Repertoire Of Chemokine Receptors Research Articles

Mapping the chemotactic landscape in NK cells reveals subset-specific synergistic migratory responses to dual chemokine receptor ligation

Natural killer (NK) cells have a unique capability of spontaneous cytotoxicity against malignant cells and hold promise for off-the-shelf cell therapy against cancer. One of the key challenges in the field is to improve NK cell homing to solid tumors. To gain a deeper understanding of the cellular mechanisms regulating trafficking of NK cells into the tumor, we used high-dimensional flow cytometry, mass cytometry, and single-cell RNA-sequencing combined with functional assays, creating a comprehensive map of human NK cell migration phenotypes. We found that the chemokine receptor repertoire of peripheral blood NK cells changes in a coordinated manner becoming progressively more diversified during NK cell differentiation and correlating tightly with the migratory response of the distinct NK cell subsets. Simultaneous ligation of CXCR1/2 and CX3CR1, synergistically potentiated the migratory response of NK cells. Analysis of 9471 solid cancers from publicly available TCGA/TARGET repositories revealed dominant chemokine patterns that varied across tumor types but with no tumor group expressing ligands for more than one chemokine receptor present on mature NK cells. The finding that chemokine stimulation can elicit a synergistic migratory response in NK cells combined with the identified lack of naturally occurring pairs of chemokines-chemokine receptors in human cancers may explain the systematic exclusion of NK cells from the tumor microenvironment and provides a basis for engineering next-generation NK cell therapies against malignancies. The Polish Ministry of Science and Higher Education, the National Science Centre, Poland, The Norwegian Cancer Society, the Norwegian Research Council, the South-Eastern Norway Regional Health Authority, The Swedish Cancer Society, the Swedish Children's Cancer Foundation, The Swedish Research Council, The Center of Excellence: Precision Immunotherapy Alliance, Knut and Alice Wallenberg Foundation and National Cancer Institute.

Inhibitory axes impacting on the activity and fate of Innate Lymphoid Cells

In neoplastic patients, an effective immune response ideally should be achieved by the coordinated action of different immune cells with tumor-suppressive functions. These include the more cytolytic members of the Innate Lymphoid Cells (ILCs) family represented by the Natural Killer (NK) cells, whose activities in cancer patients, however, can be hampered by several inhibitory signals. These are generated by membrane-bound and soluble molecules that, interacting with specific inhibitory receptors, create inhibitory axes impacting the NK cell differentiation and effector functions. These breaks, which now represent major immunotherapeutic targets, may be sensitive to interferon (IFN)-γ, whose source, in vivo, is represented by different cell types including the NK and ILC1. Since also ILCs can express receptors of the inhibitory axes like PD-1 and TIGIT, their therapeutic blockade might further amplify the IFN-γ release that, as an unwanted side effect, would promote the onset of NK cell-resistant tumor variants (NKRTV) expressing ligands involved in inhibitory axes. These variants might also arise from the activity of other cytokines such as IL-27, which can increase the expression of HLA class I and PD-Ls in different cell types, including tumor cells. Besides the amplification of membrane-bound inhibitory axes, tumors can reduce the number of infiltrating cytolytic ILCs, promote the recruitment of poorly cytolytic NK cell subsets, and manipulate to their advantage the infiltrating immune cells, which acquire tumor-promoting activities. This occurs thanks to the production of soluble factors including TGF-β1 and IL-18 that, alone or in combination, modify the activating and chemokine receptor repertoire of NK cells, and induce the ILCs differentiation towards cells ineffective in fighting cancer or, even worse, with tumor-promoting functions.The present review aims to present and discuss major inhibitory axes impacting on ILCs functions, migration, and differentiation with a major focus on tumor context.

New Insights on the Emerging Genomic Landscape of CXCR4 in Cancer: A Lesson from WHIM.

Deciphering the molecular alterations leading to disease initiation and progression is currently crucial to identify the most relevant targets for precision therapy in cancer patients. Cancers express a complex chemokine network influencing leucocyte infiltration and angiogenesis. Moreover, malignant cells also express a selective repertoire of chemokine receptors that sustain their growth and spread. At present, different cancer types have been shown to overexpress C-X-C chemokine receptor type 4 (CXCR4) and to respond to its ligand C-X-C motif chemokine 12 (CXCL12). The CXCL12/CXCR4 axis influences cancer biology, promoting survival, proliferation, and angiogenesis, and plays a pivotal role in directing migration of cancer cells to sites of metastases, making it a prognostic marker and a therapeutic target. More recently, mutations in the C-terminus of CXCR4 have been identified in the genomic landscape of patients affected by Waldenstrom’s macroglobulinemia, a rare B cell neoplasm. These mutations closely resemble those occurring in Warts, Hypogammaglobulinemia, Immunodeficiency, and Myelokathexis (WHIM) syndrome, an immunodeficiency associated with CXCR4 aberrant expression and activity and with chemotherapy resistance in clinical trials. In this review, we summarize the current knowledge on the relevance of CXCR4 mutations in cancer biology, focusing on its importance as predictors of clinical presentation and response to therapy.

