PROJECT SUMMARY Basic research has fueled a revolution in how complex diseases are treated. As our understanding of T cell activation improves, so will our ability to leverage the latest findings for translational purposes. For example, synthetic receptors could be used to redirect T cell specificity and functions to treat numerous diseases if they are able to integrate with a T cell’s intracellular signaling machinery in a way that mimics the native 5-module receptor complexes that naturally drive T cell responses to peptide antigens presented by MHC molecules [receptor module = the T cell receptor (TCR); signaling modules = CD3γε, CD3δε, CD3ζζ; coreceptor module = CD4/CD8]. We have therefore advocated for biomimetic engineering of synthetic receptors. However, our basic and translational work in this space, along with the basic research from other labs, have taught us that doing so requires a better understanding of coreceptor function. At present, controversy surrounds how the TCR-CD3 complex and CD4 work together on the outside of a CD4+ T cells to relay pMHCII-specific information across the cell membrane. And, on the inside of CD4+ T cells, the dominant paradigm that is thought to describe pMHCII-specific signal initiation is not supported by recent results of experiments that were designed to directly test this model. The current proposal will therefore interrogate the molecular mechanisms, both outside and inside of CD4+ T cells, by which CD4 contributes to pMHCII-specific responses. We will leverage advances in computational analysis of protein evolution, and co-evolution, to guide structure-function analysis of the putative docking site for CD4 and TCR-CD3-pMHCII units. We will also work to purify TCR- CD3-pMHCII-CD4 assemblies for structural characterization. Finally, we will investigate the molecular mechanisms by which motifs in CD4’s transmembrane, cytoplasmic juxtamembrane, and intracellular regions work to help initiate and regulate pMHCII-specific responses. Our results will inform our basic understanding of CD4+ T cell activation, and therefore guide biomimetic engineering of synthetic receptors for therapeutic purposes.

PROJECT NARRATIVE CD4+ T cells survey the body for evidence of microbial infection or altered self and then orchestrate long-lived protective immune responses when a problem is encountered. How they collect the relevant information from an antigen presenting cell (APC) and relay it across their cell membranes to direct their response is a matter of intense interest as this process is fundamental to their role in the immune system. This proposal will shed important new light on how CD4+ T cells relay information across their cell membrane to initiate intracellular signaling events and, in so doing, will serve as a guide for efforts to engineer synthetic receptors that mimic the form and function of their natural counterparts for therapeutic purposes. 1