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1.
Nat Commun ; 14(1): 7940, 2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-38040762

RESUMEN

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.


Asunto(s)
Quimiocinas CC , Receptores de Quimiocina , Humanos , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacología , Receptores CCR8/genética , Ligandos , Quimiocina CCL1/metabolismo , Receptores de Quimiocina/genética , Anticuerpos
2.
J Transl Med ; 21(1): 803, 2023 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-37950246

RESUMEN

BACKGROUND: Tregs are key drivers of immunosuppression in solid tumors. As an important chemokine receptor on Tregs, the regulatory effect of CCR8 on tumor immunity has received more and more attention. However, the current research on CCR8 in the immune microenvironment of ovarian cancer has not been clear. METHODS: Bioinformatics analysis was used to compare the transcriptome differences between CD4+ T cells in the peripheral circulation and infiltrated in ovarian tumor tissues. RT-PCR was used to detect the expression levels of chemokine receptor-related differential genes on CD4+ T cells in peripheral blood and ovarian tumor tissues. Multiparameter flow cytometry was used to detect the proportion and phenotypic characteristics of CD4+CCR8+ Tregs and CD4+CCR8- Tregs in different sample types. The expression level of CCR8 ligands was detected at multiple levels. To explore the important role of CCR8-CCL1 and CCR8-CCL18 axis in the migration and invasion of CD4+CCR8+ Tregs into ovarian tumor tissues by establishing a chemotaxis system in vitro. RESULTS: In this study, significantly different gene expression profiles were found between peripheral circulating CD4+ T cells and infiltrating CD4+ T cells in ovarian tumor tissues, in which chemokine-chemokine receptor signaling pathway was significantly enriched in all three groups of differential genes. The expression level of CCR8 in infiltrating CD4+ T cells of ovarian cancer tissue was significantly higher than that in peripheral blood of healthy controls and ovarian cancer patients, and high expression of CCR8 was significantly correlated with advanced tumor stage and poor differentiation. CD4+CCR8+ Tregs are the main type of infiltrating CD4+ Tregs in ovarian tumor tissues, which have stronger immunosuppressive phenotypes, secrete more inhibitory cytokines and have stronger proliferation ability. The ligands CCL1 and CCL18 corresponding to CCR8 were significantly overexpressed in ovarian tumor tissues, and the CCR8-CCL1 and CCR8-CCL18 axis played a key role in the migration and infiltration of CD4+CCR8+ Tregs into ovarian tumor tissues. CONCLUSIONS: The results of this study may help to understand the phenotypic characteristics and recruitment process of Tregs in the tumor, and provide new ideas for improving the immunosuppressive status of the ovarian cancer microenvironment.


Asunto(s)
Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Quimiotaxis , Linfocitos T , Terapia de Inmunosupresión , Receptores de Quimiocina/metabolismo , Linfocitos T Reguladores , Microambiente Tumoral , Receptores CCR8/genética , Receptores CCR8/metabolismo
3.
Proc Natl Acad Sci U S A ; 119(7)2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35140181

RESUMEN

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.


Asunto(s)
Memoria Inmunológica , Neoplasias/metabolismo , Receptores CCR8/metabolismo , Animales , Anticuerpos Monoclonales , Biomarcadores de Tumor , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Receptores CCR8/genética , Linfocitos T Reguladores
4.
Nat Commun ; 12(1): 5314, 2021 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-34493727

