P-selectin Glycoprotein Ligand-1 Research Articles

P-selectin glycoprotein ligand-1 is diversely expressed in the primary tumour and venous thrombus of clear cell renal cancer and correlates with poor overall survival

P-selectin glycoprotein ligand-1 is diversely expressed in the primary tumour and venous thrombus of clear cell renal cancer and correlates with poor overall survival

Enhanced interaction between genome-edited mesenchymal stem cells and platelets improves wound healing in mice

Impaired wound healing poses a significant burden on the healthcare system and patients. Stem cell therapy has demonstrated promising potential in the treatment of wounds. However, its clinical application is hindered by the low efficiency of cell homing. In this study, we successfully integrated P-selectin glycoprotein ligand-1 (PSGL-1) into the genome of human adipose-derived mesenchymal stem cells (ADSCs) using a Cas9-AAV6-based genome editing tool platform. Our findings revealed that PSGL-1 knock-in enhanced the binding of ADSCs to platelets and their adhesion to the injured site. Moreover, the intravenous infusion of PSGL-1-engineered ADSCs (KI-ADSCs) significantly improved the homing efficiency and residence rate at the site of skin lesions in mice. Mechanistically, PSGL-1 knock-in promotes the release of some therapeutic cytokines by activating the canonical WNT/β-catenin signaling pathway and accelerates the healing of wounds by promoting angiogenesis, re-epithelialization, and granulation tissue formation at the wound site. This study provides a novel strategy to simultaneously address the problem of poor migration and adhesion of mesenchymal stem cells (MSCs).

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.

Unraveling the Mechanisms of P-Selectin in Sickle Cell Disease Using a Microvascular-on-a-Chip Model

Individuals who suffer from Sickle Cell Disease (SCD) is known to have multifaceted complications one such being vaso-occlusive crises, which result from blockages in their blood vessels. Recently, progress has been made to get drugs to the marketplace that address this issue. Several of these drugs target p-selectin, a transmembrane protein that facilitates the adhesion of platelets, leukocytes, and activated endothelial cells. P-selectin Glycoprotein Ligand-1 (PSGL-1), a P-selectin antagonist, has been extensively researched in vitro for its ability to mediate Microvascular obstruction through leukocyte interactions, particularly leukocyte rolling. Particularly, in human patients rather than animal models, the precise role of P-selectin in the interactions between activated platelets and endothelial cells remains unclear. This study delves into this interaction using a 3D vein-on-a-chip model that can capture microvascular obstruction formation at a similar scale to the microvascular. Our data shows that P Selectin in SCD inhibition is the attenuation mainly driven by platelets with contributions from WBC rather than driven by the activated endothelium. Through the creation of a PDMS branching 32-channel microfluidic, the cell interactions and obstruction of the microvascular can be quantified. Additionally, this model serves as an analog to test the various intercellular PSGL-1 interactions. Endothelial cells are seeded into the channels and cultured under flow to create 3D structures. Once confluent, healthy, and SCD patient blood samples collected in sodium citrate can be recalcified and perfused into the endothelial network to simulate physiological conditions then endothelial activation markers like P Selectin can be tagged, imaged, and quantified. Microvascular obstruction can be imaged in live time and quantified primarily through area and accumulation. Finally, various PSGL-1 analogs can be doped into the blood before and after perfusion to simulate prophylaxis or treatment. This system allows for the study of PSGL-1's effect on the microvascular obstruction process and its effect on individual cell types through tagging [Figure 1A]. When investigating PSGL1 analogs, the system does indicate a significant reduction in microvascular obstruction, quantified by the amount of occlusion within the channels [Figure 1B & 1C]. With a cohort of n=10 SS patients on Hydroxyurea, there is a downstream effect on the amount of occlusion, specifically the percentage of the area of the channel that is occluded. Each patient is compared against itself to serve as its control, and each patient is perfused into three near-identical chips of treated and three untreated [Figure 1D]. When targeting CD41 and CD45 within the model, occlusions seem to be primarily driven by activated platelets, an indication that PSGL-1's interactions may be more involved in platelets than leukocyte dynamics. Work is ongoing to target this specific interaction. PSGL1's interaction with the activated endothelium is driving microvascular obstruction to be dominated by activated platelet leukocyte interactions. Current data shows that attenuation of P Selectin decreases vascular obstruction. PSGL-1 cell interactions and PSGL-1's effect on occlusion mitigation are quantified. By quantifying PSGL-1's interactions through a vein-on-a-chip model, SCD patient-specific drug interactions can be identified. Additionally, a greater understanding of how these drugs affect vaso-occlusive crisis and SCD.

