CC Chemokine Receptor 5 Ligands Research Articles

Abstract 2823: Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological malignancy, is characterized by abnormal thymocyte proliferation, frequently with mutations of the Notch-1 gene and has an overall 10-year survival rate of ~27%. A marker of poor prognosis is the presence of T-ALL within the central nervous system (CNS), which is treated with intrathecal chemotherapy and cranial irradiation; two toxic treatments with high morbidities. Gene-expression profiling of Notch-1-induced adhesion regulators showed that C-C chemokine receptor 7 (CCR7) was highly expressed and was associated with leukemic T-cell invasion of the CNS. Normal functions of CCR7 include lymph node homing and immune cell trafficking in response to CCR7 ligands, CC-chemokine ligand 19 (CCL19) or CCL21. In several studies CCL19 has been linked to T-cell invasion of the CNS across the blood-brain barrier (BBB). We hypothesize that antagonizing CCR7 binding to CCL19 will block migration of T-ALL into the CNS and thereby alleviate the need for toxic therapies. To test our hypothesis, we are using receptor internalization assays, calcium mobilization assays, transwell migration assays and an in vitro model of an intact human BBB. We have used flow cytometry-based CCR7 internalization assays ± CCR7 antagonist and found that we can completely block CCL19-induced receptor internalization in human CCR7-expressing CCRF-CEM, and HuT-78 T-ALL cells, and chemotaxis via β1 integrins across a fibronectin-coated transwell membrane. Our BBB employs HBEC-5i, human cerebral microvascular endothelial cells, in transwell chambers coated with gelatin and fibronectin for the adhesion of endothelial cells. HBEC-5i cells are a suitable cell line to make a 3D model of the BBB, as they form an endothelial monolayer with tight junctions (TJ), which is crucial to maintaining the integrity and inhibiting permeability of the BBB. We are establishing a transwell migration assay to assess the migration of CCRF-CEM T-ALL cells across the HBEC-5i cell monolayer. The integrity of the HBEC-5i cell monolayer is assessed by trans-epithelial electrical resistance (TEER), which measures the electrical resistance across the HBEC-5i monolayer, a measure of TJ integrity. Using TEER measurements, we observe that HBEC-5i form a monolayer that reaches an intact monolayer functioning with TJs over 72-96 hours. We are using this model system to investigate the migration of CCR7-expressing CCRF-CEM cells through the BBB ± CCL19. Subsequently, we will add the CCL19 antagonist (CCL197-77) to determine if inhibiting CCR7 is sufficient to eliminate migration of the CCRF-CEM cells through the BBB. If successful, this model will allow us to test the contribution of CCR7 signaling to integrins, the contribution of other immune cell types and the overall molecular mechanisms that mediate invasion of the CNS in a 3-D model of the BBB. Citation Format: Alondra Rodriguez, Cesar I. Cardona, Colin A. Bill, Charlotte M. Vines. Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2823.

CCR5/CXCR3 antagonist TAK-779 prevents diffuse alveolar damage of the lung in the murine model of the acute respiratory distress syndrome.

Introduction: The acute respiratory distress syndrome (ARDS), secondary to viral pneumonitis, is one of the main causes of high mortality in patients with COVID-19 (novel coronavirus disease 2019)-ongoing SARS-CoV-2 infection- reached more than 0.7 billion registered cases. Methods: Recently, we elaborated a non-surgical and reproducible method of the unilateral total diffuse alveolar damage (DAD) of the left lung in ICR mice-a publicly available imitation of the ARDS caused by SARS-CoV-2. Our data read that two C-C chemokine receptor 5 (CCR5) ligands, macrophage inflammatory proteins (MIPs) MIP-1α/CCL3 and MIP-1β/CCL4, are upregulated in this DAD model up to three orders of magnitude compared to the background level. Results: Here, we showed that a nonpeptide compound TAK-779, an antagonist of CCR5/CXCR3, readily prevents DAD in the lung with a single injection of 2.5mg/kg. Histological analysis revealed reduced peribronchial and perivascular mononuclear infiltration in the lung and mononuclear infiltration of the wall and lumen of the alveoli in the TAK-779-treated animals. Administration of TAK-779 decreased the 3-5-fold level of serum cytokines and chemokines in animals with DAD, including CCR5 ligands MIP-1α/β, MCP-1, and CCL5. Computed tomography revealed rapid recovery of the density and volume of the affected lung in TAK-779-treated animals. Discussion: Our pre-clinical data suggest that TAK-779 is more effective than the administration of dexamethasone or the anti-IL6R therapeutic antibody tocilizumab, which brings novel therapeutic modality to TAK-779 and other CCR5 inhibitors for the treatment of virus-induced hyperinflammation syndromes, including COVID-19.