RESUMEN

Adoptive T cell therapy (ACT) requires lymphodepletion preconditioning to eliminate immune-suppressive elements and enable efficient engraftment of adoptively transferred tumor-reactive T cells. As anti-CD4 monoclonal antibody depletes CD4+ immune-suppressive cells, the combination of anti-CD4 treatment and ACT has synergistic potential in cancer therapy. Here, we demonstrate a post-ACT conditioning regimen that involves transient anti-CD4 treatment (CD4post). Using murine melanoma, the combined effect of cyclophosphamide preconditioning (CTXpre), CD4post, and ex vivo primed tumor-reactive CD8+ T-cell infusion is presented. CTXpre/CD4post increases tumor suppression and host survival by accelerating the proliferation and differentiation of ex vivo primed CD8+ T cells and endogenous CD8+ T cells. Endogenous CD8+ T cells enhance effector profile and tumor-reactivity, indicating skewing of the TCR repertoire. Notably, enrichment of polyfunctional IL-18Rαhi CD8+ T cell subset is the key event in CTXpre/CD4post-induced tumor suppression. Mechanistically, the anti-tumor effect of IL-18Rαhi subset is mediated by IL-18 signaling and TCR-MHC I interaction. This study highlights the clinical relevance of CD4post in ACT and provides insights regarding the immunological nature of anti-CD4 treatment, which enhances anti-tumor response of CD8+ T cells.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos Alquilantes/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Ciclofosfamida/farmacología , Subunidad alfa del Receptor de Interleucina-18/genética , Melanoma Experimental/terapia , Neoplasias Cutáneas/terapia , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunoterapia Adoptiva/métodos , Interleucina-18/genética , Interleucina-18/inmunología , Subunidad alfa del Receptor de Interleucina-18/agonistas , Subunidad alfa del Receptor de Interleucina-18/inmunología , Activación de Linfocitos , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/mortalidad , Ratones , Ratones Endogámicos C57BL , Receptores CCR4/genética , Receptores CCR4/inmunología , Receptores CCR8/genética , Receptores CCR8/inmunología , Receptores Histamínicos H4/genética , Receptores Histamínicos H4/inmunología , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/mortalidad , Análisis de Supervivencia , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/trasplante , Carga Tumoral/efectos de los fármacos
5.
Immunology ; 163(4): 512-520, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33838058

RESUMEN

CD4+ regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8-/- mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which were abolished in Ccr8-/- mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8+ Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.


Asunto(s)
Adenocarcinoma/inmunología , Neoplasias Colorrectales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Receptores CCR8/metabolismo , Neoplasias Cutáneas/inmunología , Linfocitos T Reguladores/inmunología , Animales , Factores de Transcripción Forkhead/metabolismo , Humanos , Tolerancia Inmunológica , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR8/genética
6.
J Immunother Cancer ; 9(2)2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33589525

RESUMEN

BACKGROUND: Modulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker. METHODS: We employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models. RESULTS: We were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs. CONCLUSIONS: Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.


Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Carcinoma Pulmonar de Lewis/terapia , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Depleción Linfocítica , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores CCR8/deficiencia , Neoplasias Cutáneas/terapia , Linfocitos T Reguladores/inmunología , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/metabolismo , Terapia Combinada , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Fenotipo , Receptor de Muerte Celular Programada 1/metabolismo , RNA-Seq , Receptores CCR8/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Linfocitos T Reguladores/metabolismo
7.
Fish Shellfish Immunol ; 106: 628-639, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32853761

RESUMEN

Chemokine receptors are a superfamily of seven transmembrane domain G-coupled receptors, and they play important roles in immune surveillance, inflammation, and development. Recently, nine CC chemokine receptors (CCRs) were identified and cloned from orange-spotted grouper (Epinephelus coioides) and annotated by phylogenetic and syntenic analyses. We detected mRNA transcripts for CCRs in healthy tissues of E. coioides, and CCR genes were highly expressed in the immune-relevant tissues. Analysis of gene expression after Singapore grouper iridovirus (SGIV) infection indicated that CCR genes are regulated in a gene-specific manner. CCR8a and CCR8b were significantly upregulated in the spleen and liver of resistant fish, indicating potential roles in immunity against the pathogen. Fluorescence microscopy revealed that CCR8a and CCR8b were expressed predominantly in the cytoplasm. Overexpression of CCR8a and CCR8b in grouper cells significantly inhibited the replication of SGIV, demonstrating that they delayed the occurrence of cytopathic effects induced by SGIV infection and inhibited viral gene transcription. CCR8a and CCR8b overexpression also significantly increased the expression of interferon (IFN)-related cytokines and activated IFN response element and IFN promoter activities. These results demonstrated that CCR8a and CCR8b might have an antiviral function against SGIV infect.