Combined GM-CSF and G-CSF administration mobilizes CD4+ CD25hi Foxp3hi Treg in leukapheresis products of rhesus monkeys.

Early phase clinical trials are evaluating the feasibility, safety, and therapeutic potential of ex vivo expanded regulatory T cells (Treg) in transplantation. A limitation is the paucity of naturally occurring Treg numbers in peripheral blood. Hence, protracted ex vivo expansion is required to obtain sufficient Treg in order to meet target cell doses. Because cytokine administration has been used successfully to mobilize immune cells to the peripheral blood in experimental and clinical studies, we hypothesized that granulocyte macrophage-colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) administration would enhance Treg percentages in leukapheresis products of rhesus monkeys. Following combined GM-CSF and G-CSF administration, the incidence of Treg in peripheral blood and leukapheresis products was elevated significantly, where approximately 3.7×106 /kg CD4+ CD25hi Foxp3hi or 6.8×106 /kg CD4+ CD25hi CD127lo Treg can be collected from individual products. Mobilized Treg expressed a comparable repertoire of surface markers, chemokine receptors, and transcription factors to naïve monkey peripheral blood Treg. Furthermore, when expanded ex vivo, mobilized leukapheresis product and peripheral blood Treg exhibited similar ability to suppress autologous CD4+ and CD8+ T cell proliferation. These observations indicate that leukapheresis products from combined GM-CSF- and G-CSF-mobilized individuals are a comparatively rich source of Treg and may circumvent long-term ex vivo expansion required for therapeutic application.

Chemokine ligand\u2013receptor interactions critically regulate cutaneous wound healing

BackgroundWound healing represents a dynamic process involving directional migration of different cell types. Chemokines, a family of chemoattractive proteins, have been suggested to be key players in cell-to-cell communication and essential for directed migration of structural cells. Today, the role of the chemokine network in cutaneous wound healing is not fully understood. Unraveling the chemokine-driven communication pathways in this complex process could possibly lead to new therapeutic strategies in wound healing disorders.MethodsWe performed a systematic, comprehensive time-course analysis of the expression and function of a broad variety of cytokines, growth factors, adhesion molecules, matrixmetalloproteinases and chemokines in a murine cutaneous wound healing model.ResultsStrikingly, chemokines were found to be among the most highly regulated genes and their expression was found to coincide with the expression of their matching receptors. Accordingly, we could show that resting and activated human primary keratinocytes (CCR3, CCR4, CCR6, CXCR1, CXCR3), dermal fibroblasts (CCR3, CCR4, CCR10) and dermal microvascular endothelial cells (CCR3, CCR4, CCR6, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3) express a distinct and functionally active repertoire of chemokine receptors. Furthermore, chemokine ligand–receptor interactions markedly improved the wound repair of structural skin cells in vitro.ConclusionTaken together, we here present the most comprehensive analysis of mediators critically involved in acute cutaneous wound healing. Our findings suggest therapeutic approaches for the management of wound closure by targeting the chemokine network.

Imatinib and Nilotinib Off-Target Effects on Human NK Cells, Monocytes, and M2 Macrophages.

Tyrosine kinase inhibitors (TKIs) are used in the clinical management of hematological neoplasms. Moreover, in solid tumors such as stage 4 neuroblastomas (NB), imatinib showed benefits that might depend on both on-target and immunological off-target effects. We investigated the effects of imatinib and nilotinib on human NK cells, monocytes, and macrophages. High numbers of monocytes died upon exposure to TKI concentrations similar to those achieved in patients. Conversely, NK cells were highly resistant to the TKI cytotoxic effect, were properly activated by immunostimulatory cytokines, and degranulated in the presence of NB cells. In NB, neither drug reduced the expression of ligands for activating NK receptors or upregulated that of HLA class I, B7-H3, PD-L1, and PD-L2, molecules that might limit NK cell function. Interestingly, TKIs modulated the chemokine receptor repertoire of immune cells. Acting at the transcriptional level, they increased the surface expression of CXCR4, an effect observed also in NK cells and monocytes of patients receiving imatinib for chronic myeloid leukemia. Moreover, TKIs reduced the expression of CXCR3 (in NK cells) and CCR1 (in monocytes). Monocytes also decreased the expression of M-CSFR, and low numbers of cells underwent differentiation toward macrophages. M0 and M2 macrophages were highly resistant to TKIs and maintained their phenotypic and functional characteristics. Importantly, also in the presence of TKIs, the M2 immunosuppressive polarization was reverted by TLR engagement, and M1-oriented macrophages fully activated autologous NK cells. Our results contribute to better interpreting the off-target efficacy of TKIs in tumors and to envisaging strategies aimed at facilitating antitumor immune responses.

TGF-β1 Downregulates the Expression of CX3CR1 by Inducing miR-27a-5p in Primary Human NK Cells.

Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX3CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX3CR1. We demonstrated the functional interaction of miR-27a-5p with the 3′ untranslated region (3′UTR) of CX3CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX3CR1 3′UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX3CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX3CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX3CR1 expression.

Differences in phenotype, homing properties and suppressive activities of regulatory T cells induced by epicutaneous, oral or sublingual immunotherapy in mice sensitized to peanut

Allergen-specific immunotherapy has been proposed as an attractive strategy to actively treat food allergy using the following three different immunotherapy routes: oral (OIT), sublingual (SLIT) and epicutaneous (EPIT) immunotherapy. Regulatory T cells (Tregs) have been shown to have a pivotal role in the mechanisms of immunotherapy. The aim of this study was to compare the phenotype and function of Tregs induced in peanut-sensitized BALB/c mice using these three routes of treatment. We show that although EPIT, OIT and SLIT were all able to effectively desensitize peanut-sensitized mice, they induced different subsets of Tregs. Foxp3+ Tregs were induced by the three treatment routes but with greater numbers induced by EPIT. EPIT and OIT also increased the level of LAP+ Tregs, whereas SLIT induced IL-10+ cells. The suppressive activity of EPIT-induced Tregs did not depend on IL-10 but required CTLA-4, whereas OIT acted through both mechanisms and SLIT was strictly dependent on IL-10. Moreover, the three routes influenced the homing properties of induced Tregs differently, with a larger repertoire of chemokine receptors expressed by EPIT-induced Tregs compared with OIT- and SLIT- induced cells, resulting in different protective consequences against allergen exposure. Furthermore, whereas OIT- or SLIT-induced Tregs lost their suppressive activities after treatment was discontinued, the suppressive activities of EPIT-induced Tregs were still effective 8 weeks after the end of treatment, suggesting the induction of a more long-lasting tolerance. In summary, EPIT, OIT and SLIT mediated desensitization through the induction of different subsets of Tregs, leading to important differences in the subsequent protection against allergen exposure and the possible induction of tolerance.

Abstract A66: Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation

Abstract Tumor-recruited myeloid cells represent a significant portion of inflammatory cells within the tumor microenvironment and influence nearly all steps of tumor progression. Although tumor-associated neutrophils (TANs) are present in large numbers, the role of TANs in cancer progression remains unclear and has only been recently investigated in murine models. The goal of this study was to provide a phenotypic and functional characterization of TANs in freshly harvested surgically resected lung cancer samples. Tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. Importantly, fresh lung tumors from patients were processed within 20 minutes of removal from the patient. We developed an approach to prepare human lung tumors for immunological studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This approach retrieved all major tumor-associated cell populations with high cell yield and maintained the expression of cell surface markers and the functions of isolated cells. In multicolor tracings, TANs were defined as CD15hi/CD66b+/CD11b+/MPO+/Arg1+/IL-5Ra-/ cells. We found that TANs comprised 5—25% of cells isolated from all digested human lung tumors (n=42). Compared to blood neutrophils, TANs displayed an activated phenotype (CD62Llo/CD54hi) and a novel repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. They also produced substantial quantities of the pro-inflammatory factors MCP-1, IL-8, MIP-1a, and IL-6, as well as the anti-inflammatory IL-1R antagonist. TANs were not hypofunctional cells, since they were able to phagocytize bacteria and produce reactive oxygen species. Understanding the role of TANs in regulating T cell responses in cancer patients is particularly important because cytotoxic T lymphocytes are the chief effector cells mediating antigen-driven anti-tumor immunity. We found that TANs were able to stimulate antigen non-specific T cell proliferation and IFN-γ release. Crosstalk between TANs and activated T cells led to substantial up-regulation of CD54, CD86, OX40L, and 4-1BBL co-stimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive feedback loop. This stimulatory activity of TANs appeared to be related to tumor size, as TANs from the majority of the smaller tumors (< 3cm) were more stimulatory than those from larger tumors. We also identified a novel subset of TANs with a hybrid “composite” phenotype of neutrophils and antigen-presenting cells (MPO+CD66b+CD14+HLA-DR+CCR7+CD86+). We found that this hybrid subset had the ability to: 1) dramatically stimulate antigen non-specific T cell proliferation, 2) induce the proliferation of allogeneic T cells in mixed lymphocyte reaction and 3) trigger antigen-specific memory T cell response to a mixture of T cell epitopes from human CMV, Epstein-Barr, flu viruses, as well as tetanus toxoid from all the common HLA types. Thus, our data suggest that in patients with early-stage lung cancer, TANs do not significantly contribute to inhibition of T cell responses. Rather, the majority of neutrophils recruited into the tumor microenvironment undergo phenotypic and functional changes that result in the formation of cells that resemble the murine anti-tumor “N1 TANs” and could potentially augment and support T cells responses. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Tom Stephen, Anjana Ranganathan, Charuhas Deshpande, Tatiana Akimova, Anil Vachani, Leslie Litzky, Wayne Hancock, Jose Conejo-Garcia, Michael Feldman, Sunil Singhal, Steven Albelda. Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A66.

