P-selectin Glycoprotein Ligand-1 Expression Research Articles

Correlation between plasma PSGL-1 and FIGO stage, tumor metastasis, and survival in epithelial ovarian cancer.

Plasma circulating P-selectin glycoprotein ligand-1 (PSGL-1) levels and its clinical correlation in patients with epithelial ovarian cancer (EOC) are unknown. The study determined plasma PSGL-1 levels in EOC patients and investigated its relationship with clinicopathological factors and prognosis. Plasma PSGL-1 levels were measured using ELISA in 69 patients with EOC, 34 patients with benign ovarian cystadenoma, and 36 healthy controls. Subsequently, the relationship between PSGL-1 levels and clinicopathological characteristics of patients, as well as the prognosis of EOC patients, was examined. Additionally, the specificity and sensitivity of plasma PSGL-1 were assessed through ROC curve analysis. Plasma PSGL-1 was upregulated in EOC patients compared with healthy subjects and/or patients with benign ovarian cystadenoma (p < 0.01). Elevated levels of PSGL-1 in the plasma were positively associated with advanced FIGO stage (p < 0.001), tumor size (p = 0.001), tumor metastasis (p = 0.036), and tumor recurrence (p = 0.013), while was negatively correlated with residual tumor size (p < 0.001). Kaplan-Meier survival analysis showed that high plasma PSGL-1 levels were associated with progression-free survival (p = 0.0345). In univariate and multivariate Cox regression analyses, PSGL-1 (HR = 1.456, p = 0.009) was an independent prognostic marker. Plasma PSGL-1 levels distinguished EOC patients and healthy individuals (AUC = 0.905), patients at late and early FIGO stages (AUC = 0.886), and metastatic and non-metastatic EOC (AUC = 0.722). The expression of plasma PSGL-1 is significantly increased in patients with EOC, serving as a reliable biomarker to differentiate between healthy individuals and those with EOC. Furthermore, PSGL-1 in patients is correlated with prognostic indicators, such as advanced FIGO stage, tumor lymph node metastasis, and progression-free survival.

Antibody targeting of surface P-selectin glycoprotein ligand 1 leads to lymphoma apoptosis and tumorigenesis inhibition.

Lymphomas are a heterogeneous group of diseases that originate from T, B or natural killer cells. Lymphoma treatment is based on chemotherapy, radiotherapy, and monoclonal antibody (mAb) or other immunotherapies. The P-selectin glycoprotein ligand 1 (PSGL-1) is expressed at the surface of hematological malignant cells and has been shown to have a pro-oncogenic role in multiple myeloma and lymphoma. Here, we investigated the expression and therapeutic potential of PSGL-1 in T and B cell lymphomas. By flow cytometry analysis, we found that PSGL-1 was expressed in both T and B cell-derived lymphoma cell lines but generally at higher levels in T cell lymphoma cell lines. For most T and B cell-derived lymphoma cell lines, in vitro targeting with the PL1 mAb, which recognizes the PSGL-1 N-terminal extracellular region and blocks functional interactions with selectins, resulted in reduced cell viability. The PL1 mAb pro-apoptotic activity was shown to be dose-dependent, to be linked to increased ERK kinase phosphorylation, and to be dependent on the MAP kinase signaling pathway. Importantly, anti-PSGL-1 treatment of mice xenografted with the HUT-78 cutaneous T-cell lymphoma cell line resulted in decreased tumor growth, had no effect on in vivo proliferation, but increased the levels of apoptosis in tumors. Anti-PSGL-1 treatment of mice xenografted with a Burkitt lymphoma cell line that was resistant to anti-PSGL-1 treatment in vitro, had no impact on tumorigenesis. These findings show that PSGL-1 antibody targeting triggers lymphoma cell apoptosis and substantiates PSGL-1 as a potential target for lymphoma therapy.

