Increased Protein Stability and Interleukin-2 Production of a LAT(G131D)Variant With Possible Implications for T Cell Anergy

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-gamma 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-gamma activation and calcium influx generation. Interestingly, the LAT(G131D)mutant has a slower protein turnover while being equally sensitive to Fas-mediated 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

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

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex.This research was funded by Consejeria de Salud de Andalucia, Junta de Andalucia (grant PI-0055-2017 to E.A.), and Fundacion Biomedica Cadiz Proyectos INIBICA 2019 (grant LI19/I14NCO15 to E.A. and M.M.A.-E.)

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

International audienceThe LAT transmembrane adaptor is essential to transduce intracellular signals triggered by the TCR. Phosphorylation of its four C-terminal tyrosine residues (136, 175, 195, and 235 in mouse LAT) recruits several proteins resulting in the assembly of the LAT signalosome. Among those tyrosine residues, the one found at position 136 of mouse LAT plays a critical role for T cell development and activation. The kinetics of phosphorylation of this residue is delayed as compared to the three other C-terminal tyrosines due to a conserved glycine residue found at position 135. Mutation of this glycine into an aspartate residue (denoted LAT G135D ) increased TCR signaling and altered antigen recognition in human Jurkat T cells and ex vivo mouse T cells. Here, using a strain of LAT G135D knockin mice, we showed that the LAT G135D mutation modifies thymic development, causing an increase in the percentage of CD4+CD8+ double-positive cells, and a reduction in the percentage of CD4+ and CD8+ single-positive cells. Interestingly, the LAT G135D mutation alters thymic development even in a heterozygous state. In the periphery, the LAT G135D mutation reduces the percentage of CD8+ T cells and results in a small increment of γδ T cells. Remarkably, the LAT G135D mutation dramatically increases the percentage of central memory CD8+ T cells. Finally, analysis of the proliferation and activation of T lymphocytes shows increased responses of T cells from mutant mice. Altogether, our results reinforce the view that the residue preceding Tyr136 of LAT constitutes a crucial checkpoint in T cell development and activation

A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling

The adaptor protein linker for activation of T cells (LAT) has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT–Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL) in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance (p = 0.051). Nevertheless, downstream signals such as Ca2+ influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT–Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex