The prognosis for patients with metastatic melanoma is poor and treatment options are limited. Genetically-engineered T cell therapy targeting chondroitin sulfate proteoglycan 4 (CSPG4), however, represents a promising treatment option, especially as both primary melanoma cells as well as metastases uniformly express CSPG4. Aiming to prevent off-tumor toxicity while maintaining a high cytolytic potential, we combined a chimeric co-stimulatory receptor (CCR) and a CSPG4-directed second-generation chimeric antigen receptor (CAR) with moderate potency. CCRs are artificial receptors similar to CARs, but lacking the CD3ζ activation element. Thus, T cells expressing solely a CCR, do not induce any cytolytic activity upon target cell binding, but are capable of boosting the CAR T cell response when both CAR and CCR engage their target antigens simultaneously. Here we demonstrate that co-expression of a CCR can significantly enhance the anti-tumor response of CSPG4-CAR T cells in vitro as well as in vivo. Importantly, this boosting effect was not dependent on co-expression of both CCR- and CAR-target on the very same tumor cell, but was also achieved upon trans activation. Finally, our data support the idea of using a CCR as a powerful tool to enhance the cytolytic potential of CAR T cells, which might open a novel therapeutic window for the treatment of metastatic melanoma.

The MC148 CC chemokine from the human poxvirus molluscum contagiosum (MCV) was probed in parallel with viral macrophage inflammatory protein (vMIP)-II encoded by human herpesvirus 8 (HHV8) in 16 classified human chemokine receptors. In competition binding using radiolabeled endogenous chemokines as well as radiolabeled MC148, MC148 bound with high affinity only to CCR8. In calcium mobilization assays, MC148 had no effect on its own on any of the chemokine receptors, but in a dose-dependent manner blocked the stimulatory effect of the endogenous I-309 chemokine on CCR8 without affecting chemokine-induced signaling of any other receptor. In contrast, vMIP-II acted as an antagonist on 10 of the 16 chemokine receptors, covering all four classes: XCR, CCR, CXCR, and CX(3)CR. In chemotaxis assays, MC148 specifically blocked the I-309-induced response but, for example, not stromal cell-derived factor 1alpha, monocyte chemoattractant protein 1, or interleukin 8-induced chemotaxis. We thus concluded that the two viruses choose two different ways to block the chemokine system: HHV8 encodes the broad-spectrum chemokine antagonist vMIP-II, whereas MCV encodes a highly selective CCR8 antagonist, MC148, conceivably to interfere with monocyte invasion and dendritic cell function. Because of its pharmacological selectivity, the MC148 protein could be a useful tool in the delineation of the role played by CCR8 and its endogenous ligand, I-309.

Tumor epithelial cells (TEpCs) and spindle-shaped stromal cells, not associated with the vasculature, of patients with early breast cancer express osteoprotegerin (OPG), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor kappa B ligand, stromal cell derived factor-1, interleukin-6, macrophage colony stimulating factor, chemokine (C-C motif) ligand-2 (CCL-2) and their receptors at significantly higher levels compared with non-neoplastic breast tissues. We evaluated the clinicopathological significance of these ligands and receptors in TEpC and spindle-shaped stromal cells, not associated with the vasculature, to determine their impact on prognosis of patients with early-stage breast cancer.

The Kaposi's sarcoma-related herpesvirus (KSHV), also designated human herpesvirus 8, is the presumed etiologic agent of Kaposi's sarcoma and certain lymphomas. Although KSHV encodes several chemokine homologues (viral macrophage inflammatory protein [vMIP]-I, -II, and -III), only vMIP-II has been functionally characterized. We report here that vMIP-I is a specific agonist for the CC chemokine receptor (CCR)8 that is preferentially expressed on Th2 T cells. Y3 cells transfected with CCR8 produced a calcium flux in response to vMIP-I and responded vigorously in in vitro chemotaxis assays. In competition binding experiments, the interaction of vMIP-I with CCR8 was shown to be specific and of high affinity. In contrast to its agonist activity at CCR8, vMIP-I did not interact with CCR5 or any of 11 other receptors examined. Furthermore, vMIP-I was unable to inhibit CCR5-mediated HIV infection. These findings suggest that expression of vMIP-I by KSHV may influence the Th1/Th2 balance of the host immune response.

