Abstract 1550: Single-cell RNA-Seq identified an immunosuppressive IL-17 producing tumor-infiltrating T cells in murine gastric tumor model
Authors: Shirai Masataka , Nagaoka Koji , Taniguchi Kiyomi , Sun Changbo , Hosoi Akihiro , Kakimi Kazuhiro 
Published: September 2020

Abstract
BACKGROUND: YTN2 and YTN16 cells are chemically induced gastric cancer cell lines. YTN2 is spontaneously rejected in C57BL/6 mice in CD8+ T cell-dependent manner, while YTN16 grows progressively. To investigate the interaction of tumor microenvironment and T cells, bulk RNA-Seq of total tumor RNA and single-cell RNA-Seq of tumor-infiltrating T cells were performed in YTN2 and YTN16 gastric tumor-bearing mice.
METHOD: Tumor cells (5×106) were subcutaneously inoculated into C57BL/6 mice. On day 7, tumors were harvested, and total tumor RNA and tumor-infiltrating lymphocytes were isolated. CD8+ and CD4+ cells were sorted from YTN2 and YTN16 tumor tissues. Single-cell RNA-sequencing analysis was performed using vertical flow array chips (VAFC) for 44 T cell-related genes. IL-17 production in tumor-infiltrating T cells was evaluated by intracellular cytokine staining. Anti-IL-17(clone 17F3) and anti-PD-1(RMP1-14) mAbs were injected into YTN16 tumor-bearing mice on days 3, 6, 9, and 12 and days 6, 9, and 12, respectively.
RESULT: Bulk RNA-Seq revealed that higher gene expression of immune-related genes, including cytokines, chemokines, their receptors and checkpoint molecules, were detected in YTN2 tumors than YTN16. IL-17 expression was not detected in YTN2 or YTN16. We analyzed 1142 tumor infiltrating CD4+ and CD8+ T cells isolated from YTN2 and YTN16 tumor tissues. Unsupervised clustering of all T cells using the Louvain method identified 7 clusters, including 3 clusters (clusters 1, 4, 5) for CD8+ and 3 clusters (clusters 2, 3, 6, 7) for CD4+ T cells. Clusters 1-6 included T cells from both YTN2 and YTN16. Cluster 7 consisted of CD4+ T cells only from YTN16. CD8+ T cells in cluster 5 highly expressed Slc2a1, Ifng, Tnf, Fasl, Cxcr3, Il2rb, Il2rg, Il7r, Pdcd1, Tigit, Eomes, Bcl2, and Tbx21. In contrast, these genes were scarcely expressed in cluster 4. Cluster 1 was an intermediate phenotype of these two clusters. CD8+ T cells from YTN16, but not YTN2 in cluster 5, expressed Il17. CD4+ T cells in cluster 2 expressed Slc2a1, Tnfrsf9, Pdcd1, Clta4, Tigit, Hif-1a and Icos at high level. Cluster 2 CD4+ T cells from YTN2 expressed more Havcr2 and Tbx21 and less Il17 than those from YTN16. T cells in cluster 3 did not express these genes. Cluster 7 was characterized by high Il17 expression. By single-cell RNA-Seq analysis, IL-17-producing cells were only detected in YTN16 tumors. Consistent with single-cell analysis, we detected IL-17 producing cells in the YTN16 tumor by flow cytometry. To elucidate the function of IL-17 in the tumor, we used anti-IL-17 blocking mAb and showed that blocking of IL-17 suppressed YTN16 tumor growth. Combination therapy of anti-IL-17 and anti-PD-1 enhanced the anti-tumor effect.
CONCLUSION: Single-cell RNA-sequencing analysis, but not bulk RNA-Seq, identified IL-17-producing CD4+ T cells in YTN16 tumors and revealed an immunosuppressive role of IL-17 in progressive cancer.
Citation Format: Masataka Shirai, Koji Nagaoka, Kiyomi Taniguchi, Changbo Sun, Akihiro Hosoi, Kazuhiro Kakimi. Single-cell RNA-Seq identified an immunosuppressive IL-17 producing tumor-infiltrating T cells in murine gastric tumor model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1550.