Population Pharmacokinetic Modeling of Different Formulations of Inclacumab Using Phase 1 Data in Healthy Participants

Introduction: P-selectin is a cell adhesion molecule that contributes to vaso-occlusive crises (VOCs) in sickle cell disease (SCD). Inclacumab is a recombinant, fully human, immunoglobulin G4 monoclonal antibody that selectively binds to P-selectin to prevent binding with P-selectin glycoprotein ligand-1. Inclacumab was previously evaluated by Roche in clinical trials with healthy participants and patients with cardiovascular disease and is now being developed as a potential treatment to reduce the frequency of VOCs in individuals with SCD. Changes in the manufacturing of inclacumab have resulted in 3 formulations/drug materials: Roche v0.1, Roche v0.2, and Global Blood Therapeutics (GBT). GBT-manufactured inclacumab utilizes a similar formulation and the same cell line as Roche v0.2 (Roche v0.1 utilizes a different cell line), and analytical and nonclinical comparability between Roche v0.2 and GBT inclacumab have been demonstrated (Mihaila et al. Expert Opin Biol Ther 2022). Here we report results from a population pharmacokinetic (PopPK) model developed to compare the pharmacokinetics (PK) of different formulations using data from 4 phase 1 clinical studies in healthy participants administered Roche v0.1, Roche v0.2, or GBT inclacumab. The objectives were to develop a PopPK model to describe inclacumab PK in healthy adult participants and to quantify potential differences in inclacumab PK associated with different formulations/drug materials. Methods: A PopPK model was developed using single- and multiple-dose PK data from healthy participants who received intravenous (IV) inclacumab across 4 studies (BP21112 [Roche v0.1, single ascending dose (SAD)], BP21617 [Roche v0.1, multiple ascending dose], BP28134 [Roche v0.2, SAD], and GBT2104-111 [GBT, SAD]). Evaluable participants had ≥1 measurable postdose concentration with an associated sample time, relevant dosing information prior to each measurable concentration, and no protocol violation that could confound PK analyses. A nonlinear mixed effects model was fit to the inclacumab plasma concentration-time data using first-order conditional estimation with eta-epsilon interaction in NONMEM ® (v7.3). Model selection was based on the plausibility of parameter estimates, goodness-of-fit plots, and the objective function value. The final model was evaluated using a prediction-corrected visual predictive check (pcVPC). Results: The PopPK dataset comprised 145 healthy participants, of which 2622 inclacumab plasma concentrations (84.9%) from 143 participants were evaluable (Roche v0.1: n=68 [47.6%]; Roche v0.2: n=60 [42.0%]; GBT: n=15 [10.5%]). Median (range) age and body weight were 42 (21-63) years and 73.2 (45.8-109) kg, respectively. Nominal doses ranged from 0.03 to 40 mg/kg IV; 23.8% of participants received 3 mg/kg and 29.4% received 20 mg/kg. A 3-compartment disposition model with parallel linear clearance (CL=0.125 L/d) and nonlinear (concentration-dependent; V max=1.19 mg/d; K m=0.784 μg/mL) clearance from the central compartment described the plasma concentration-time data. The effect of drug material was incorporated a priori as relative bioavailability term F in the base model using exploratory analyses of observed exposures. Dosing of Roche v0.1 and Roche v0.2 inclacumab resulted in 23% and 2% higher exposure than GBT inclacumab, respectively. Between-subject variability was estimated for linear clearance, nonlinear clearance, and all volume parameters, and residual error was described using a proportional model (Table). Of the tested covariates, baseline body weight was a statistically significant covariate effect and was included on all linear clearance and volume terms. Model qualification by pcVPC showed good predictive performance of the final model. Conclusions: The PopPK of IV inclacumab in healthy participants was described by a 3-compartment disposition model with parallel linear and nonlinear clearances. Estimated relative exposure of Roche v0.2 and GBT-manufactured inclacumab was similar, indicating comparability in drug exposure. Understanding the relationship between exposure and dose for different formulations facilitates more accurate assessment of safety data across multiple clinical studies. These data provide support for the ongoing phase 3 clinical development in SCD using GBT-manufactured inclacumab without further clinical comparability testing.

