CXCR4 Research Articles

ACKR3 agonism induces heterodimerization with chemokine receptor CXCR4 and attenuates platelet function.

Platelet receptors ACKR3 and CXCR4 play a crucial role in a variety of cardiovascular diseases. Like most chemokine receptors, CXCR4 is a G protein coupled receptor that induces platelet activation. In contrast, the atypical chemokine receptor 3 (ACKR3) lacks the ability to activate heterotrimeric G proteins and its activation leads to platelet inhibition and attenuates thrombus formation. In nucleated cells, heterodimerization of ACKR3 with CXCR4 regulates CXCL12-dependent signalling. The aim of our study was to investigate the formation of ACKR3/CXCR4 heterodimers in platelets and the subsequent consequences for platelet function. Using a proximity ligation assay (PLA, Duolink®) to screen for CXCR4/ACKR3 heterodimerization inducing compounds, we found that ACKR3 agonism but not conventional platelet agonists or endogen ligands lead to heterodimer formation. To further characterize the formation of ACKR3/CXCR4 heterodimers, we studied the CXCL12-dependent platelet activation via CXCR4. Both, CXCL12-dependent platelet aggregation and collagen-dependent exvivo thrombus formation were significantly downregulated by ACKR3 agonism. Moreover, platelet intracellular calcium and Akt signalling were increased by CXCL12 and again suppressed by ACKR3-specific agonists. Previously, CXCL12 was shown to decrease platelet cAMP levels via CXCR4. Treatment with a specific ACKR3 agonist counteracted this CXCL12/CXCR4-dependent cAMP decrease. Our results reveal that the formation of platelet ACKR3/CXCR4 heterodimers is dependent on ACKR3 rather than CXCR4. Furthermore, ACKR3 agonism induced heterodimerization is associated with mitigating CXCL12/CXCR4-dependent platelet activation possibly by modulating CXCR4-dependent G protein signalling. Our results indicate possible ACKR3 agonist functions and reinforce the potential therapeutic applications of ACKR3 agonists.

8439 Targeting Interferon Gamma-Mediated CXCL16-CXCR6 Chemokine-Receptor Interactions As A Strategy To Prevent Immune Checkpoint Inhibitor Autoimmune Diabetes

Abstract Disclosure: S. Wang: None. N. Huang: None. J.G. Ortega: None. J. Lee: None. K. Kimbrell: None. J. Nguyen: None. J. Olay: None. E. McCarthy: None. E. Peluso: None. H. Chen: None. M.G. Lechner: None. Autoimmune diabetes mellitus is a rare but life-threatening side effect of immune checkpoint inhibitor (ICI) cancer therapy. Checkpoint inhibitors increase immune activation by blocking regulatory molecules on T cells, including programmed death protein (PD1). As a result of this heightened immune activation, patients can develop autoimmunity in healthy tissues, known as immune related adverse events (IRAEs). ICI-associated diabetes mellitus (ICI-T1DM) is an IRAE that results in autoimmune destruction of insulin-producing pancreatic beta-cells. Patients with ICI-T1DM require life-long insulin therapy and more than half present initially with diabetic ketoacidosis. Unfortunately, the mechanism of this IRAE is not fully understood. Anti-PD1 antibody treatment (10mg/kg/dose, twice weekly, i.p.) of male and female non-obese diabetic (NOD) mice leads to autoimmune infiltrates in multiple tissues, including accelerated insulitis and DM. Single cell RNA sequencing of islet infiltrating immune cells from ICI-treated mice previously showed a role for CD8+ T cells and cytokine interferon gamma (IFNγ) in the development of ICI-T1DM. Using CellChat to perform receptor-ligand interaction analysis of these data, we identified both CD8+ and CD4+ T cells as the primary source of IFNγ in immune infiltrates and islet myeloid cells as the primary target. Furthermore, CellChat analysis showed increased receptor-ligand interactions between T cell receptor CXCR6 and its chemokine CXCL16, as well as CXCR3 and chemokines CXCL9 and CXCL10. In vitro IFNγ stimulation of myeloid cells induced strong expression of CXCL16, 9, and 10, suggesting a potential mechanism by which effector T cells are recruited to islets during ICI therapy. Immunophenotyping of islet-infiltrating immune cells indeed showed increased IFNγ+CXCR6+ effector CD8+ cells in anti-PD1 treated mice compared to isotype-treated controls. Moreover, these IFNγ+CXCR6+CD8+ T cells stained positively for the NRP-V7 mimotope tetramer, a known antigen for CD8+ T cells in spontaneous autoimmune diabetes in NOD mice, suggesting this as an antigen-specific population contributing to the immunopathogenesis of ICI-T1DM. Finally, genetic deletion of CXCR6 in NOD mice delayed the onset of autoimmune diabetes during anti-PD1 treatment in female (p=0.0162) but not male mice (p=0.3656). In summary, these data further elucidate the cellular mechanisms by which IFNγ and chemokine signaling pathways drive the development of ICI-T1DM and identify the CXCR6-CXCL16 axis as a potential therapeutic target to prevent this IRAE in checkpoint inhibitor-treated cancer patients. Presentation: 6/2/2024

An overview of the role of chemokine CX3CL1 (Fractalkine) and CX3C chemokine receptor 1 in systemic sclerosis.

Systemic sclerosis (SSc) is a complex autoimmune disease characterized by fibrosis, vascular damage, and immune dysregulation. Fractalkine or chemokine (C-X3-C motif) ligand 1 (CX3CL1), a chemokine and adhesion molecule, along with its receptor CX3CR1, have been implicated in the inflammatory processes of SSc. CX3CL1 functions as both a chemoattractant and an adhesion molecule, guiding immune cell trafficking. This systematic review examines the role of CX3CL1 and its receptor CX3CR1 in the pathogenesis of SSc, with a focus on pulmonary and vascular complications. A systematic literature search was conducted across databases including PubMed, Scopus, and Web of Science from inception to November 2020. The search focused on studies investigating the CX3CL1/CX3CR1 axis in the context of SSc. The review identified elevated CX3CL1 expression in SSc patients, particularly in the skin and lungs, where CX3CR1 is expressed on infiltrating immune cells. Higher levels of CX3CL1 were correlated with the severity of interstitial lung disease in SSc patients, indicating a potential predictive marker for disease progression. CX3CR1-positive monocytes and NK cells were recruited to inflamed tissues, contributing to fibrosis and tissue damage. Animal studies showed that inhibition of the CX3CL1/CX3CR1 axis reduced fibrosis and improved vascular function. The CX3CL1/CX3CR1 axis plays a key role in immune cell recruitment and fibrosis in SSc. Elevated CX3CL1 levels are associated with lung and vascular complications, making it a potential biomarker for disease progression and a promising therapeutic target.

CXCL13/CXCR5 axis facilitates TFH expansion and correlates with disease severity in adults with immune thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) is an autoimmune bleeding disorder defined by a diminished platelet count. ITP pathogenesis involves intricate changes to both cellular and humoral immunity. The pivotal roles of follicular helper T (TFH) cells in the maturations of B cells and the production of antibodies are well-established. However, the specific role of TFH to the immunopathogenesis of ITP remain incompletely understood. This study aimed to clarify the association of CXCL13/CXCR5 axis with TFH in adults with ITP. MethodsA total of 97 ITP patients and 41 healthy controls were enrolled. CD4+CXCR5+ TFH, CD4+CXCR5+PD-1+ TFH, CD4+CXCR5+Foxp3+ follicular regulatory T cells (TFR), and desialylated platelets in peripheral blood were measured by flow cytometry. Plasma cytokines were assessed by enzyme-linked immunosorbent assay. CD4+ T cells cocultured with chemokine CXCL13 in vitro was performed for the measurement of TFH proliferation. Intracellular production of reactive oxygen species (ROS) was examined by dichlorodihydrofluorescein diacetate (DCFH-DA) probe staining. ResultsWe observed a significant increase in circulating TFH and a marked decrease in circulating TFR in the entire ITP cohort. The ratio of TFH/TFR was elevated, accompanied by heightened levels of platelet desialylation, cytokines BAFF, HMGB1, and IL-21, while levels of IL-10 were downregulated in adults with ITP. Notably, patients with ITP exhibiting platelet count below 50 × 109/L had dramatically elevated levels in both chemokine CXCL13 and its receptor CXCR5+ TFH compared to those with platelet count above 100 × 109/L. High frequencies of TFH correlated with poor therapeutic response. Furthermore, in vitro CD4+ T cell proliferation assay demonstrated a CXCL13 dose-dependent increase in the frequencies in both CD4+CXCR5+ TFH and CD4+CXCR5+PD-1+ TFH from ITP patients. Intriguingly, DCFH-DA assay illustrated a significant enhancement in intracellular ROS generation in CXCR5+ T cell subsets, especially in CD4+CXCR5+PD-1+ TFH from 4 patients with ITP. ConclusionsThese results underscore the pivotal role of CXCL13/CXCR5 axis-drived TFH expansion in the pathogenesis of ITP, providing a potential disease severity biomarker.

Neutrophils with low production of reactive oxygen species are activated during immune priming and promote development of arthritis

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease mediated by immune cell dysfunction for which there is no universally effective prevention and treatment strategy. As primary effector cells, neutrophils are important in the inflammatory joint attack during the development of RA. Here, we used single-cell sequencing technology to thoroughly analyze the phenotypic characteristics of bone marrow-derived neutrophils in type II collagen (COL2)-induced arthritis (CIA) models, including mice primed and boosted with COL2. We identified a subpopulation of neutrophils with high expression of neutrophil cytoplasmic factor 1 (NCF1) in primed mice, accompanied by a characteristic reactive oxygen species (ROS) response, and a decrease in Ncf1 expression in boosted mice with the onset of arthritis. Furthermore, we found that after ROS reduction, arthritis occurred in primed mice but was attenuated in boosted mice. This bidirectional effect of ROS suggested a protective role of ROS during immune priming. Mechanistically, we combined functional assays and metabolomics identifying Ncf1-deficient neutrophils with enhanced migration, chemotactic receptor CXCR2 expression, inflammatory cytokine secretion, and Th1/Th17 differentiation. This alteration was mainly due to the metabolic reprogramming of Ncf1-deficient neutrophils from an energy supply pathway dominated by gluconeogenesis to an inflammatory immune pathway associated with the metabolism of histidine, glycine, serine, and threonine signaling, which in turn induced arthritis. In conclusion, we have systematically identified the functional and inflammatory phenotypic characteristics of neutrophils under ROS regulation, which provides a theoretical basis for understanding the pathogenesis of RA, to further improve prevention strategies and identify novel therapeutic targets.

Low dose methotrexate impaired T cell transmigration through down-regulating CXCR4 expression in rheumatoid arthritis (RA)

