Search

Significance: Our findings are of interest to readers because they bring new evidence that small-molecule CCR4 antagonists may be an alternative therapeutic strategy to target CCR4+ CTCL cells. They may inhibit CCR4 function but not eradicate cells, so the side effects may be avoided or minimized., (©2024 The Authors; Published by the American Association for Cancer Research.)

Aims: Acute lymphoblastic leukemia (ALL) is the most common type of pediatrics cancer. Chemokines exert different roles in leukemia process through leukocyte recruitment and regulation of disease severity. Due to the prominent roles of chemokine/receptor axes, this study aimed to measure the blood expression levels of CCR4 and their ligands in pediatrics with B-cell ALL (B-ALL). We also evaluated the impact of cytotoxic chemotherapy on this axis., Material and Method: Thirty children suffering from B-ALL were included in the study and followed up for 30 days after completion of a chemotherapy course. The blood sampling was performed before and after chemotherapy. 30 healthy donors have also entered the study as control subjects. The mRNA expression of CCL17, CCL22 and CCR4 genes was determined by quantitative real-time PCR. The frequency of the peripheral blood mononuclear cells expressing CCR4 (CCR4 + PBMCs) was also evaluated by the flow cytometry method. Moreover, we evaluated the association of the CCL17/CCL22-CCR4 axis with some diagnostic, prognostic and predictive biomarkers in ALL patients., Results: There was overexpression of the CCL17/CCL22-CCR4 axis along with lactate dehydrogenase (LDH) in pediatrics with B-ALL compared to healthy controls. After induction of chemotherapy, the blood expression levels of the CCL17/CCL22-CCR4 axis have reached the levels of healthy controls. The findings for the blood expression levels of CCR4 were also confirmed using flow cytometry., Conclusion: The CCL17/CCL22-CCR4 axis can be used as a novel predictive and prognostic biomarker in B-ALL., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high-grade glioma and human glioblastomas share many molecular similarities, including the accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford to target the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic glioma model. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma., Methods: We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine., Results: We established a flow cytometry gating strategy for identifying and isolating FOXP3 + Tregs in dogs. The canine CD4 + CD25 high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines, and expression increased when exposed to Tregs but not CD4 + helper T-cells., Conclusion: Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma., (© 2024. The Author(s).)

Objective: To study the expressions of C-C class chemokine 17 (CCL17), C-C class chemokine 22 (CCL22), and C-C chemokine receptor 4 (CCR4) in newly diagnosed multiple myeloma (NDMM) for analyzing their correlations with clinical features and to preliminarily explore their roles in the development of NDMM. Methods: The study included 40 patients with NDMM and 20 healthy volunteers from the Department of Hematology of the Affiliated Hospital of Zunyi Medical University from July 2020 to December 2022. Peripheral blood, bone marrow, and bone marrow biopsy tissue samples were collected from the two groups. The expression levels of CCL17, CCL22, and CCR4 in patients with NDMM were analyzed using real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. The mRNA expression levels of CCL17, CCL22, and CCR4 in the bone marrow mononuclear cell (BMMNC) of patients with NDMM were analyzed to assess their correlations with clinical indicators. Results: The mRNA expression levels of CCL17, CCL22, and CCR4 in BMMNC were higher in patients with NDMM than in controls (all P <0.05). The protein expression levels of CCL17 and CCL22 in peripheral blood supernatants and bone marrow supernatants were higher in patients with NDMM than in controls (all P <0.05). The expression levels of CCL17, CCL22, and CCR4 in bone marrow biopsy tissues were higher in patients with NDMM than in controls (all P <0.05). The mRNA expression level of CCL17 was increased in NDMM patients with combined anemia, bone damage, renal damage, and M protein level ≥30 g/L (all P <0.05). The mRNA expression level of CCL22 was increased in NDMM patients with combined anemia, bone damage, and renal damage (all P <0.05). The mRNA expression level of CCR4 was increased in NDMM patients with combined anemia and renal damage (all P <0.05) . Conclusion: CCL17, CCL22, and CCR4 were highly expressed in clinical samples from patients with NDMM compared to those from controls, and they may be involved in the occurrence and development of NDMM.

Objectives: Predicting the efficacy of biological disease-modifying antirheumatic drugs is challenging. In this study, we aimed to explore markers that predict the efficacy of abatacept in rheumatoid arthritis (RA) patients., Methods: Thirty RA patients receiving abatacept were recruited, and peripheral blood mononuclear cells from the participants were subjected to DNA microarray analysis. The expression of CC chemokine receptor 4 (CCR4), which was selected by the result of DNA microarray, was determined by flow cytometry in 16 newly diagnosed treatment-naïve RA patients. CCR4 expression on each helper T-cell subset was also measured., Results: CCR4 was upregulated in the abatacept responder. The expression levels of CCR4 were significantly correlated with the improvement of the Clinical Disease Activity Index. CCR4 expression was predominantly observed in CD4+ T cells in peripheral blood mononuclear cells. The percentage of CCR4-expressing CD4+ T cells was significantly higher in RA patients than in healthy individuals. Interestingly, Th17 and Treg cells expressed high levels of CCR4 compared to non-Th17-related helper T cells., Conclusions: CCR4 is a Th17- and Treg-related gene, and the high CCR4 expression in peripheral blood samples may predict the efficacy of abatacept in RA., (© Japan College of Rheumatology 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There is a regional preference around lymph nodes (LNs) for adipose beiging. Here, we show that local LN removal within inguinal white adipose tissue (iWAT) greatly impairs cold-induced beiging, and this impairment can be restored by injecting M2 macrophages or macrophage-derived C-C motif chemokine (CCL22) into iWAT. CCL22 injection into iWAT effectively promotes iWAT beiging, while blocking CCL22 with antibodies can prevent it. Mechanistically, the CCL22 receptor, C-C motif chemokine receptor 4 (CCR4), within eosinophils and its downstream focal adhesion kinase/p65/interleukin-4 signaling are essential for CCL22-mediated beige adipocyte formation. Moreover, CCL22 levels are inversely correlated with body weight and fat mass in mice and humans. Acute elevation of CCL22 levels effectively prevents diet-induced body weight and fat gain by enhancing adipose beiging. Together, our data identify the CCL22-CCR4 axis as an essential mediator for LN-controlled adaptive thermogenesis and highlight its potential to combat obesity and its associated complications.

Purpose: Hepatocellular carcinoma (HCC) is a critical global health concern, with existing treatments benefiting only a minority of patients. Recent findings implicate the chemokine ligand 17 (CCL17) and its receptor CCR4 as pivotal players in the tumor microenvironment (TME) of various cancers. This investigation aims to delineate the roles of CCL17 and CCR4 in modulating the tumor's immune landscape, assessing their potential as therapeutic interventions and prognostic markers in HCC., Methods: 873 HCC patients post-radical surgery from 2008 to 2012 at Zhongshan Hospital, Fudan University were retrospectively examined. These individuals were stratified into a training cohort (n = 354) and a validation cohort (n = 519). Through immunohistochemical analysis on HCC tissue arrays, the expressions of CCL17, CCR4, CD73, CD47, HHLA2, and PD-L1 were quantified. Survival metrics were analyzed using the Cox model, and a prognostic nomogram was devised via R software., Results: The investigation confirmed the presence of CCL17 and CCR4 within the cancerous and stromal compartments of HCC tissues, associating their heightened expression with adverse clinical markers and survival outcomes. Notably, the interplay between CD73 and CCR4 expression in tumor stroma highlighted a novel cellular entity, CCR4 + CD73 + stromal cells, impacting overall and relapse-free survival. A prognostic nomogram amalgamating these immunological markers and clinical variables was established, offering refined prognostic insights and aiding in the management of HCC. The findings suggest that reduced CCR4 and CCR4 + CD73 + cell prevalence may forecast improved outcomes post-TACE., Conclusion: This comprehensive evaluation of CCR4, CCL17, and associated markers introduces a nuanced understanding of the HCC immunological milieu, proposing CCR4 + CD73 + stromal cells as critical to HCC pathogenesis and patient stratification., (© 2024. The Author(s).)