Helicobacter pylori and pro-inflammatory protein biomarkers in myocardial infarction with and without obstructive coronary artery disease

Abstract Introduction Myocardial infarction (MI) with (MI-CAD) and without (MINOCA) obstructive coronary artery disease affects different populations and may have separate pathophysiological mechanisms with greater inflammatory activity in MINOCA compared to MI-CAD. Helicobacter pylori (Hp) is the most common chronic infection globally. It can cause systemic inflammation and has been associated with cardiovascular disease (CVD). We aimed to investigate whether Hp infection is associated with concentrations of protein biomarkers of inflammation and CVD. Methods In a case-control study patients with MINOCA (n=99) in Sweden were included, complemented by subjects with MI-CAD (n=99) and controls (n=100) matched by age and sex. Protein biomarkers were measured with a proximity extension assay in plasma samples collected 3 months after MI. Seroprevalence of Hp and cytotoxin associated gene A (CagA) was determined using ELISA. The associations between protein levels and Hp status were studied with linear regression. Results The prevalence of Hp was 20.2%, 19.2% and 16.0% for MINOCA, MI-CAD and controls, respectively (p=0.73). Seven proteins were significantly associated with Hp in an adjusted model: tissue plasminogen activator (tPA), interleukin-6 (IL-6), myeloperoxidase (MPO), TNF-related activation-induced cytokine (TRANCE), pappalysin-1 (PAPPA), soluble urokinase plasminogen activator receptor (suPAR) and P-selectin glycoprotein ligand 1 (PSGL-1). Conclusion Hp infection was present in one in five patients with MI, irrespective of the presence of obstructive CAD. Proteins previously associated with MINOCA and MI-CAD were elevated in Hp positive subjects, suggesting that Hp might promote an inflammatory response and contribute to the development of MI with and without CAD.Forest plot of regression coefficients

Safety Profile of Crizanlizumab 5.0 Mg/Kg in Sickle Cell Disease: Pooled Data from Clinical Trials