Asunto(s)
Proteínas de Peces/inmunología , Perciformes/inmunología , Receptores CCR8/inmunología , Animales , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Iridovirus , Perciformes/genética , Receptores CCR8/genética
8.
Mol Carcinog ; 59(8): 897-907, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32319143

RESUMEN

Lactate dehydrogenase isozyme (LDH) is a tetramer constituted of two isoforms, LDHA and LDHB, the expression of which is associated with cell metabolism and cancer progression. Our previous study reveals that CC-chemokine ligand-18 (CCL18) is involved in progression of prostate cancer (PCa).This study aims to investigate how CCL18 regulates LDH isoform expression, and therefore, contributes to PCa progression. The data revealed that the expression of LDHA was upregulated and LDHB was downregulated in PCa cells by CCL18 at both messenger RNA and protein levels. The depletion of CCR8 reduced the ability of CCL18 to promote the proliferation, migration, and lactate production of PCa cells. Depletion of a CCR8 regulated transcription factor, ARNT, significantly reduced the expression of LDHA. In addition, The Cancer Genome Atlas dataset analyses revealed a positive correlation between CCR8 and ARNT expression. Two dimension difference gel electrophoresis revealed that the LDHA/LDHB ratio was increased in the prostatic fluid of patients with PCa and PCa tissues. Furthermore, increased LDHA/LDHB ratio was associated with poor clinical outcomes of patients with PCa. Together, our results indicate that the CCR8 pathway programs LDH isoform expression in an ARNT dependent manner and that the ratio of LDHA/LDHB has the potential to serve as biomarkers for PCa diagnosis and prognosis.


Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Biomarcadores de Tumor/metabolismo , Quimiocinas CC/metabolismo , Regulación Neoplásica de la Expresión Génica , L-Lactato Deshidrogenasa/metabolismo , Neoplasias de la Próstata/patología , Receptores CCR8/metabolismo , Apoptosis , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Biomarcadores de Tumor/genética , Proliferación Celular , Quimiocinas CC/genética , Humanos , Isoenzimas , L-Lactato Deshidrogenasa/genética , Masculino , Pronóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores CCR8/genética , Tasa de Supervivencia , Células Tumorales Cultivadas
9.
J Exp Med ; 216(12): 2763-2777, 2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31537642

RESUMEN

Group 2 innate lymphoid cells (ILC2s) possess indispensable roles during type 2-mediated inflammatory diseases. Although their physiological and detrimental immune functions seem to depend on the anatomical compartment they reside, their tissue tropism and the molecular and immunological processes regulating the self-renewal of the local pool of ILC2s in the context of inflammation or infection are incompletely understood. Here, we analyzed the role of the CC-chemokine receptor CCR8 for the biological functions of ILC2s. In vitro and in vivo experiments indicated that CCR8 is in comparison to the related molecule CCR4 less important for migration of these cells. However, we found that activated mouse and human ILC2s produce the CCR8 ligand CCL1 and are a major source of CCL1 in vivo. CCL1 signaling to ILC2s regulates their proliferation and supports their capacity to protect against helminthic infections. In summary, we identify a novel chemokine receptor-dependent mechanism by which ILC2s are regulated during type 2 responses.


Asunto(s)
Quimiocina CCL1/metabolismo , Inmunidad Innata , Inflamación/etiología , Inflamación/metabolismo , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Receptores CCR8/metabolismo , Animales , Comunicación Autocrina , Biomarcadores , Movimiento Celular/genética , Movimiento Celular/inmunología , Citocinas/metabolismo , Expresión Génica , Helmintos/inmunología , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunología , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Ratones , Ratones Noqueados , Receptores CCR8/genética
10.
PLoS One ; 14(7): e0218944, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31314754

