Project Summary/Abstract CD4+ helper T (Th) cells are responsible for host immunity to phagosomal pathogens and augment antibody and cytotoxic T cell-mediated responses. They also cause autoimmunity and can participate in immune responses to tumors. Th cells carry out these functions by using antigen receptors (TCRs) to recognize epitopes consisting of peptides (p) bound to major histocompatibility complex class II molecules (MHCII) displayed on various host cells. Fluorochrome-labeled p:MHCII tetramers and flow cytometry have been critical tools for studying immune responses by epitope-specific polyclonal Th cells. It has become clear, however, that p:MHCII tetramers do not detect TCRs at the low end of the relevant affinity spectrum. This limitation has created a knowledge gap about the roles that Th cells with low-affinity TCRs play in immune responses to infections and cancer. We hypothesized that p:MHCII tetramers underperform because their stalk regions do not bind to CD4 molecules on Th cells. We used molecular evolution to select an MHCII molecule with improved CD4 binding (MHCII-4E) and found that p:MHCII-4E tetramers are superior to conventional reagents at detecting Th cells with low affinity TCRs. We will use these innovative reagents to identify the roles that Th cells with low affinity TCRs play in immune responses to persistent and chronic infections, and an autoimmune disease of the central nervous system. We will also test a p:MHCII-4E-based vaccine in a B cell leukemia model. Completion of these aims will fill basic knowledge gaps about the composition of Th repertoires for self and foreign epitopes and provide proof of principle for new therapies targeted to low affinity Th cells.