Abstract 4121: Heterogenous cellular responses to GITR and TIGIT immunotherapy in the human gastrointestinal tumor microenvironment
Authors: Sathe Anuja , Ayala Carlos , Bai Xiangqi , Grimes Sue M. , Shelton Andrew , Lee Byrne , Kin Cindy , Poultsides George , Ji Hanlee P. 
Published: April 2023

Abstract
Many new immunotherapy agents fail in clinical trials. Major gaps remain in our understanding of their precise mechanism of action, with findings from preclinical studies that do not translate into the clinic. We identified the cellular and molecular responses to co-stimulatory GITR and co-inhibitory TIGIT immunotherapy by using an experimental approach that overcomes these challenges. We investigated these responses in a patient-derived ex vivo system that maintains the original tumor microenvironment (TME) in its near-native state. Using single-cell RNA sequencing (scRNA-seq) and paired single-cell TCR sequencing (scTCR-seq), we identified treatment induced transcriptional reprogramming in different cell lineages in the TME. We established ex vivo slice cultures from ten surgical resections of colorectal or gastric cancer. These cultures maintained all epithelial, stromal, and immune cell types and transcriptional cell states found in the original tissue sample. All tumors responded with activation of CD8, CD4 and T regulatory (Treg) cells following PMA/Ionomycin treatment, confirming the viable and functional status of the model system. GITR agonist led to a modest increase in cytotoxic gene expression in CD8 T cells in four out of nine patients. Transcriptional non-responders had significantly increased dysfunctional phenotype at baseline compared to responders. Ex vivo analysis confirmed that only cells with an effector non-exhausted cell state responded to GITR agonist, while exhausted cells did not. GITR agonist did not induce significant changes in expression in regulatory T (Treg) or T follicular helper-like (TFh-like) cells. TIGIT antagonist increased TCR signaling and activation in all five patients. These responses were observed in both effector and exhausted cells. A reduced transcriptional response to TIGIT in CD8 T cells correlated with the increased expression of GZMK, TXNIP and RGS1 together with reduced expression of metallothionine family genes. TIGIT antagonist also increased activation specifically in expanded TCR clonotypes, which are indicative of tumor antigen reactive cells. TIGIT antagonist further increased activation of TFh-like cells together with increased expression of B cell attracting chemokine CXCL13. Tregs responded with a reduced expression of CTLA4 and TNFRSF4 (OX40), indicative of modulation of immunosuppressive cell state.Our approach successfully identified heterogenous cellular and patient responses to GITR stimulation and TIGIT inhibition in gastrointestinal cancers. TIGIT antagonist led to a wider reprogramming of the TME compared to the limited effects of GITR agonist. Our strategy identified mechanisms of action of immunotherapy and factors associated with response or resistance, which can aid in prioritization of targets and their clinical translation.
Citation Format: Anuja Sathe, Carlos Ayala, Xiangqi Bai, Sue M. Grimes, Andrew Shelton, Byrne Lee, Cindy Kin, George Poultsides, Hanlee P. Ji. Heterogenous cellular responses to GITR and TIGIT immunotherapy in the human gastrointestinal tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4121.