BackgroundCXC chemokine CXCL12 is involved in the pathological development of rheumatoid arthritis (RA) through abnormal migration of peripheral immune cells in the joint. Although low dose methotrexate (MTX) is clinically used to treat RA patients, CXCL12 signaling responses to MTX-mediated treatments is still not well understood.MethodsIn this study, we examined the expression of CXCR4 (cognatic receptor for CXCL12) in peripheral T cells from RA patients and arthritis mice models received from low dose MTX therapies. The effects of low dose MTX on CXCR4 were further determined via both in vitro CD3+ T cells and Cxcr4 conditional knockout (CKO) arthritis mice models.ResultsOur clinical data shows that low dose MTX treatment was clinically associated with down-regulated expression of chemokine receptor CXCR4 on patient peripheral T cells. In vitro, low dose MTX significantly decreased cell transmigration through down-regulated CXCR4’s expression in CD3+ T cells. Consistently, CD3+ T cells treated with low dose MTX demonstrated an increased genomic hypermethylation across the promoter region of Cxcr4 gene. Furthermore, our preclinical studies showed that low dose MTX-mediated downregulation of CXCR4 significantly improved the pathological development in mouse arthritis models. Conditional disruption of the Cxcr4 gene in peripheral immune cells potentially alleviated inflammation of joints and lung tissue in the arthritis mice, though genetic modification itself overall did not change their clinical scores of arthritis, except for a significant improvement on day 45 in CXCR4 CKO arthritis mice models during the recovery phase.ConclusionOur findings suggest that the effect of low dose MTX treatment could serve to eliminate inflammation in RA patients through impairment of immune cell transmigration mediated by CXCR4.

Enhancing T-cell recruitment in renal cell carcinoma with cytokine-armed adenoviruses

ABSTRACT Immunotherapy has emerged as a promising approach for cancer treatment, with oncolytic adenoviruses showing power as immunotherapeutic agents. In this study, we investigated the immunotherapeutic potential of an adenovirus construct expressing CXCL9, CXCL10, or IL-15 in clear cell renal cell carcinoma (ccRCC) tumor models. Our results demonstrated robust cytokine secretion upon viral treatment, suggesting effective transgene expression. Subsequent analysis using resistance-based transwell migration and microfluidic chip assays demonstrated increased T-cell migration in response to chemokine secretion by infected cells in both 2D and 3D cell models. Flow cytometry analysis revealed CXCR3 receptor expression across T-cell subsets, with the highest percentage found on CD8+ T-cells, underscoring their key role in immune cell migration. Alongside T-cells, we also detected NK-cells in the tumors of immunocompromised mice treated with cytokine-encoding adenoviruses. Furthermore, we identified potential immunogenic antigens that may enhance the efficacy and specificity of our armed oncolytic adenoviruses in ccRCC. Overall, our findings using ccRCC cell line, in vivo humanized mice, physiologically relevant PDCs in 2D and patient-derived organoids (PDOs) in 3D suggest that chemokine-armed adenoviruses hold promise for enhancing T-cell migration and improving immunotherapy outcomes in ccRCC. Our study contributes to the development of more effective ccRCC treatment strategies by elucidating immune cell infiltration and activation mechanisms within the tumor microenvironment (TME) and highlights the usefulness of PDOs for predicting clinical relevance and validating novel immunotherapeutic approaches. Overall, our research offers insights into the rational design and optimization of viral-based immunotherapies for ccRCC.

Crosstalk between androgen signaling and the chemokine receptor CXCR4: a novel strategy to promote myelin regeneration.

Crosstalk between androgen signaling and the chemokine receptor CXCR4: a novel strategy to promote myelin regeneration.

Chemokine receptor CXCR4 based positron emission tomography imaging in systemic sclerosis-related interstitial lung disease.

To assess chemokine receptor CXCR4 expression in lung parenchyma and on peripheral immune cells in systemic sclerosis-related interstitial lung disease (SSc-ILD) patients. SSc-ILD patients underwent 68Ga- CPCR4 Trifluoroacetate positron emission tomography (PET) scan, SUVmean in different lung regions and architecturally abnormal areas, and receiver operating characteristic (ROC) curves were analyzed. CXCR4 expression on peripheral immune cells using flow cytometer was studied and correlated with the different lung regions. In addition, subset analysis of CXCR4 expression by clinical subset (early, progressive, stable), ILD pattern and anti-Scl-70 positivity were done. On PET, SSc-ILD patients showed higher median SUVmean uptake of CXCR4 in the whole lung (0.56; p< 0.0001), different lung regions and architecturally abnormal areas than controls. Highest area under curve (AUC) were observed in dorsobasal regions (AUC-0.91; p< 0.0001) and reticular with architecturally distorted areas (AUC-0.95; p< 0.0001). Progressive subset had higher whole lung median SUVmean (0.73) than early (0.49; p< 0.0001) and stable (0.45; p< 0.0001) subsets, and AUC than early and stable subsets. Usual interstitial pneumonia pattern ILD showed higher CXCR4 uptake compared with non-specific interstitial pneumonia (p= 0.0032). Additionally, a trend for higher uptake was noted in anti-Scl70 positive patients as compared with anti-Scl70 negative ones. SSc-ILD patients had higher CD4+CXCR4+T cells (p= 0.0003), and CD8+CXCR4+T cells (p= 0.0013), and showed moderate to strong association on correlation with the lung parenchymal regions. In SSc-ILD, CXCR4 expression is upregulated in both lung parenchyma and peripheral T cells, significantly in progressive and UIP subsets. CXCR4 expression is a potential tool for activity assessment and prognostication.