Impact of cancer-associated mutations in CC chemokine receptor 2 on receptor function and antagonism

CC chemokine receptor 2 (CCR2), a G protein-coupled receptor, plays a role in many cancer-related processes such as metastasis formation and immunosuppression. Since ∼ 20 % of human cancers contain mutations in G protein-coupled receptors, ten cancer-associated CCR2 mutants obtained from the Genome Data Commons were investigated for their effect on receptor functionality and antagonist binding. Mutations were selected based on either their vicinity to CCR2′s orthosteric or allosteric binding sites or their presence in conserved amino acid motifs. One of the mutant receptors, namely S101P2.63 with a mutation near the orthosteric binding site, did not express on the cell surface. All other studied mutants showed a decrease in or a lack of G protein activation in response to the main endogenous CCR2 ligand CCL2, but no change in potency was observed. Furthermore, INCB3344 and LUF7482 were chosen as representative orthosteric and allosteric antagonists, respectively. No change in potency was observed in a functional assay, but mutations located at F1163.28 impacted orthosteric antagonist binding significantly, while allosteric antagonist binding was abolished for L134Q3.46 and D137N3.49 mutants. As CC chemokine receptor 2 is an attractive drug target in cancer, the negative effect of these mutations on receptor functionality and drugability should be considered in the drug discovery process.

CCR4 plays a pivotal role in Th17 cell recruitment and expansion in a mouse model of rheumatoid arthritis.

T helper 17 (Th17) cells express CC chemokine receptor 4 (CCR4) and secrete cytokines such as interleukin-17A (IL-17A) and granulocyte macrophage colony-stimulating factor (GM-CSF), while dendritic cells (DCs) produce CC chemokine ligand 22 (CCL22), a CCR4 ligand, upon stimulation with GM-CSF. Th17 cells are known to play a critical role in the pathogenesis of rheumatoid arthritis (RA). CCL22 has also been shown to be up-regulated in the synovial tissues of RA patients. Here, we investigated the role of CCR4 in collagen-induced arthritis (CIA), a mouse model of RA. DBA/1J mice efficiently developed CIA as shown by erythema, paw swelling, joint rigidity, and joint destruction. Th17 cells were increased in the arthritic joints and regional lymph nodes (LNs) of CIA mice. A fraction of Th17 cells were also shown to produce GM-CSF. On the other hand, we observed no significant increases of Th2 cells or Treg cells, the T cell subsets also known to express CCR4, in these tissues. We further observed clusters of CCR4-expressing memory Th17 cells and CCL22-producing DCs in the regional LNs of CIA mice, supporting the role of the CCR4-CCL22 axis in the expansion of Th17 cells in the regional LNs. Compound 22, a CCR4 inhibitor, ameliorated the disease severity with reduction of Th17 cells in the arthritic joints and regional LNs and Th17-DC clusters in the regional LNs. We further confirmed that CCR4-deficient mice in the C57BL/6J background were highly resistant to CIA induction compared with wild-type mice. Collectively, CCR4 contributes to the pathogenesis of CIA and may thus represent a new therapeutic target for RA.

Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR2.

Fluorescently labeled ligands are versatile molecular tools to study G protein-coupled receptors (GPCRs) and can be used for a range of different applications, including bioluminescence resonance energy transfer (BRET) assays. Here, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a drug target in oncology and inflammation. Starting from previously reported intracellular CCR2 antagonists, several tetramethylrhodamine (TAMRA)-labeled CCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CCR2. By means of these studies, we developed 14 as a fluorescent CCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a non-isotopic and high-throughput manner. Further, we show that 14 can be used as a tool for fragment-based screening approaches. Thus, our small-molecule-based fluorescent CCR2 ligand 14 represents a promising tool for future studies of CCR2 pharmacology.

