor volumes were recorded every 3?d, and tumor growth curves were plotted during treatment ( Figure?2B ). With the anti-PD-1 treatment, the tumor growth was significantly inhibited, indicating this model is suitable for studying the irAEs. After the treatment of anti-PD-1 for 9 days, mucus in the stool was observed in some mice, indicating that the intestinal canal became dysfunctional. In addition, pruritus and convulsion were also common phenomena during drug treatment. By day 18, the mice were of poor health status. Consequently, the mice were sacrificed, and the intestine were harvested for analysis. Severe hemorrhages were observed in the intestine from mice treated with anti-PD-1, but not in the control group ( Figure?2C ). Compared with the control group, Haematoxylin and eosin (H&E) analysis in intestine showed a significant increase in immune cells infiltrated into the intestine from anti-PD-1 group ( Figure?2D ). Immunochemistry analysis of CD31 in intestine demonstrated that the new blood vessels were formed ( Figure?2E ). To characterize the structural and morphological of blood vessels in intestine, the area of CD31+ staining was carefully measured at each mouse ( Figure?2F ). The area of CD31+ staining was significantly increased in intestine from anti-PD-1 treatment group, which is in line with the phenomena of gastrointestinal hemorrhage. Open in a separate window Figure?2 Establishment of irAEs mouse model. (A) Representative diagram showing experimental scheme of establishment of irAEs mouse model. Immune competent C57BL/6 mice were subcutaneously inoculated with 1106 SMM103 melanoma cells on day 0. When tumor volume achieved 80 mm3, mice were treated with saline or anti-PD-1, respectively. Details of treatment are provided in the Methods section. (B) The average tumor growth curves of mice treated with saline and anti-PD-1, respectively. The results are presented as mean SD (n=5), *** 0.001. (C) Representative intestine from each group after euthanizing the mice C11orf81 on day 22. (D) Lymphatic infiltration in intestine detected by staining with hematoxylin and eosin (H&E). Scale bar 200 m (E) The formation of new blood vascular was visualized by CD31 IHC staining. Scale bar 200 Lycorine chloride m. (F) Quantitative results of CD31 positive area. Data was expressed as mean SD (n=4), **** 0.0001. Increase in CD8+ T Cells and Activation of mTOR Pathways During Anti-PD-1 Treatment Initial bioinformatic analysis of scRNA-seq data showed that, compared with healthy donors, the striking increase of CD8+ effector T cells in the colon was obvious in checkpoint inhibition therapy treated melanoma patients with colitis. To investigate whether the increase of CD8+ T cells can be.