RESUMEN

Embryoid bodies (EBs) are three dimensional aggregates of pluripotent stem cells primarily used to investigate morphogenesis and cell toxicity, are also attractive tools in regenerative medicine. While embryonic stem cells (ESCs) and induced pluripotent cells (IPSCs) have been shown to form EBs in mouse, primate and humans, EB formation have not been previously demonstrated in mesenchymal stem cells (MSCs). Here we show that rat MSCs form EBs; which express regulatory T cell (Treg) marker Foxp3 and CC chemokine CCL1 receptor CCR8. We show a novel method for formation of EBs from MSCs under stress and demonstrate that the induction of FoxP3+ CCR8+ EBs is dependent upon CCL1 gradients which mediate cell proliferation, migration and invasion of mTregs. The identification of EBs and novel FoxP3+ CCR8+ regulatory T cells (mTregs) for selective conversion and isolation of bone marrow derived MSCs offers novel avenues for research, diagnosis and treatment.


Asunto(s)
Quimiocina CCL1/genética , Cuerpos Embrioides/metabolismo , Factores de Transcripción Forkhead/genética , Receptores CCR8/genética , Animales , Quimiotaxis/genética , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratas , Linfocitos T Reguladores/metabolismo , Células Th2
11.
Sci Rep ; 9(1): 9608, 2019 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-31270368

RESUMEN

Allergic enteritis (AE) is a gastrointestinal form of food allergy. This study aimed to elucidate cellular and molecular mechanisms of AE using a murine model. To induce AE, BALB/c wild type (WT) mice received intraperitoneal sensitization with ovalbumin (an egg white allergen) plus ALUM and feeding an egg white (EW) diet. Microarray analysis showed enhanced gene expression of CC chemokine receptor (CCR) 8 and its ligand, chemokine CC motif ligand (CCL) 1 in the inflamed jejunum. Histological and FACS analysis showed that CCR8 knock out (KO) mice exhibited slightly less inflammatory features, reduced eosinophil accumulation but accelerated neutrophil accumulation in the jejunums, when compared to WT mice. The concentrations of an eosinophil chemoattractant CCL11 (eotaxin-1), but not of IL-5, were reduced in intestinal homogenates of CCR8KO mice, suggesting an indirect involvement of CCR8 in eosinophil accumulation in AE sites by inducing CCL11 expression. The potential of CCR8 antagonists to treat allergic asthma has been discussed. However, our results suggest that CCR8 blockade may promote neutrophil accumulation in the inflamed intestinal tissues, and not be a suitable therapeutic target for AE, despite the potential to reduce eosinophil accumulation. This study advances our knowledge to establish effective anti-inflammatory strategies in AE treatment.


Asunto(s)
Enteritis/etiología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Hipersensibilidad/complicaciones , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptores CCR8/genética , Animales , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Enteritis/metabolismo , Enteritis/patología , Femenino , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , Receptores CCR8/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo
12.
Mol Med Rep ; 19(3): 1678-1686, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30592282

RESUMEN

Increased expression of CCL18 has been observed in various malignancies and in the urine samples of patients with bladder cancer (BC). However, the roles of CCL18 in the development, progression and metastasis of BC remain unclear. The present study demonstrated that CCL18 expression was significantly associated with advanced clinical stages of BC. Furthermore, exogenous CCL18 promoted cell invasion and migration, and induced cell epithelial­mesenchymal transition (EMT) in BC cells. Western blotting demonstrated that E­cadherin, an epithelial marker, was decreased, whereas matrix metalloproteinase (MMP)­2 and vascular endothelial growth factor (VEGF)­C were increased in CCL18­treated cells. Blocking CCR8 via a small molecule inhibitor or short hairpin (sh)RNA mitigated the decrease in E­cadherin, and increase in MMP­2 and VEGF­C, caused by human recombinant (r)CCL18. CCR8 knockdown by shRNA reversed rCCL18­induced cancer cell invasion, migration and EMT. In conclusion, these data suggested that CCL18 may promote migration, invasion and EMT by binding CCR8 in BC cells. Inhibition of CCL18 activity by blocking CCR8 could be a potential therapeutic strategy for preventing the progression of BC.