Introduction: Crizanlizumab is a humanized monoclonal antibody that binds to P-selectin and blocks its interaction with its ligands, including P-selectin glycoprotein ligand 1. It is approved to reduce the frequency of or prevent recurrent vaso-occlusive crises (VOCs) in patients aged ≥16 years with sickle cell disease (SCD). This pooled analysis assesses the safety of the recommended dosage of crizanlizumab (5.0 mg/kg monthly) in treated patients. Methods: Safety was assessed based on two clinical program data pools. Pool 1 comprised data from the crizanlizumab-treated groups from 3 Phase 2 studies (SUSTAIN [NCT01895361], SOLACE-adults [NCT03264989], and the pediatric SOLACE-Kids [NCT03474965]) and 1 Phase 3 study (STAND [NCT03814746]) in patients with SCD, with a history of VOCs leading to a healthcare visit. Pool 2 included data from the SUSTAIN and STAND treatment groups and the corresponding placebo group. Adverse events (AEs) were summarized by preferred term /system organ class and seriousness/severity per MedDRA version 23.1, with severity assessed based on CTCAE v5. Based on both its mechanism of action and the fact that crizanlizumab is a mAb, AEs of special interest (AESIs) included infections, infusion-related reactions (IRRs, events typically occurring within 24 hours of the recent crizanlizumab infusion), effects on hemostasis, and immunogenicity (antidrug antibodies [ADAs]). Pooled incidences of treatment-related AEs (TRAEs), suspected to be related to study treatment, were also evaluated. Results: In Pool 1, 245 patients received crizanlizumab 5.0 mg/kg (median age: 24 years [range,12-65]; 54.3% female), with 66.1% having 1 to 4 VOCs and 33.1% having ≥5 VOCs (VOC data were missing for 0.8% patients) at baseline. Pool 2 had 150 patients in the 5.0 mg/kg treatment group (median age: 27 years [range, 12-64]) and 147 patients in the placebo group (median age: 26 years [range, 12-68]). Median crizanlizumab exposure was 87.4 weeks (range, 2-228) in Pool 1, 54.1 weeks (range, 51.9-93.9) in Pool 2 treatment group and 53.3 weeks (range: 50.3-86.0) in the Pool 2 placebo group. The use of crizanlizumab in combination with hydroxyurea/hydroxycarbamide did not result in meaningful differences in safety profile. Most frequently reported AE in Pool 1 were headache (25.7%), pyrexia (22.9%), arthralgia (18.4%), nausea (16.7%), and COVID-19 (15.9%). In Pool 2 (treatment group vs. placebo), the most common AEs were headache (21.3% vs. 17.7%), pyrexia (19.3% vs. 17.7%), and nausea (17.3% vs. 10.2%). Infection events were observed in 62.4% and 56.7% of patients in the Pool 1 and 2 treatment groups, respectively, and in 55.1% in the placebo group (Table 1). The most common infections in Pool 1 were COVID-19 (15.9%), upper respiratory tract infections (12.7%), and urinary tract infections (11.8%). No increased risk or severity of infection was found in patients treated with crizanlizumab. IRRs occurred in 38.8% of Pool 1, 36.7% of the Pool 2 treatment group, and 27.9% of the placebo group. Most common IRRs were headache and nausea (Table 1). IRRs with grade ≥3 occurred in 3.3% of patients in Pool 1, with no grade 4 or 5 events. Bleeding-related events were observed in 16.7% of Pool 1 patients; epistaxis (3.7%) was the most reported. Treatment-induced ADA were reported in 0.8% (n=2) of patients in Pool 1, confirming the low immunogenic potential of crizanlizumab. TRAEs were observed in 31.8% patients in Pool 1, 34.7% in the Pool 2 treatment group, and 24.5% in the placebo group. Among the patients in Pool 1, 10 (4.1%) experienced grade 3 or higher TRAEs. The most reported TRAEs are listed in Table 1. Treatment-related serious AEs occurred in 10 patients (4.1%), with 7 (2.9%) experiencing ≥ grade 3 AEs in Pool 1. Treatment discontinuation due to AEs occurred in 3.3% of patients in Pool 1, 2.7% in the Pool 2 treatment group, and 3.4% in the placebo group. Five on-treatment deaths were reported across the four studies and were considered not related to crizanlizumab by the investigator (myocardial infarction, multiple organ dysfunction, endocarditis, meningitis bacterial, and sepsis [1 patient each]). Conclusion: This pooled analysis of crizanlizumab 5.0 mg/kg in patients with SCD and a history of VOCs showed that crizanlizumab was well tolerated, with a favorable safety profile. Most AEs were mild to moderate, and the discontinuations due to AEs were infrequent. No new safety concerns were identified.

The roles of P-selectin in cancer cachexia.

P-selectin, a cell adhesion molecule of the selectin family, is expressed on the surface of activated endothelial cells (ECs) and platelets. Binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) supports the leukocytes capture and rolling on stimulated ECs and increases the aggregation of leukocytes and activated platelets. Cancer cachexia is asystemic inflammation disorder characterized by metabolic disturbances, reduced body weight, loss ofappetite, fat depletion, and progressive muscle atrophy. Cachexia status is associated with increased pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), which activates ECs to release P-selectin. Single-nucleotide polymorphisms (SNPs) loci of P-selectin encoding gene SELP are associated with higher level of plasma P-selectin and increase the susceptibility to cachexia in cancer patients. Elevated P-selectin expression has been observed in the hypothalamus, liver, and gastrocnemius muscle in animal models with cancer cachexia. Increased P-selectin may cause excessive inflammatory processes, muscle atrophy, and blood hypercoagulation, thus facilitating the development of cancer cachexia. In this review, physiological functions of P-selectin and its potential roles in cancer cachexia have been summarized. We also discuss the therapeutic potential of P-selectin inhibitors for the treatment of cancer cachexia.

