Inhibitory CD161 Receptor Identified in Glioma-infiltrating T cells by Single Cell Analysis

T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA-seq to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with IDH-wildtype glioblastoma and IDH-mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several NK cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets. We profiled and characterized the types, programs, and states of glioma-infiltrating T cells isolated from fresh tumor samples using full-length or 5’end single-cell RNA-seq (scRNA-seq) in 31 adult patients with either IDH-wildtype glioblastoma (GBM) or IDH-mutant glioma (IDH-G). We use this tumor T cell atlas to identify new pathways regulating T cell function in the tumor microenvironment across GBM and IDH-G cases. We focus on subsets of cytotoxic glioma-infiltrating T cells that co-express several NK cell genes, and may serve as potential effectors of anti-tumor immunity. Analysis of clonally expanded T cells in tumors supports the NK gene KLRB1 (encoding CD161), as a potential inhibitory receptor. We reasoned that CD161 may represent an attractive therapeutic target, as it is a cell surface molecule expressed by both CD8 and CD4 T cell subsets, and a larger fraction of T cells express CD161 than the PD-1 protein. Additionally, CLEC2D, the ligand for CD161, is a surface molecule expressed by myeloid cells and malignant cells, suggesting a ligand-receptor pathway for immunotherapy. Accordingly, functional experiments where KLRB1 was inactivated in primary human T cells using CRISPR/Cas9 showed that CD161 inhibits the anti-tumor function of T cells in vitro and in vivo. Inhibition of ligand binding with a CD161 blocking mAb showed similar anti-tumor efficacy in vitro. Generalizing our observations, we find that KLRB1, and the gene expression signature of a KLRB1 expressing T cell, is shared across diverse cancer types. Our comprehensive atlas of T cell expression programs across the major classes of diffuse gliomas thus identifies the CD161-CLEC2D pathway as a potential target for immunotherapy of diffuse gliomas and other human cancers. *** Due to patient privacy concerns, the submitter declares that patient data will be submitted to the Data Use Oversight System (https://duos.broadinstitute.org/). ***