Pharmacological Inhibition of CCR2 Signaling Exacerbates Exercise-Induced Inflammation Independently of Neutrophil Infiltration and Oxidative Stress

Although exercise-induced humoral factors known as exerkines benefit systemic health, the role of most exerkines has not been investigated. Monocyte chemoattractant protein-1 (MCP-1) is a representative chemokine whose circulating concentrations increase after exercise, and it is one of the exerkines. MCP-1 is a ligand for CC chemokine receptor 2 (CCR2), which is expressed on monocytes, macrophages, and muscle cells. However, there is no information on the role of CCR2 signaling in exercise. Therefore, to investigate the research question, we administrated CCR2 antagonist or PBS to mice to inhibit CCR2 signaling before and after exercise. Our results showed that CCR2 signaling inhibition promoted exercise-induced macrophage infiltration and inflammation 24 h after exercise in muscle. CCR2 signaling inhibition also exacerbated exercise-induced inflammation immediately after exercise in muscle. However, neutrophil infiltration and oxidative stress had no contribution to exercise-induced inflammation by CCR2 signaling inhibition. CCR2 signaling inhibition also exacerbated exercise-induced inflammation immediately after exercise in kidney, liver, and adipose tissues. To summarize, pharmacological inhibition of CCR2 signaling exacerbated exercise-induced inflammation independently of neutrophil infiltration and oxidative stress.

Recombinant CCL17-dependent CCR4 activation alleviates neuroinflammation and neuronal apoptosis through the PI3K/AKT/Foxo1 signaling pathway after ICH in mice

BackgroundIntracerebral hemorrhage (ICH), a devastating subtype of stroke, is associated with high mortality and morbidity. Neuroinflammation is an important factor leading to ICH-induced neurological injuries. C-C Chemokine Receptor 4 (CCR4) plays an important role in enhancing hematoma clearance after ICH. However, it is unclear whether CCR4 activation can ameliorate neuroinflammation and apoptosis of neurons following ICH. The aim of the present study was to examine the effects of recombinant CCL17 (rCCL17)-dependent CCR4 activation on neuroinflammation and neuronal apoptosis in an intrastriatal autologous blood injection ICH model, and to determine whether the PI3K/AKT/Foxo1 signaling pathway was involved.MethodsTwo hundred twenty-six adult (8-week-old) male CD1 mice were randomly assigned to sham and ICH surgery groups. An intrastriatal autologous blood injection ICH model was used. rCCL17, a CCR4 ligand, was delivered by intranasal administration at 1 h, 3 h, and 6 h post-ICH. CCL17 antibody was administrated by intraventricular injection at 1 h post-ICH. C021, a specific inhibitor of CCR4 and GDC0068, an AKT inhibitor were delivered intraperitoneally 1 h prior to ICH induction. Brain edema, neurobehavioral assessments, western blotting, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunofluorescence staining were conducted.ResultsEndogenous expression of CCL17 and CCR4 were increased following ICH, peaking at 5 days post-induction. CCR4 was found to co-localize with microglia, neurons, and astrocytes. rCCL17 treatment decreased brain water content, attenuated short- and long-term neurological deficits, deceased activation of microglia/macrophages and infiltration of neutrophils, and inhibited neuronal apoptosis in the perihematomal region post-ICH. Moreover, rCCL17 treatment post-ICH significantly increased the expression of CCR4, PI3K, phosphorylated AKT, and Bcl-2, while Foxo1, IL-1β, TNF-α, and Bax expression were decreased. The neuroprotective effects of rCCL17 were reversed with the administration of C021 or GDC0068.ConclusionsrCCL17-dependent CCR4 activation ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the PI3K/AKT/Foxo1 signaling pathway after ICH. Thus, activation of CCR4 may provide a promising therapeutic approach for the early management of ICH.

Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

Covalently acting inhibitors constitute a large and growing fraction of approved small-molecule therapeutics as well as useful tools for a variety of in vitro and in vivo applications. Here, we aimed to develop a covalent antagonist of CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a therapeutic target in inflammation and immuno-oncology. Based on a known intracellularly binding CCR2 antagonist, several covalent derivatives were synthesized and characterized by radioligand binding and functional assays. These studies revealed compound 14 as an intracellular covalent ligand for CCR2. In silico modeling followed by site-directed mutagenesis confirmed that 14 forms a covalent bond with one of three proximal cysteine residues, which can be engaged interchangeably. To our knowledge, compound 14 represents the first covalent ligand reported for CCR2. Due to its unique properties, it may represent a promising tool for ongoing and future studies of CCR2 pharmacology.