Platelet-derived circulating soluble P-selectin is sufficient to induce hematopoietic stem cell mobilization

BackgroundGranulocyte colony-stimulating factor (G-CSF)-mediated mobilization of hematopoietic stem cells (HSCs) is a well-established method to prepare HSCs for transplantation nowadays. A sufficient number of HSCs is critical for successful HSC transplantation. However, approximately 2–6% of healthy stem cell donors are G-CSF-poor mobilizers for unknown reasons; thus increasing the uncertainties of HSC transplantation. The mechanism underlining G-CSF-mediated HSC mobilization remains elusive, so detailed mechanisms and an enhanced HSC mobilization strategy are urgently needed. Evidence suggests that P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) are one of the cell–cell adhesion ligand–receptor pairs for HSCs to keep contacting bone marrow (BM) stromal cells before being mobilized into circulation. This study hypothesized that blockage of PSGL-1 and P-selectin may disrupt HSC-stromal cell interaction and facilitate HSC mobilization.MethodsThe plasma levels of soluble P-selectin (sP-sel) before and after G-CSF administration in humans and male C57BL/6J mice were analyzed using enzyme-linked immunosorbent assay. Male mice with P-selectin deficiency (Selp−/−) were further employed to investigate whether P-selectin is essential for G-CSF-induced HSC mobilization and determine which cell lineage is sP-sel derived from. Finally, wild-type mice were injected with either G-CSF or recombinant sP-sel to investigate whether sP-sel alone is sufficient for inducing HSC mobilization and whether it accomplishes this by binding to HSCs and disrupting their interaction with stromal cells in the BM.ResultsA significant increase in plasma sP-sel levels was observed in humans and mice following G-CSF administration. Treatments of G-CSF induced a decrease in the level of HSC mobilization in Selp−/− mice compared with the wild-type (Selp+/+) controls. Additionally, the transfer of platelets derived from wild-type mice can ameliorate the defected HSC mobilization in the Selp−/− recipients. G-CSF induces the release of sP-sel from platelets, which is sufficient to mobilize BM HSCs into the circulation of mice by disrupting the PSGL-1 and P-selectin interaction between HSCs and stromal cells. These results collectively suggested that P-selectin is a critical factor for G-CSF-induced HSC mobilization.ConclusionssP-sel was identified as a novel endogenous HSC-mobilizing agent. sP-sel injections achieved a relatively faster and more convenient regimen to mobilize HSCs in mice than G-CSF. These findings may serve as a reference for developing and optimizing human HSC mobilization in the future.

Helicobacter pylori and Pro-Inflammatory Protein Biomarkers in Myocardial Infarction with and without Obstructive Coronary Artery Disease.

Myocardial infarction (MI) with obstructive coronary artery disease (MI-CAD) and MI in the absence of obstructive coronary artery disease (MINOCA) affect different populations and may have separate pathophysiological mechanisms, with greater inflammatory activity in MINOCA compared to MI-CAD. Helicobacter pylori (Hp) can cause systemic inflammation and has been associated with cardiovascular disease (CVD). We aimed to investigate whether Hp infection is associated with concentrations of protein biomarkers of inflammation and CVD. In a case-control study, patients with MINOCA (n = 99) in Sweden were included, complemented by matched subjects with MI-CAD (n = 99) and controls (n = 100). Protein biomarkers were measured with a proximity extension assay in plasma samples collected 3 months after MI. The seroprevalence of Hp and cytotoxin-associated gene A (CagA) was determined using ELISA. The associations between protein levels and Hp status were studied with linear regression. The prevalence of Hp was 20.2%, 19.2%, and 16.0% for MINOCA, MI-CAD, and controls, respectively (p = 0.73). Seven proteins were associated with Hp in an adjusted model: tissue plasminogen activator (tPA), interleukin-6 (IL-6), myeloperoxidase (MPO), TNF-related activation-induced cytokine (TRANCE), pappalysin-1 (PAPPA), soluble urokinase plasminogen activator receptor (suPAR), and P-selectin glycoprotein ligand 1 (PSGL-1). Hp infection was present in one in five patients with MI, irrespective of the presence of obstructive CAD. Inflammatory proteins were elevated in Hp-positive subjects, thus not ruling out that Hp may promote an inflammatory response and potentially contribute to the development of CVD.