Structural insights into CXCR4 modulation and oligomerization.

Activation of the chemokine receptor CXCR4 by its chemokine ligand CXCL12 regulates diverse cellular processes. Previously reported crystal structures of CXCR4 revealed the architecture of an inactive, homodimeric receptor. However, many structural aspects of CXCR4 remain poorly understood. Here, we use cryo-electron microscopy to investigate various modes of human CXCR4 regulation. CXCL12 activates CXCR4 by inserting its N terminus deep into the CXCR4 orthosteric pocket. The binding of US Food and Drug Administration-approved antagonist AMD3100 is stabilized by electrostatic interactions with acidic residues in the seven-transmembrane-helix bundle. A potent antibody blocker, REGN7663, binds across the extracellular face of CXCR4 and inserts its complementarity-determining region H3 loop into the orthosteric pocket. Trimeric and tetrameric structures of CXCR4 reveal modes of G-protein-coupled receptor oligomerization. We show that CXCR4 adopts distinct subunit conformations in trimeric and tetrameric assemblies, highlighting how oligomerization could allosterically regulate chemokine receptor function.

Interaction and dynamics of chemokine receptor CXCR4 binding with CXCL12 and hBD-3

Chemokine receptor CXCR4 is involved in diverse diseases. A comparative study was conducted on CXCR4 embedded in a POPC lipid bilayer binding with CXCL12 in full and truncated forms, hBD-3 in wildtype, analog, and mutant forms based on in total 63 µs all-atom MD simulations. The initial binding structures of CXCR4 with ligands were predicted using HADDOCK docking or random-seed method, then μs-long simulations were performed to refine the structures. CXCR4&ligand binding structures predicted agree with available literature data. Both kinds of ligands bind stably to the N-terminus, extracellular loop 2 (ECL2), and ECL3 regions of CXCR4; the C2-C3 (K32-R38) region and occasionally the head of hBD-3 bind stably with CXCR4. hBD-3 analogs with Cys11-Cys40 disulfide bond can activate CXCR4 based on the Helix3-Helix6 distance calculation, but not other analogs or mutant. The results provide insight into understanding the dynamics and activation mechanism of CXCR4 receptor binding with different ligands.

The Role of the CX3CR1-CX3CL1 Axis in Respiratory Syncytial Virus Infection and the Triggered Immune Response.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes respiratory illnesses, ranging from mild symptoms to severe lower respiratory tract infections in infants and older adults. This virus is responsible for one-third of pneumonia deaths in the pediatric population; however, there are currently only a few effective vaccines. A better understanding of the RSV-host relationship at the molecular level may lead to a more effective management of RSV-related symptoms. The fractalkine (CX3CL1) receptor (CX3CR1) is a co-receptor for RSV expressed by airway epithelial cells and diverse immune cells. RSV G protein binds to the CX3CR1 receptor via a highly conserved amino acid motif (CX3C motif), which is also present in CX3CL1. The CX3CL1-CX3CR1 axis is involved in the activation and infiltration of immune cells into the infected lung. The presence of the RSV G protein alters the natural functions of the CX3CR1-CX3CL1 axis and modifies the host's immune response, an aspects that need to be considered in the development of an efficient vaccine and specific pharmacological treatment.