RAGE in circulating immune cells is fundamental for hippocampal inflammation and cognitive decline in a mouse model of latent chronic inflammation

BackgroundLatent chronic inflammation has been proposed as a key mediator of multiple derangements in metabolic syndrome (MetS), which are increasingly becoming recognized as risk factors for age-related cognitive decline. However, the question remains whether latent chronic inflammation indeed induces brain inflammation and cognitive decline. MethodsA mouse model of latent chronic inflammation was constructed by a chronic subcutaneous infusion of low dose lipopolysaccharide (LPS) for four weeks. A receptor for advanced glycation end products (RAGE) knockout mouse, a chimeric myeloid cell specific RAGE-deficient mouse established by bone marrow transplantation and a human endogenous secretory RAGE (esRAGE) overexpressing adenovirus system were utilized to examine the role of RAGE in vivo. The cognitive function was examined by a Y-maze test, and the expression level of genes was determined by quantitative RT-PCR, western blot, immunohistochemical staining, or ELISA assays. ResultsLatent chronic inflammation induced MetS features in C57BL/6J mice, which were associated with cognitive decline and brain inflammation characterized by microgliosis, monocyte infiltration and endothelial inflammation, without significant changes in circulating cytokines including TNF-α and IL-1β. These changes as well as cognitive impairment were rescued in RAGE knockout mice or chimeric mice lacking RAGE in bone marrow cells. P-selectin glycoprotein ligand-1 (PSGL-1), a critical adhesion molecule, was induced in circulating mononuclear cells in latent chronic inflammation in wild-type but not RAGE knockout mice. These inflammatory changes and cognitive decline induced in the wild-type mice were ameliorated by an adenoviral increase in circulating esRAGE. Meanwhile, chimeric RAGE knockout mice possessing RAGE in myeloid cells were still resistant to cognitive decline and brain inflammation. ConclusionsThese findings indicate that RAGE in inflammatory cells is necessary to mediate stimuli of latent chronic inflammation that cause brain inflammation and cognitive decline, potentially by orchestrating monocyte activation via regulation of PSGL-1 expression. Our results also suggest esRAGE-mediated inflammatory regulation as a potential therapeutic option for cognitive dysfunction in MetS with latent chronic inflammation.

Abstract P3027: Potasium Adaptation Down-regulates PSGL-1 And I-CAM-1 Gene Expressions In Salt -Induced Hypertensive Sprague Dawley Rats

Background: There is an increasing prevalence of hypertension and dietary salt intake has been linked as its risk factor. Although many studies have elucidated this association, the mechanisms by which the increase in salt intake leads to development hypertension have been postulated but yet inconclusive. P - Selectin glycoprotein ligand-1(PSGL-1) and Intercellular adhesion molecule (I-CAM-1) have been implicated in the development of salt sensitive hypertension through vascular inflammation and injury. The role of potassium in blood pressure regulation has been severally reported. Scarce reports of the association of potassium adaptation with high blood pressure development form the basis of this study. We aimed at defining the role of potassium adaptation in the development of salt -induced hypertension in Sprague Dawley rats. Methods: This study was divided into two phases: the induction and confirmation of hypertension and the effect of potassium adaptation. The test group received high salt diet (8% NaCl) for 6 weeks while the control animals were fed with standard rat chow and given water ad libitum. Phase II comprised of pre-treated animals with KCl before induction of hypertension and post induction treatment group with KCl. Blood pressures were monitored as well as blood samples taken for analysis of the gene expressions of I-CAM-1 and PSGL-1 using Standard Laboratory methods Results: We observed a spontaneous significant increases in the blood pressures of the test animals as from the 4 th week compared with the control (P&lt;.05, respectively), the potassium -adapted group and KCl post treated groups exhibited relatively stable blood pressure compared with the control. There were significant increases in the expression of PSGL -1 and I-CAM-1 protein in all the test groups but lower in the potassium adapted and post-treated animal groups when compared with hypertensive group. The potassium adapted group did not develop hypertension throughout the period. Conclusion: Raised arterial blood pressure is inducible by high salt diet. This is associated with an increased expressions of PSGL-1 and ICAM-1. Potassium adaptation inhibited the development of hypertension and down-regulate PSGL-1 expression significantly.

NMR indicates the N-termini of PSGL1 and CCR7 bind competitively to the chemokine CCL21

Chemokines are from a family of secreted cytokines that direct the trafficking of immune cells to coordinate immune responses. Chemokines are involved in numerous disease states, including responding to infections, autoimmune disorders, and cancer metastasis. Ther are chemokines, like CCL21, that signal for cellular migration through the activation of G protein-coupled receptors, like CCR7, through interaction with the receptor's extracellular N-terminus, loops, and core of the receptor. CCL21 is involved in routine immune surveillance but can also attract metastasizing cancer cells to lymph nodes. P-selectin glycoprotein ligand 1 (PSGL1) has a role in cellular adhesion during chemotaxis and is a transmembrane signaling molecule. PSGL1 expression enhances chemotactic responses of T cells to CCL21. Here NMR studies indicate the binding sites on CCL21 for the N-termini or PSGL1 and CCR7 overlap, and binding of the N-termini of PSGL1 and CCR7 is competitive.

