Class II MHC Signaling and Antigen-Presenting Bifunctional Receptors (SABRs) for CD4+ T Cell Antigen Discovery and Regulatory T Cell Engineering

ABSTRACT

This thesis details the engineering of cells using MHC class II Signaling and Antigenpresenting Bifunctional Receptors (SABRs) for both antigen discovery and as a novel strategy to restore tolerance in type 1 diabetes. SABRs are a chimeric receptor comprised of an extracellular peptide covalently linked to MHC in addition to an intracellular signaling domains CD28-CD3. The first demonstration of this molecule is for T cell antigen discovery in the context of type 1 diabetes, a disease where T cells target insulin producing beta cells in the pancreas. We hypothesized that SABRs could be used to identify the antigen specificities of CD4+ T cells infiltrating the pancreatic islets of non-obese diabetic (NOD) mice. To that that end we performed single cell RNA sequencing on T cells isolated from the islets of NOD mice from 6-, 8-, and 10- weeks of age. We identified the top 35 clonally expanded CD4+ T cell clones and reconstructed them for expression in Jurkat cells. In parallel, we constructed SABR libraries in the I-Ag7 allele then screened each of the TCRs. This process allowed us to identify the antigen specificities of 11 of the top expanded CD4+ T cell clones, including the discovery of reactivity to a novel hybrid insulin peptide. Furthermore, SABR libraries have allowed us to identify populations of islet infiltrating T cells which recognize putative hybrid insulin peptides comprised of insulin B-chain fusions with other beta cell secretory granule proteins. The second half of this thesis focuses on leveraging SABRs as a therapeutic modality in autoimmunity. Specifically, we have expressed SABRs in CD4+ regulatory T cells (Tregs). SABR expression by a Treg allows for recognition of the SABR by a cognate CD4+ T cell. This cognate interaction activates Tregs via the CD28-CD3 v intracellular signaling domains. We have demonstrated that SABR-Tregs can suppress CD4+ T cells in vitro in an antigen specific manner. In vivo studies have indicated that SABR-Tregs can prevent induced autoimmune diabetes and may prevent spontaneous diabetes in NOD mice. All in all, SABRs are a potent tool for T cell antigen discovery and cell based therapeutic.