The importance of neuroinflammation during the ischemic stroke has been extensively studied. The role of CD4+CD25+ regulatory T (Treg) cells during the recovery phase have shown infarct size reduction and functional improvement, possibly through the mitigation of inflammatory immune responses. We aimed to investigate the molecular factors involved in microglia-Treg cell communication that result in Treg trafficking. First, we observed the migration patterns of CD8+ (cytotoxic) T cells and Treg cells and then searched for chemokines released by activated microglia in an oxygen-glucose deprivation (OGD) model. The transwell migration assay showed increased migration into OGD media for both cell types, in agreement with the increase in chemokines involved in immune cell trafficking from the mouse chemokine profiling array. MSCV retrovirus was transduced to overexpress CCR4 in Treg cells. CCR4-overexpressed Treg cells were injected into the mouse transient middle cerebral artery occlusion (tMCAO) model to evaluate the therapeutic potential via the tetrazolium chloride (TTC) assay and behavioral tests. A general improvement in the prognosis of animals after tMCAO was observed. Our results suggest the increased mobility of CCR4-overexpressed Treg cells in response to microglia-derived chemokines in vitro and the therapeutic potential of Treg cells with increased mobility in cellular therapy., (© 2024. The Author(s).)

Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or 'mixed tailing', has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4-NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4-NOT stalls at the 0, -1 and -2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability., (© 2024. The Author(s).)

Cutaneous drug reactions (CDRs) are common drug-induced allergic reactions that cause severe consequences in HIV/AIDS patients. The CCL17/CCR4 axis is involved in the immune mechanism of allergic diseases, but its role in the CDRs has not been determined. Here, we aimed to determine the role of the CCL17/CCR4 axis and the underlying mechanism involved in CDRs. In this study, the serum cytokine levels in patients with CDR and healthy controls were measured. The CCL17-triggered allergic profile was screened via a PCR array. Apoptosis of keratinocytes cocultured with CCL17-stimulated Th2 cells was analyzed by flow cytometry. An NVP-induced rat CDR model was established, and dynamic inflammatory factor levels and Th2 cells in the peripheral blood of the rats were measured. Rat skin lesions and signaling pathways in Th2 cells were also analyzed. We showed that the serum CCL17 level was significantly upregulated in CDR patients (P = 0.0077), and the Th2 cell subgroup was also significantly elevated in the CDR rats. The CCL17/CCR4 axis induces Th2 cells to release IL-4 and IL-13 via the ERK/STAT3 pathway. The CCR4 antagonist compound 47 can alleviate rash symptoms resulting from NVP-induced drug eruption, Th2 cell subgroup, IL-4, and IL-13 and inhibit keratinocyte apoptosis. Taken together, these findings indicate that the CCL17/CCR4 axis mediates CDR via the ERK/STAT3 pathway in Th2 cells and type 2 cytokine-induced keratinocyte apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss-of-function screen in primary human fibroblasts, we here identify the host CCR4-NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro-viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome-wide host responses in CCR4-NOT-disrupted cells. By profiling poly(A)-tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)-tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4-NOT disruption. Our data establish that mRNA deadenylation by host CCR4-NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti-HCMV target., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

A challenge when targeting T-cell lymphoma with chimeric antigen receptor (CAR) T-cell therapy is that target antigens are often shared between T cells and tumor cells, resulting in fratricide between CAR T cells and on-target cytotoxicity on normal T cells. CC chemokine receptor 4 (CCR4) is highly expressed in many mature T-cell malignancies, such as adult T-cell leukemia/lymphoma (ATLL) and cutaneous T-cell lymphoma (CTCL), and has a unique expression profile in normal T cells. CCR4 is predominantly expressed by type-2 and type-17 helper T cells (Th2 and Th17) and regulatory T cells (Treg), but it is rarely expressed by other T helper (Th) subsets and CD8+ cells. Although fratricide in CAR T cells is generally thought to be detrimental to anticancer functions, in this study, we demonstrated that anti-CCR4 CAR T cells specifically depleted Th2 and Tregs, while sparing CD8+ and Th1 T cells. Moreover, fratricide increased the percentage of CAR+ T cells in the final product. CCR4-CAR T cells were characterized by high transduction efficiency, robust T-cell expansion, and rapid fratricidal depletion of CCR4-positive T cells during CAR transduction and expansion. Furthermore, mogamulizumab-based CCR4-CAR T cells induced superior antitumor efficacy and long-term remission in mice engrafted with human T-cell lymphoma cells. In summary, CCR4-depleted anti-CCR4 CAR T cells are enriched in Th1 and CD8+ T cells and exhibit high antitumor efficacy against CCR4-expressing T-cell malignancies., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

NOT1, NOT10, and NOT11 form a conserved module in the CCR4-NOT complex, critical for post-transcriptional regulation in eukaryotes, but how this module contributes to the functions of the CCR4-NOT remains poorly understood. Here, we present cryo-EM structures of human and chicken NOT1:NOT10:NOT11 ternary complexes to sub-3 Å resolution, revealing an evolutionarily conserved, flexible structure. Through biochemical dissection studies, which include the Drosophila orthologs, we show that the module assembly is hierarchical, with NOT11 binding to NOT10, which then organizes it for binding to NOT1. A short proline-rich motif in NOT11 stabilizes the entire module assembly., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Chemokines are critical players in the local immune responses to tumors. CCL17 (thymus and activation-regulated chemokine, TARC) and CCL22 (macrophage-derived chemokine, MDC) can attract CCR4-bearing cells involving the immune landscape of cancer. However, their direct roles and functional states in tumors remain largely unclear., Methods: We analyzed the lymphoma-related scRNA-seq and bulk RNA-seq datasets and identified the CCL17/CCL22-CCR4 axis as the unique participant of the tumor microenvironment. Then we edited the A20 lymphoma cell line to express CCL17 and CCL22 and assessed their function using three mouse models (Balb/C mouse, Nude mouse, and NSG mouse). In addition, we retrospectively checked the relationship between the CCL17/CCL22-CCR4 axis and the survival rates of cancer patients., Results: The active CCL17/CCL22-CCR4 axis is a distinctive feature of the Hodgkin lymphoma microenvironment. CCR4 is widely expressed in immune cells but highly exists on the surface of NK, NKT, and Treg cells. The tumor model of Balb/C mice showed that CCL17 acts as an anti-tumor chemokine mediated by activated T cell response. In addition, the tumor model of Nude mice showed that CCL17 recruits NK cells for inhibiting lymphoma growth and enhances the NK-cDC1 interaction for resisting IL4i1-mediated immunosuppression. Interestingly, CCL17-mediated antitumor immune responses depend on lymphoid lineages but not mainly myeloid ones. Furthermore, we found CCL17/CCL22-CCR4 axis cannot be regarded as biomarkers of poor prognosis in most cancer types from the TCGA database., Conclusion: We provided direct evidence of antitumor functions of CCL17 mediated by the recruitment of conventional T cells, NKT cells, and NK cells. Clinical survival outcomes of target gene (CCL17, CCL22, and CCR4) expression also identified that CCL17/CCL22-CCR4 axis is not a marker of poor prognosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In response to the host environment, the human pathogen Cryptococcus neoformans must rapidly reprogram its translatome from one which promotes growth to one which is responsive to host stress. In this study, we investigate the two events which comprise translatome reprogramming: the removal of abundant, pro-growth mRNAs from the translating pool, and the regulated entry of stress-responsive mRNAs into the translating pool. Removal of pro-growth mRNAs from the translating pool is controlled primarily by two regulatory mechanisms, repression of translation initiation via Gcn2, and decay mediated by Ccr4. We determined that translatome reprogramming in response to oxidative stress requires both Gcn2 and Ccr4, whereas the response to temperature requires only Ccr4. Additionally, we assessed ribosome collision in response to host-relevant stress and found that collided ribosomes accumulated during temperature stress but not during oxidative stress. The phosphorylation of eIF2α that occurred as a result of translational stress led us to investigate the induction of the integrated stress response (ISR). We found that eIF2α phosphorylation varied in response to the type and magnitude of stress, yet all tested conditions induced translation of the ISR transcription factor Gcn4. However, Gcn4 translation did not necessarily result in canonical Gcn4-dependent transcription. Finally, we define the ISR regulon in response to oxidative stress. In conclusion, this study begins to reveal the translational regulation in response to host-relevant stressors in an environmental fungus which is capable of adapting to the environment inside the human host. IMPORTANCE Cryptococcus neoformans is a human pathogen capable of causing devastating infections. It must rapidly adapt to changing environments as it leaves its niche in the soil and enters the human lung. Previous work has demonstrated a need to reprogram gene expression at the level of translation to promote stress adaptation. In this work, we investigate the contributions and interplay of the major mechanisms that regulate entry of new mRNAs into the pool (translation initiation) and the clearance of unneeded mRNAs from the pool (mRNA decay). One result of this reprogramming is the induction of the integrated stress response (ISR) regulon. Surprisingly, all stresses tested led to the production of the ISR transcription factor Gcn4, but not necessarily to transcription of ISR target genes. Furthermore, stresses result in differential levels of ribosome collisions, but these are not necessarily predictive of initiation repression as has been suggested in the model yeast.

Natural killer (NK) cells are a subset of lymphocytes that offer great potential for cancer immunotherapy due to their natural anti-tumor activity and the possibility to safely transplant cells from healthy donors to patients in a clinical setting. However, the efficacy of cell-based immunotherapies using both T and NK cells is often limited by a poor infiltration of immune cells into solid tumors. Importantly, regulatory immune cell subsets are frequently recruited to tumor sites. In this study, we overexpressed two chemokine receptors, CCR4 and CCR2B, that are naturally found on T regulatory cells and tumor-resident monocytes, respectively, on NK cells. Using the NK cell line NK-92 as well as primary NK cells from peripheral blood, we show that genetically engineered NK cells can be efficiently redirected using chemokine receptors from different immune cell lineages and migrate towards chemokines such as CCL22 or CCL2, without impairing the natural effector functions. This approach has the potential to enhance the therapeutic effect of immunotherapies in solid tumors by directing genetically engineered donor NK cells to tumor sites. As a future therapeutic option, the natural anti-tumor activity of NK cells at the tumor sites can be increased by co-expression of chemokine receptors with chimeric antigen receptors (CAR) or T cell receptors (TCR) on NK cells can be performed in the future.

Regulatory T-cells (Tregs) play a major role in suppressing anti-tumor immune responses. Mogamulizumab, an anti-CC chemokine receptor type 4 (CCR4) monoclonal antibody, depletes effector Tregs (eTregs). However, the clinical efficacy of mogamulizumab was limited in phase Ia/Ib studies for solid tumors (NCT01929486); the finding suggests that mogamulizumab may also deplete beneficial CCR4 + CD8 + T-cells in patients. Therefore, we focused on CTLs and aimed to identify a way to protect CCR4 + CTLs. Here, we evaluated the association of CCR4 expression in cytotoxic T-lymphocytes (CTLs) with antigen and cytokine stimulations and kinase inhibition using cytomegalovirus antigen instead of tumor antigen. CCR4 expression in CTLs was induced by antigen stimulation (mean 3.14-29.0%), enhanced by transforming growth factor-β1 (TGF-β1) (mean 29.0-51.2%), and downregulated by trametinib with (mean 51.2-11.4%) or without TGF-β1 treatment (mean 29.0-6.98%). Phosphorylation of ERK in CD8 + T-cells was suppressed by trametinib. Regarding the effect on immunological function of CTL, trametinib reduced cytokine production but not affected cytotoxicity. Importantly, trametinib alleviated CTL reduction by anti-CCR4 antibody without affecting eTreg depletion because CCR4 expression in eTregs was not downregulated. In conclusion, combination therapy with trametinib may improve the clinical efficacy of mogamulizumab., (© 2022. The Author(s).)

Several studies have suggested that fibroblast-like synoviocytes (FLSs) and miRNAs are implicated in the pathogenesis of rheumatoid arthritis (RA). This study was aimed to evaluate the function of miR-6089 in the regulation of RA-FLSs. The levels of miR-6089 were detected to be significantly lower in the synovial tissues and FLSs of RA than in the healthy synovial tissues and FLSs. The miR-6089 up-regulation in RA-FLSs significantly inhibited the proliferation and promoted cell apoptosis accompany with an increase protein expression of cleaved-Caspase-3, -8 and -9. Furthermore, CCR4 was determined to target miR-6089 directly, and its expression was significantly increased in the synovial tissues of RA than in the healthy synovial tissues. The overexpression of CCR4 reversed the effect of miR-6089 on proliferation and apoptosis in RA-FLSs effectively. In conclusion, our study suggests that the miR-6089 may be a potential target for prevention and treatment of RA.

GW182 family proteins are a key component of microRNA-protein complex eliciting translational repression and/or degradation of microRNA-targets. The microRNAs in complex with Argonaute proteins bind to target mRNAs, and GW182 proteins are recruited by association with Argonaute proteins. The GW182 protein acts as a scaffold that links the Argonaute protein to silencing machineries including the CCR4-NOT complex which accelerates deadenylation and inhibits translation. The carboxyl-terminal effector domain of GW182 protein, also called the silencing domain, has been shown to bind to the subunits of the CCR4-NOT complex, the CNOT1 and the CNOT9. Here we show that a small region within the amino-terminal Argonaute-binding domain of human GW182/TNRC6A can associate with the CCR4-NOT complex. This region resides between the two Argonaute-binding sites and contains reiterated GW/WG-motifs. Alanine mutation experiments showed that multiple tryptophan residues are required for the association with the CCR4-NOT complex. Furthermore, co-expression and immunoprecipitation assays suggested that the CNOT9 subunit of the CCR4-NOT complex is a possible binding partner of this region. Our work, taken together with previous studies, indicates that the human GW182 protein contains multiple binding interfaces to the CCR4-NOT complex., (© 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Background: Chemokines and their receptors regulate inflammatory processes in major depressive disorder (MDD). Here, we characterize the expression pattern of the C-C chemokine receptor 4 (CCR4) and its ligands CCL17 and CCL22 in MDD and its clinical relevance in predicting disease severity., Methods: Expression of CCR4 on peripheral blood lymphocytes and serum CCL17/CCL22 levels were measured using multiparameter flow cytometry and multiplex assays in 33 depressed inpatients at baseline (T0) and after 6-week multimodal treatment (T1) compared with 21 healthy controls (HC). Using stratified and correlation analysis, we examined the associations of CCR4-CCL17/CCL22 expression with depression severity and symptoms according to standard clinical rating scales and questionnaires. Additionally, we assessed whether polygenic risk score (PRS) for psychiatric disorders and chronotype are associated with disease status or CCR4-CCL17/CCL22 expression. Regression analysis was performed to assess the capacity of CCR4 and PRS in predicting disease severity., Results: Compared with HC, MDD patients showed significantly decreased CCR4 expression on T cells (T0 and T1), whereas CCL17/CCL22 serum levels were increased. Stratified and correlation analysis revealed an association of CCR4 expression on CD4 + T cells with depression severity as well as Beck Depression Inventory-II items including loss of pleasure, agitation and cognitive deficits. CCR4 expression levels on CD4 + T cells together with cross-disorder and chronotype PRS significantly predicted disease severity., Limitations: This exploratory study with small sample size warrants future studies., Conclusions: This newly identified CCR4-CCL17/CCL22 signature and its predictive capacity for MDD severity suggest its potential functional involvement in the pathophysiology of MDD., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Among all the cancer-related deaths globally, pancreatic ductal adenocarcinoma (PDAC) accounts for the seventh leading cause of mortality. A dysregulated immune system disrupts anti-tumor immunity by abnormal accumulation of myeloid-derived suppressor cells (MDSCs), but the underlying mechanisms are still inconclusive. To gain new insights into the role of MDSCs in tumor settings, we aimed to determine the mechanism of expansion of various subsets of MDSCs in PDAC patients and their role in promoting invasiveness. We assessed the load of MDSCs, chemokines responsible for the recruitment of MDSCs in PDAC patients by flow cytometry. We investigated the chemokine profile of tumor tissue using qRT-PCR and the status of epithelial-mesenchymal transition (EMT) related markers E-Cadherin, N-Cadherin, Snail, and ZEB1 by qRT-PCR and immunohistochemistry. We found a higher frequency of tumor infiltrated MDSCs in PDAC patients. Chemokine ligands CCL2 and the receptor CCR4 were markedly elevated in the PDAC tumor, while CCR4 + monocytic MDSCs (M-MDSCs) were found significantly elevated in peripheral blood and tumor tissue. In tumor tissue, expression of E-Cadherin was significantly reduced, while N-Cadherin, Snail, and ZEB1 were markedly raised. The frequency of CCR4 + M-MDSCs significantly correlated with the expression of mesenchymal transition markers N-Cadherin, Snail, and ZEB1. Collectively, these results suggest that the CCL2-CCR4 axis plays a crucial role in driving the recruitment of M-MDSCs, which is associated with increased invasiveness in PDAC. This study sheds light on the expansion mechanism of MDSCs, which can serve as a crucial target of future anti-cancer strategies to inhibit tumor cell invasiveness., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The CC chemokine receptor type-4 (CCR4) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of T cells and its malignancy. Two CCR4 ligands (CCL17 and CCL22) bind to CCR4 that mediate physiological and pathological functions of T cell immune responses. Anti-CCR4 monoclonal antibody (mAb) mogamulizumab is approved for adult T cell leukemia/lymphoma and cutaneous T cell lymphomas. In addition, mogamulizumab can deplete regulatory T cells, implying the application to solid tumors as an immunomodulator. Therefore, the development of sensitive mAbs for CCR4 has been desired for basic research, diagnosis, and therapy. In this study, a specific, and sensitive anti-mouse CCR4 (mCCR4) mAb, C 4 Mab-1 (rat IgG 1 , kappa), was established using N-terminal peptide immunization. C 4 Mab-1 reacted with mCCR4-overexpressed Chinese hamster ovary (CHO)-K1 cells, P388 (mouse lymphoid neoplasm), and J774-1 (mouse macrophage-like) cells in flow cytometry. Kinetic analyses using flow cytometry showed that K D s of C 4 Mab-1 for CHO/mCCR4, P388, and J774-1 cells were 4.2 × 10 -9 M, 5.4 × 10 -7 M, and 1.1 × 10 -6 M, respectively. C 4 Mab-1 could be a valuable tool for elucidating mCCR4-related biological responses.

N6-methyladenosine (m6A) is one of the most significant modifications in human mRNAs. Emerging evidence indicates that m6A participates in the initiation and development of malignant tumors. Nevertheless, the biological roles and mechanism of m6A in osteosarcoma (OS) remain unclear. The purpose of this study was to investigate the role and mechanism of the methylation recognition protein-YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in OS. The YTHDF1 expression in OS was detected by qRT-PCR and Western blot assay. M6A quantification was utilized to measure the methylation level of OS. Cell counting kit-8 (CCK8), 5-Ethynyl-2'-deoxyuridine (EdU) assay and transwell experiments were conducted to confirm the biological effects of YTHDF1 on OS cells. The bioinformatics websites and in vitro assays were conducted to analyze the downstream targets of YTHDF1 was upregulated in OS tissues at mRNA and protein level. The results showed that the expression level of YTHDF1 might be closely associated with the poor prognosis for OS patients. Inhibition of YTHDF1 could suppress the proliferation, migration and invasion of the OS cells. Moreover, we found that CCR4-NOT transcription complex subunit 7 (CNOT7) might be the potential target of YTHDF1, which was upregulated in OS tissues. YTHDF1 could recognize the m6A sites of CONT7 and promote its expression in an m6A manner. Moreover, methyltransferase-like 3 (METTL3) could promote the m6A level of CONT7. YTHDF1 was upregulated in OS and could promote cell proliferation, migration and invasion. The METTL3-CONT7-YTHDF1 regulatory axis might be the potential target for the prognosis and therapy of OS.

Infiltrating T-regulatory cells in the tumor microenvironment is a key impediment to immunotherapy and is linked to a poor prognosis. We found that tumor-infiltrating Tregs express a higher expression of the chemokine receptor CCR4 than peripheral Tregs in breast cancer patients. CCL22 and CCL17 are released by tumor cells and tumor-associated macrophages, attracting CCR4 + Tregs to the tumor site. The Treg lineage-specific transcription factor FOXP3 changes the CCR4 promoter epigenetically in conjunction with HAT1 to provide a space for FOXP3 binding and activation of the CCR4 gene. To increase CCR4 expression in Tregs, the FOXP3/HAT1 axis is required for permissive (K23 and K27) or repressive (K14 and K18) acetylation of histone-3. In murine breast and melanoma tumor models, genetic ablation of FOXP3 reduced CCR4 + Treg infiltration and tumor size while also restoring anti-tumor immunity. Overexpression of FOXP3, on the other hand, increased CCR4 + Treg infiltration, resulting in a decreased anti-tumor immune response and tumor progression. These findings point to FOXP3 playing a new role in the tumor microenvironment as a transcriptional activator of CCR4 and a regulator of Treg infiltration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sarkar, Dhar, Chakraborty, Pati, Bose, Panda, Basak, Chakraborty, Mukherjee, Guin, Jana, Sarkar and Sa.)

Inflammatory pain, such as hypersensitivity resulting from surgical tissue injury, occurs as a result of interactions between the immune and nervous systems with the orchestrated recruitment and activation of tissue-resident and circulating immune cells to the site of injury. Our previous studies identified a central role for Ly6C low myeloid cells in the pathogenesis of postoperative pain. We now show that the chemokines CCL17 and CCL22, with their cognate receptor CCR4, are key mediators of this response. Both chemokines are up-regulated early after tissue injury by skin-resident dendritic and Langerhans cells to act on peripheral sensory neurons that express CCR4. CCL22, and to a lesser extent CCL17, elicit acute mechanical and thermal hypersensitivity when administered subcutaneously; this response abrogated by pharmacological blockade or genetic silencing of CCR4. Electrophysiological assessment of dissociated sensory neurons from naïve and postoperative mice showed that CCL22 was able to directly activate neurons and enhance their excitability after injury. These responses were blocked using C 021 and small interfering RNA (siRNA)-targeting CCR4. Finally, our data show that acute postoperative pain is significantly reduced in mice lacking CCR4, wild-type animals treated with CCR4 antagonist/siRNA, as well as transgenic mice depleted of dendritic cells. Together, these results suggest an essential role for the peripheral CCL17/22:CCR4 axis in the genesis of inflammatory pain via direct communication between skin-resident dendritic cells and sensory neurons, opening therapeutic avenues for its control., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Background & Aims: Regulatory T cell (Treg) depletion increases antitumor immunity. However, severe autoimmunity can occur following systemic loss of Tregs, which could be avoided by selectively depleting intratumoral Tregs. Herein, we aimed to investigate the role of tumor-infiltrating CCR4 + Tregs in hepatocellular carcinoma (HCC) and to provide a potential target strategy for immunotherapy., Methods: CCR4 + Tregs were analyzed by flow cytometry in murine models and clinical samples. The function of tumor-infiltrating and induced CCR4 + Tregs was interrogated by genetic and epigenetic approaches. To block CCR4 + Treg chemotaxis, we developed an N-terminus recombinant protein of CCR4 (N-CCR4-Fc) as a neutralizing pseudo-receptor that effectively bound to its ligand CCL22. The efficacy of CCR4 antagonism as an immunotherapeutic agent was evaluated by tumor weights, growth kinetics and survival curves., Results: CCR4 + Tregs were the predominant type of Tregs recruited to hepatitis B-associated HCC (HBV + HCC), correlating with sorafenib resistance and HBV load titers. Compared with CCR4 - Tregs, CCR4 + Tregs exhibited increased IL-10 and IL-35 expression, and enhanced functionality in suppressing CD8 + T cells. CCR4 + Tregs also displayed PD-1 + TCF1 + stem-like properties. ATAC-seq data revealed substantial chromatin remodeling between tumor-infiltrating Tregs (TIL-Tregs) and induced Tregs, suggesting that long-term chromatin reprogramming accounted for the acquisition of enhanced immunosuppressive stem-like specificity by CCR4 + TIL-Tregs. Treatment with a CCR4 antagonist or N-CCR4-Fc blocked intratumoral Treg accumulation, overcame sorafenib resistance, and sensitized tumors to PD-1 checkpoint blockade., Conclusions: Intratumoral stem-like CCR4 + Tregs orchestrated immunosuppressive resource cells in the tumor microenvironment. CCR4 could be targeted to enhance antitumor immunity by specifically blocking infiltration of Tregs into the tumor microenvironment and inhibiting maintenance of the TIL-Treg pool., Lay Summary: Targeting regulatory T cells is a promising approach in cancer immunotherapy; however, severe autoimmunity can occur following systemic regulatory T cell loss. This could be avoided by selectively depleting intratumoral regulatory T cells. Herein, targeting intratumoral stem-like CCR4 + regulatory T cells helped to overcome sorafenib resistance and sensitize tumors to immune checkpoint blockade in mouse models of liver cancer. This approach could have wide clinical applicability., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

CCR4-NOT complex-mediated mRNA deadenylation serves critical functions in multiple biological processes, yet how this activity is regulated is not fully understood. Here, we show that osmotic stress induces MAPKAPK-2 (MK2)-mediated phosphorylation of CNOT2. Programmed cell death is greatly enhanced by osmotic stress in CNOT2-depleted cells, indicating that CNOT2 is responsible for stress resistance of cells. Although wild-type (WT) and non-phosphorylatable CNOT2 mutants reverse this sensitivity, a phosphomimetic form of CNOT2, in which serine at the phosphorylation site is replaced with glutamate, does not have this function. We also show that mRNAs have elongated poly(A) tails in CNOT2-depleted cells and that introduction of CNOT2 WT or a non-phosphorylatable mutant, but not phosphomimetic CNOT2, renders their poly(A) tail lengths comparable to those in control HeLa cells. Consistent with this, the CCR4-NOT complex containing phosphomimetic CNOT2 exhibits less deadenylase activity than that containing CNOT2 WT. These data suggest that CCR4-NOT complex deadenylase activity is regulated by post-translational modification, yielding dynamic control of mRNA deadenylation.

Regulatory T (Treg) cells are important negative regulators of immune homeostasis, but in cancers they tone down the anti-tumor immune response. They are distinguished by high expression levels of the chemokine receptor CCR4, hence their targeting by the anti-CCR4 monoclonal antibody mogamulizumab holds therapeutic promise. Here we show that despite a significant reduction in peripheral effector Treg cells, clinical responses are minimal in a cohort of patients with advanced CCR4-negative solid cancer in a phase Ib study (NCT01929486). Comprehensive immune-monitoring reveals that the abundance of CCR4-expressing central memory CD8 + T cells that are known to play roles in the antitumor immune response is reduced. In long survivors, characterised by lower CCR4 expression in their central memory CD8 + T cells possessed and/or NK cells with an exhausted phenotype, cell numbers are eventually maintained. Our study thus shows that mogamulizumab doses that are currently administered to patients in clinical studies may not differentiate between targeting effector Treg cells and central memory CD8 + T cells, and dosage refinement might be necessary to avoid depletion of effector components during immune therapy., (© 2021. The Author(s).)

The CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon HDAC inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified RNF219 as an acetylation-regulated cofactor. We demonstrate that RNF219 is an active RING-type E3 ligase which stably associates with CCR4-NOT via NOT9 through a short linear motif (SLiM) embedded within the C-terminal low-complexity region of RNF219. By using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 inhibits deadenylation through the direct interaction of the α-helical SLiM with the NOT9 module. Transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, with differential requirement of its RING domain. Our results establish RNF219 as an inhibitor of CCR4-NOT-mediated deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover., (© 2021. The Author(s).)

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) along with their blood lipid-lowering effect exhibit anti-inflammatory and immunomodulatory activity. We studied the effects of long-term (72-h or longer) exposure of human T lymphocytes in culture to atorvastatin and rosuvastatin (5-80 nM) on their functional activity. Treatment with statins inhibited PHA/IL-2-induced proliferation of CD4 + T lymphocytes isolated from the peripheral blood of healthy donors. This was accompanied by a decrease in the relative content of cells expressing active caspase-3. Addition of mevalonate or fetal bovine serum simultaneously with statins restored proliferative activity of cells. Culturing of CD4 + T lymphocytes with statins in the presence of IL-2 did not significantly affect the expression of chemokine receptors CCR4, CCR5, CXCR3, and CXCR4. Pretreatment with statins suppressed spontaneous and SDF-1-stimulated migration of CD4 + T lymphocytes, but little changed the content of intracellular phosphorylated protein kinases Akt, p38 and p42/44 (ERK1/2). The cellular effects of "lipophilic" atorvastatin were observed at lower concentrations compared to "hydrophilic" rosuvastatin., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The pathogenesis of allergic contact dermatitis (ACD) requires the activation of Ag-specific T cells, including effector and regulatory T cells. The differentiation and function of these T cells is epigenetically regulated through DNA methylation and histone modifications. However, the roles of altered histone H3K27 methylation in T cells in the development of ACD remain unknown. Two types of histone H3K27 demethylases, Utx and Jmjd3, have been reported in mammals. To determine the role of the histone H3K27 demethylase expression of T cells in the development of ACD, we generated T cell-specific, Utx -deficient ( Utx KO) mice or Jmjd3 -deficient ( Jmjd3 KO) mice. Unlike control mice, Utx KO mice had severer symptoms of ACD, whereas Jmjd3 KO mice showed symptoms identical to those in control mice. In Utx KO mice with ACD, the massive infiltration of myeloid cells, including neutrophils and dendritic cells, has been observed. In addition, the expression of proinflammatory cytokines in CD4 + T cells of the draining lymph nodes (LNs) and in CD8 + T cells of the skin was increased in Utx KO mice, whereas the ratio of Foxp3 + regulatory CD4 + T cells to Foxp3 - conventional CD4 + T cells was decreased in both the draining LNs and the skin of Utx KO mice with ACD. Furthermore, Foxp3 + regulatory CD4 + T cells of Utx KO mice with ACD expressed a decreased level of CCR4 (a skin-tropic chemokine receptor) in comparison with control. Thus, in CD4 + T cells, Utx could potentially be involved in the regulation of the pathogenesis of ACD., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Regulatory T cells (Tregs) are impaired in human systemic lupus erythematosus (SLE) and involved in disease pathogenesis. However, the mechanisms responsible for the Treg dysfunction in SLE remain unclear. In this study, we aimed to investigate the chemotaxis of Treg response to inflammatory stimulation. Sixty two patients were enrolled, and chemokine receptors, including CCR4, CCR5, CCR6, CCR8 and CXCR3 on CD4+Foxp3+Tregs and non-Treg CD4 T cells, were analysed using FACS. The expression of CCR4 and CCR6 on Tregs of SLE patients decreased, while the expression of CCR4 on non-Treg CD4 T cells increased, as compared with those of age- and sex-matched healthy donors. In parallel, in SLE, the chemotactic capacity of non-Treg CD4 T cells response to CCR4 and CCR6 ligands dramatically increased, while that of Tregs significantly decreased. Moreover, we found that cytokines IL-6 and IL-10 positively and negatively modulate the expression of those receptors respectively. IL-6, the significantly increased cytokine in active SLE, dramatically elevated CCR4 and CCR6 expression on non-Treg CD4 T cells. However, as for Tregs, these cells produced more IL-10 than non-Treg CD4 T cells upon IL-6 stimulation, and these IL-10 led to the inhibition of CCR4 and CCR6. In sum, our data provided new evidence suggesting a functional deficiency of Tregs in SLE. It may suggest that those dysfunctional Tregs have less access to the inflammation locus to exert inhibitory capacity., (© 2021 The Scandinavian Foundation for Immunology.)

Primary cutaneous lymphomas are heterogenous lymphoproliferative disorders. Some patients show rapid progression and the need for treatment of advanced disease is still unmet. The frequency of each subtype of cutaneous lymphoma varies among different ethnic groups, as do the medical systems found in different countries. It is important to know the differences in clinical guidelines in different areas of the world. Although current monochemotherapy with gemcitabine or pegylated liposomal doxorubicin is temporarily effective for mycosis funogides (MF) and Sézary syndrome (SS)-representative types of cutaneous lymphomas-the duration of response is usually limited. Therefore, treatment strategies targeting tumor-specific molecules have been developed. Molecular targets for MS/SS are currently CD30, CCR4, CD25, CD52, and histone deacetylases, most of which are surface molecules specifically expressed on tumor cells. As a result of advances in research techniques, different kinds of genomic alterations in MF/SS have been revealed. Molecular targets for MS/SS in the near future would be CD158k, JAK, PIK3, the mammalian target of rapamycin, and microRNAs, most of which mediate intracellular signaling pathways. Personalized therapy based on the detection of the genetic signatures of tumors and inhibition of the most suitable target molecules constitutes a future treatment strategy for MF/SS.

The cellular response to environmental exposures, such as nutrient shifts and various forms of stress, requires the integration of the signaling apparatus that senses these environmental changes with the downstream gene regulatory machinery. Delineating this molecular circuitry remains essential for understanding how organisms adapt to environmental flux, and it is critical for determining how dysregulation of these mechanisms causes disease. Ccr4-Not is a highly conserved regulatory complex that controls all aspects of the gene expression process. Recent studies in budding yeast have identified novel roles for Ccr4-Not as a key regulator of core nutrient signaling pathways that control cell growth and proliferation, including signaling through the mechanistic target of rapamycin complex 1 (TORC1) pathway. Herein, I will review the current evidence that implicate Ccr4-Not in nutrient signaling regulation, and I will discuss important unanswered questions that should help guide future efforts to delineate Ccr4-Not's role in linking environmental signaling with the gene regulatory machinery. Ccr4-Not is highly conserved throughout eukaryotes, and increasing evidence indicates it is dysregulated in a variety of diseases. Determining how Ccr4-Not regulates these signaling pathways in model organisms such as yeast will provide a guide for defining how it controls these processes in human cells., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nox2 is a ROS-generating enzyme, deficiency of which increases suppression by Tregs in vitro and in an in vivo model of cardiac remodeling. As Tregs have emerged as a candidate therapy in autoimmunity and transplantation, we hypothesized that Nox2 deficiency in Tregs in recipient mice may improve outcomes in a heart transplant model. We generated a potentially novel B6129 mouse model with Treg-targeted Nox2 deletion (Nox2fl/flFoxP3Cre+ mice) and transplanted with hearts from CB6F1 donors. As compared with those of littermate controls, Nox2fl/flFoxP3Cre+ mice had lower plasma levels of alloantibodies and troponin-I, reduced levels of IFN-γ in heart allograft homogenates, and diminished cardiomyocyte necrosis and allograft fibrosis. Single-cell analyses of allografts revealed higher absolute numbers of Tregs and lower CD8+ T cell infiltration in Nox2-deficient recipients compared with Nox2-replete mice. Mechanistically, in addition to a greater suppression of CD8+CD25- T effector cell proliferation and IFN-γ production, Nox2-deficient Tregs expressed higher levels of CCR4 and CCR8, driving cell migration to allografts; this was associated with increased expression of miR-214-3p. These data indicate that Nox2 deletion in Tregs enhances their suppressive ability and migration to heart allografts. Therefore, Nox2 inhibition in Tregs may be a useful approach to improve their therapeutic efficacy.

Psoriasis is a chronic skin disease associated with T helper (Th)17-mediated inflammation. Because CCR4 is a major chemokine receptor expressed on Th17 cells, we investigated the role of CCR4 in a modified imiquimod-induced psoriasis model that showed enhanced skin infiltration of Th17 cells. CCR4-deficient mice had less severe skin disease than wild-type mice. Th17 cells were decreased in the skin lesions and regional lymph nodes of CCR4-deficient mice. In the regional lymph nodes of wild-type mice, CD44 + memory Th17 cells expressing CCR4 were found to be clustered with dendritic cells expressing CCL22, a ligand for CCR4. Such dendritic cell‒Th17 cell clusters were significantly decreased in CCR4-deficient mice. Similar results were obtained using the IL-23‒induced psoriasis model. In vitro, compound 22, a CCR4 antagonist, significantly reduced the expansion of Th17 cells in the coculture of CD11c + dendritic cells and CD4 + T cells separately prepared from the regional lymph nodes of wild-type mice with psoriasis. In vivo, compound 22 ameliorated the psoriasis-like skin disease in wild-type mice with significant decreases of Th17 cells in the regional lymph nodes and skin lesions. Collectively, CCR4 is likely to play a role in the pathogenesis of psoriasis through the expansion of Th17 cells., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Recognition of cell-surface receptors and co-receptors is a crucial molecular event towards the establishment of HIV infection. HIV exists as several variants that differentially recognize the principal co-receptors, CCR5 and CXCR4, in different cell types, known as HIV co-receptor-tropism. The relative levels of these variants dynamically adjust to the changing host selection pressures to infect a vast repertoire of cells in a stage-specific manner. HIV infection sets in through immune cells such as dendritic cells, macrophages, and T-lymphocytes in the acute stage, while a wide range of other cells, including astrocytes, glial cells, B-lymphocytes, and epithelial cells, are infected during chronic stages. A change in tropism occurs during the transition from acute to a chronic phase, termed as co-receptor switching marked by a change in disease severity. The cellular and molecular events leading to co-receptor switching are poorly understood. This review aims to collate our present understanding of the dynamics of HIV co-receptor-tropism vis-à-vis host and viral factors, highlighting the cellular and molecular events involved therein. We present the possible correlations between virus entry, cell tropism, and co-receptor switching, speculating its consequences on disease progression, and proposing new scientific pursuits to help in an in-depth understanding of HIV biology.

Development of lymphocytes is precisely regulated by various mechanisms. In addition to transcriptional rates, post-transcriptional regulation of mRNA abundance contributes to differentiation of lymphocytes. mRNA decay is a post-transcriptional mechanism controlling mRNA abundance. The carbon catabolite repression 4 (CCR4)-negative on TATA-less (NOT) complex controls mRNA longevity by catalyzing mRNA deadenylation, which is the rate-limiting step in the mRNA decay pathway. mRNA decay, regulated by the CCR4-NOT complex, is required for differentiation of pro-B to pre-B cells and V(D)J recombination in pro-B cells. In this process, it is likely that the RNA-binding proteins, ZFP36 ring finger protein like 1 and 2, recruit the CCR4-NOT complex to specific target mRNAs, thereby inducing cell quiescence of pro-B cells. A recent study showed that the CCR4-NOT complex participates in positive selection of thymocytes. Mechanistically, the CCR4-NOT deadenylase complex inhibits abnormal apoptosis by reducing the expression level of mRNAs encoding pro-apoptotic proteins, which are otherwise up-regulated during positive selection. We discuss mechanisms regulating CCR4-NOT complex-dependent mRNA decay in lymphocyte development and selection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Akiyama and Yamamoto.)

New treatments are needed for patients with cutaneous T-cell lymphoma (CTCL), particularly for advanced mycosis fungoides (MF) and Sezary syndrome (SS). The immunopathology of MF and SS is complex, but recent advances in tumor microenvironment understanding have identified CCR4 as a promising therapeutic target. CCR4 is widely expressed on malignant T cells and Tregs in the skin and peripheral blood of patients with MF and SS. The interaction of CCR4 with its dominant ligands CCL17 and CCL22 plays a critical role in the development and progression of CTCL, facilitating the movement into, and accumulation of, CCR4-expressing T cells in the skin, and recruiting CCR4-expressing Tregs into the tumor microenvironment. Expression of CCR4 is upregulated at all stages of MF and in SS, increasing with advancing disease. Several CCR4-targeted therapies are being evaluated, including "chemotoxins" targeting CCR4 via CCL17, CCR4-directed chimeric antigen receptor-modified T-cell therapies, small-molecule CCR4 antagonists, and anti-CCR4 monoclonal antibodies. Only one is currently approved: mogamulizumab, a defucosylated, fully humanized, anti-CCR4, monoclonal antibody for the treatment of relapsed/refractory MF and SS. Clinical trial da1ta confirm that mogamulizumab is an effective and well-tolerated treatment for relapsed/refractory MF or SS, demonstrating the clinical value of targeting CCR4., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Objective: To compare immune cell phenotype and function in psoriatic arthritis (PsA) versus psoriasis in order to better understand the pathogenesis of PsA., Methods: In-depth immunophenotyping of different T cell and dendritic cell subsets was performed in patients with PsA, psoriasis, or axial spondyloarthritis and healthy controls. Subsequently, we analyzed cells from peripheral blood, synovial fluid (SF), and skin biopsy specimens using flow cytometry, along with high-throughput transcriptome analyses and functional assays on the specific cell populations that appeared to differentiate PsA from psoriasis., Results: Compared to healthy controls, the peripheral blood of patients with PsA was characterized by an increase in regulatory CD4+ T cells and interleukin-17A (IL-17A) and IL-22 coproducing CD8+ T cells. One population specifically differentiated PsA from psoriasis: i.e., CD8+CCR10+ T cells were enriched in PsA. CD8+CCR10+ T cells expressed high levels of DNAX accessory molecule 1 and were effector memory cells that coexpressed skin-homing receptors CCR4 and cutaneous lymphocyte antigen. CD8+CCR10+ T cells were detected under inflammatory and homeostatic conditions in skin, but were not enriched in SF. Gene profiling further revealed that CD8+CCR10+ T cells expressed GATA3, FOXP3, and core transcriptional signature of tissue-resident memory T cells, including CD103. Specific genes, including RORC, IFNAR1, and ERAP1, were up-regulated in PsA compared to psoriasis. CD8+CCR10+ T cells were endowed with a Tc2/22-like cytokine profile, lacked cytotoxic potential, and displayed overall regulatory function., Conclusion: Tissue-resident memory CD8+ T cells derived from the skin are enhanced in the circulation of patients with PsA compared to patients with psoriasis alone. This may indicate that aberrances in cutaneous tissue homeostasis contribute to arthritis development., (© 2021 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3' untranslated region (3'-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3'-UTR reporter transgene to Ca 2+ , the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3'-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca 2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.

