Biochemical Basis of T Cell Activation

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Engagement of multicomponent immunoreceptors such as the T cell antigen receptor results in rapid recruitment and activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate many enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane adapter protein. We have performed studies to characterize how LAT is phosphorylated and then binds many critical signaling molecules, thus bringing other adapter molecules and enzymes in multimolecular complexes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, microscopic and genetic techniques are currently employed to study the characteristics of LAT-based signaling complexes and the enzyme pathways that are coupled to and activated at LAT complexes. Previously we demonstrated that LAT-based structures known as microclusters, as they are visible microscopically, are the site of molecular complex formation occurring with T cell activation. We have used state-of-the-art microscopy including super-resolution analysis to study the molecular organization of these microclusters. We demonstrated that there are two spatially separate domains in the microclusters. One consists of the TCR and associated ZAP-70 PTK and the other contains LAT and LAT-bound signaling molecules. A kinetic analysis of the formation of the microclusters revealed that molecules are recruited in a step-wise fashion beginning with ZAP-70 recruitment to the TCR followed by LAT and other signaling proteins and then by molecules involved in microcluster dissociation. In the past year we have completed a study that reports our efforts to use this information to generate improved chimeric antigen receptor proteins (CARs). Such proteins contain an antigen binding domain, a transmembrane domain and an intracellular region consisting of the TCR zeta chain. These CAR molecules have proven to be useful, when expressed in a patient's lymphocytes, for targeting and destroying tumor cells in patients with cancer. Based on our imaging studies, above, we reasoned that replacing the TCR zeta chain with a more distal signaling element such as the ZAP-70 protein tyrosine kinase or the adapter protein LAT would be more effective in clearing tumors. We found that linking the ZAP-70 kinase domain instead of the TCR zeta chain to the CAR tumor recognition domains made an effective construct. When expressed in human T cells, our CAR effectively recognized tumor cells in vitro and in vivo murine models. This new construct successfully targeted and killed tumors, and the T cells bearing the CAR demonstrated properties (for example, prolonged efficacy) that are superior to T cells expressing the commonly used CAR constructs. .