CD28 and TCR In-Situ Biophysical Analyses Inform T Cell Immunity Mechanisms

This work investigates two receptors on T lymphocytes that shape immunity, the T cell receptor (TCR) and cluster of differentiation 28 (CD28). T cells coordinate adaptive immunity, but how signaling via TCR and CD28 interactions with peptide-major compatibility complex (pMHC) and B7 family ligands on antigen presenting cells govern T cell function and differentiation remains poorly understood. In-situ biophysical measurements on live T cell surfaces suggest both B7 family ligands form monomeric bonds with CD28. This work demonstrated CD28 catch bonds with B7 family ligands. Catch bonds refer to a counter-intuitive phenomenon where force prolongs bond lifetime contrasted with the more intuitive slip bond where force shortens bond lifetime. Although TCR–pMHC catch bonds on splenic T cells characterize a well-established TCR mechanosensing mechanism, the same interaction on hepatic T cells showed slip bonds correlating with a more activated state among liver T cells. We also analyzed both short- and long-term memory effects from the same molecular interactions. Short-term (within seconds) memory analyses found that bond formation increased bond formation likelihood but not dissociation in the immediate future. Long-term (~5 minutes) memory analyses found that splenic T cells became more activated by repeated ligand engagement and receptor tension resulting in TCR–pMHC catch bond elimination. Our sensitive assay also revealed subtle T cell activation by piconewton-level T cell pushing and pulling forces as well as changes in short-term memory. This work suggests biophysical instrumentation employed in-situ can reveal information about dynamic processes mediating important immunological functions. The findings within this work provide insights into mechanistically how co-stimulation works at a single molecule level as well as how signaling overlap between TCR and CD28 influence receptor localization, mechanosensing, and triggering. These insights answer longstanding mechanistic questions about how T cells function and provide foundations for future investigations.Ph.D