ABSTRACT T cells constitute an essential arm of the adaptive immune system, protecting against pathogens and tumors, while tolerating self-tissues. Dysregulation of T cell responses can lead to infectious diseases, cancers, or autoimmune disorders. The key to the effectiveness of T cells is their exquisite antigenic specificity, which can be harnessed to combat diseases. T cells use their surface T cell receptor (TCR) to recognize peptide epitopes on Major Histocompatibility Complex (MHC) molecules (pMHC). While signaling through the TCR and its consequences have been studied extensively, pMHC is conventionally seen as merely a ‘flag’ on target cells for recognition by T cells. MHC molecules do not have canonical intracellular signaling domains, and therefore do not elicit any function into the cells presenting them, making TCR-pMHC interaction a ‘one-way street’ in terms of functional response. This presents a unique engineering opportunity: can TCR-pMHC interactions become ‘two-way streets’? Here, we hypothesize that if pMHC complexes are augmented with signaling domains, they can elicit signaling cascades, leading to expression of response genes that will cause cell-intrinsic functional changes, ultimately leading to cell-extrinsic functional changes. To that end, we will use the engineering platform developed by my group: Signaling and Antigen-presenting Bifunctional Receptors (SABRs). SABRs consist of extracellular full-length MHC complexes with genetically (and hence covalently) linked epitopes, fused with intracellular signaling domains. SABRs can present epitopes to T cells and elicit intracellular signaling upon successful recognition, thereby converting TCR-pMHC interactions into ‘two-way streets. In this proposal, we aim to wield SABRs to impart novel functional capabilities to immune and non-immune cells, thereby opening a new frontier of immune engineering and synthetic immunology. We will first lay out a framework for developing SABRs as a cellular engineering platform to empower combinatorial engineering of immune and non-immune cells to achieve desired immune outcome. We will describe three immune applications of SABRs: 1) engineering of cytotoxic CD8+ T cells or Natural Killer (NK) cells to eliminate autoreactive CD4+ T cells, 2) engineering professional Antigen-Presenting Cells (APCs) to modulate self-reactive or anti-tumor CD8+ T cell responses, 3) engineering CD8+ T cells to sense endogenous immunity to specific antigens and induce a secondary function, leading to a ‘read-and-react’ molecular circuit. These studies will create cellular therapeutic modalities, immune monitoring and perturbation tools, experimental model systems and uncover new immune phenomena. These studies will have profound implications on study and treatment of a wide range of diseases – autoimmune disorders, infectious diseases, cancers, and organ transplantations. 1

NARRATIVE T cells play a crucial role in protecting against pathogens and cancers, while tolerating normal self-tissues by recognizing antigens and responding to them. The specificity of T cells towards their antigens is key to their function. In this project, we create new tools that will enable us to modulate T cell responses in various disease contexts. These studies will lead to new therapies and diagnostic tools that will impact a wide range of diseases including cancers, autoimmune disorders, infectious diseases, and organ transplants. 1