Biology subjects

Mor, A.

Publications and source records attributed to Mor, A..

4 recordsLinked to original sources

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↗

bioRxiv2023

PD-1 signaling uncovers a pathogenic subset of T cells in inflammatory arthritis

BackgroundPD-1 is an immune checkpoint on T cells and interventions to block this receptor result in T cell activation and enhanced immune response to tumors. Paired to that, and despite a decade of research, approaches to treat autoimmunity with PD-1 agonists still need to be more successful. To resolve this, new methods must be developed to augment PD-1 function beyond engaging the receptor. MethodsWe conducted a flow cytometry analysis of T cells isolated from the peripheral blood and synovial fluid of patients with rheumatoid arthritis. In addition, we performed a genome-wide CRISPR/Cas9 screen to identify genes associated with PD-1 signaling. We further analyzed genes involved in PD-1 signaling using publicly available bulk and single-cell RNA sequencing datasets. ResultsOur screen confirmed known regulators in proximal PD-1 signaling and, importantly, found an additional 1,112 unique genes related to PD-1 ability to inhibit T cell functions. These genes were strongly associated with the response of cancer patients to PD-1 blockades and with high tumor immune dysfunction and exclusion scores, confirming their role downstream of PD-1. Functional annotation revealed that more significant genes uncovered were those associated with known immune regulation processes. Remarkably, these genes were considerably downregulated in T cells isolated from patients with inflammatory arthritis, supporting their overall inhibitory functions. A study of rheumatoid arthritis single-cell RNA sequencing data demonstrated that five genes, KLRG1, CRTAM, SLAMF7, PTPN2, and KLRD1, were downregulated in activated and effector T cells isolated from synovial fluids. Back-gating these genes to canonical cytotoxic T cell signatures revealed PD-1+ HLA-DRHIGH KLRGLOW T cells as a novel inflammatory subset of T cells. ConclusionWe concluded that PD-1+ HLA-DRHIGH KLRGLOW T cells are a potential target for future PD-1 agonists to treat inflammatory diseases. Our study uncovers new genes associated with PD-1 downstream functions and, therefore, provides a comprehensive resource for additional studies that are much needed to characterize the role of PD-1 in the synovial subset of T cells.

immunology↗

bioRxiv2023

SAP expressing T peripheral helper cells identify systemic lupus erythematosus patients with lupus nephritis

IntroductionT follicular (TFH) and peripheral helper (TPH) cells have been increasingly recognized as a pathogenic subset of CD4 T cells in systemic lupus erythematosus (SLE). The SLAM Associated Protein (SAP) regulates TFH and TPH function by binding to the co -stimulatory signaling lymphocyte activation molecule family (SLAMF) receptors that mediate T cell - B cell interactions. SAP and SLAMF are critical for TPH dependent B cell maturation into autoantibody-producing plasma cells that characterize SLE pathogenesis. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated using density gradient separation from whole blood. Cells were stained for cell surface markers, followed by permeabilization and staining of intracellular SAP for spectral flow cytometry analysis. Additionally, we analyzed SAP expression from renal infiltrating lupus nephritis (LN) T cells using the publicly available single-cell RNA sequencing (sc-RNA seq) Accelerated Medicines Partnership (AMP) in SLE dataset. ResultsPBMCs from 30 patients with SLE (34{+/-}10 years old, 83% female), including 10 patients with LN, were analyzed. We found an increase in total SAP-positive CD4 and CD8 T cells in SLE compared with controls (55.5{+/-}2.6 vs. 41.3{+/-}3.4, p=0.007 and 52.5{+/-}3.0 vs. 39.2{+/-}2.8, p=0.007 respectively). In CD4 T cells, the highest SAP expression was in the TPH subset. The frequency of SAP+TPH in circulation correlated with disease activity, SLE patients with renal disease had higher levels of circulating SAP+TPH that remained significant after adjusting for age, sex, race, low complements, and elevated anti-dsDNA (p=0.014). scRNA-seq data of renal infiltrating T cells identified increased SAP from LN compared with control kidney biopsy samples (p=0.03), including an expansion of SAP-positive TFH-like subsets in the LN kidneys. Increased SAP expression in LN was associated with the differential expression of SLAMF3 and SLAMF7 as well as granzyme K and EOMES. The existence of two predominant SAP-expressing subsets, the TFH-like CD4 T cells and granzyme K positive effector CD8 T cells, was verified using scRNA-seq data from a human transcriptomic atlas of fifteen major organs. ConclusionThe expansion of SAP expressing T helper cells was associated with lupus nephritis in our cohort and verified using scRNA-seq data of renal infiltrating T cells. Improved understanding of SLAM/SAP signaling can identify new therapeutic targets in LN.

immunology↗

bioRxiv2023

Exclusion of PD-1 from the immune synapse: a novel strategy to modulate T cell function

Targeting immune checkpoint receptors on T cells is a common cancer treatment strategy. Frequently, this is accomplished through antibodies targeting the ligand of inhibitory co-receptors. Blocking the immune checkpoint PD-1 binding to its ligands PD-L1 and PD-L2 prevents downstream signaling and enhances anti-tumor T cell responses. This approach improved cancer patients outcome. However, only one-third of the patients respond to these treatments. To better understand the mechanism of anti-PD-1 antibodies, we explored the location of PD-1 within the immune synapse. Surprisingly, we discovered that anti-PD-1 antibodies, besides blocking the interaction between PD-1 and its ligands, also removed PD-1 from the synapse. We demonstrated a correlation between removing PD-1 from the synapse by anti-PD-1 antibodies and the extent of T cell activation. Interestingly, a short version of the anti-PD-1 antibody, F(ab)2, failed to remove PD-1 from the synapse and activate T cells. Using syngeneic tumor model, we showed a superior anti-tumor effect to anti-PD-1 antibody over the shorter version of the antibody. Our data indicates that anti-PD-1 antibodies activate T cells by removing PD-1 away from the synapse and changing the location of PD-1 or other immune receptors within immune synapse could serve as an alternative, efficient approach to treat cancer.

immunology↗