ACKR3 promotes CXCL12/CXCR4-mediated migration of lymphoma cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE169144 | GEO | 2022/10/13
REPOSITORIES: GEO
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Project description:CXCL12 is a chemokine that binds to its cognate receptors CXCR4 and ACKR3 (CXCR7). An increase in CXCL12 concentrations has been used as a pharmacodynamic biomarker to assess ACKR3 antagonism in healthy adults. Furthermore, increased CXCL12 concentrations have been observed in various human pathologies. To date, CXCL12 concentrations have typically been quantified using antibody-based assays with overlapping or unclear specificity for the various existing CXCL12 proteoforms. Only the N-terminal full-length CXCL12 proteoform is biologically active and can engage CXCR4 and ACKR3. However, this proteoform could so far not be quantified in healthy adults. Here, we describe a new and validated fit-for-purpose immunoaffinity mass spectrometry (IA-MS) biomarker assay for specific measurement of five CXCL12α proteoforms in human plasma, including the biologically active CXCL12α proteoform. The assay was employed in a Phase 1 clinical study with the ACKR3 antagonist ACT-1004-1239 to quantify CXCL12α proteoforms. At baseline levels, 1.00 nM total CXCL12α and 0.10 nM biologically active CXCL12α was quantified in placebo treated adults. The concentrations of both proteoforms increased up to two-fold in healthy adults following drug administration. At all doses, 10% of CXCL12α was biologically active and the simultaneous increase of all proteoforms suggests that a new equilibrium has been reached 24 h following dosing. Hence, this IA-MS biomarker assay can be used to specifically measure active CXCL12 proteoform concentrations in clinical trials. Specific quantification of active chemokines can support decision making in clinical trial and thus successful drug development.
2024-01-27 | PXD038814 | Pride
Project description:Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and non-peptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key-factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine-triphosphate (ATP) and uridine-triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin. In vivo, pre-incubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Gαi) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5â-triphosphatases (GTPase) Rac2 and downstream effectors Rho GTPaseâactivated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the migration of HSCs and their homing to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases. Experiment Overall Design: Highly purified CD34+ cells from 6 healthy donors were seeded at 1000000 cells/ml in serum free medium (EX vivo 15) w/o cytokines and treated with 10 mM UTP, 150ng/ml CXCL12, or 10 mM UTP plus 150ng/ml CXCL12 respectively for 24 hours. As a control, CD34+ untreated cells were maintained in the same culture conditions at the same time.
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Project description:Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet, the mechanisms of lymphocyte transit remain poorly defined. We find that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression on effector CD8+ T cells. Only high affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells exit the tumor, thereby limiting tumor control. CXCR4 inhibition and loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. Strategies that limit T cell egress, therefore, provide a new tool to boost the response to immunotherapy.
2023-01-20 | GSE218455 | GEO
Project description:The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in various tumors promotes cell proliferation and invasiveness. The intracellular cellular pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by characterizing the the interactome of recombinant ACKR3 expressed in HEK293T cells. Among the ACKR3-interacting proteins identified, we focused on the gap junction protein Connexin 43 (Cx43).
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Project description:Coordinated migration of B cells within and between secondary lymphoid tissues is required for robust antibody responses to infection or vaccination. Secondary lymphoid tissues normally expose B cells to a low O2 (hypoxic) environment. Recently, we have shown that human B cell migration is modulated by an O2-dependent molecular switch, centrally controlled by the hypoxia-induced (transcription) factor-1a (HIF1A), which can be disrupted by the immunosuppressive calcineurin inhibitor, cyclosporine A (CyA). However, the mechanisms by which low O2 environments attenuate B cell migration remain poorly defined. Proteomics analysis has linked CXCR4 chemokine receptor signaling to cytoskeletal rearrangement. We now hypothesize that the pathways linking the O2 sensing molecular switch to chemokine receptor signaling and cytoskeletal rearrangement would likely contain phosphorylation events, which are typically missed in traditional transcriptomic and/or proteomic analyses. Hence, we have performed a comprehensive phosphoproteomics analysis of human B cells treated with CyA after engagement of the chemokine receptor CXCR4 with CXCL12. Statistical analysis of the separate and synergistic effects of CyA and CXCL12 revealed 116 proteins whose abundance is driven by a synergistic interaction between CyA and CXCL12. Among these 116 proteins, Lymphocyte Specific Protein 1 (LSP1) was most interesting due to its original identification as a protein modulating neutrophil migration. We used our previously described algorithm BONITA to reveal a critical role for LSP1 in cytoskeletal rearrangement. Validating these modeling results, we show experimentally that LSP1 levels in B cells increase with low O2 exposure, and CyA treatment results in decreased LSP1 protein levels. This correlates with the increased chemotactic activity observed after CyA treatment. Lastly, we directly link LSP1 levels to chemotactic capacity, as shRNA knock-down of LSP1 results in significantly increased B cell chemotaxis at low O2 levels. These results directly link the CyA to LSP1-dependent cytoskeletal regulation, demonstrating a previously unrecognized mechanism by which CyA modulates human B cell migration.
