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

BACKGROUND: PD-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 and pathogens. Reciprocally, despite a decade of research, approaches to treat autoimmunity with PD-1 agonists have only had limited successful. To resolve this, new methods must be developed to augment PD-1 function beyond engaging the receptor. METHODS: We 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. RESULTS: Our screen confirmed known regulators in proximal PD-1 signaling and, importantly, identified an additional 1112 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 the most 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. Backgating these genes to canonical cytotoxic T cell signatures revealed PD-1+ HLA-DRHIGH KLRG1LOW T cells as a novel inflammatory subset of T cells. CONCLUSIONS: We concluded that PD-1+ HLA-DRHIGH KLRG1LOW 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.

SLAMF6 clustering is required to augment T cell activation.

The signaling lymphocytic activation molecule (SLAM) family is comprised of nine distinct receptors that are expressed exclusively on hematopoietic cells. Most of these transmembrane receptors are homotypic by nature and downstream signaling occurs when cells that express the same SLAM receptor interact. Previous studies have determined that anti-SLAMF6 antibodies can have a therapeutic effect in autoimmunity and cancer. However, little is known about the role of SLAMF6 in the adaptive immune responses and in order to utilize SLAMF6 interventional approaches, a better understanding of the biology of this receptor in T cell is warranted. Accordingly, the objective of our study was to investigate both functionally and structurally the role of SLAMF6 in T cell receptor (TCR) mediated responses. Biochemical and genetic experiments revealed that SLAMF6 was required for productive TCR downstream signaling. Interestingly, SLAMF6 ectodomain was required for its function, but not for its recruitment to the immunological synapse. Flow-cytometry analysis demonstrated that tyrosine 308 of the tail of SLAMF6 was crucial for its ability to enhance T cell function. Imaging studies revealed that SLAMF6 clustering, specifically with the TCR, resulted in dramatic increase in downstream signaling. Mechanistically, we showed that SLAMF6 enhanced T cell function by increasing T cell adhesiveness through activation of the small GTPase Rap1. Taken together SLAMF6 is an important regulator of T cell activation where both its ectodomain and its endodomain contribute differentially to T cell functions. Additional studies are underway to better evaluate the role of anti-SLAMF6 approaches in specific human diseases.

Biomechanical characterization of TIM protein-mediated Ebola virus-host cell adhesion.

Since the most recent outbreak, the Ebola virus (EBOV) epidemic remains one of the world's public health and safety concerns. EBOV is a negative-sense RNA virus that can infect humans and non-human primates, and causes hemorrhagic fever. It has been proposed that the T-cell immunoglobulin and mucin domain (TIM) family proteins act as cell surface receptors for EBOV, and that the interaction between TIM and phosphatidylserine (PS) on the surface of EBOV mediates the EBOV-host cell attachment. Despite these initial findings, the biophysical properties of the TIM-EBOV interaction, such as the mechanical strength of the TIM-PS bond that allows the virus-cell interaction to resist external mechanical perturbations, have not yet been characterized. This study utilizes single-molecule force spectroscopy to quantify the specific interaction forces between TIM-1 or TIM-4 and the following binding partners: PS, EBOV virus-like particle, and EBOV glycoprotein/vesicular stomatitis virus pseudovirion. Depending on the loading rates, the unbinding forces between TIM and ligands ranged from 40 to 100 pN, suggesting that TIM-EBOV interactions are mechanically comparable to previously reported adhesion molecule-ligand interactions. The TIM-4-PS interaction is more resistant to mechanical force than the TIM-1-PS interaction. We have developed a simple model for virus-host cell interaction that is driven by its adhesion to cell surface receptors and resisted by membrane bending (or tension). Our model identifies critical dimensionless parameters representing the ratio of deformation and adhesion energies, showing how single-molecule adhesion measurements relate quantitatively to the mechanics of virus adhesion to the cell.

EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity.

Programmed cell death 1 (PD-1) is a major coinhibitory receptor and a member of the immunological synapse (IS). To uncover proteins that regulate PD-1 recruitment to the IS, we searched for cytoskeleton-related proteins that also interact with PD-1 using affinity purification mass spectrometry. Among these proteins, EF hand domain family member D2 (EFHD2), a calcium binding adaptor protein, was functionally and mechanistically analyzed for its contribution to PD-1 signaling. EFHD2 was required for PD-1 to inhibit cytokine secretion, proliferation, and adhesion of human T cells. Interestingly, EFHD2 was also required for human T cell-mediated cytotoxicity and for mounting an antitumor immune response in a syngeneic murine tumor model. Mechanistically, EFHD2 contributed to IS stability, lytic vesicles trafficking, and granzyme B secretion. Altogether, EFHD2 is an important regulator of T cell cytotoxicity and further studies should evaluate its role in T cell-mediated inflammation.

The SLAM family receptors: Potential therapeutic targets for inflammatory and autoimmune diseases.

The signaling lymphocytic activation molecule (SLAM) family is comprised of nine distinct receptors (SLAMF1 through SLAMF9) that are expressed on hematopoietic cells. All of these receptors, with the exception of SLAMF4, are homotypic by nature as downstream signaling occurs when hematopoietic cells that express the same SLAM receptor interact. The SLAM family receptor function is largely controlled via SLAM associated protein (SAP) family adaptors. The SAP family adaptors consist of SAP, Ewing sarcoma associated transcript (EAT)-2, and EAT-2-related transducer (ERT). These adaptors associate with the cytoplasmic domain of the SLAM family receptors through phosphorylated tyrosines. Defects in SLAM family members and SAP adaptors have been implicated in causing immune deficiencies. This is exemplified in patients with X-linked lymphoproliferative (XLP) disease, where SAP undergoes a loss of function mutation. Furthermore, evidence has been accumulating that SLAM family members are potential targets for inflammatory and autoimmune diseases. This review will discuss the structure and function of the SLAM family receptors and SAP family adaptors, their role in immune regulation, and potential approaches to target this family of receptors therapeutically.

Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets.

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease characterized by autoantibody-induced platelet clearance. Some ITP patients are refractory to standard immunosuppressive treatments such as intravenous immunoglobulin (IVIg). These patients often have autoantibodies that target the ligand-binding domain (LBD) of glycoprotein Ibα (GPIbα), a major subunit of the platelet mechanoreceptor complex GPIb-IX. However, the molecular mechanism of this Fc-independent platelet clearance is not clear. Here, we report that many anti-LBD monoclonal antibodies such as 6B4, but not AK2, activated GPIb-IX in a shear-dependent manner and induced IVIg-resistant platelet clearance in mice. Single-molecule optical tweezer measurements of antibodies pulling on full-length GPIb-IX demonstrated that the unbinding force needed to dissociate 6B4 from the LBD far exceeds the force required to unfold the juxtamembrane mechanosensory domain (MSD) in GPIbα, unlike the AK2-LBD unbinding force. Binding of 6B4, not AK2, induced shear-dependent unfolding of the MSD on the platelet, as evidenced by increased exposure of a linear sequence therein. Imaging flow cytometry and aggregometry measurements of platelets and LBD-coated platelet-mimetic beads revealed that 6B4 can sustain crosslinking of platelets under shear, whereas 6B4 Fab and AK2 cannot. These results suggest a novel mechanism by which anti-LBD antibodies can exert a pulling force on GPIb-IX via platelet crosslinking, activating GPIb-IX by unfolding its MSD and inducing Fc-independent platelet clearance.

Dual Regulation of L-Selectin-Mediated Leukocyte Adhesion by Endothelial Surface Glycocalyx.

Endothelial surface glycocalyx (ESG) is a carbohydrate-rich, gel-like layer found on vascular endothelium, serving critical functions in mechanotransduction of blood flows, maintenance of the endothelial permeability, and the control of leukocyte adhesion and inflammation. This study aimed to clarify the role of ESG in the adhesion between leukocytes and Human Umbilical Vein Endothelial Cells (HUVECs) under resting or inflammatory conditions. Using an atomic force microscopy-based single-cell adhesion assay, we directly quantified the detachment force and work perpendicular to the cell membrane. Detachment force and work were measured for every separation event of a leukocyte from a HUVEC with ESG, or with the major ESG glycosaminoglycan components, heparan sulfate (HS) and hyaluronic acid (HA) removed. For the resting HUVECs, when HS and/or HA were removed, the detachment force and work increased dramatically. For the HUVECs activated by inflammatory cytokine tumor necrosis factor alpha, we observed increases in the detachment force and work compared to the resting HUVECs, and removal of HS and/or HA resulted in significant decreases in the detachment force and work. The results demonstrate that the ESG layer serves a dual function: (1) on resting endothelium, it prevents leukocyte adhesion, and (2) under inflammatory conditions, it participates in endothelial-leukocyte interactions with molecules such as selectins.

Mechanotransduction of the endothelial glycocalyx mediates nitric oxide production through activation of TRP channels.

The endothelial surface glycocalyx (ESG) is a carbohydrate-rich layer found on the vascular endothelium, serving critical functions in the mechanotransduction of blood flow-induced forces. One of the most important protective functions of the ESG is to mediate the production of nitric oxide (NO) in response to blood flow. However, the detailed mechanism underlying ESG's mechanotransduction of the production of NO has not been completely identified. Herein, using the cultured rat brain microvascular endothelial cells (bEnd.3) as a model system, we have implemented a combined atomic force and fluorescence microscopy approach to show that the ESG senses and transduces vertical mechanical stretch to produce NO. This rapid NO production is dependent on the presence of both heparan sulfate (HS) and hyaluronic acid (HA) in ESG, as the removal of HS and/or HA leads to a significant decrease in NO production. Moreover, the production of NO is dependent on the intake of Ca2+ via endothelial transient receptor potential (TRP) channels. Together, our results demonstrate the molecular mechanism of rapid production of NO in response to vertical mechanical stretch.

Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor.

Mechanisms by which blood cells sense shear stress are poorly characterized. In platelets, glycoprotein (GP)Ib-IX receptor complex has been long suggested to be a shear sensor and receptor. Recently, a relatively unstable and mechanosensitive domain in the GPIbα subunit of GPIb-IX was identified. Here we show that binding of its ligand, von Willebrand factor, under physiological shear stress induces unfolding of this mechanosensory domain (MSD) on the platelet surface. The unfolded MSD, particularly the juxtamembrane 'Trigger' sequence therein, leads to intracellular signalling and rapid platelet clearance. These results illustrate the initial molecular event underlying platelet shear sensing and provide a mechanism linking GPIb-IX to platelet clearance. Our results have implications on the mechanism of platelet activation, and on the pathophysiology of von Willebrand disease and related thrombocytopenic disorders. The mechanosensation via receptor unfolding may be applicable for many other cell adhesion receptors.