PROJECT SUMMARY T cells respond to peptide antigens presented by MHC molecules (pMHC). They are driven by 5-module macrocomplexes, composed of one receptor module [the T cell receptor (TCR)], three signaling modules (CD3δε, CD3γε, CD3ζζ), and a CD4 or CD8 coreceptor module, that allow T cells to respond to a single agonist pMHC and direct differentiation to cytotoxic (CTL), helper (Th), regulatory (Treg), or memory (Tm) cell phenotypes that are essential for productive immunity. Importantly, T cells also pose the risk of pathogenic responses if they are specific to self or transplant antigens and are not controlled by peripheral tolerance mechanisms. The macrocomplexes that drive T cell activity are therefore interesting both from an engineering standpoint, as they serve as an ideal framework upon which to design novel chimeric receptors for redirected T cell therapy, and from a targeting standpoint for therapies aimed at mitigating T cell-mediated pathologies when tolerance breaks down. This proposal will test the efficacy of using a novel 5-module chimeric antigen receptor system (5MCAR), which has been engineered to operate according to the mechanistic principles that govern the TCR-CD3-pMHC-CD4/CD8 macrocomplexes, to redirect CTLs to target pathogenic T cells. Our goals are to: use 5MCAR-CTLs to eliminate pMHC-specific T cell populations, including pathogenic T cells, via a surgical strike in order to induce tolerance in mouse models; and, engineer and test humanized 5MCARs in a humanized mouse model system. When completed, the work will provide a blueprint for using 5MCAR-CTL therapy to induce tolerance to defined pMHC and mitigate T cell-mediated pathologies.