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

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 (LAT G131D) increases phosphorylation of Tyr132, PLC-γ activation and calcium influx generation. Interestingly, the LAT G131D mutant has a slower protein turnover while being equally sensitive to Fasmediated protein cleavage by caspases. Moreover, J.CaM2 cells expressing LAT G131D 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 LAT G131D 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.

Mutation of the glycine residue preceding the sixth tyrosine of the LAT adaptor severely alters T cell development and activation

“…As seen in Figure 1C , when thymocytes are stimulated with a concentration of 0.62 µg/ml the phosphorylation of PLC-γ1 and LAT tyrosine 136 are increased in mutant mice relative to wild type. This supports previous data generated by our group and others in Jurkat cells ( 12 , 15 ). However, Erk phosphorylation ( Figure 1C ) and Ca 2+ influx generation ( Supplementary Figure 3C ) do not appear to be increased in thymocytes from LAT-G135D mice.…”

“…It has been previously shown that proper integration of calcium signals with other signaling pathways results in full T cell activation, while unopposed calcium signaling leads to anergy ( 25 , 26 ). Although we have not been able to demonstrate an increase in calcium influxes, previous data obtained in Jurkat cells show that the LAT G135D mutation potentiates PLC-γ1 activation and Ca 2+ responses ( 12 , 15 ). Therefore, it was of interest to analyze whether the LAT G135D mutation increased the percentage of anergic T cells.…”

“…To address in vivo the significance of the residue preceding the sixth tyrosine found at position 136 of mouse LAT, we generated KI mice with a mutation that replaced the glycine residue found at position 135 with an aspartate residue (G135D). It has been previously demonstrated that the LAT G135D mutation is able to increase and accelerate phosphorylation of the neighboring LAT-Y136 tyrosine residue ( 12 , 15 ). LAT G135D KI mice were generated using a CRISPR/Cas9 nuclease-based approach on a C57BL/6 background (see Material and Methods).…”

A Novel, LAT/Lck Double Deficient T Cell Subline J.CaM1.7 for Combined Analysis of Early TCR Signaling

“…This cell line may constitute a new tool to untangle molecular events related to the TCR signaling cassette and biology of T cells. Through the lentiviral transfection of J.CaM2 cells, we have recently shown that a mutation of LAT glycine 131 to aspartate increases IL-2 production in response to CD3/CD28 engagement [64]. The new J.CaM1.7 cells may be useful to confirm these data and verify the role of Lck domains or motifs in this behavior.…”

Adapting T Cell Receptor Ligand Discrimination Capability via LAT

“…This slow phosphorylation of LAT Y132 is a feature that had been previously noted (96) but with an unclear basis or significance. Interestingly, the simple substitution of an acidic residue, glutamate or aspartate, at the -1 position (i.e., G131E or G131D) increased the phosphorylation rates of LAT Y132, the recruitment, phosphorylation and activation of PLCg1, and the magnitude and the rate of calcium elevation (50,97). The augmented LAT-PLCg1-calcium pathway resulted in an increase in T cell responses (50).…”