Dendritic cells (DCs) in peripheral tissues may have a unique role to regulate innate and adaptive immune responses to antigens that enter the tissues. Peritoneal cavity is the body compartment surrounding various tissues and organs and housing diverse immune cells. Here, we investigated the specialized features of classical DC (cDC) subsets following the intraperitoneal injection of a model antigen ovalbumin (OVA). Peritoneal cDC1s were superior to cDC2s in activating OVA-specific CD8 T cells, while both cDCs were similar in stimulating OVA-specific CD4 T cells. Each peritoneal cDC subset differentially regulated the homing properties of CD8 T cells. CD8 T cells stimulated by cDC1s displayed a higher level of lung-homing receptor CCR4, whereas those stimulated by cDC2s prominently expressed various homing receptors including gut-homing molecules CCR9 and α4β7. Also, we found that cDC1s played a dominating role over cDC2s in controlling the overall gene expression of CD8 T cells. Soluble factor(s) emanating from CD8 T cells stimulated by peritoneal cDC1s were responsible for mediating this dominance of cDC1s, and we identified IL-2 as a soluble factor regulating the global gene expression of T cells. Collectively, our study indicates that different peritoneal cDC subsets effectively diversify T cell responses by altering the level of cytokines, such as IL-2, in the milieu., Competing Interests: CP is employed by GENUV Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sohn, Na, Shin, Ryu, Park, In, Choi, Park, Hwang, Chu and Park.)

FoxP3 + regulatory T cells (Tregs) play crucial roles in peripheral immune tolerance. In addition, Tregs that reside or accumulate in nonlymphoid tissues, called tissue Tregs, exhibit tissue-specific functions and contribute to the maintenance of tissue homeostasis and repair. In an experimental mouse model of crescentic glomerulonephritis induced by an anti-glomerular basement membrane antibody, Tregs started to accumulate in the kidney on day 10 of disease onset and remained at high levels (~30-35% of CD4 + T cells) during the late stage (days 21-90), which correlated with stable disease control. Treg depletion on day 21 resulted in the relapse of renal dysfunction and an increase in Th1 cells, suggesting that Tregs are essential for disease control during the convalescence stage. The Tregs that accumulated in the kidney showed tissue Treg phenotypes, including high expression of GATA3, ST2 (the IL33 receptor subunit), amphiregulin (Areg), and PPARγ. Although T-bet + Tregs and RORγt + Tregs were observed in the kidney, GATA3 + Tregs were predominant during the convalescence stage, and a PPARγ agonist enhanced the accumulation of GATA3 + Tregs in the kidney. To understand the function of specific genes in kidney Tregs, we developed a novel T cell transfer system to T cell-deficient mice. This experiment demonstrates that ST2, Areg, and CCR4 in Tregs play important roles in the accumulation of GATA3 + Tregs in the kidney and in the amelioration of renal injury. Our data suggest that GATA3 is important for the recruitment of Tregs into the kidney, which is necessary for convalescence after renal tissue destruction.

Chemokines interact with chemokine receptors in a promiscuous network, such that each receptor can be activated by multiple chemokines. Moreover, different chemokines have been reported to preferentially activate different signalling pathways via the same receptor, a phenomenon known as biased agonism. The human CC chemokine receptors (CCRs) CCR4, CCR7 and CCR10 play important roles in T cell trafficking and have been reported to display biased agonism. To systematically characterize these effects, we analysed G protein- and β-arrestin-mediated signal transduction resulting from stimulation of these receptors by each of their cognate chemokine ligands within the same cellular background. Although the chemokines did not elicit ligand-biased agonism, the three receptors exhibited different arrays of signaling outcomes. Stimulation of CCR4 by either CC chemokine ligand 17 (CCL17) or CCL22 induced β-arrestin recruitment but not G protein-mediated signaling, suggesting that CCR4 has the potential to act as a scavenger receptor. At CCR7, both CCL19 and CCL21 stimulated G protein signaling and β-arrestin recruitment, with CCL19 consistently displaying higher potency. At CCR10, CCL27 and CCL28(4-108) stimulated both G protein signaling and β-arrestin recruitment, whereas CCL28(1-108) was inactive, suggesting that CCL28(4-108) is the biologically relevant form of this chemokine. These comparisons emphasize the intrinsic abilities of different receptors to couple with different downstream signaling pathways. Comparison of these results with previous studies indicates that differential agonism at these receptors may be highly dependent on the cellular context.

Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad coregulation of target mRNAs through the PUM-CNOT repression mechanism. Functional dissection of the domains of PUM1&2 identified a conserved amino-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 amino-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway., (© 2021 Enwerem et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Exposure to moderate doses of ionizing radiation (IR), which is sufficient for causing skin injury, can occur during radiation therapy as well as in radiation accidents. Radiation-induced skin injury occasionally recovers, although its underlying mechanism remains unclear. Moderate-dose IR is frequently utilized for bone marrow transplantation in mice; therefore, this mouse model can help understand the mechanism. We had previously reported that bone marrow-derived cells (BMDCs) migrate to the epidermis-dermis junction in response to IR, although their role remains unknown. Here, we investigated the role of BMDCs in radiation-induced skin injury in BMT mice and observed that BMDCs contributed to skin recovery after IR-induced barrier dysfunction. One of the important mechanisms involved the action of CCL17 secreted by BMDCs on irradiated basal cells, leading to accelerated proliferation and recovery of apoptosis caused by IR. Our findings suggest that BMDCs are key players in IR-induced skin injury recovery.

Chimeric antigen receptor (CAR) T cell therapy faces a number of challenges for the treatment of non-small-cell lung carcinoma (NSCLC), and efficient migration of circulating CAR T cells plays an important role in anti-tumor activity. In this study, a CAR specific for tumor antigen mesothelin (Msln-CAR) was co-expressed with cell chemokine receptors CCR2b or CCR4. Findings showed that CCR2b and CCR4 enhanced the migration of Msln-CAR T cell in vitro by transwell assay. When incubated with mesothelin-positive tumor cells, Msln-CCR2b-CAR and Msln-CCR4-CAR T cell specifically exerted potent cytotoxicity and produced high levels of proinflammatory cytokines, including IL-2, IFN-γ, and TNF-α. Furthermore, NSCLC cell line-derived xenograft (CDX) model was constructed by implanting subcutaneously modified A549 into NSG mice. Compared to conventional Msln-CAR T cells, living imaging indicated that Msln-CCR2b-CAR T cells displayed superior anti-tumor function due to enhanced migration and infiltration into tumor tissue shown by immunohistochemistry (IHC) analysis. In addition, histopathological examinations of mice organs showed that no obvious organic damages were observed. This is the first time that CAR T cell therapy combined with chemokine receptor is applied to NSCLC treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Wang, Yang, Yang, Lu, Zhao, Li, Pan, Jiang, Shen, Liang, Liang and Zhu.)

Background: Intracerebral 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., Methods: Two 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., Results: Endogenous 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., Conclusions: rCCL17-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.