Low P-Selectin Glycoprotein Ligand-1 Expression in Neutrophils Associates with Disease Activity and Deregulated NET Formation in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the generation of anti-DNA autoantibodies due to exposure of immune cells to excessive amounts of extracellular DNA. Lack of P-selectin in mice induces the development of a lupus-like syndrome and patients with cutaneous lupus have reduced P-selectin expression in skin vessels. Using flow cytometry we analyzed in healthy donors and patients the expression of P-selectin Glycoprotein Ligand-1 (PSGL-1) in circulating neutrophils and the implication of PSGL-1/P-selectin interaction in neutrophil extracellular traps (NETs) generation. We found a statistical significance that neutrophils from active SLE patients have a reduced expression of PSGL-1 and low levels of PSGL-1 in neutrophils from SLE patients associated with the presence of anti-dsDNA antibodies, clinical lung involvement, Raynaud's phenomenon, and positive lupus anticoagulant. PSGL-1 is present along the DNA in the NET. In healthy donors, neutrophil interaction with immobilized P-selectin triggers Syk activation, increases the NETs percentage and reduces the amount of DNA extruded in the NETs. In active SLE patients, neutrophil interaction with P-selectin does not activate Syk or reduce the amount of DNA extruded in the NETs, that might contribute to increase the extracellular level of DNA and hence, to disease pathogenesis.

PSGL-1 is a novel tumor microenvironment prognostic biomarker with cervical high-grade squamous lesions and more.

Macrophages secrete many cytokines and chemokines, which can provoke either an anti-tumor or pro-tumor immune response. P-selectin glycoprotein ligand-1 (PSGL-1) is expressed in macrophages and plays a vital role in synergizing for a more robust anti-tumor response. However, there are few studies about PSGL-1 expression status and clinical value of biological function in cervical cancer. In this study, 565 participants were enrolled. PSGL-1 mRNA was detected by real-time quantitative PCR (qPCR) with cervical cytology specimens. The relationship between PSGL-1 and cervical intraepithelial neoplasia in two grades and more (CIN2+) was analyzed, and the optimal cut-off values of PSGL-1 to predict CIN2+ were calculated. In addition, the clinical significance of PSGL-1 in cervical cancer was determined by Kaplan-Meier Cox regression based on the database. The mean PSGL-1 increased significantly with cervical lesion development, especially compared with CIN2+ (p<0.05). Moreover, the expression of PSGL-1 increased significantly in HPV-16/18 positive and HPV-18 positive, but not in HPV-16 positive and other HR-HPV positive. And then, it demonstrated that the area under the receiver operating characteristic curve (AUC) of PSGL-1 was 0.820, and an optimal cut-off 0.245. Furthermore, the PSGL-1 had the highest odds ratio and highest OR (OR= 8.707; 95% CI (.371-19.321)) for the detection of CIN 2+. In addition, our result also indicated that higher PSGL-1 expression was significantly related to a better prognosis in cervical cancer due to immune cell infiltration. PSGL-1≥0.245 in cervical cytology specimens is a new auxiliary biomarker of CIN2+, and it may be a promising prognosis predictor and potential immunotherapy target linked with immune infiltration of cervical cancer.

QiShen YiQi and its components attenuate acute thromboembolic stroke and carotid thrombosis by inhibition of CD62P/PSGL-1-mediated platelet-leukocyte aggregate formation