Chemokine Receptors CC Chemokine Receptor 5 and C-X-C Motif Chemokine Receptor 4 Are New Therapeutic Targets for Brain Recovery after Traumatic Brain Injury.

Recently, chemokine receptor CC chemokine receptor 5 (CCR5) was found to be a negative modulator of learning and memory. Its inhibition improved outcome after stroke and traumatic brain injury (TBI). To better understand its role after TBI and establish therapeutic strategies, we investigated the effect of reduced CCR5 signaling as a neuroprotective strategy and of the temporal changes of CCR5 expression after TBI in different brain cell types. To silence CCR5 expression, ccr5 short hairpin RNA (shRNA) or dsred shRNA (control) was injected into the cornu ammonis (CA) 1 and CA3 regions of the hippocampus 2 weeks before induction of closed-head injury in mice. Animals were then monitored for 32 days and euthanized at different time points to assess lesion area, inflammatory components of the glial response (immunohistochemistry; IHC), cytokine levels (enzyme-linked immunosorbent array), and extracellular signal-regulated kinase (ERK) phosphorylation (western blot). Fluorescence-activated cell sorting (FACS) analysis was performed to study post-injury temporal changes of CCR5 and C-X-C motif chemokine receptor 4 (CXCR4) expression in cortical and hippocampal cell populations (neurons, astrocytes, and microglia). Phosphorylation of the N-methyl-d-aspartate subunit 1 (NR1) subunit of N-methyl-d-aspartate (western blot) and cAMP-response-element-binding protein (CREB; IHC) were also assessed. The ccr5 shRNA mice displayed reduced lesion area, dynamic alterations in levels of inflammation-related CCR5 ligands and cytokines, and higher levels of phosphorylated ERK. The ccr5 shRNA also reduced astrocytosis in the lesioned and sublesioned cortex. FACS analysis revealed increased cortical CCR5 and CXCR4 expression in CD11b-positive cells, astrocytes, and neurons, which was most evident in cells expressing both receptors, at 3 and 11 days post-injury. The lowest levels of phosphorylated NR1 and phosphorylated CREB were found at day 3 post-injury, suggesting that this is the critical time point for therapeutic intervention.

Tumors establish resistance to immunotherapy by regulating Treg recruitment via CCR4

BackgroundCheckpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some...

CCR4 antagonist (C021) influences the level of nociceptive factors and enhances the analgesic potency of morphine in a rat model of neuropathic pain

Neuropathic pain is a chronic condition which significantly reduces the quality of life and serious clinical issue that is in general resistant to available therapies. Therefore looking for new analgesics is still critical issue. Recent, studies have indicated that chemokine signaling pathways are crucial for the development of neuropathy; however, the role of CC chemokine receptor 4 (CCR4) in this process has not yet been studied. Therefore, the aim of our research was to investigate the influence of C021 (a CCR4 antagonist) and CCR4 CC chemokine ligands 17 and 22 (CCL17 and CCL22) on the development of hypersensitivity and the effectiveness of morphine induced analgesia in naive animals and/or animals exposed to chronic constriction injury (CCI) of the sciatic nerve. Firstly, we demonstrated that the intrathecal administration of CCL17 and CCL22 induced pain-related behavior in naive mice. Secondly, we revealed that the intrathecal injection of C021 significantly reduced CCI-induced hypersensitivity after nerve injury. In parallel, C021 reduced microglia/macrophages activation and the level of some pronociceptive interleukins (IL-1beta; IL-18) in the spinal cord 8 days after CCI. Moreover, C021 not only attenuated mechanical and thermal hypersensitivity but also enhanced the analgesic properties of morphine. Our research indicates that CCR4 ligands might be important factors in the early stages of neuropathy, when we observe intense microglia/macrophages activation. Moreover, pharmacological blockade of CCR4 may serve as a potential new target for better understanding the mechanisms of neuropathic pain development.

CCR5 blockade inflames antitumor immunity in BAP1-mutant clear cell renal cell carcinoma

BackgroundPatients with BRCA1-associated protein 1 (BAP1)-mutant clear cell renal cell carcinoma (ccRCC) have worse prognosis. C-C chemokine receptor 5 (CCR5) plays an important role in ccRCC development and its expression...

