De novo identification of CD4+ T cell epitopes

ABSTRACT CD4+ T cells recognize peptide antigens presented on class II Major Histocompatibility Complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor (TCR) sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of Signaling and Antigen-presenting Bifunctional Receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Here, we demonstrate that SABR-IIs libraries presenting endogenous and post-translationally modified epitopes can be used for antigen discovery. Using SABR-II libraries in conjunction with single cell RNA sequencing, we de-convoluted multiple highly expanded TCRs from pancreatic islets of Non-Obese Diabetic (NOD) mice. We compounded antigen discovery by incorporating computational TCR similarity prediction metrics followed by experimental validation. Finally, we showed SABR-IIs presenting epitopes in class II HLA alleles can be used for antigen discovery for human CD4+ T cells. Taken together, we have developed a rapid, flexible, scalable, and versatile approach for the de novo identification of CD4+ T cell ligands from single cell RNA sequencing data using experimental and computational approaches.