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<.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.

Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation

Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.

Isolation and functional analysis of phage-displayed antibody fragments targeting the staphylococcal superantigen-like proteins.

Staphylococcus aureus produces numerous virulence factors that manipulate the immune system, helping the bacteria avoid phagocytosis. In this study, we are investigating three immune evasion molecules called the staphylococcal superantigen-like proteins 1, 5, and 10 (SSL1, SSL5, and SSL10). All three SSLs inhibit vital host immune processes and contribute to S. aureus immune evasion. This study aimed to identify single-chain variable fragment (scFvs) antibodies from synthetic antibody phage libraries, which can recognize either of the three SSLs and could block the interaction between the SSLs and their respective human targets. The antibodies were isolated after three rounds of panning against SSL1, SSL5, and SSL10, and their ability to bind to the SSLs was studied using a time-resolved fluorescence-based immunoassay. We successfully obtained altogether 44 unique clones displaying binding activity to either SSL1, SSL5, or SSL10. The capability of the SSL-recognizing scFvs to inhibit the SSLs' function was tested in an MMP9 enzymatic activity assay, a P-selectin glycoprotein ligand 1 competitive binding assay, and an IgG1-mediated phagocytosis assay. We could show that one scFv was able to inhibit SSL1 and maintain MMP9 activity in a concentration-dependent manner. Finally, the structure of this inhibiting scFv was modeled and used to create putative scFv-SSL1-complex models by protein-protein docking. The complex models were subjected to a 100-ns molecular dynamics simulation to assess the possible binding mode of the antibody.

PSGL-1 deficiency re-wires T cell signaling to support development of stem cell like exhausted T cells that control PD-1 blockade resistant melanoma tumor growth

Abstract We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates CD8 +T cell effector functions and promotes exhaustion in responses to chronic viral infection and melanoma tumors. In this study, we investigated cellular and molecular mechanisms by which intrinsic PSGL-1 signaling regulates the differentiation of exhausted T cells (T EX). We show that PSGL-1 localizes with the TCR upon co-ligation and find that PSGL-1 associates with Zap70 and the Zap70 inhibitor, Sts-1. In the absence of PSGL-1, expression of Sts-1 is reduced allowing for greater activation of Zap70 and the downstream signaling molecules Akt and Erk1/2. In addition, PSGL-1-deficiency empowers CD8 +T cells to respond to low affinity TCR ligands. In vivo, PSGL-1 deficiency leads to growth inhibition of a PD-1-blockade resistant melanoma. Notably, PSGL-1-deficiency in T cells enables retention of effector T cell (T EFF) functions, and an increased representation of stem cell-like T cells (T SC) that express TCF-1 and low levels of TOX, which are associated with sustained effector responses and responsiveness to ICB. We now show that concomitant PSGL-1 signaling with repeated TCR stimulation of human T EFFelicits functional exhaustion. Importantly, blockade of PSGL-1 recapitulates PSGL-1 deficiency in anti-tumor as well as antiviral responses, and its distinct mechanism of T cell inhibition underscores the translational potential of targeting PSGL-1 by ICB. Supported by grants from NIH (R01 AI106895, R21 CA249353, R21 CA216678, R03 CA252144) Melanoma Research Alliance MRA 696326, Department of Defense W81XWH-20-1-0324, Sanford Burnham Prebys NCI-Designated Cancer Center Support Grant, P30 CA030199 American Cancer Society Postdoctoral Fellowship (PF-20-113-01-LIB)