2022-08-24 | PXD036167 | Pride
Project description:We here reported that HDAC3 inhibition induces the infiltration of NK cells in lymphoma and enhances their cytotoxicity by promoting the secretion of CXCL12 from lymphoma cells. RNA-seq analysis of EL4 cells treated with or without RGFP966 showed that chemokine signaling was significantly enriched in the HDAC3 inhibitor-treated group. And the expression of CXCL12 in mRNA and protein level were increased. ATF3 overexpression decreased the mRNA level of CXCL12 in lymphoma cells and reduced CXCL12 transcription can be reversed by HDAC3 inhibition in H9 cells. To sum up, targeting HDAC3 promoted the secretion of CXCL12 by inhibiting ATF3’s transcriptional activity.
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Project description:The chemokine CXCL12 and its receptor CXCR4 play important roles in signaling and migration of T-cells, but little is known about the transcriptional events involved in CXCL12-mediated T-cell migration. In this study we performed microarray analysis on CXCL12- treated T-cells, and found that the Wnt family of proteins was significantly upregulated during CXCL12 treatment. Confirmation of these results by real-time PCR and Western analysis indicated that the non-canonical Wnt pathway was specifically upregulated during CXCL12 treatment. In vitro and in vivo knockdown studies confirm that b-catenin (the key mediator of canonical Wnt signaling) is not involved in the CXCL12-mediated migration of T-cells. However, Wnt5A, a non-canonical Wnt protein, increases signaling through the CXCL12/ CXCR4 axis via Protein Kinase C (PKC). Our results demonstrated that CXCL12 required Wnt5A to mediate T-cell migration, and the treatment of T-cells with recombinant Wnt5A sensitized T-cells to CXCL12 induced migration. Additionally, Wnt5A expression was required for the sustained expression of CXCR4, both transcriptionally and translationally. These results could be translated in vivo, using EL4 thymoma metastasis as a model of T-cell migration. Taken together our data indicate, for the first time, that Wnt5A is a critical mediator of the CXCL12/ CXCR4 signaling axis. Keywords: Wnt5A, CXCL12, CXCL12, CXCR4, T-cell Migration
2009-01-01 | GSE12449 | GEO
Project description:Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and non-peptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key-factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine-triphosphate (ATP) and uridine-triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin. In vivo, pre-incubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Gαi) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5’-triphosphatases (GTPase) Rac2 and downstream effectors Rho GTPase–activated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the migration of HSCs and their homing to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases. Keywords: treatment comparison
2006-10-04 | GSE5168 | GEO
Project description:The marginal zone (MZ) of the spleen contains a specialized set of innate-like B cells that are in direct contact with the blood. Here we show that expression of the atypical chemokine receptor 3 (ACKR3) defines two phenotypically, transcriptionally and functionally distinct, equal-sized populations of mouse MZ B cells (MZBs). The ACKR3-expressing population is required for optimal development and function of the MZ and for the differentiation and positioning of all MZBs. In the absence of ACKR3 expression on B cells the MZ is distorted and the MZBs fail to deliver blood-borne antigens to follicular dendritic cells (FDCs), leading to a reduced early humoral immune response. Effective reconstitution of MZ deficient CD19ko mice with either of the two MZB subsets shows that ACKR3- MZBs can differentiate into the more mature ACKR3+ MZBs, but not vice versa. The phenotype of CD19ko mice lacking a MZ is readily rescued by the adoptive transfer of ACKR3-sufficient, and to a much lower degree by ACKR3-deficient follicular B cells (FoBs), indicating that ACKR3 expression is crucial for the establishment of the MZ developmental niche. Similarly, the inability of CD19ko mice to respond to T-independent antigen is rescued when ACKR3-proficient, but not ACKR3-deficient FoBs are adoptively transferred. Accordingly, ACKR3-deficient FoBs are able to reconstitute the MZ if the niche is pre-established by ACKR3 proficient MZBs. Our data uncover a new unexpected role of ACKR3 on B cells for forming the splenic microarchitecture.
2020-06-19 | GSE126860 | GEO
Project description:Recent data indicate that CD38, a cell surface enzyme and receptor, is part of the complex network of signals underlying the development, maintenance and progression of chronic lymphocytic leukemia (CLL). Here we show that CD38 signals directly facilitate short- (ERK1/2 phosphorylation) and long-term (chemotaxis) effects induced by CXCL12 exposure. Use of i) a specific enzyme inhibitor, ii) an agonistic anti-CD38 monoclonal antibody (mAb) and iii) microarray studies led to the conclusion that the receptor-like functions of CD38 are responsible for the observed effects. Interactions between CD38 and its non substrate ligand CD31 define a genetic signature characterized by modulation of a significant number of genetic pathways, involved in proliferation and migration. Blocking CD38 with domain-specific mAbs significantly lowers CXCL12 effects, in terms of ERK1/2 phosphorylation and chemotaxis. The molecular explanation behind the blocking activity of these mAbs lies in physical proximity between CD38 and the CXCL12 receptor CXCR4, as demonstrated by i) co-immunoprecipitation, ii) confocal microscopy and iii) direct measurement of CXCL12 binding to its receptor. Lastly, blocking anti-CD38 mAbs were successfully used in vivo to interfere with CLL cell recirculation from blood to the lymphoid organs, an event critically controlled by CXCL12. These results represent a first step towards the validation of anti-CD38 mAbs as potential therapeutics for certain CLL patients. Keywords: gene expression profiling by microarray, CLL, CD31, CD38
2009-12-22 | GSE14063 | GEO