P2861Elevated cc chemokine receptor 2 expression and higher migratory activity of monocytes in atrial fibrillation patients with progressive structural remodeling

Abstract Background Inflammation in atrial tissue underlies structural remodeling of left atrium, which is a hallmark of atrial fibrillation (AF). Activated monocytes mediate inflammation; however, the role of monocytes in AF pathogenesis has not been extensively examined. In this study, we thus investigated the association between structural remodeling of left atrium, represented by left atrial dimension (LAD), and characteristics of peripheral monocytes in patients with AF. Methods Blood samples were collected from patients undergone catheter ablation between July 2017 and October 2018, including AF patients (n=152) and paroxysmal supraventricular tachycardia (PSVT) patients, which serves as a control non-AF group (n=22). AF patients were further divided into two groups by the median of LAD (normal LAD group: LAD <40 mm, n=77, large LAD group: LAD ≥40 mm, n=75). Peripheral blood mononuclear cells (PBMC) were isolated to analyze monocyte subsets by flow cytometry. In a subset of patients, we further isolated monocytes from PBMC by using magnetic bead-based negative selection method then gene products associated with inflammation or monocyte functions were evaluated. We also examined migratory activity of monocytes toward monocyte chemotactic protein-1, a ligand for CC chemokine receptor 2 (CCR2), using a modified Boyden chamber method. Finally, we performed immunofluorescence staining of monocytes and macrophages in left atrial appendages resected from patients underwent coronary bypass graft surgery (CABG) complicated by AF. Results There were no differences in age, body mass index and high-sensitivity C-reactive protein levels among three groups, including non-AF, normal LAD and large LAD groups, except that more female subjects were included in non-AF group. We found that proportions of classical CD14++CD16- and nonclassical CD14+CD16++ monocytes were higher (non-AF: 71.2±7.3% vs. AF: 75.5±8.3%, p<0.05) and lower (non-AF: 16.4±5.9% vs. AF: 13.2±5.5%, p<0.05), respectively, in all AF patients compared with those in non-AF group, while no significant difference was observed between normal and large LAD groups. In monocytes from large LAD group, mRNA levels of CCR2, a receptor to mediate monocyte chemotaxis, were significantly higher compared to those in normal LAD group (Figure A, p<0.05). Furthermore, monocytes isolated from large LAD group exhibited higher migratory capacity compared to normal LAD group (Figure B, p<0.01). Finally, higher monocyte/macrophage infiltrations to left atrial appendages were implicated in patients with large LAD, shown by immunofluorescence staining. Conclusions Monocytes in AF patients with enlarged left atrium expressed higher CCR2 mRNA and were more active in chemotaxis to MCP-1, suggesting the proactive roles of activated monocytes in the pathogenesis of arterial remolding in AF.

Abstract 4849: CAP-100: first-in-class anti-CCR7 antibody for CLL

Abstract The homeostatic CC-chemokine receptor 7 (CCR7) is highly expressed in many hematological malignancies including chronic lymphocytic leukemia (CLL). CCR7 surface expression levels are much higher than those of CD20, and are correlated with poor prognosis. Upon engagement by its ligands (CCL19 and CCL21), CCR7 controls trafficking of CLL cells to locations where these chemokines are expressed, such as the lymph node (LN). In this protective microenvironment CCR7 ligands contribute to CLL cell survival and proliferation. Indeed, high CCR7-mediated in vitro migration of patient CLL cells correlates with LN involvement and adverse prognostic factors. Thus, strategies targeting CCR7 could provide an additional therapeutic approach for CLL. Therefore, we have generated CAP-100, the first humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand-mediated signaling from both CCL19 and CCL21. The antibody effectively inhibited CLL cells migration and survival. Furthermore, in in vivo pre-clinical studies, CAP-100 was shown to inhibit entry of CCR7-expressing cells to LNs. The antibody showed potent cell killing activity against CLL cells while preserving normal blood cells. This Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) clearly outperformed standard-of-care in CLL, such as rituximab. ADCC and migration inhibition were both independent of prognostic markers for high risk CLL, such as TP53 mutation including 17p deletion and relapse/refractoriness to various standard therapies, including BTK-inhibitors. Finally, when given as monotherapy in disseminated NHL xenograft tumors in SCID mice, CAP-100 exhibited tumor growth inhibition in established tumors and provided significant survival. Collectively, our results demonstrate that CAP-100, the first-in-class anti-CCR7 mAb, is a potent antagonist with biological activity in CLL primary cells, including those from relapsed/refractory patients. Moreover, these results highlight the relevance of the CCR7-CCL19/CCL21 pathway as a therapeutic target in CLL. CAP-100’s unique propensity to block migration of CLL cells to the LN, in combination with its potent cell killing activity provides the biological rationale for use of CAP-100 in CLL, either as monotherapy or in combination with novel agents such as BTK-inhibitors. Clinical trials in CLL and other CCR7-expressing NHL will be initiated soon. Citation Format: Carlos Cuesta-Mateos, Cecilia Muñoz-Calleja, Javier Loscertales, Fernando Terron, Wim Mol. CAP-100: first-in-class anti-CCR7 antibody for CLL [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 4849.

