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.

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.