Elimination of viral infections is dependent on rapid recruitment of leucocytes to infected areas. Chemokines constitute a class of cytokines that regulate migration of leucocytes to sites of infection. In this work, the expression and function of CC chemokine receptor (CCR)1 and CCR5 and their ligands during a generalized herpes simplex virus type 2 (HSV-2) infection in mice were studied. Many CCR1 and CCR5 ligands were expressed in infected organs after intraperitoneal infection. In particular, CC chemokine expression in the liver preceded the expression of CCR1 and CCR5, suggesting recruitment of cells bearing these receptors, which correlated with a decrease in viral titres. Administration of Met-RANTES, a CCR1 and CCR5 antagonist, led to impaired antiviral response with significantly higher viral titre in the liver on days 1 and 6 after infection. This observation was accompanied by a decreased and shortened recruitment of natural killer cells to the peritoneum of infected mice treated with the antagonist. Despite this reduced recruitment of antiviral leucocytes in mice receiving Met-RANTES, peritoneal cells from these mice produced markedly enhanced levels of pro-inflammatory cytokines. Altogether, the results suggest that CCR1 and/or CCR5 are important for both viral clearance and eventual control of the immune response.

Isolated peripheral blood CD4 cells from allergic individuals express CC chemokine receptor (CCR)3 and CCR4 after expansion in vitro. In addition, human T helper type 2 (Th2) cells polarized in vitro selectively express CCR3 and CCR4 at certain stages of activation/differentiation and respond preferentially to the ligands eotaxin and monocyte-derived chemokine (MDC). However, controversy arises when the in vivo significance of this distinct expression is discussed. To address the functional role of CCR3/eotaxin and CCR4/MDC during the in vivo recruitment of Th2 cells, we have transferred effector Th cells into naive mice to induce allergic airway disease. Tracking of these cells after repeated antigen challenge has established that both CCR3/eotaxin and CCR4/MDC axes contribute to the recruitment of Th2 cells to the lung, demonstrating the in vivo relevance of the expression of these receptors on Th2 cells. We have shown that involvement of the CCR3/eotaxin pathway is confined to early stages of the response in vivo, whereas repeated antigen stimulation results in the predominant use of the CCR4/MDC pathway. We propose that effector Th2 cells respond to both CCR3/eotaxin and CCR4/MDC pathways initially, but that a progressive increase in CCR4-positive cells results in the predominance of the CCR4/MDC axis in the long-term recruitment of Th2 cells in vivo.

In view of the previously described anti-inflammatory effects of insulin, we investigated the potential suppressive effect of insulin on plasma concentrations and expression of the chemokines, monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T-cell expressed and secreted (RANTES) and their receptors, chemokine receptor (CCR)-2 and CCR-5, in mononuclear cells (MNCs). We also investigated the effect of insulin on other chemokines.

Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation.

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1alpha, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1alpha led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8alpha(1) T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection-induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell-specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine-biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major-resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major-susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.

Wound healing is a multi-phased pathophysiological process requiring chemoattractant receptor-dependent accumulation of myeloid cells in the lesion. Two G protein-coupled formylpeptide receptors Fpr1 and Fpr2 mediate rapid neutrophil infiltration in the liver of Listeria-infected mice by sensing pathogen-derived chemotactic ligands. These receptors also recognize host-derived chemotactic peptides in inflammation and injury. Here we report the capacity of Fprs to promote the healing of sterile skin wound in mice by initiating neutrophil infiltration. We found that in normal miceneutrophils rapidly infiltrated the dermis in the wound before the production of neutrophil-specific chemokines by the injured tissue. In contrast, rapid neutrophil infiltration was markedly reduced with delayed wound closure in mice deficient in both Fprs. In addition, we detected Fpr ligand activity that chemoattracted neutrophils into the wound tissue. Our study thus demonstrates that Fprs are critical for normal healing of the sterile skin wound by mediating the first wave of neutrophil infiltration.

Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated by binding of chemoattractants to interleukin (IL) 8 receptors RA and RB (IL8RA/B) C-C chemokine receptors (CCR) 2 and 5 expressed on neutrophil and monocyte/macrophage membranes. Endothelial cells (ECs) derived from rat-induced pluripotent stem cells (RiPS) were transduced with adenovirus containing cDNA of IL8RA/B and/or CCR2/5. We hypothesized that RiPS-ECs overexpressing IL8RA/B (RiPS-IL8RA/B-ECs), CCR2/5 (RiPS-CCR2/5-ECs), or both receptors (RiPS-IL8RA/B+CCR2/5-ECs) will inhibit inflammatory responses and neointima formation in balloon-injured rat carotid artery. Twelve-week-old male Sprague-Dawley rats underwent balloon injury of the right carotid artery and intravenous infusion of (a) saline vehicle, (b) control RiPS-Null-ECs (ECs transduced with empty virus), (c) RiPS-IL8RA/B-ECs, (d) RiPS-CCR2/5-ECs, or (e) RiPS-IL8RA/B+CCR2/5-ECs. Inflammatory mediator expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 hours postinjury by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Neointima formation was assessed at 14 days postinjury. RiPS-ECs expressing the IL8RA/B or CCR2/5 homing device targeted the injured arteries and decreased injury-induced inflammatory cytokine expression, neutrophil/macrophage infiltration, and neointima formation. Transfused RiPS-ECs overexpressing IL8RA/B and/or CCR2/5 prevented inflammatory responses and neointima formation after vascular injury. Targeted delivery of iPS-ECs with a homing device to inflammatory mediators in injured arteries provides a novel strategy for the treatment of cardiovascular diseases. Stem Cells Translational Medicine 2017;6:1168-1177.

Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in children. Treatment outcomes, particularly for relapsed/refractory or metastatic disease, have not improved in decades. The current lack of novel therapies and low tumor mutational burden suggest that chimeric antigen receptor (CAR) T-cell therapy could be a promising approach to treating RMS. Previous work identified FGF receptor 4 (FGFR4, CD334) as being specifically upregulated in RMS, making it a candidate target for CAR T cells. We tested the feasibility of an FGFR4-targeted CAR for treating RMS using an NSG mouse with RH30 orthotopic (intramuscular) tumors. The first barrier we noted was that RMS tumors produce a collagen-rich stroma, replete with immunosuppressive myeloid cells, when T-cell therapy is initiated. This stromal response is not seen in tumor-only xenografts. When scFV-based binders were selected from phage display, CARs targeting FGFR4 were not effective until our screening approach was refined to identify binders to the membrane-proximal domain of FGFR4. Having improved the CAR, we devised a pharmacologic strategy to augment CAR T-cell activity by inhibiting the myeloid component of the T-cell-induced tumor stroma. The combined treatment of mice with anti-myeloid polypharmacy (targeting CSF1R, IDO1, iNOS, TGFbeta, PDL1, MIF, and myeloid misdifferentiation) allowed FGFR4 CAR T cells to successfully clear orthotopic RMS tumors, demonstrating that RMS tumors, even with very low copy-number targets, can be targeted by CAR T cells upon reversal of an immunosuppressive microenvironment.

Adoptive T-cell therapy aims to achieve lasting tumor clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T-cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilizing nonnative dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilizing the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimicked the cytokine receptor heterodimerization, with transcriptomic signatures like those obtained by activation of the native IL2 receptor. Moreover, we found that this dimerization structure was agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs coexpressed with a clinically relevant solid tumor-specific GD2-specific chimeric antigen receptor (CAR). Based on this characterization, we suggest that the coexpression of either the common β-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T-cell expansion, engraftment, and efficacy. Our results demonstrate how Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T-cell therapies, as well as therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T-cell therapies.

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.

The present study was designed to investigate the effect of bacterial lipopolysaccharide (LPS) on C-C chemokine receptors (CCR) expressed in human mononuclear phagocytes. LPS caused a rapid and drastic reduction of CCR2 mRNA levels, which binds MCP-1 and -3. CCR1 and CCR5 mRNAs were also reduced, though to a lesser extent, whereas CXCR2 was unaffected. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced from 1.5 h to 45 min. As expected, LPS-induced inhibition of CCR2 mRNA expression was associated with a reduction of both MCP-1 binding and chemotactic responsiveness. The capacity to inhibit CCR2 expression in monocytes was shared by other microbial agents and cytokines (inactivated Streptococci, Propionibacterium acnes, and to a lesser extent, IL-1 and TNF-alpha). In contrast, IL-2 augmented CCR2 expression and MCP-1 itself had no effect. These results suggest that, regulation of receptor expression in addition to agonist production is likely a crucial point in the regulation of the chemokine system.

Listeria monocytogenes (Listeria) causes opportunistic infection in immunocompromised hosts with high mortality. Resistance to Listeria depends on immune responses and recruitment of neutrophils of the immune system into infected sites is an early and critical step. Mouse neutrophils express two G protein-coupled formylpeptide receptor subtypes Fpr1 and Fpr2 that recognize bacterial and host-derived chemotactic molecules including Listeria peptides for cell migration and activation. Here we report deficiency in Fprs exacerbated the severity of the infection and increased the mortality of infected mice. The mechanism involved impaired early neutrophil recruitment to the liver with Fpr1 and Fpr2 being sole receptors for neutrophils to sense Listeria chemoattractant signals and for production of bactericidal superoxide. Thus, Fprs are essential sentinels to guide the first wave of neutrophil infiltration in the liver of Listeria-infected mice for effective elimination of the invading pathogen.

Bone marrow-derived mesenchymal stem cells (BMSCs) are increasingly being used in bone marrow transplantation (BMT) to enable homing of the allogeneic hematopoietic stem cells and suppress acute graft versus host disease (aGVHD). The aim of this study was to optimize the labelling of BMSCs with superparamagnetic iron oxide particles (SPIOs), and evaluate the impact of the SPIOs on the biological characteristics, gene expression profile and chemotaxis function of the BMSCs. The viability and proliferation rates of the SPIO-labeled BMSCs were analyzed by trypan blue staining and CCK-8 assay respectively, and the chemotaxis function was evaluated by the transwell assay. The expression levels of chemokine receptors were measured by RT-PCR and flow cytometry. The SPIOs had no effect on the viability of the BMSCs regardless of the labelling concentration and culture duration. The labelling rate of the cells was higher when cultured for 48 h with the SPIOs. Furthermore, cells labeled with 25 µg/ml SPIOs for 48 h had the highest proliferation rates, along with increased expression of chemokine receptor genes and proteins. However, there was no significant difference between the chemotaxis function of the labeled and unlabeled BMSCs. To summarize, labelling BMSCs with 25 µg/ml SPIOs for 48h did not affect their biological characteristics and chemotaxis function, which can be of significance for in vivo applications.