Placental chemokine compartmentalisation: A novel mammalian molecular control mechanism.

Atypical chemokine receptor 2 (ACKR2) is a chemokine-scavenging receptor. ACKR2–/–embryos display a reduction in size of a novel, to our knowledge, embryonic skin macrophage population referred to as ‘intermediate’ cells. CC chemokine receptor 2 (CCR2)–/–embryos display an identical phenotype, indicating that these cells require CCR2 to enable them to populate embryonic skin. Further analysis revealed that ACKR2–/–embryos have higher circulating concentrations of the CCR2 ligand, CC ligand 2 (CCL2); thus, ACKR2 regulates intraembryonic CCL2 levels. We show that ACKR2 is strongly expressed by trophoblasts and that it blocks movement of inflammatory chemokines, such as CCL2, from the maternal decidua into the embryonic circulation. We propose that trophoblastic ACKR2 is responsible for ensuring chemokine compartmentalisation on the maternal decidua, without which chemokines enter the embryonic circulation, disrupting gradients essential for directed intraembryonic cell migration. Overall, therefore, we describe a novel, to our knowledge, molecular mechanism whereby maternal decidual chemokines can function in a compartmentalised fashion without interfering with intraembryonic leukocyte migration. These data suggest similar functions for other atypical chemokine receptors in the placenta and indicate that defects in such receptors may have unanticipated developmental consequences.

Low CCL17 expression associates with unfavorable postoperative prognosis of patients with clear cell renal cell carcinoma

BackgroundChemokine (C–C motif) ligand 17 (CCL17) is a chemokine mainly produced by myeloid dendritic cells. It is a ligand for CC chemokine receptor 4 (CCR4) and CC chemokine receptor 8 (CCR8). The aim of this study was to investigate prognostic values of CCL17 expression in patients with clear cell renal cell carcinoma (ccRCC).MethodsThe study included 286 patients with ccRCC. CCL17 expression was analyzed by immunohistochemistry on tissue microarrays. Prognostic values of CCL17 expression and patients’ clinical outcomes were evaluated.ResultsKaplan-Meier method showed that low CCL17 expression was associated with worse patient overall survival (OS) and recurrence-free survival (RFS) (OS, P = 0.002; RFS, P = 0.007). Low CCL17 expression was an adverse independent risk factor for OS and RFS in multivariate analyses (OS, P = 0.006, P = 0.011 for bootstrap; RFS, P = 0.002, P = 0.025 for bootstrap). We constructed two nomograms incorporating parameters derived from multivariate analyses to predict patients’ OS and RFS (OS, c-index 0.799; RFS, c-index 0.787) and they performed better than existed integrated models.ConclusionLow CCL17 expression is a potential independent adverse prognostic biomarker for recurrence and survival of patients with ccRCC after nephrectomy. Established nomograms based on this information could help predict ccRCC patients’ OS and RFS.