CSF1R Ligands Expressed by Murine Gliomas Promote M-MDSCs to Suppress CD8+ T Cells in a NOS-Dependent Manner.

Glioblastoma (GBM) is the most common malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a subset of myeloid cells, expressing monocytic (M)-MDSC markers and dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate the TME. This study evaluated the mechanism of CCR2+/CX3CR1+ M-MDSC differentiation and T cell suppressive function in murine glioma models. We determined that bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Glioma-secreted CSF1R ligands M-CSF and IL-34 were identified as key drivers of M-MDSC differentiation while adenosine and iNOS pathways were implicated in the M-MDSC suppression of T cells. Mining a human GBM spatial RNAseq database revealed a variety of different pathways that M-MDSCs utilize to exert their suppressive function that is driven by complex niches within the microenvironment. These data provide a more comprehensive understanding of the mechanism of M-MDSCs in glioblastoma.

IS4-FAM, a fluorescent tool to study CXCR4 affinity and competitive antagonism in native cancer cells.

The ability to accurately measure drug-target interaction is critical for the discovery of new therapeutics. Classical pharmacological bioassays such as radioligand or fluorescent ligand binding assays can define the affinity or Kd of a ligand for a receptor with the lower the Kd, the stronger the binding and the higher the affinity. However, in many drug discovery laboratories today, the target of interest if often artificially upregulated by means of transfection to modify the host cell's genetic makeup. This then potentially invalidates the assumptions of classical pharmacology affinity calculations as the receptor of interest is no longer at normal physiological densities. The CXCR4 receptor is expressed on many different cancer cell types and is associated with metastasis and poor prognosis. Therefore, the CXCR4 receptor is a desirable target for novel therapeutics. In this study, we explore the applicability of the newly developed fluorescently tagged CXCR4 antagonists, IS4-FAM as an investigative tool to study CXCR4 affinity and competitive antagonism in native, non-transfected cancer cells using confocal microscopy and flow cytometry. IS4-FAM directly labels CXCR4 in several cell lines including high CXCR4 expressing SK-MEL-28 (malignant melanoma) and PC3 (metastatic prostate cancer) and lower CXCR4 expressing THP-1 (acute monocytic leukemia) and was competitive with the established CXCR4 antagonist, AMD3100. This highlights the potential of IS4-FAM as a pharmacological tool for drug discovery in native cells lines and tissues.

Immune response and protection efficacy of formalin-killed vaccines against Streptococcus iniae in four-finger threadfin Eleutheronema tetradactylum.

Four-finger threadfin, Eleutheronema tetradactylum farming in southern Taiwan has been facing disease problems caused by Streptococcus iniae since 2018. The development of a vaccine against infectious S. iniae in the cultured threadfin industry is necessary. Thus, this study aimed to examine the efficacy of threadfin immunized formalin-killed cells (FKC) from S. iniae GSI-111 for 42 days post-vaccination (dpv) using two doses of FKC alone (a booster at 14 dpv) as group A, and FKC mixed with ISA763A adjuvant using a single dose as group B or double doses as group C. Immunoglobulin (Ig)-M was purified from threadfin, and rabbit anti-threadfin IgM polyclonal antibodies were used to detect antibody level in immunized fish; the vaccinated group A displayed higher levels at 3 dpv and all vaccinated treatments demonstrated high antibody levels between 14 and 42 dpv. All vaccine groups showed significantly higher values of lysozyme activity at 42 dpv compared with the control group; the vaccinated A group peaked at 14 dpv. The expression profiles of pro-inflammatory and immune-related genes, TNF-α, IL-12A, and C2 were upregulated at 3 dpv, while CD8A and chemokine receptor CXCR4 were upregulated at 42 dpv. Finally, the threadfins were challenged with S. iniae at 42 dpv. The average relative percent survival was 96% for vaccination A and B treatments, and 100% for vaccination C treatment. In summary, this study demonstrated that FKC vaccines whether formulated with an adjuvant could stimulate immune response and effective protect threadfins against S. iniae infection.

