Increased Protein Stability and Interleukin-2 Production of a LATG131D Variant With Possible Implications for T Cell Anergy.

Mikel M Arbulo-Echevarria,Isaac Narbona-Sánchez,Inmaculada Vico-Barranco,Enrique Aguado,Francisco García-Cózar,Arkadiusz Miazek

doi:10.3389/fcell.2020.561503

Abstract

The adaptor LAT plays a crucial role in the transduction of signals coming from the TCR/CD3 complex. Phosphorylation of some of its tyrosines generates recruitment sites for other cytosolic signaling molecules. Tyrosine 132 in human LAT is essential for PLC-γ activation and calcium influx generation. It has been recently reported that a conserved glycine residue preceding tyrosine 132 decreases its phosphorylation kinetics, which constitutes a mechanism for ligand discrimination. Here we confirm that a LAT mutant in which glycine 131 has been substituted by an aspartate (LATG131D) increases phosphorylation of Tyr132, PLC-γ activation and calcium influx generation. Interestingly, the LATG131D mutant has a slower protein turnover while being equally sensitive to Fas-mediated protein cleavage by caspases. Moreover, J.CaM2 cells expressing LATG131D secrete greater amounts of interleukin-2 (IL-2) in response to CD3/CD28 engagement. However, despite this increased IL-2 secretion, J.CaM2 cells expressing the LATG131D mutant are more sensitive to inhibition of IL-2 production by pre-treatment with anti-CD3, which points to a possible role of this residue in the generation of anergy. Our results suggest that the increased kinetics of LAT Tyr132 phosphorylation could contribute to the establishment of T cell anergy, and thus constitutes an earliest known intracellular event responsible for the induction of peripheral tolerance.

Highlights

After the specific recognition of a peptide antigen bound to an MHC molecule on the surface of an Antigen Presenting Cell, a cascade of intracellular signaling events are triggered in T lymphocytes (Malissen and Bongrand, 2015; Alcover et al, 2018; Courtney et al, 2018)
LATG131D Mutation Favors Anergy contribution of some of the nine conserved LAT tyrosines to the T cell receptor (TCR)/CD3 signaling cascade (Finco et al, 1998; Zhang et al, 2000; Lin and Weiss, 2001; Paz et al, 2001). Those early works showed that the three most distal tyrosines (171, 191 and 226 in the human form of LAT) are necessary for Grb2-SOS and GadsSLP76 binding and Erk activation and that the sixth tyrosine residue (132 in human LAT) is essential for PLC-γ1 binding and activation and calcium influx generation
The phenotype of LAT-knockout (KO) mice revealed the essential role of this molecule for the transduction of intracellular signals emanating from the pre-TCR, since thymic development was completely blocked at the CD4-CD8- Double Negative (DN) stage (Zhang et al, 1999)

Summary

INTRODUCTION

After the specific recognition of a peptide antigen bound to an MHC molecule on the surface of an Antigen Presenting Cell, a cascade of intracellular signaling events are triggered in T lymphocytes (Malissen and Bongrand, 2015; Alcover et al, 2018; Courtney et al, 2018). The analysis of those mice strains revealed for the first time that the LAT adaptor acts as a transducer of activation signals, and constitutes a negative regulator of TCR signaling and T-cell homeostasis One of these strains of mice had a Tyr to Phe mutation in tyrosine 136 of LAT (mouse ortholog of human tyrosine 132, LATY136F KI), and presented a paradoxical phenotype with a lymphoproliferative disorder of polyclonal CD4 T cells along with high Th2 cytokine production, despite a reduction in thymic development (Aguado et al, 2002; Sommers et al, 2002). J.CaM2 cells expressing the LATG131D mutant are more sensitive to inhibition of IL-2 production by pre-treatment with anti-CD3, which points to a possible role of this residue in the generation of anergy

METHOD

RESULTS AND DISCUSSION

DATA AVAILABILITY STATEMENT