The CCL2-CCR2 Axis in Lymph Node Metastasis From Oral Squamous Cell Carcinoma: An Immunohistochemical Study

Cytokine or chemokine networks involve lymphatic and distant metastasis of various malignancies, including oral squamous cell carcinoma (OSCC). Immunohistochemical analysis was used to investigate the contribution of the axis of the CC chemokine receptor-2 (CCR2) and the CC chemokine receptor-2 ligand (CCL2) to lymphatic metastasis, particularly the relation between primary OSCC and marginal sinus histiocytosis in regional lymph nodes. Thirteen metastasis-free cases, 15 metastatic cases at resection of primary tumor resection, and 13 postresection metastasis cases were examined. No patient was treated with radiotherapy or chemotherapy before neck dissection. Samples were fixed in formalin, embedded inparaffin, and subjected to immunohistochemical analysis using antibodies against CCL2, CCR2, podoplanin, and α-smooth muscle actin (α-SMA). Marginal sinus histiocytosis was frequently observed in metastatic cases. CCL2 was expressed in tumor-associated neutrophils (TANs) and moderately or poorly differentiated SCC was detected atprimary tumor sites. TANs expressing CCL2 flowed into the marginal sinus in the lymph nodes. CCR2-positive macrophages and mesenchymal cells infiltrated the tumor stroma and were seen within the carcinoma nests. They were predominantly present in the marginal sinus of metastatic cases. In small metastatic foci, α-SMA-positive spindle cells resembling carcinoma-associated fibroblasts (CAFs) were observed adjacent to the macrophages. The CCL2-CCR2 axis is associated with lymphatic metastasis. To clarify the mechanism of lymphatic metastasis from OSCC, further functional analyses of the CCL2-positive TANs, CCR2-positive macrophages, and CAF-like cells detected in this study are recommended.

CCR5-CCL Axis in PDL during Orthodontic Biophysical Force Application

Tooth movement by application of orthodontic biophysical force primarily reflects the role of soluble molecules released from the periodontal ligament (PDL). Thus far, many factors have been reported to be involved in orthodontic tooth movement (OTM), but key molecules that orchestrate responses of periodontal tissues to biophysical force are still enigmatic. In this in vivo study, in which the upper first molars in rats were moved, differential display–polymerase chain reaction revealed that CC chemokine receptor 5 (CCR5) level was differentially increased during OTM. Strong immunoreactivity for CCR5 was found in the PDL undergoing force application. Moreover, the in vitro compression or tension force application to primary cultured human PDL cells increased the expression of CCR5 and CCR5 ligands. In vitro tension force on human PDL cells did not induce RANKL, an osteoclastogenesis-inducing factor, but did induce the upregulation of IL12, an osteoclast inhibitory factor, and osteoblast differentiation factors, including Runx2, which was attenuated under tension by CCR5 gene silencing whereas augmented with CCR5 ligands. In contrast, in vitro compression force did not induce the expression of osteoprotegerin, a decoy receptor for RANKL and Runx2, but did induce the upregulation of RANKL, which was attenuated under compression by CCR5 gene silencing. These results suggest that the CCR5–CCR5 ligands axis in PDL cells may play a crucial role in the remodeling of periodontal tissues and can be a therapeutic target for achieving efficient OTM.

A gain-of-function CCR4 mutations in adult T-cell leukemia/lymphoma (ATL) enhance the chemotactic abilities and P13K/AKT activation

High expression of the CC chemokine receptor 4 (CCR4) has been identified as a hallmark gene in ATL-an aggressive peripheral T-cell neoplasm. CCR4 is a chemokine receptor, which has a critical role in immune cell trafficking including Th2, T-regs and skin homing memory T-cells. CCR4 ligands, CCL17 and CCL22 are produced in lymph nodes and skin from dendritic cells, macrophages and Langerhans cells. Most ATL cases express surface CCR4 (90%) and infiltrate lymph nodes and skin. We performed RNA-Seq on two primary ATL cases and discovered recurrent nonsense mutations in CCR4. Through an extended analysis using Sanger sequencing, CCR4 mutations were detected in 14/53 ATL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxyl-terminus. We hypothesized that the mutant CCR4 isoforms might enhance chemotaxis of the affected cells to CCR4 ligands. A chemotaxis assay using 32D beta, a mouse myeloid cell line and ED40515 (+), an ATL cell line, clarified that the ectopic expression of CCR4-Q330* but not CCR4-WT transduced cells enhanced the chemotactic ability of the transduced cells toward CCL17 and CCL22. To define the mechanism of the enhanced chemotactic ability, we examined the change in surface CCR4 levels after CCL22 exposure in CCR4-WT-and CCR4-Q330*-reconstituted ED40515 (+) cells. Compared with CCR4-WT, CCR4 internalization in CCR4-Q330*-reconstituted cells was significantly impaired. Thus, the ATL CCR4 mutants manifested impaired desensitization by ligand, which likely contributed to the enhanced chemotaxis of cells bearing these mutants. We explored the influence of the ATL CCR4 mutants on PI(3) kinase (PI3K)-dependent activation of AKT since it has been reported that binding of CCL22 to CCR4 activates AKT in CEM leukemic T-cells and in human Th2 cells. CCR4-Q330* reconstituted ED40414 (+) cells showed strong activation of AKT with CCL22 ligation compared with CCR4-WT reconstituted cells. The AKT activation was inhibited by the pan-P13K inhibitor, BKM120, indicating that the CCR4-mediated AKT activation was PI3K dependent. Lastly, we tested whether the acquisition of CCR4 mutations by ATL cells imparts a selective growth advantage relative to cells with wild-type CCR4. CCR4-Q330*-reconstituted cells had a selective growth advantage in the presence of CCL22, supporting, at least in part, the hypothesis that CCR4 mutations provided the affected cells with a positive selection pressure through CCL22 ligation that contributed to the ATL pathogenesis. Our findings provide a rationale for a clinical trial to test whether inhibition of CCR4 signaling might have a therapeutic potential for patients with ATL.

