Balagopalan, L. | bioRxiv Science

bioRxiv2025

Weak SLP-76-PLC-γ1 interaction in the LAT-nucleated multi-protein complex fine-tunes TCR signal strength to optimize T cell responsiveness

Upon TCR engagement several protein tyrosine kinases are recruited and activated, and adapter proteins and enzymes are phosphorylated on tyrosine residues, leading to further events characterizing activated T cells. Phosphorylation of the LAT adapter protein enables binding of the enzyme PLC-{gamma}1 and of a dimer of two additional adapter proteins Gads and SLP-76, forming a tetrameric structure. Within this heterotetramer there is a weak interaction between SLP-76 and PLC-{gamma}1, and the relevant binding sites of SLP-76 and PLC-{gamma}1 are highly conserved in vertebrates. To address the biological relevance of this weak interaction, we introduced a mutation in the SLP-76 that enhanced its affinity for PLC-{gamma}1 and found that this mutation increased PLC-{gamma}1 activity and altered thymocyte development and peripheral T cell responses due to enhanced TCR signal strength. The conserved weak SLP-76-PLC-{gamma}1 interaction is critical for the controlled activation of PLC-{gamma}1, thus fine-tuning TCR signal strength to optimize T cell-mediated immunity.

immunology↗

bioRxiv2022

Generation of anti-tumor chimeric antigen receptors incorporating T cell signaling motifs

Chimeric antigen receptors (CAR) T cells have been successfully used to treat lymphoma, leukemia, and multiple myeloma, but adverse effects due to cytokine secretion, CAR-T cell exhaustion, and loss of target antigen have limited their potential. Furthermore, while CARs have been designed to harness T Cell Receptor (TCR) signaling, they are significantly less sensitive than TCRs, resulting in suboptimal signaling. We have developed novel Chimeric Adapter Proteins (CAPs) that are designed to trigger signaling downstream of the TCR{zeta} chain. CAPs are chimeric molecules that contain adapter domains in tandem with the kinase domain of ZAP70, fused to an extracellular targeting domain. We hypothesized that CAPs would be more potent than CARs because kinetic proofreading steps that define the signaling threshold and the inhibitory regulation of upstream molecules are bypassed. Indeed, second generation CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19-28{zeta} CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28{zeta}-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.

immunology↗