ABSTRACT CD8+ T cells play a critical role in antitumor immunity and recent therapeutic strategies targeting CD8+ T cells have shown unprecedented efficacy in treating a growing number of cancer types. One leading strategy involves the use of immune checkpoint inhibitor (ICI) therapies. ICI therapy works by interfering with inhibitory receptors expressed on the surface of dysfunctional T cells in the tumor microenvironment (TME) to enhance and restore T cell functionality. LAG3 is one such inhibitory receptor that promotes T cell dysfunction during periods of prolonged antigenic challenge by restricting T cell activation. Until recently, the underlying mechanism by which LAG3 restrains T cell activation was unclear. Using a combination of LAG3 mutant plasmids and super resolution microscopy, our lab demonstrated that the conserved EP motif within the LAG3 cytoplasmic tail disrupts the association between LCK and the co-receptors CD4 and CD8 thereby limiting T cell receptor (TCR) signaling and downstream activation. Interestingly, in addition to limiting activation, recent evidence from our lab has revealed that LAG3 can also restrict T cell motility. In a series of in vitro studies using time-lapse microscopy, we observed that LAG3 deficient CD8+ T cells consistently moved at a higher average velocity and covered a significantly greater distance compared to their WT counterparts. These results may have important implications for CD8+ T cell functionality in the context of anti-LAG3 ICI therapy. However, there are a number of unanswered questions, and it remains to be determined, 1.) how LAG3 functions to reduce T cell motility and 2.) whether LAG3 impacts CD8+ T cell infiltration into the TME or cell-cell interactions within the TME. I hypothesize that the LAG3 cytoplasmic EP motif restricts motility by limiting the phosphorylation and activation of focal adhesion kinases. In addition, I hypothesize that greater motility in the absence of LAG3 will facilitate enhanced CD8+ T cell infiltration into tumors. I will use a series of in vitro motility assays involving LAG3 mutant plasmids paired with detailed phosphorylation analyses to investigate the mechanism by which LAG3 impacts T cell motility. In addition, I will examine the impact of LAG3 on live CD8+ T cells in situ using multiphoton intravital imaging approaches. If successful, these studies will provide a greater mechanistic understanding of LAG3 that could be used to enhance current cancer immunotherapeutic approaches.

PROJECT NARRATIVE LAG3 is an inhibitory receptor expressed on T cells that interferes with effective immune mediated tumor clearance. The goal of this project is to investigate the underlying mechanisms by which LAG3 restrains T cell activity to improve current cancer immunotherapies and extend the benefits to a greater number of patients.