BackgroundQiShen YiQi (QSYQ) dropping pill, a component-based Chinese medicine consisting of benefiting Qi (YQ) and activating blood (HX) components, has been reported to exert a beneficial effect on cerebral ischemia-induced stroke. However, its efficacy and pharmacological mechanism on acute thromboembolic stroke is not clear. PurposeThis study is to explore the preventative effect and pharmacological mechanism of QSYQ and its YQ/HX components on the formation of platelet-leukocyte aggregation (PLA) in acute thromboembolic stroke. Study design and methodsIn vivo thromboembolic stroke model and FeCl3-induced carotid arterial occlusion models were used. Immunohistochemistry, Western blot, RT-qPCR, and flow cytometry experiments were performed to reveal the pharmacological mechanisms of QSYQ and its YQ/HX components. ResultsIn thromboembolic stroke rats, QSYQ significantly attenuated infarct area, improved neurological recovery, reduced PLA formation, and inhibited P-selection (CD62P)/ P-selectin glycoprotein ligand-1 (PSGL-1) expressions. The YQ component preferentially down-regulated PSGL-1 expression in leukocyte, while the HX component preferentially down-regulated CD62P expression in platelet. In carotid arterial thrombosis mice, QSYQ and its YQ/HX components inhibited thrombus formation, prolonged vessel occlusion time, reduced circulating leukocytes and P-selectin expression. PLA formation and platelet/leukocyte adhesion to endothelial cell were also inhibited by QSYQ and its YQ/HX components in vitro. ConclusionQSYQ and YQ/HX components attenuated thromboembolic stroke and carotid thrombosis by decreasing PLA formation via inhibiting CD62P/PSGL-1 expressions. This study shed a new light on the prevention of thromboembolic stroke.

Myeloid Mineralocorticoid Receptor Transcriptionally Regulates P-Selectin Glycoprotein Ligand-1 and Promotes Monocyte Trafficking and Atherosclerosis.

MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity. These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation.

Constitutive PSGL-1 Correlates with CD30 and TCR Pathways and Represents a Potential Target for Immunotherapy in Anaplastic Large T-Cell Lymphoma.

Simple SummaryP-selectin glycoprotein ligand-1 (PSGL-1), coded by the SELPLG gene, is the major ligand of selectins and plays a pivotal role in tethering, rolling and extravasation of immune cells. PSGL-1 involvement in core molecular programs, such as SYK, PLCγ2, PI3Kγ or MAPK pathways, suggests additional functions beyond the modulation of cell trafficking. Recently, several studies identified a novel mechanism responsible for PSGL-1-mediated immune suppression in the tumor microenvironment and proved a novel concept of PSGL-1 as a critical checkpoint molecule for tumor immunotherapy. The immunotherapeutic approach has gained an ever-growing interest in the treatment of several hematological malignancies, and in particular, novel targets for immunotherapy are still highly sought-after in T-cell lymphomas. Based on our results obtained through gene expression profiling and immunohistochemical analysis, PSGL-1, already suggested as a potential target in multiple myeloma humoral immunotherapy, could be considered noteworthy among the candidates.Due to the high expression of P-selectin glycoprotein ligand-1 (PSGL-1) in lymphoproliferative disorders and in multiple myeloma, it has been considered as a potential target for humoral immunotherapy, as well as an immune checkpoint inhibitor in T-cells. By investigating the expression of SELPLG in 678 T- and B-cell samples by gene expression profiling (GEP), further supported by tissue microarray and immunohistochemical analysis, we identified anaplastic large T-cell lymphoma (ALCL) as constitutively expressing SELPLG at high levels. Moreover, GEP analysis in CD30+ ALCLs highlighted a positive correlation of SELPLG with TNFRSF8 (CD30-coding gene) and T-cell receptor (TCR)-signaling genes (LCK, LAT, SYK and JUN), suggesting that the common dysregulation of TCR expression in ALCLs may be bypassed by the involvement of PSGL-1 in T-cell activation and survival. Finally, we evaluated the effects elicited by in vitro treatment with two anti-PSGL-1 antibodies (KPL-1 and TB5) on the activation of the complement system and induction of apoptosis in human ALCL cell lines. In conclusion, our data demonstrated that PSGL-1 is specifically enriched in ALCLs, altering cell motility and viability due to its involvement in CD30 and TCR signaling, and it might be considered as a promising candidate for novel immunotherapeutic approaches in ALCLs.

