Functional relationships among lck kinase and the lat and grb2 adaptors in the intracellular signaling cascade of the tcr/cd3 complex: implications in thymic development and lymphocyte activation

Abstract

T cells are an important part of the adaptive immune system, as they are able to specifically recognize foreign agents and generate responses based on the nature of the recognized agent. In addition, they are responsible for the maintenance of cellular tolerance and homeostasis. T lymphocytes need to undergo processes of development, differentiation and activation in order to perform their functions, and for this purpose, they express the TCR receptor in their membrane, which upon activation triggers a very complex intracellular signaling. Failures in these intracellular signals can lead to the loss of tolerance or to the development of deficient T lymphocytes. In this work, we attempt to address the role of two essential proteins in early TCR signaling, the LAT adaptor and Grb2. In the first chapter, we describe a new cell line (termed J.CaM1.7 cells) derived from J.CaM1.6 cells that happens to be doubly deficient in LAT and Lck. These cells require simultaneous expression of LAT and Lck to recover intracellular signals upon stimulation of the TCR/CD3 complex. In the second chapter we delve into the role of the LAT adaptor in TCR signaling. It has been shown in vitro, that glycine preceding Y132 of LAT, makes it a worse substrate for ZAP70 by reducing the rate of phosphorylation with respect to the other LAT tyrosines. This mechanism could constitute an essential step in the kinetic proofreading model for ligand discrimination. To elucidate the biological function of this glycine in vivo, thymic and peripheral T lymphocyte populations from LATG135D knock-in mice were analyzed. These mice express LAT protein with a mutation in glycine 135 by an aspartate, which causes an increase in Y136 phosphorylation kinetics. LATG135D mice showed alterations in DP and SP populations of thymocytes and peripheral CD4+ or CD8+ T lymphocytes. Data demonstrating the role of G135 of LAT for ligand discrimination. Finally, we analyzed the role of Grb2 in TCR signaling in the Jurkat cell line by suppression of Grb2 expression using CRISPR/Cas9n. Signaling assays revealed that Grb2 is essential for LAT signalosome formation. Surprisingly, after a preliminary proteomics assay it was found that these cells also fail to express THEMIS, making this new cell line a good study model for Grb2 interactions with ligand proteins to regulate intracellular signaling.