Chemokine-mediated F-actin dynamics, polarity, and migration in B lymphocytes depend on WNK1 signaling.

Ligand-engaged chemokine receptors trigger nucleotide exchange in heterotrimeric Gαi proteins, which stimulates cytoskeletal reorganization and cell polarity changes. To better understand the signaling events responsible for these cellular changes, we focused on early changes in F-actin dynamics after engagement of the chemokine receptor CXCR5 in murine splenic B cells. Within 10 seconds of exposure to the CXCR5 ligand CXCL13, three-dimensional lamellar-like pseudopods and F-actin-rich ridges appeared. The transient F-actin increase depended on Gαi2/3 signaling, the PI3K/AKT pathway, ERK activation, phospholipase C activity, and Rac1/2 activation mediated by Dock2 (dedicator of cytokinesis 2). Immunoblot analyses identified the kinase WNK1 (with no lysine kinase 1) as a potential early AKT effector. Treating B cells with specific WNK inhibitors disrupted F-actin dynamics and impaired B cell polarity, motility, and chemotaxis. These changes were mimicked in a murine B cell line by CRISPR-Cas9 gene editing of Wnk1, which also suggested that WNK1 contributed to B cell proliferation. Administration of a single dose of a WNK inhibitor transiently reduced B cell motility and polarity in the lymph nodes of live mice. These results indicate that WNK1 signaling maintains B cell responsiveness to CXCL13 and suggest that pharmacological inhibition of WNK1, which is involved in cancer progression and blood pressure regulation, may affect humoral immunity.

[68 Ga]Ga-CXCR4 PET/CT imaging in high-grade glioma for assessment of CXCR4 receptor expression

PurposeGliomas account for 75 % of primary malignant CNS tumors. High-grade glioma (CNS WHO grades 3 and 4) have an unfavorable treatment response and poor outcome. CXCR4 is a G protein-coupled receptor that plays an important part in the signaling pathway between cancer cells and tumor microenvironment. CXCR4 overexpression has been shown in a variety of cancers. In this study, we evaluate the potential value of [68Ga]Ga-Pentixafor as a PET/CT CXCR4-probe for in vivo assessment of CXCR4 expression in patients with high-grade glioma and its correlation with tumor grade. Materials and Methods[68Ga]Ga-CXCR4 PET/CT was performed in the prospective single-center study in treatment-naïve biopsy-proven patients with high-grade glioma. The acquired images were analyzed qualitatively and semi-quantitatively. ResultA total of 26 patients (mean age: 53.3±14.4 years, 11 women, 15 men) were enrolled. CNS WHO grade 3 pathology was seen in 19 % (5/26) of the sample. The patient-based sensitivity of 68Ga-CXCR4 was 96.2 %. Overall, 28 pathologic lesions were detected, leading to a lesion-based sensitivity of 96.4 %. The median (IQR) SUVmax of grade 4 lesions was substantially greater than the grade 3(3.03(2.5–3.7) vs. 1.51(1.2–1.8), p = 0.0145).). The highest tracer activity of organs −beside bladder as the main excretion reservoir-was in lymphoid tissue of Waldeyer’s ring (mean SUVmax: 7.41), and spleen (mean SUVmax: 6.62). ConclusionIn conclusion, this new application for [68Ga]Ga-Pentixafor PET tracer exhibits excellent visual and semi-quantitative diagnostic properties. Further studies are warranted.

CXCR3-Expressing T Cells in Infections and Autoimmunity.

