Project Summary/Abstract The long-term goal of this work is to develop immunotherapeutic agents that can prevent T cells becoming dysfunctional within the tumor microenvironment (TME), primarily for use as co-therapies with vaccine and adoptive cellular therapies. The specific goal of this project is to use a systems biology approach to identify and rank novel candidate regulators of T cell dysfunction for immunotherapeutic targeting. Functional tumor- specific CD8 T become dysfunctional on trafficking from the blood to the TME. Checkpoint inhibitors that act on T cells that already have a dysfunctional phenotype have shown remarkable efficacy in a subset of tumors. We reason that the pathways by which functional T cells progress to dysfunction within the TME should present new targets for immunotherapeutic interventions. Yet these pathways are poorly understood, in part because they are only transiently expressed on the road to failure. Snapshot analyses of TILs show us dysfunctional T cells and bystander trafficking T cells, but don't capture T cells in the act of failing. In this project we utilize a novel reagent that is a chimera between a tumor-specific antibody and a peptide- MHC, that takes bystander T cells of a known specificity in the TME and essentially turns them into tumor- specific cells. We administer the reagent iv and simultaneously initiate TCR signaling in a large number of CD8 T cells. In the first specific aim, we analyze the cells by flow cytometry for phenotype, function and numbers, comparing TCR-activated cells in the TME with non-targeted bystander cells. We will identify the specific consequences of TCR activation in the TME, describe the eventual phenotypes (e.g. death, exhaustion), and importantly, identify the time frame over which the progression occurs. In the second specific aim, we will use RNA-seq, first to identify changes in transcriptome induced by trafficking into the TME in the absence of TCR ligation. We will then monitor transcriptional changes in response to TCR ligation, from very early timepoints to the time identified in SA1 at which final stable phenotypes or death occur. We will identify induced pathways and networks, and rank putative regulators for validation and investigation as potential therapeutic targets. We predict that transcriptional changes will occur in waves, and that early, transiently expressed regulators will provide novel targets for immunotherapy. Finally, in a third specific aim, we will investigate the expression of our candidate regulators in a series of models to test the generality of our findingsi, and interrogate databases of human TIL transcriptomes. This will serve to refine our ranking of putative targets for immunotherapy.