Asunto(s)
Quimiocinas CC/genética , Receptores CCR8/genética , Neoplasias de la Vejiga Urinaria/genética , Factor C de Crecimiento Endotelial Vascular/genética , Cadherinas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Quimiocinas CC/antagonistas & inhibidores , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasa 2 de la Matriz/genética , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Unión Proteica , ARN Interferente Pequeño/farmacología , Receptores CCR8/antagonistas & inhibidores , Transducción de Señal/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias de la Vejiga Urinaria/patología
13.
Immunity ; 49(3): 449-463.e6, 2018 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-30170811

RESUMEN

The migration of mature dendritic cells (DCs) into the draining lymph node (dLN) is thought to depend solely on the chemokine receptor CCR7. CD301b+ DCs migrate into the dLN after cutaneous allergen exposure and are required for T helper 2 (Th2) differentiation. We found that CD301b+ DCs poorly upregulated CCR7 expression after allergen exposure and required a second chemokine signal, mediated by CCR8 on CD301b+ DCs and its ligand CCL8, to exit the subcapsular sinus (SCS) and enter the lymph node (LN) parenchyma. After allergen exposure, CD169+SIGN-R1+ macrophages in interfollicular regions produced CCL8, which synergized with CCL21 in a Src-kinase-dependent manner to promote CD301b+ DC migration. In CCR8-deficient mice, CD301b+ DCs remained in the SCS and were unable to enter the LN parenchyma, resulting in defective Th2 differentiation. We have defined a CCR8-dependent stepwise mechanism of DC-subset-specific migration through which LN CD169+SIGN-R1+ macrophages control the polarization of the adaptive immune response.


Asunto(s)
Células Dendríticas/fisiología , Hipersensibilidad/inmunología , Ganglios Linfáticos/inmunología , Receptores CCR7/metabolismo , Receptores CCR8/metabolismo , Animales , Antígenos CD/metabolismo , Movimiento Celular , Células Cultivadas , Quimiocina CCL8/metabolismo , Modelos Animales de Enfermedad , Femenino , Cadenas alfa de Integrinas/metabolismo , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR8/genética
14.
PLoS One ; 13(7): e0200765, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30024927

RESUMEN

Following positive selection, thymocytes migrate into the medulla where they encounter diverse self-antigens that induce central tolerance. Thymocytes expressing T cell receptors (TCRs) with high affinity for self-antigens displayed by medullary antigen presenting cells (APCs) undergo either negative selection or diversion to the regulatory T cell (Treg) lineage, thus ensuring maturation of non-autoreactive T cells. Because many self-antigens are expressed by only a small percentage of medullary thymic epithelial cells, thymocytes must enter the medulla and efficiently scan APCs therein to encounter the full array of self-antigens that induce central tolerance. Chemokine receptors play a critical role in promoting medullary entry and rapid motility of post-positive selection thymocytes. We found that the chemokine receptor CCR8 is expressed by post-positive selection CD4+ single positive (SP) thymocytes in mice, while the corresponding chemokine ligands are expressed by medullary APCs, and thus hypothesized that CCR8 would promote thymocyte medullary entry and/or rapid motility to induce negative selection. However, despite a subtle decline in thymocyte medullary accumulation and the presence of autoantibodies in aged CCR8-deficient mice, CCR8 was not required for thymocyte differentiation, rapid motility, or negative selection.


Asunto(s)
Antígenos CD4/metabolismo , Receptores CCR8/metabolismo , Timocitos/metabolismo , Animales , Células Presentadoras de Antígenos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores CCR8/genética
15.
J Biol Chem ; 291(31): 16208-20, 2016 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-27226537

RESUMEN

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.


Asunto(s)
Quimiocina CCL1/química , Quimiocinas CC/química , Disulfuros/química , Inositol 1,4,5-Trifosfato/química , Receptores CCR8/química , Proteínas Virales/química , Animales , Células COS , Quimiocina CCL1/metabolismo , Quimiocinas CC/metabolismo , Chlorocebus aethiops , Disulfuros/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Receptores CCR8/genética , Receptores CCR8/metabolismo , Proteínas Virales/metabolismo
16.
J Immunol ; 195(1): 96-104, 2015 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-26002980

