Molecular Mechanisms of T Cell Receptor Signaling Regulation by THEMIS

Abstract ID 129542 Poster Board 355 Chimeric Antigen Receptor (CAR) T-cell therapy is a revolutionary immunotherapy used to treat hematological cancers. CAR T-Cell therapy functions by engineering T cell receptors (TCRs) to recognize tumor-associated antigens on cancer cells, leading to their death by T cells. Thymocyte Expressed Molecule Involved in Selection (THEMIS) modulates signaling through the TCR and is essential for the development of mature T cells. This occurs through protein-protein interactions (PPIs) with SHP phosphatases, which alter their enzymatic activities and phosphorylation in the TCR pathway. THEMIS is of therapeutic interest because it also regulates response to CAR T-Cell therapy. However, the molecular mechanisms by which THEMIS regulates TCR and CAR T-cell signaling are unclear. Previous research suggests that the complex that regulates signaling is comprised of THEMIS, SHP1 phosphatase, and the adaptor protein GRB2. It is unknown exactly how these proteins form the complex and how they regulate each other. Our main goal is to use a variety of biophysical and biochemical methods to develop assays with which to understand the architecture of this complex and how these PPIs play a role in modulating phosphatase function. In conjunction with AlphaFold predictions, we aim to provide a structural rationale for our experimental observations. We conducted Ni-NTA and GST pulldown assays with a variety of purified proteins to assess the PPIs between THEMIS, SHP1, and GRB2. We found that recombinant THEMIS and GRB2 readily interact. We are actively working on isolating complexes with SHP1 to investigate the molecular mechanism by which THEMIS regulates phosphatase function in conjunction with fluorescence-based enzymatic assays. Resolving these molecular interaction details will enable effective protein engineering of THEMIS to enhance or fine tune CAR T-cell cancer immunotherapies. We acknowledge Emory University for startup funding.