Abstract 6373: P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro

Abstract P-selectin glycoprotein ligand-1 (PSGL-1) is a type I transmembrane protein expressed on the surface of most hematopoietic cells. PSGL-1 can engage multiple ligands (e.g., selectins, VISTA, Siglec-5, versican, CCL19, and CCL21). Apart from being a key adhesion molecule involved in immune cell trafficking, PSGL-1 has been shown to function as a negative immune checkpoint receptor in both T cells and macrophages. PSGL-1 is highly expressed in tumor-infiltrating T cells (TILs) and in tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). PSGL-1 signaling in TILs induces development of T-cell exhaustion and, in TAMs, it promotes immunosuppressive activity of macrophages. In the TME, PSGL-1 is also highly expressed on the surface of myeloid-derived suppressor cells, regulatory T cells, and some types of cancer cells. We hypothesized that PSGL-1 may also act as a ligand to modulate the effector functions of human T cells and macrophages in the TME. In the present study, flow cytometry analysis showed that monocytes and granulocytes had a higher PSGL-1 expression than CD4+ and CD8+ T cells isolated from normal human blood cells. To mimic the cell-expressing PSGL-1, a recombinant human PSGL-1-Fc protein (rhPSGL-1-Fc, R&D Systems) was coated onto 96-well plates overnight. Human peripheral blood mononuclear cells (PBMCs), purified human CD4+ T cells, or CD8+ T cells were cultured in the PSGL-1-coated plates for 3 days in the presence of anti-CD3 antibody. In some experiments, a commercial tool PSGL-1 monoclonal antibody (mAb) with the ability to block PSGL-1/P-selectin interaction was added into the culture system. Cytokine secretion in the culture supernatants was measured using Meso Scale Discovery assays. Similar experiments were also conducted with commercially supplied human M1 and M2 macrophages. The results demonstrated that plate-bound rhPSGL-1-Fc dose-dependently inhibited IFN-γ, IL-2, and TNF-α cytokine production in human PBMCs, CD4+ T cells, and in CD8+ T cells following T-cell receptor stimulation. The tool PSGL-1 mAb was able to partially rescue the suppression of cytokine production. Plate-bound rhPSGL-1-Fc also inhibited TNF-α secretion from human M1 macrophage, and the PSGL-1 mAb significantly enhanced TNF-α production in both human M1 and M2 macrophages treated with plate-bound PSGL-1-Fc. Together, these data indicate that PSGL-1 expressed on immune cells in TME may act as both an inhibitory ligand and receptor to impact on T-cell and macrophage function. Acknowledgements: Thanks to Patrick Mayes and Ricardo Macarron for scientific input and thanks to Qian Wang Yao and Lynn Leffet for technical assistance. Trial Registration: N/A Ethics Approval: N/A Citation Format: Jun Guan, Sundee Dees, Tony Chadderton, Alejandro Amador Arjona. P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6373.

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.

Monocyte migration profiles define disease severity in acute COVID-19 and unique features of long COVID.

COVID-19 is associated with a dysregulated immune response but it is unclear how immune dysfunction contributes to the chronic morbidity persisting in many COVID-19 patients during convalescence (long COVID). We assessed phenotypical and functional changes of monocytes in COVID-19 patients during hospitalisation and up to 9 months of convalescence following COVID-19, respiratory syncytial virus or influenza A. Patients with progressive fibrosing interstitial lung disease were included as a positive control for severe, ongoing lung injury. Monocyte alterations in acute COVID-19 patients included aberrant expression of leukocyte migration molecules, continuing into convalescence (n=142) and corresponding with specific symptoms of long COVID. Long COVID patients with unresolved lung injury, indicated by sustained shortness of breath and abnormal chest radiology, were defined by high monocyte expression of C-X-C motif chemokine receptor 6 (CXCR6) (p<0.0001) and adhesion molecule P-selectin glycoprotein ligand 1 (p<0.01), alongside preferential migration of monocytes towards the CXCR6 ligand C-X-C motif chemokine ligand 16 (CXCL16) (p<0.05), which is abundantly expressed in the lung. Monocyte CXCR6 and lung CXCL16 were heightened in patients with progressive fibrosing interstitial lung disease (p<0.001), confirming a role for the CXCR6-CXCL16 axis in ongoing lung injury. Conversely, monocytes from long COVID patients with ongoing fatigue exhibited a sustained reduction of the prostaglandin-generating enzyme cyclooxygenase 2 (p<0.01) and CXCR2 expression (p<0.05). These monocyte changes were not present in respiratory syncytial virus or influenza A convalescence. Our data define unique monocyte signatures that define subgroups of long COVID patients, indicating a key role for monocyte migration in COVID-19 pathophysiology. Targeting these pathways may provide novel therapeutic opportunities in COVID-19 patients with persistent morbidity.

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.