RESUMEN

The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. The expression of CCR8 during naive T cell activation is controlled by skin-specific factors derived from epidermal keratinocytes and not by resident dendritic cells. In this study, we show that the CCR8-inducing factors are heat stable and protease resistant and include the vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 and PGE2. The effect of either metabolite alone on CCR8 expression was weak, whereas their combination resulted in robust CCR8 expression. Elevation of intracellular cAMP was essential because PGE2 could be substituted with the adenylyl cyclase agonist forskolin, and CCR8 expression was sensitive to protein kinase A inhibition. For effective induction, exposure of naive T cells to these epidermal factors needed to occur either prior to or during T cell activation even though CCR8 was only detected 4-5 d later in proliferating T cells. The importance of tissue environments in maintaining cellular immune surveillance networks within distinct healthy tissues provides a paradigm shift in adaptive immunity. Epidermal-derived vitamin D3 metabolites and PGs provide an essential cue for the localization of CCR8(+) immune surveillance T cells within healthy human skin.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Calcitriol/metabolismo , Dinoprostona/metabolismo , Epidermis/inmunología , Queratinocitos/inmunología , Inmunidad Adaptativa , Adenilil Ciclasas/genética , Adenilil Ciclasas/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Calcitriol/farmacología , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Dinoprostona/farmacología , Células Epidérmicas , Epidermis/efectos de los fármacos , Femenino , Regulación de la Expresión Génica , Calor , Humanos , Vigilancia Inmunológica , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica , Receptores CCR8/genética , Receptores CCR8/inmunología , Transducción de Señal
17.
J Am Soc Nephrol ; 26(9): 2105-17, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25762060

RESUMEN

ANCA-associated vasculitis is the most frequent cause of crescentic GN. To define new molecular and/or cellular biomarkers of this disease in the kidney, we performed microarray analyses of renal biopsy samples from patients with ANCA-associated crescentic GN. Expression profiles were correlated with clinical data in a prospective study of patients with renal ANCA disease. CC chemokine ligand 18 (CCL18), acting through CC chemokine receptor 8 (CCR8) on mononuclear cells, was identified as the most upregulated chemotactic cytokine in patients with newly diagnosed ANCA-associated crescentic GN. Macrophages and myeloid dendritic cells in the kidney were detected as CCL18-producing cells. The density of CCL18(+) cells correlated with crescent formation, interstitial inflammation, and impairment of renal function. CCL18 protein levels were higher in sera of patients with renal ANCA disease compared with those in sera of patients with other forms of crescentic GN. CCL18 serum levels were higher in patients who suffered from ANCA-associated renal relapses compared with those in patients who remained in remission. Using a murine model of crescentic GN, we explored the effects of the CCL18 murine functional analog CCL8 and its receptor CCR8 on kidney function and morphology. Compared with wild-type mice, Ccr8(-/-) mice had significantly less infiltration of pathogenic mononuclear phagocytes. Furthermore, Ccr8(-/-) mice maintained renal function better and had reduced renal tissue injury. In summary, our data indicate that CCL18 drives renal inflammation through CCR8-expressing cells and could serve as a biomarker for disease activity and renal relapse in ANCA-associated crescentic GN.


Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/complicaciones , Quimiocinas CC/sangre , Glomerulonefritis/etiología , Glomerulonefritis/metabolismo , Anciano , Animales , Biomarcadores/sangre , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Quimiocinas CC/análisis , Células Dendríticas/química , Femenino , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Humanos , Macrófagos/química , Masculino , Ratones , Persona de Mediana Edad , Estudios Prospectivos , Análisis por Matrices de Proteínas , Receptores CCR8/genética , Receptores CCR8/metabolismo , Regulación hacia Arriba
18.
Int Immunol ; 27(4): 169-81, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25344933