Abstract 3936: Frequent gain-of-function CCR4 mutations in adult T-cell leukemia/lymphoma (ATLL)

Abstract High expression of CC chemokine receptor 4 (CCR4) has been identified as a hallmark gene in ATLL, an aggressive peripheral T-cell neoplasm. CCR4 is a chemokine receptor, which has a critical role in immune cell trafficking. CCR4 ligands, CCL17 and CCL22, were produced in lymph nodes and skin from dendritic cells, macrophages and Langerhans cells. Most ATLL cases express surface CCR4 (90%) and infiltrate to lymph nodes and skin. These observations suggest that CCR4 could have a role in ATLL biology, but it is still unclear whether dysregulation of CCR4 function contributes to ATLL pathogenesis. We performed RNA-Seq for two primary ATLL cases and discovered recurrent non-sense mutations in CCR4. Though an extended analysis using Sanger sequencing, CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. In 5 cases for which paired normal DNA was available, three different CCR4 mutations were detected only in the ATLL cells (Q330*; Q330 frameshift; Y331*), demonstrating that they were acquired somatically during malignant transformation or progression. Chemotaxic assay using 32Dβ, a mouse myeloid cell line, and ED40515(+), an ATLL cell line, clarified that the ectopic expression of CCR4-Q330* enhanced the chemotactic ability of the transduced cells toward CCL17 and CCL22 rather than CCR4-WT transduced cells. To understand the mechanism of this enhanced chemotactic ability, we studied the change in surface CCR4 levels after CCL22 exposure in CCR4-WT-and CCR4-Q330*-reconstituted ED40515(+) cells. Compared with CCR4-WT, CCR4 internalization in CCR4-Q330*-reconstituted cells was significantly impaired. Thus, the ATLL CCR4 mutants impair desensitization by ligand, which likely contributes to the enhanced chemotaxis of cells bearing these mutants. We explored the influence of the ATLL CCR4 mutants on PI3K/AKT signaling by immunoblot analysis and phosphor-flow analysis. CCR4-Q330*-reconstituted ED40515(+) showed strong activation of AKT with CCL22 ligation compared with CCR4-WT-reconstituted cell. Furthermore, we tested whether the acquisition of CCR4 mutations by ATLL cells imparts a selective growth advantage relative to cells with wild type CCR4. CCR4-Q330*-reconstituted cells had a selective growth advantage in the presence of CCL22, supporting at least in part the hypothesis that CCR4 mutation are able to provide the affected cells a positive selection pressure through CCL22 ligation and contributes to ATLL pathogenesis. Our findings implicate somatic gain-of-function CCR4 mutations in the pathogenesis of ATLL and suggest that inhibition of CCR4 signaling might have therapeutic potential in this refractory malignancy. Citation Format: Masao Nakagawa, Roland Schmitz, Wenming Xiao, Carolyn K. Goldman, Weihong Xu, Yandan Yang, Xin Yu, Thomas A. Waldmann, Louis M. Staudt. Frequent gain-of-function CCR4 mutations in adult T-cell leukemia/lymphoma (ATLL). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3936. doi:10.1158/1538-7445.AM2015-3936