The presence of immunosuppressive molecules and cells in the tumor microenvironment (TME) can lead to T cell dysfunction. CD8-positive cytotoxic T cells (CTLs) are ineffective in killing tumor cells primarily due to upregulated expression of inhibitory checkpoint molecules and decreased production of effective cytokines. In addition, immune suppressive cell types such as regulatory T cells (Tregs) and tumor associated macrophages (TAMs) are recruited to the TME, further establishing a suppressive immune environment. In this study, we evaluated expression profiles of key immunosuppressive molecules and cell types by applying RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, and dual ISH-IHC staining. First, we evaluated CD8-positive cell infiltration in TME of archived human tissues from non-small cell lung cancer and ovarian cancer. Selected tissues with either high or low CD8-positive cell number (CD8-high or CD8-low) were evaluated for (1) the presence of Tregs (FOXP3+CD4+) and TAMs (CD163+, including IL10 and CCL22), (2) the expression of immune checkpoint molecules including PD1, PD-L1, TIM3, and LAG3, (3) the expression of immune suppressive molecules IDO1 and TGFβ, and (4) IFNγ expression in CD8-positive subsets. Examination of immune inhibitory molecules expressed in single cells in the tumor and stromal microenvironment revealed that in general, CD8-high tissues expressed higher level of immune checkpoint molecules, often co-expressed in the same individual cells in the same TME, while the expression of IDO1 and TGFβ was independent of CD8-positive cell inflammation. Tregs and TAMs often co-existed with IFNγ-positive CTLs in the same TME. Unexpectedly, all investigated inhibitory molecules were expressed in both tumor cells and stromal/immune cells in some tumors. Beyond PD-L1, immune checkpoint molecules PD1, TIM3, and LAG3 were frequently expressed in tumor cells but at lower level than in immune cells. Expression of IDO1 and TGFβ was observed in many cell types, including tumor cells, with various expression levels in each tumor cell. The single-cell tumor expression profiles suggest a potential tumor-intrinsic mechanism of expression for these inhibitory molecules. This exploratory study highlights the potential of RNAscope® ISH to better understand the cellular and molecular suppressive mechanisms associated with T cell dysfunction and exhaustion in the TME. The robust RNAscope® ISH platform is well suited for evaluating critical secreted factors and other key molecules in a highly sensitive and cell type-specific manner. As multiple therapeutic approaches to augment the CTL function are being developed, the presented method may facilitate the identification and development of key biomarkers to stratify patients based on their specific tumor and immune cell states.

Citation Format: Annelies Laeremans, Na Li, Jeff Kim, Xiao-Jun Ma, Emily Park. Evaluation of potential factors contributing to the exhaustion of T lymphocytes in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1022.