P-selectin glycoprotein ligand 1 promotes T cell lymphoma development and dissemination

P-selectin glycoprotein ligand-1 (PSGL-1) is a membrane-bound glycoprotein expressed in lymphoid and myeloid cells. It is a ligand of P-, E- and L-selectin and is involved in T cell trafficking and homing to lymphoid tissues, among other functions. PSGL-1 expression has been implicated in different lymphoid malignancies, so here we aimed to evaluate the involvement of PSGL-1 in T cell lymphomagenesis and dissemination. PSGL-1 was highly expressed at the surface of human and mouse T cell leukemia and lymphoma cell lines. To assess its impact on T cell malignancies, we stably expressed human PSGL-1 (hPSGL-1) in a mouse thymic lymphoma cell line, which expresses low levels of endogenous PSGL-1 at the cell surface. hPSGL-1-expressing lymphoma cells developed subcutaneous tumors in athymic nude mice recipients faster than control empty vector or parental cells. Moreover, the kidneys, lungs and liver of tumor-bearing mice were infiltrated by hPSGL-1-expressing malignant T cells. To evaluate the role of PSGL-1 in lymphoma cell dissemination, we injected intravenously control and hPSGL-1-expressing lymphoma cells in athymic mice. Strikingly, PSGL-1 expression facilitated disease infiltration of the kidneys, as determined by histological analysis and anti-CD3 immunohistochemistry. Together, these results indicate that PSGL-1 expression promotes T cell lymphoma development and dissemination to different organs.

P-Selectin Glycoprotein Ligand-1 Deficiency Protects Against Aortic Aneurysm Formation Induced by DOCA Plus Salt.

P-selectin glycoprotein ligand-1 (PSGL-1) acts as a crucial regulator for the inflammatory cells infiltration by mediating the adhesion of leukocytes. However, the role of PSGL-1 in aortic aneurysm remains elusive. Here, we investigated the role of PSGL-1 in aortic aneurysm (AA) development. We first detected PSGL-1 expression in samples from aortic aneurysm patients and mouse AA models via western blotting, immunofluorescence, and flow cytometry, and then we used global PSGL-1 knockout mice and their wild type controls to establish an aortic aneurysm model induced by deoxycorticosterone acetate (DOCA) plus high salt (HS). The incidence, fatality rates, and the pathological changes of aortic aneurysm were analyzed in each group. The inflammation, adhesion molecules expression, and PSGL-1 mediated leukocyte-endothelial adhesion and their underlying mechanisms were explored further. Increased PSGL-1 levels were observed in human and mouse aortic aneurysm, and on leukocytes of mice treated with DOCA+HS. PSGL-1 deficiency reduced the incidence and severity of aortic aneurysm significantly, as well as decreased elastin fragmentation, collagen accumulation, and smooth muscle cells degeneration. Mechanistically, the protective effect of PSGL-1 inhibition was mediated by the reduced adhesion molecules, and the subsequently reduced leukocyte-endothelial adhesion through the NF-κB pathway, which finally led to reduced inflammatory cells infiltration and decreased inflammatory factors expression. PSGL-1 deficiency is protective against inflammatory cells migration and recruitment in the condition of AA through attenuation of leukocyte-endothelial adhesion. Inhibition of PSGL-1 may be a potential therapeutic target for the prevention and treatment of human AA.

PSGL-1 Inhibits the Incorporation of SARS-CoV and SARS-CoV-2 Spike Glycoproteins into Pseudovirions and Impairs Pseudovirus Attachment and Infectivity.

P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-γ (INF-γ)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks pseudovirus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1+ cells

P-selectin glycoprotein ligand 1 deficiency prevents development of acute pancreatitis by attenuating leukocyte infiltration.

BACKGROUNDAcute pancreatitis (AP) is rapid-onset pancreatic inflammation that causes local and systemic inflammatory response syndrome (SIRS) with high morbidity and mortality, but no approved therapies are currently available. P-selectin glycoprotein ligand 1 (PSGL-1) is a transmembrane glycoprotein to initiate inflammatory responses. We hypothesized that PSGL-1 may be involved in the development of AP and would be a new target for the treatment of AP.AIMTo investigate the role and mechanism of PSGL-1 in the development of AP.METHODSThe PSGL-1 expression on leukocytes was detected in peripheral blood of AP patients and volunteers. Pancreatic injury, inflammatory cytokines expression, and inflammatory cell infiltration was measured in AP mouse models induced with PSGL-1 knockout (PSGL-1-/-) and wild-type (PSGL-1+/+) mice. Leukocyte-endothelial cell adhesion was measured in a peripheral blood mononuclear cell (PBMC)-endothelial cell coculture system.RESULTSThe expression of PSGL-1 on monocytes and neutrophils was significantly increased in AP patients. Compared with PSGL-1+/+ mice, PSGL-1-/- AP mice induced by caerulein exhibited lower serum amylase, less Interleukin-1beta (IL-1beta) and Interleukin-6 (IL-6) expression, less neutrophil and macrophage infiltration, and reduced peripheral neutrophil and monocyte accounts. PSGL-1 deficiency alleviated leukocyte-endothelial cell adhesion via IL-6 but not IL-1beta.CONCLUSIONPSGL-1 deficiency effectively inhibits the development of AP by preventing leukocyte-endothelial cell adhesion via IL-6 stimulation and may become a potential therapeutic target for treating AP.

