Abstract 5217: Defining the balance of anti-viral and anti-tumor T cell responses to oncolytic virus therapy using single cell approaches
Authors: Burman Bharat , Ceglia Nicholas , Hirschhorn Daniel , Budhu Sadna , Mangarin Levi , Oseledchyk Anton , Bykov Yonina , McPherson Andrew , Shah Sohrab , Wolchok Jedd , Merghoub Taha , Zamarin Dmitriy 
Published: June 2022

Abstract
Preclinical and clinical studies have shown that intratumoral oncolytic viruses (OVs) can potentiate host anti-tumor immunity and overcome resistance to immune checkpoint blockade, although clinical responses to OVs have been modest to date. While T cell infiltration of tumors is frequently cited as a measure of OV immunogenicity, this measure is non-specific as OVs elicit a strong virus-directed T cell response, and it remains unknown what proportion of T cells expanded by OV therapy are virus-specific vs. tumor-specific or how these T cells distribute across virus-treated and distant tumors. We employed oncolytic Newcastle disease virus (NDV) in a bilateral flank melanoma mouse model to identify and phenotypically characterize anti-viral and anti-tumor T cells using single cell (sc) RNA and T cell receptor (TCR) sequencing. Intratumoral NDV therapy to a single flank tumor resulted in increased infiltration of CD4+ and CD8+ T cells in the injected (enestic) and distant (non-enestic) tumors and increased the breadth of the TCR repertoire at both sites with preferential expansion of several dominant clonotypes. There was substantial expansion of the proportion of T cell clonotypes shared between enestic and nonenestic tumors as well as the spleen, indicative of inter-tumor TCR repertoire normalization. In both treated and distant tumors, we observed a significant increase in the frequency of convergent TCR clonotypes, i.e. TCRs encoded by different nucleotide sequences that converge on the same amino acid sequence, implying that the presence of these TCRs in tumors is non-random. Using scRNA and paired TCR sequencing, we demonstrate that NDV therapy leads to expansion of unique clusters of terminally differentiated and activated CD4+ and CD8+ T cells associated with distinct TCR-based clonotypes. Notably, the predominant phenotypic clusters were distinct between the enestic and non-enestic tumors. Enestic tumors were dominated by CD8+ T cells exhibiting a signature associated with terminal dysfunction (PDCD1, LAG3, TOX), while the predominant expanded CD8+ T cells in non-enestic tumors exhibited an activation signature associated with high expression of cytolytic markers. While phenotypic states were conserved for the dominant TCR clones shared across the enestic and non-enestic tumors, TCRs unique to the non-enestic tumor were predominantly associated with an activated T cell state. Taken together, these studies highlight that T cells expanded by OV therapy exhibit unique functional states and TCRs in treated and distant tumors and imply that virus- and tumor-specific T cells may be identified by distinct TCR repertoires and phenotypes. Understanding the balance between virus- and tumor-directed T cells elicited by OV therapy will be key to engineering OVs and designing combination strategies that drive stronger tumor-directed T cell response.
Citation Format: Bharat Burman, Nicholas Ceglia, Daniel Hirschhorn, Sadna Budhu, Levi Mangarin, Anton Oseledchyk, Yonina Bykov, Andrew McPherson, Sohrab Shah, Jedd Wolchok, Taha Merghoub, Dmitriy Zamarin. Defining the balance of anti-viral and anti-tumor T cell responses to oncolytic virus therapy using single cell approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5217.