Elucidating the Role of CD8+ TCR Attributes in Cancer Immunotherapy

Abstract public domain (NIH RePORTER)

PROJECT ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with the lowest 5-year survival rate among all solid tumors. Adoptive cell transfer (ACT) of tumor neoepitope specific CD8+ T cells has shown therapeutic promise, but current approaches rely on peptide-MHC tetramers to select high-affinity T cell receptors (TCRs), potentially overlooking functionally superior clones. Our preliminary data reveal that low- affinity TCRs—particularly those undetectable by tetramers—can mediate potent antitumor responses, suggesting that TCR bond lifetime under mechanical force, rather than affinity alone, may dictate therapeutic efficacy. To address this, I developed a novel panel of T cell receptor exchange (TRex) mice expressing PDA- specific TCRs spanning a spectrum of affinities for the same tumor neoantigen (CB101-109:H2-Db). Strikingly, the lowest-affinity TCR (TCR5.1) demonstrated enhanced tumor control compared to higher-affinity counterparts, challenging conventional TCR selection paradigms. This proposal will define how TCR biophysical properties influence CD8+ T cell fate and function in PDA through two specific aims. Aim 1, will employ a biomembrane force probe (BFP) to quantify TCR-pMHC bond lifetimes and correlate these with ACT outcomes in orthotopic PDA models, using high-resolution ultrasound and multimodal T cell profiling. In collaboration with Brian Evavold’s lab at the University of Utah, I will use BFP analysis to quantify each TCRs affinity, bond lifetime, and ideal force conditions. I will then evaluate the effector functions of each clonotype ex vivo using titrated peptide stimulation assays and assess tumor clearance in vivo through ACT in immunocompetent orthotopic PDA models. Aim 2 will dissect competitive fitness of TCRs during immunotherapy by co-transferring T cell clones into immunocompetent hosts, combining scRNAseq and spatial analyses to uncover mechanisms driving TCR-dependent persistence. By “parking” naïve TRex CD8+ T cells in mice prior to orthotopic tumor implantation, I will identify the impact of competition between TCRs of varying affinities on the anti-tumor response during immune checkpoint blockade or CD40 agonist therapies. Tracking these clonal populations through single-cell RNA sequencing and spatial analyses will allow for the identification of the molecular and cellular mechanisms that drive TCR-dependent persistence and stem-like maintenance. Together, these studies will test the central hypothesis that low-affinity, long-lived TCR interactions promote the development of durable, stem-like T cell populations that are resistant to exhaustion - a potential key to achieving sustained antitumor responses. The successful completion of this research will provide transformative insights into TCR- intrinsic determinants of therapeutic efficacy, with direct implications for the design of improved ACT protocols for PDA and other solid tumors.

Public health relevance public domain (NIH RePORTER)

PROJECT NARRATIVE Pancreatic ductal adenocarcinoma (PDA) remains a highly lethal malignancy with a 5-year overall survival of only 13%. Here, we aim to identify traits of tumor-specific T cell receptors (TCRs) that impart prolonged CD8+ T cell persistence, function, and antitumor activity, thereby informing the development of immune based strategies for PDA patient treatment. Our work will establish fundamental principles of TCR mechanobiology that determine T cell fate in solid tumors, with immediate applications for optimizing adoptive cell therapies in PDA and broader implications for overcoming resistance mechanisms across multiple cancer types.