Moore, E. K. | bioRxiv Science

bioRxiv2024

PAG orchestrates T cell immune synapse function by binding to actin

Many immunotherapies impact T cell function by impacting the immune synapse. While immunotherapy is extremely successful in some patients, in many others, it fails to help or causes complications, including immune-related adverse events. Phosphoprotein Associated with Glycosphingolipid Rich Microdomains 1 (PAG) is a transmembrane scaffold protein with importance in T cell signaling. PAG has 10 tyrosine phosphorylation sites where many kinases and phosphatases bind. PAG is palmitoylated, so it localizes in lipid rafts of the membrane, and contains a C-terminal PDZ domain to link to the actin cytoskeleton. As a link between signaling-protein-rich membrane regions and the actin cytoskeleton, PAG is an exciting and novel target for manipulating immune function. Here, we sought to determine if PAG works with actin to control T cell synapse organization and function. We found that PAG and actin dynamics are tightly coordinated during synapse maturation. A PDZ domain mutation disrupts the PAG-actin interaction, significantly impairing synapse formation, stability, and function. To assess the impact of the PDZ mutation functionally in vivo, we employed a mouse model of type IV hypersensitivity and an OVA-tumor mouse model. In both systems, mice with T cells expressing PDZ-mutant PAG had diminished immune responses, including impaired cytotoxic function. These findings highlight the importance of the PAG-actin link for effective T cell immune synapse formation and function. The results of our study suggest that targeting PAG is a promising approach for modulating immune responses and treating immune-related diseases. One Sentence SummaryAdaptor protein PAG links to the actin cytoskeleton, and this link is essential for T cell synapse formation and cytotoxic function.

immunology↗

bioRxiv2022

The CD58:CD2 axis is co-regulated with PD-L1 via CMTM6 and governs anti-tumor immunity

The cell autonomous balance of immune-inhibitory and -stimulatory signals is a critical yet poorly understood process in cancer immune evasion. Using patient-derived co-culture models and humanized mouse models, we show that an intact CD58:CD2 interaction is necessary for anti-tumor immunity. Defects in this axis lead to multi-faceted immune evasion through impaired CD2-dependent T cell polyfunctionality, T cell exclusion, impaired intra-tumoral proliferation, and concurrent protein stabilization of PD-L1. We performed genome-scale CRISPR-Cas9 and CD58 coimmunoprecipitation mass spectrometry screens identifying CMTM6 as a key stabilizer of CD58, and show that CMTM6 is required for concurrent upregulation of PD-L1 in CD58 loss. Single-cell RNA-seq analysis of patient melanoma samples demonstrates that most TILs lack expression of primary costimulatory signals required for response to PD-1 blockade (e.g. CD28), but maintain strong CD2 expression, thus providing an opportunity to mobilize a so far therapeutically untapped pool of TILs for anti-tumor immunity. We identify two potential therapeutic avenues, including rescued activation of human CD2-expressing TILs using recombinant CD58 protein, and targeted disruption of PD-L1/CMTM6 interactions. Our work identifies an underappreciated yet critical axis at the nexus of cancer immunity and evasion, uncovers a fundamental mechanism of co-inhibitory and -stimulatory signal balancing, and provides new approaches to improving cancer immunotherapies.

cancer biology↗