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.

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.

Activation through the T cell receptor (TCR) initiates a signaling cascade in T cells that induces extensive molecular and cellular changes. The adaptor protein Linker for Activation of T cells (LAT) plays an essential role in transducing activation and regulatory signals downstream of the TCR. Phosphorylation of LAT tyrosine residues recruits multiple signaling proteins, leading to the assembly of the LAT signalosome, which is crucial for relaying signals that regulate T cell development and function. We previously showed that substitution of a negatively charged amino acid segment preceding the fifth tyrosine residue of LAT (Tyr127 in humans or Tyr132 in mouse LAT) enhances some early TCR signaling events, whereas downstream responses, such as Ca²⁺ influx and Erk phosphorylation, are partially inhibited. To investigate the physiological relevance of this segment in vivo, we generated a new LAT knock-in mouse strain (Lat^NIL) in which the negatively charged segment was replaced with a non-charged sequence. Unexpectedly, this mutation led to an alternative splicing event in the Lat gene that excluded exons 6 and 7, resulting in a frameshift, a premature stop codon at residue 145, and the loss of the six C-terminal tyrosine residues of LAT. Homozygous Lat^NIL/NIL mice showed a phenotype similar to that of LAT-knockout and Lat^4YF mice (in which the four C-terminal tyrosines had been mutated to phenylalanine). Interestingly, homozygous Lat^NIL/NIL mice exhibited a distinct population of γδ T cells in lymphoid organs, which has not been observed in LAT-KO or Lat^4YF mice. These γδ T cells expressed higher levels of CD27 compared to those in wild-type and LAT-KO mice, suggesting altered activation or differentiation states. Together, these data highlight how subtle alterations in LAT structure can profoundly impact T cell signaling and lineage composition.

T lymphocytes are key players in adaptive immune responses through the recognition of peptide antigens through the T Cell Receptor (TCR). After TCR engagement, a signaling cascade is activated, leading to T cell activation, proliferation, and differentiation into effector cells. Delicate control of activation signals coupled to the TCR is needed to avoid uncontrolled immune responses involving T cells. It has been previously shown that mice deficient in the expression of the adaptor NTAL (Non-T cell activation linker), a molecule structurally and evolutionarily related to the transmembrane adaptor LAT (Linker for the Activation of T cells), develop an autoimmune syndrome characterized by the presence of autoantibodies and enlarged spleens. In the present work we intended to deepen investigation into the negative regulatory functions of the NTAL adaptor in T cells and its potential relationship with autoimmune disorders. For this purpose, in this work we used Jurkat cells as a T cell model, and we lentivirally transfected them to express the NTAL adaptor in order to analyze the effect on intracellular signals associated with the TCR. In addition, we analyzed the expression of NTAL in primary CD4+ T cells from healthy donors and Rheumatoid Arthritis (RA) patients. Our results showed that NTAL expression in Jurkat cells decreased calcium fluxes and PLC-γ1 activation upon stimulation through the TCR complex. Moreover, we showed that NTAL was also expressed in activated human CD4+ T cells, and that the increase of its expression was reduced in CD4+ T cells from RA patients. Our results, together with previous reports, suggest a relevant role for the NTAL adaptor as a negative regulator of early intracellular TCR signaling, with a potential implication in RA.

Hepatitis C virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. When expressed in CD4⁺ T lymphocytes, it can induce modifications in several essential cellular and biological networks. To shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed HCV-core subcellular localization and its associations with host proteins in Jurkat T cells. In order to investigate the intracellular localization of Hepatitis C virus core protein, we have used a lentiviral system to transduce Jurkat T cells and subsequently localize the protein using immunoelectron microscopy techniques. We found that in Jurkat T cells, Hepatitis C virus core protein mostly localizes in the nucleus and specifically in the nucleolus. In addition, we performed pull-down assays combined with Mass Spectrometry Analysis, to identify proteins that associate with Hepatitis C virus core in Jurkat T cells. We found proteins such as NOLC1, PP1γ, ILF3, and C1QBP implicated in localization and/or traffic to the nucleolus. HCV-core associated proteins are implicated in RNA processing and RNA virus infection as well as in functions previously shown to be altered in Hepatitis C virus core expressing CD4⁺ T cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. Thus, in the current work, we show the ultrastructural localization of Hepatitis C virus core and the first profile of HCV core associated proteins in T cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of Hepatitis C virus core protein. Thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying HCV core mediated alterations that had been described in relevant biological processes in CD4⁺ T cells.

The adapter protein GRB2 is central to T-cell receptor (TCR) signal transduction, yet its precise role in setting activation thresholds remains controversial. To clarify its functions, we generated stable GRB2-knockout (KO) Jurkat T-cell lines using CRISPR/Cas9 gene editing. Systematic analysis of TCR/CD3-stimulated GRB2-KO cells revealed widespread signaling hyper-activation: proximal ZAP-70 phosphorylation was drastically increased, and downstream signaling was markedly enhanced, evidenced by elevated ERK and PLC-γ1 phosphorylation along with heightened intracellular Ca²⁺ influx. In contrast, LAT phosphorylation was significantly decreased in GRB2-KO cells, indicating that GRB2 acts downstream or in parallel to constrain activation even when LAT phosphorylation is blunted. Intriguingly, loss of GRB2 was accompanied by reduced THEMIS protein expression, suggesting a potential link between GRB2 and THEMIS levels in human T cells. These findings support a role for GRB2 as an important negative gatekeeper of TCR signaling whose absence promotes hyperactivation despite reduced LAT phosphorylation, while to enhanced activation pathways while highlighting an intriguing association between GRB2 and THEMIS. Together, our results provide new mechanistic insights into the regulatory constraints governing human T-cell activation.