RESUMEN

Allergic contact dermatitis (ACD) is a typical occupational disease in industrialized countries. Although various cytokines and chemokines are suggested to be involved in the pathogenesis of ACD, the roles of these molecules remain to be elucidated. CC chemokine receptor 8 (CCR8) is one such molecule, of which expression is up-regulated in inflammatory sites of ACD patients. In this study, we found that Ccr8(-/-) mice developed severer contact hypersensitivity (CHS) responses to 2,4-dinitrofluorobenzene, a murine model of ACD, compared with wild-type mice. T cells from Ccr8(-/-) mice showed enhanced proliferative recall responses and Th1 and Th17 cell populations were expanded in these mice. However, CHS responses were similar between SCID mice adoptively transferred with Ccr8(-/-) and wild-type T cells, suggesting that CCR8 in T cells is not responsible for the exacerbation of CHS. Notably, skin-resident dendritic cells (DCs), such as Langerhans cells and dermal DCs, and inflammatory DCs were highly accumulated in lymph nodes (LNs) of Ccr8(-/-) mice after sensitization. Consistent with this, Ccr8(-/-) antigen-presenting cells readily migrated from the skin to the draining LNs after sensitization. These observations suggest that CCR8 negatively regulates migration of cutaneous DCs from the skin to the draining LNs in CHS by keeping these cells in the skin.


Asunto(s)
Movimiento Celular/inmunología , Dermatitis por Contacto/inmunología , Células de Langerhans/inmunología , Ganglios Linfáticos/citología , Receptores CCR8/inmunología , Traslado Adoptivo , Animales , Proliferación Celular , Dermatitis por Contacto/genética , Dinitrofluorobenceno , Inflamación/inmunología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Receptores CCR8/biosíntesis , Receptores CCR8/genética , Linfocitos T/inmunología , Linfocitos T/trasplante , Células TH1/inmunología , Células Th17/inmunología
19.
Appl Immunohistochem Mol Morphol ; 23(8): 580-9, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25390351

RESUMEN

Anaplastic large cell lymphoma (ALCL) is one of the most common T-cell non-Hodgkin lymphomas and has 2 main subtypes: an anaplastic lymphoma kinase (ALK)-positive subtype characterized by ALK gene rearrangements and an ALK-negative subtype that is poorly understood. We recently identified recurrent rearrangements of the DUSP22 locus on 6p25.3 in both primary cutaneous and systemic ALK-negative ALCLs. This study aimed to determine the relationship between these rearrangements and expression of the chemokine receptor gene, CCR8. CCR8 has skin-homing properties and has been suggested to play a role in limiting extracutaneous spread of primary cutaneous ALCLs. However, overexpression of CCR8 has also been reported in systemic ALK-negative ALCLs. As available antibodies for CCR8 have shown lack of specificity, we examined CCR8 expression using quantitative real-time PCR in frozen tissue and RNA in situ hybridization (ISH) in paraffin tissue. Both approaches showed higher CCR8 expression in ALCLs with DUSP22 rearrangements than in nonrearranged cases (PCR: 19.5-fold increase, P=0.01; ISH: 3.3-fold increase, P=0.0008). CCR8 expression was not associated with cutaneous presentation, cutaneous biopsy site, or cutaneous involvement during the disease course. These findings suggest that CCR8 expression in ALCL is more closely related to the presence of DUSP22 rearrangements than to cutaneous involvement and that the function of CCR8 may extend beyond its skin-homing properties in this disease. This study also underscores the utility of RNA-ISH as a paraffin-based method for investigating gene expression when reliable antibodies for immunohistochemical analysis are not available.


Asunto(s)
Fosfatasas de Especificidad Dual/genética , Linfoma Anaplásico de Células Grandes/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Receptores CCR8/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto Joven
20.
PLoS One ; 9(4): e94445, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24714157

RESUMEN

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8-/-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.


Asunto(s)
Citocinas/biosíntesis , Macrófagos Peritoneales/metabolismo , Receptores CCR8/metabolismo , Animales , Quimiocina CCL1/antagonistas & inhibidores , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Colitis/genética , Colitis/inmunología , Colitis/metabolismo , Modelos Animales de Enfermedad , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Espacio Intracelular/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Noqueados , Transporte de Proteínas , Receptores CCR8/antagonistas & inhibidores , Receptores CCR8/deficiencia , Receptores CCR8/genética , Transducción de Señal/efectos de los fármacos , Receptores Toll-Like/metabolismo
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