The chemokine receptor CXCR3 and its ligands (MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11) play a central role in the generation of cellular inflammation, both in the protective responses to invading pathogens, and in different pathological conditions associated with autoimmunity. It is worth noting that CXCR3 is highly expressed on innate and adaptive lymphocytes, as well as on various cell subsets that are localized in non-immune organs and tissues. Our review focuses exclusively on CXCR3-expressing T cells, including Th1, Th17.1, Tfh17, Tfh17.1, CXCR3+ Treg cells, and Tc1 CD8+ T cells. Currently, numerous studies have highlighted the role of CXCR3-dependent interactions in the coordination of inflammation in the peripheral tissues, both to increase recruitment of CD4+ and CD8+ T cells that upregulate inflammation, and also for recruitment of CXCR3+ T regulatory cells to dampen overexuberant responses. Understanding the role of CXCR3 and its ligands might help to apply them as new and effective therapeutic targets in a wide range of diseases.

Perinatal liver inflammation is associated with persistent elevation of CXCL10 and its canonical receptor CXCR3 on common myeloid progenitors.

Biliary atresia (BA) is a leading cause of liver failure in infants. Despite effective surgical drainage, patients with BA exhibit attenuated immune responses to childhood vaccines, suggesting there are long-lasting alterations to immune function. The perinatal liver is home to hematopoietic stem and progenitor cells (HSPCs) and serves as the epicenter for rapidly progressive and significantly morbid inflammatory diseases like BA. We have previously established the role of neonatal myeloid progenitors in the pathogenesis of perinatal liver inflammation (PLI) and hypothesize that PLI leads to long-term changes to HSPCs in mice that recovered from PLI. To test this hypothesis, we compared the changes that occur to HSPCs and mature myeloid populations in the bone marrow of adult mice during homeostasis and during PLI. Our results demonstrate that HSPCs from animals that recover from PLI ("PLI-recovered") undergo long-term expansion with a reduced proliferative capacity. Notably, PLI leads to persistent activation of common myeloid progenitors through the involvement of CXCL10 and its canonical receptor, CXCR3. Our data suggests that the CXCR3-CXCL10 axis may mediate the changes in HSPCs that lead to altered immune function observed in BA, providing support for a targetable pathway to mitigate the detrimental long-term immune effects observed in patients with BA.

Characterization of a CXCR4 antagonist TIQ-15 with dual tropic HIV entry inhibition properties.

The chemokine co-receptors CXCR4 and CCR5 mediate HIV entry and signal transduction necessary for viral infection. However, to date only the CCR5 antagonist maraviroc is approved for treating HIV-1 infection. Given that approximately 50% of late-stage HIV patients also develop CXCR4-tropic virus, clinical anti-HIV CXCR4 antagonists are needed. Here, we describe a novel allosteric CXCR4 antagonist TIQ-15 which inhibits CXCR4-tropic HIV-1 infection of primary and transformed CD4 T cells. TIQ-15 blocks HIV entry with an IC50 of 13 nM. TIQ-15 also inhibits SDF-1α/CXCR4-mediated cAMP production, cofilin activation, and chemotactic signaling. In addition, TIQ-15 induces CXCR4 receptor internalization without affecting the levels of the CD4 receptor, suggesting that TIQ-15 may act through a novel allosteric site on CXCR4 for blocking HIV entry. Furthermore, TIQ-15 did not inhibit VSV-G pseudotyped HIV-1 infection, demonstrating its specificity in blocking CXCR4-tropic virus entry, but not CXCR4-independent endocytosis or post-entry steps. When tested against a panel of clinical isolates, TIQ-15 showed potent inhibition against CXCR4-tropic and dual-tropic viruses, and moderate inhibition against CCR5-tropic isolates. This observation was followed by a co-dosing study with maraviroc, and TIQ-15 demonstrated synergistic activity. In summary, here we describe a novel HIV-1 entry inhibitor, TIQ-15, which potently inhibits CXCR4-tropic viruses while possessing low-level synergistic activities against CCR5-tropic viruses. TIQ-15 could potentially be co-dosed with the CCR5 inhibitor maraviroc to block viruses of mixed tropisms.