Comprehensive EBV-specific T cell profiling and multi-omics analysis of nasopharyngeal carcinoma CD8 T cells reveals novel cancer-associated phenotypes

Abstract

Nasopharyngeal carcinoma (NPC) is a high mortality, Epstein-Barr virus (EBV)-driven cancer with limited treatment options. Although EBV is highly antigenic and detectable in nearly all NPC tumor cells, it remains unclear why NPC tumors evade immune targeting despite high levels of immune infiltration. Here, we investigate the phenotypes of peripheral EBV-specific T cells with the purpose of identifying clinically relevant, cancer-associated EBV-specific T cell phenotypes. We developed a 34-marker mass cytometry and multiplexed MHC-I tetramer panel to detect and phenotype CD8 T cells of up to 55 antigen-specificities, including EBV and other known viral and non-viral epitopes. We used this approach to profile peripheral blood mononuclear cells (PBMCs) from treatment-naïve NPC patients (n=51). Compared to other antigen-specific T cells detected in NPC patients and EBV-specific T cells in healthy individuals, EBV-specific T cells in NPC patients expressed high levels of activation markers (CD38, HLA-DR), co-inhibitory markers (CD39, TIGIT, PD-1), and migratory markers (ITB7, CD103). Using unsupervised clustering, we found that the frequency of EBV-specific T cells in a phenotypic cluster characterized by high CD103, CD39, CD71, HLA-DR, and CD38 expression positively correlated with plasma EBV-DNA titers and gross tumor volume. Remarkably, we observed that regardless of antigen specificity, NPC patients had a higher frequency of total CD8 T cells with this activated/exhausted cluster phenotype, and CD8 frequencies of this cluster were also positively correlated with plasma EBV-DNA titers stage (TNM), and EBV early antigen antibody titers. To investigate the gene expression profiles and TCR clonal diversity of this cancer-associated phenotype, we performed single-cell multi-omics analysis on peripheral CD8 T cells from NPC patients (n=17). We identified that cell subsets within this phenotype had distinct gene expression patterns indicative of potential associations with the tumor and we also assessed the clonality of these subpopulations. Finally, we assess connections between the NPC tumor microenvironment using multiplexed immunohistochemistry (mIHC) and bulk tumor gene expression and TCR sequencing. We found that peripheral activated/exhausted T cell phenotypes are correlated with mIHC tumor PD-1 scores, neutrophil levels identified using gene expression data, and have high TCR clonal sharing with NPC tumors. Overall, this research identified unique cancer-associated peripheral EBV-specific T cell phenotypes in the context of nasopharyngeal carcinoma that are positively correlated with advanced disease. Frequencies of peripheral T cells with this phenotype could also be useful as a blood-based biomarker for NPC.