Expression of 6 Biomarkers in Liver Grafts After Pediatric Liver Transplantation: Correlations with Histology, Biochemistry, and Outcome.

BackgroundSubclinical graft inflammation and fibrosis after pediatric liver transplantation (LT) are common. Biomarkers are needed that precede and are associated with these changes and graft outcome.Material/MethodsWe evaluated immunohistochemical expression of 6 biomarkers [α-smooth muscle actin (α-SMA), collagen I, decorin, vimentin, P-selectin glycoprotein ligand-1 (PSGL-1), and CD34] in biopsies taken intraoperatively at LT (baseline) (n=29) and at 11.3 years after LT (first follow-up) (n=51). Liver biochemistry and graft histology were assessed at the first follow-up and at final assessment (19.6 years after LT) (n=48). Second follow-up biopsies for histology were available from 24 patients. The immunostainings were correlated with liver histology, biochemistry, and outcome at these time-points.ResultsBaseline levels of the biomarkers were unrelated to presence of fibrosis at follow-up. Increased α-SMA, collagen I levels, decorin, and vimentin were associated with simultaneous fibrosis at the first follow-up (p=0.001–0.027). Increased SMA, collagen I, decorin, vimentin, PSGL-1, and CD34 expression at first follow-up were associated with simultaneous portal inflammation (p=0.001–0.025). α-SMA, decorin, and vimentin expression were increased in patients without fibrosis at the first follow-up but who developed fibrosis in second follow-up (p=0.014 p=0.024 and p=0.024). Significant fibrosis (F2) and markedly increased α-SMA, collagen I, decorin, and vimentin levels at first follow-up were associated with suboptimal liver status at the final assessment (p=0.002–0.042).ConclusionsThe expression of the biomarkers at LT was unrelated to later development of graft fibrosis. α-SMA, decorin, and vimentin were associated with later graft fibrosis and suboptimal liver status.

Abstract 5525: VISTA/PSGL1 axis as a dominant immunomodulatory pathway in human non-small cell lung cancer

Abstract Introduction. VISTA/PD-1H is an Ig domain-containing type I transmembrane protein able to suppress T-cell activation and is being evaluated as a candidate anti-cancer immunotherapy target. VISTA can bind P-selectin glycoprotein ligand-1 (PSGL1) and V-set and immunoglobulin domain containing 3 (VSIG3) suggesting these interactions could mediate VISTA's immunomodulatory effect. We studied the tissue distribution of VISTA, PSGL1 and VSIG3 in human non-small cell lung cancer (NSCLC). Methods We used multiplexed quantitative immunofluorescence (QIF) to simultaneously measure DAPI, cytokeratin (CK), VISTA, PSGL1 and VSIG3 in 48 paired tumor/normal lung samples and in 850 stage I-IV NSCLCs from four independent cohorts represented in tissue microarray format. The first 2 NSCLC cohorts (Cohorts #1, n=382 and #2, n=282) included cases treated with standard of care non-immunotherapy. Cohort #3 included 137 lung adenocarcinomas with analysis of mutant oncogenic drivers; and Cohort #4 included 49 NSCLC cases treated with PD-1 axis blockers. The targets were selectively measured in CK+ tumor cells and CK-negative stromal cells. We also determined the spatial association between VISTA and its binding partners by fluorescence signal co-localization. The association between target levels, clinicopathologic/molecular variables, tumor infiltrating lymphocytes (TILs), PD-L1 expression and survival was established. Results PSGL1 was located predominantly in stromal/immune cells and VSIG3 was detected in both tumor and stromal compartments. Both targets were expressed at higher levels in NSCLC than in non-tumor lung tissue and showed a positive association with VISTA in cancer tissues. Using the visual detection threshold PSGL1 and VSIG3 expression were detected in &amp;gt;90% of cases from cohorts #1 and #2 and showed positive association with CD3+ TILs and PD-L1 levels. PSGL1 was higher in lung adenocarcinomas harboring EGFR mutations than in tumors with KRAS variants or cases lacking mutations in both oncogenes. Elevated VISTA/PSGL1 co-localization was significantly associated with longer 5-year overall survival in cases not treated with immunostimulatory therapy (Cohorts #1 and #2). However, an opposite association was seen in cases treated with PD-1 axis blockers, where elevated VISTA/PSGL1 co-expression was associated with shorter survival. Conclusions PSGL1 and VSIG3 are frequently expressed in human NSCLC. Expression of PSGL1 is associated with increased tumor immune infiltration and activating EGFR mutations. High baseline VISTA/PSGL1 co-expression is associated with adverse outcome after PD-1 axis blockers. The latter suggest VISTA/PSGL1 as a dominant immune evasion pathway independent from PD-1/PD-L1 axis in a subset of human NSCLC. Validation of these findings is ongoing. Citation Format: Alicia Ding, Franz Villarroel-Espindola, Adam Ducler, Brian S. Henick, Shruti Desai, Nicole Gianino, Jon Zugazagoitia, David L. Rimm, Alain Robert, Francisco Cruzalegui, Pierre Ferré, Roy Herbst, Miguel Sanmamed, Lieping Chen, Kurt A. Schalper. VISTA/PSGL1 axis as a dominant immunomodulatory pathway in human non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5525.

PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1-mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1-related monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action.

Nox2 Regulates Platelet Activation and NET Formation in the Lung.

The mortality rate of patients with critical illness has decreased significantly over the past two decades, but the rate of decline has slowed recently, with organ dysfunction as a major driver of morbidity and mortality. Among patients with the systemic inflammatory response syndrome (SIRS), acute lung injury is a common component with serious morbidity. Previous studies in our laboratory using a murine model of SIRS demonstrated a key role for NADPH oxidase 2 (Nox2)-derived reactive oxygen species in the resolution of inflammation. Nox2-deficient (gp91phox−/y) mice develop profound lung injury secondary to SIRS and fail to resolve inflammation. Alveolar macrophages from gp91phox−/y mice express greater levels of chemotactic and pro-inflammatory factors at baseline providing evidence that Nox2 in alveolar macrophages is critical for homeostasis. Based on the lung pathology with increased thrombosis in gp91phox−/y mice, and the known role of platelets in the inflammatory process, we hypothesized that Nox2 represses platelet activation. In the mouse model, we found that platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) and CXCL7 were increased in the bronchoalveolar fluid of gp91phox−/y mice at baseline and 24 h post intraperitoneal zymosan-induced SIRS consistent with platelet activation. Activated platelets interact with leukocytes via P-selectin glycoprotein ligand 1 (PSGL-1). Within 2 h of SIRS induction, alveolar neutrophil PSGL-1 expression was higher in gp91phox−/y mice. Platelet-neutrophil interactions were decreased in the peripheral blood of gp91phox−/y mice consistent with movement of activated platelets to the lung of mice lacking Nox2. Based on the severe lung pathology and the role of platelets in the formation of neutrophil extracellular traps (NETs), we evaluated NET production. In contrast to previous studies demonstrating Nox2-dependent NET formation, staining of lung sections from mice 24 h post zymosan injection revealed a large number of citrullinated histone 3 (H3CIT) and myeloperoxidase positive cells consistent with NET formation in gp91phox−/y mice that was virtually absent in WT mice. In addition, H3CIT protein expression and PAD4 activity were higher in the lung of gp91phox−/y mice post SIRS induction. These results suggest that Nox2 plays a critical role in maintaining homeostasis by regulating platelet activation and NET formation in the lung.

Abstract 1548: Acidic pH selective binding of VISTA to PSGL-1 and anti-tumor activity of combined VISTA and PD-1 blockade