In bovine placentomes, the inflammatory response is considered important for the detachment of the fetal membrane from the caruncle after parturition. Glucocorticoids, a trigger of the onset of parturition, facilitate functional maturation of placentomes via prostaglandin (PG) and estrogen production in cattle. This study investigated how exogeneous glucocorticoids, which exert immunosuppressive effects, affect placental inflammation at parturition. Placentomes were collected immediately after spontaneous or induced parturition. Parturition was conventionally induced using PGF2α or dexamethasone or with a combination of triamcinolone acetonide and high-dose betamethasone (TABET treatment). Polymerase chain reaction (PCR) array analysis indicated that 9/13 C-C motif chemokine ligands (CCLs) were upregulated > two-fold in spontaneous parturition, with CCL2 and CCL8 being highly expressed. The expressions of CCL2, CCL8, C-C motif chemokine receptor 1 (CCR1), and CCR5 in caruncles were significantly higher in spontaneous parturition than in induced parturition. Although the clinical dose of dexamethasone did not influence the expression of these CCLs and CCRs, TABET treatment increased CCR1 expression. CCL8, CCR1, CCR2, and CCR5 were localized in the caruncular epithelial cells. CCR2 was also localized in the epithelial cells of the cotyledonary villi. This study is the first report to reveal the disruption in CCL and CCR expression in bovine placentomes at induced parturition. Enhanced glucocorticoid exposure for the induction of parturition may upregulate CCR1 expression in placentomes, but the treatment does not adequately promote CCL expression. Additionally, immunohistochemistry suggested that the CCL-CCR system is involved in the functional regulation of maternal and fetal epithelial cells in placentomes at parturition.

Chemokines are crucial regulators of cell mobilization for development, homeostasis, and immunity. Chemokines signal through binding to chemokine receptors, a superfamily of seven-transmembrane domain G-coupled receptors. In the present study, eleven CC chemokine receptors (CCRs) and seven CXC chemokine receptors (CXCRs) were identified from turbot genome. Phylogenetic and syntenic analyses were performed to annotate these genes, indicating the closest relationship between the turbot chemokine receptors and their counterparts of Japanese flounders (Paralichthys olivaceus). Evolutionary analyses revealed that the tandem duplications of CCR8 and CXCR3, the whole genome duplications of CCR6, CCR9, CCR12, and CXCR4, and the teleost-specific CCR12 led to the expansion of turbot chemokine receptors. In addition, turbot chemokine receptors were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in spleen, gill, and head kidney. Moreover, most turbot chemokine receptors were significantly differentially expressed in spleen and gill after Aeromonas salmonicida infection, and exhibited general down-regulations at early time points and then gradually up-regulated. Finally, protein-protein interaction network (PPI) analyses indicated that chemokine receptors interacted with a few immune-related genes such as interleukins, Grk genes, CD genes, etc. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokine receptors in turbots.

Chemokines are vital regulators of cell mobilization for immune surveillance, inflammation, and development. Chemokines signal through binding to their receptors that are a superfamily of seven-transmembrane domain G-coupled receptors. Recently, a complete repertoire of both CC and CXC chemokines have been identified in channel catfish, but nothing is known about their receptors. In this study, a set of 29 CC chemokine receptor (CCR) genes and 8 CXC chemokine receptor (CXCR) genes were identified and annotated from the channel catfish genome. Extensive phylogenetic and comparative genomic analyses were conducted to annotate these genes, revealing fish-specific CC chemokine receptors, and lineage-specific tandem duplications of chemokine receptors in the teleost genomes. With 29 genes, the channel catfish genome harbors the largest numbers of CC chemokine receptors among all the genomes characterized. Analysis of gene expression after bacterial infections indicated that the chemokine receptors were regulated in a gene-specific manner. Most differentially expressed chemokine receptors were up-regulated after Edwardsiella ictaluri and Flavobacterium columnare infection. Among which, CXCR3 and CXCR4 were observed to participate in immune responses to both bacterial infections, indicating their potential roles in catfish immune activities. In addition, CXCR3.2 was significantly up-regulated in ESC-susceptible fish, and CXCR4b was mildly induced in ESC-resistant fish, further supporting the significant roles of CXCR3 and CXCR4 in catfish immune responses. CXCR4b and CCR9a were both up-regulated not only after bacterial infection, but also after hypoxia stress, providing the linkage between bacterial infection and low oxygen stresses. These results should be valuable for comparative immunological studies and provide insights into their roles in disease and stress responses.