Th139 - Engineering Optimized Autoreactive T Cells for the Study of Type 1 Diabetes Pathogenesis

A hallmark histopathological feature of Type 1 Diabetes (T1D) is infiltration of β-cell specific T cells into the insulin producing Islets of Langerhans. These cells are key mediators of disease, but due to scarcity in circulation there remains a paucity of information regarding their phenotype and function.  This has been circumvented by the generation of antigen specific T cells through lentiviral vector-mediated gene transfer, though the main caveat to this approach is the potential for T cell receptor (TCR) chain mispairing. The consequences of chain mispairing can range from reduced antigen specific functionality to off-target reactivity, thus complicating functional testing, and ultimately reducing therapeutic potential in the case of antigen-specific regulatory T cell (Treg) adoptive cell therapy applications. Our lab has extensive experience generating GAD-specific Tregs and IGRP-specific CD8⁺ T cells by lentiviral transduction. With the goal of extending this work, we deleted the endogenous TCR a-chain gene (TRAC) via CRISPR/cas9 in these cell subsets.  We observed enhanced antigen specificity as shown by increased peptide MHC-dextramer staining, as well as CD69 and CD25 upregulation after stimulation with cognate antigen. TRAC KO in CD8⁺ T cells increased antigen-specific killing of b-cell lines when compared to unmodified cells. Importantly, TRAC KO Tregs maintained purity and epigenetic stability. Hence, we present a TCR-gene editing protocol that results in efficient deletion of endogenous TCR and robust enhancement of antigen-specific T cell function, which could be used for the study of autoreactive T cells or downstream development of antigen-specific Treg therapeutics with enhanced antigen affinity.