Abstract Background: Therapeutic blockade of the immune checkpoints CTLA-4 and PD-1/PD-L1 has provided durable survival benefits in multiple malignancies. However, additional treatment options are often required to maximally reverse immune dysfunction. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an orphan B7 family ligand that is highly expressed on immunosuppressive myeloid cells and has been shown to inhibit T-cell responses in vitro and in preclinical models of cancer. Here we report that VISTA, a ligand for the receptor P-selectin glycoprotein ligand-1 (PSGL-1), uses a histidine-rich interface to engage PSGL-1 and suppress immune responses selectively in acidic environments, such as tumor beds. Methods: Recombinant VISTA multimers were used to assess binding to cells and recombinant PSGL-1 over a range of pH values (6.0-7.4). Antibodies against human and mouse VISTA were used to map binding and functional epitopes. Acidic pH receptor-based ligand capture was used to identify PSGL-1 as a VISTA receptor. X-ray crystallography was used to resolve the VISTA structure in complex with an anti-VISTA antigen-binding fragment. The MC38 mouse tumor model was used to assess the effects of VISTA deficiency and the effects of VISTA antibody blockade alone and combined with anti-PD-1 in vivo. Results: Recombinant VISTA bound leukocytes at pH 6.0 but was not detectable at pH 7.4. Antibodies in a single epitope bin blocked VISTA binding and reversed VISTA suppression of T cells. VISTA-mediated inhibition of T cells was detectable at pH 7.4 but was more pronounced below pH 7.0, suggesting that VISTA functions selectively in acidic conditions. VISTA’s structure was resolved at 1.6 Å and characterized by a histidine-rich extension of the immunoglobulin V domain central β-sheet. VISTA blocking antibodies, but not nonblocking antibodies, bound this β-sheet region. Engineered antibodies could distinguish this epitope in its active and inactive states at acidic and neutral pH, respectively. Receptor capture on T cells at acidic pH identified PSGL-1 as a VISTA receptor. T-cell PSGL-1 CRISPR ablated VISTA binding, whereas PSGL-1 expression on CHO cells conferred VISTA binding at acidic pH. Thus, an antibody that blocks mouse VISTA binding to mouse T cells at acidic pH combined with a PD-1 blocking antibody was shown to enhance anti-tumor T-cell responses and drive MC38 tumor rejection in vivo. Conclusions: VISTA is a highly pH-selective ligand for PSGL-1. VISTA antibody blockade reverses immune suppression in vitro and in vivo, especially when combined with PD-1 antibody blockade. Our results identify acidic pH as a direct regulator of VISTA engagement with PSGL-1 and suggest new strategies to enhance anti-tumor T-cell responses. Citation Format: Robert J. Johnston, Linhui Julie Su, Jason Pinckney, David Critton, Arathi Krishnakumar, Martin Corbett, Andrew Rankin, Rose A. DiBella, Lynne Campbell, Xiaodi Deng, Haibin Chen, Alexander Kozhich, Jim Holloway, Zheng Yang, Ginger Rakestraw, Michael Quigley, Alan J. Korman. Acidic pH selective binding of VISTA to PSGL-1 and anti-tumor activity of combined VISTA and PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1548.

P-Selectin Glycoprotein Ligand-1 (PSGL-1) Expressing CD4 T Cells Contribute Plaque Instability in Acute Coronary Syndrome.

Adhesion molecules have essential roles in the development of atherosclerosis. We investigated whether P-selectin glycoprotein ligand-1 (PSGL-1)-expressing CD4 T cells contribute to plaque instability in acute coronary syndrome (ACS).Methods and Results:We studied the adhesion molecules on CD4 T cells from consecutive patients with ACS treated with thrombus-aspirating device and compared them with healthy controls (n=48 each). Blood, thrombi, and plaque samples from the culprit coronary arteries were collected by thrombus aspiration performed during emergency coronary artery angiography. According to flow cytometry results, peripheral CD4 T cells from ACS patients strongly expressed PSGL-1 and integrin β2 (P<0.05 for both) more than those from controls; culprit coronary arteries contained an abundance of PSGL-1+(P<0.001) but not integrin β2+CD4 T cells. In addition, immunohistochemical analysis of the thrombus-aspirating device samples revealed numerous PSGL-1+CD4 T cells in plaques from the culprit lesions. Results from the selectin-binding assay demonstrated that activated PSGL-1+CD4 T cells from ACS patients bound to P- or E-selectin after triggering the T-cell receptor, and adhered to endothelial cells under laminar flow conditions (P<0.05 and P<0.05, respectively), inducing their apoptosis (P<0.01) via activated caspase-3, which correlated with PSGL-1 expression (R=0.788, P=0.021) and was suppressed by application of a PSGL-1-specific antibody (P<0.05). PSGL-1 contributed to cytotoxic CD4 T cell homing to the culprit coronary artery and promoted plaque instability in ACS.