Human liver-resident CD56bright/CD16neg NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways

RationaleThe liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. ObjectivesWe took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. FindingsNearly 50% of the entire liver NK cell population is composed of functionally relevant CD56bright lr-NK cells that localize within hepatic sinusoids. CD56bright lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids express CCL3pos Kupffer cells, CXCL16pos endothelial cells and CCL5pos T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56bright NK cells that constitutively express CCR5 and CXCR6. CD56bright lr-NK cells co-exist with CD56dim conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56dim c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. ConclusionOur findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56bright lr-NK cells will require modification of hepatic CCR5 and CXCR6.

Polyphenol administration impairs T-cell proliferation by imprinting a distinct dendritic cell maturational profile.

Currently little is known as to how nutritionally derived compounds may affect dendritic cell (DC) maturation and potentially prevent inappropriate inflammatory responses that are characteristic of chronic inflammatory syndromes. Previous observations have demonstrated that two polyphenols quercetin and piperine delivered through reconstituted oil bodies (ROBs-QP) can influence DC maturation in response to LPS leading to a modulated inflammatory response. In the present study, we examined the molecular effects of ROBs-QP exposure on DC differentiation in mice and identified a unique molecular signature in response to LPS administration that potentially modulates DC maturation and activity in inflammatory conditions. Following LPS administration, ROBs-QP-exposed DCs expressed an altered molecular profile as compared with control DCs, including cytokine and chemokine production, chemokine receptor repertoire, and antigen presentation ability. In vivo ROBs-QP administration suppresses antigen-specific T-cell division in the draining lymph nodes resulting from a reduced ability to create stable immunological synapse. Our data demonstrate that polyphenols exposure can drive DCs toward a new anti-inflammatory molecular profile capable of dampening the inflammatory response, highlighting their potential as complementary nutritional approaches in the treatment of chronic inflammatory syndromes.

The Jekyll and Hyde story of IL17-Producing γδT Cells.

In comparison to conventional αβT cells, γδT cells are considered as specialized T cells based on their contributions in regulating immune response. γδT cells sense early environmental signals and initiate local immune-surveillance. The development of functional subtypes of γδT cells takes place in the thymus but they also exhibit plasticity in response to the activating signals and cytokines encountered in the extrathymic region. Thymic development of Tγδ1 requires strong TCR, CD27, and Skint-1 signals. However, differentiation of IL17-producing γδT cells (Tγδ17) is independent of Skint-1 or CD27 but requires notch signaling along with IL6 and TGFβ cytokines in the presence of weak TCR signal. In response to cytokines like IL23, IL6, and IL1β, Tγδ17 outshine Th17 cells for early activation and IL17 secretion. Despite expressing similar repertoire of lineage transcriptional factors, cytokines, and chemokine receptors, Tγδ17 cells differ from Th17 in spatial and temporal fashion. There are compelling reasons to consider significant role of Tγδ17 cells in regulating inflammation and thereby disease outcome. Tγδ17 cells regulate mobilization of innate immune cells and induce keratinocytes to secrete anti-microbial peptides thus exhibiting protective functions in anti-microbial immunity. In contrast, dysregulated Tγδ17 cells inhibit Treg cells, exacerbate autoimmunity, and are also known to support carcinogenesis by enhancing angiogenesis. The mechanism associated with this dual behavior of Tγδ17 is not clear. To exploit, Tγδ17 cells for beneficial use requires comprehensive analysis of their biology. Here, we summarize the current understanding on the characteristics, development, and functions of Tγδ17 cells in various pathological scenarios.

Chemokine-mediated inflammation in the degenerating retina is coordinated by Müller cells, activated microglia, and retinal pigment epithelium.

BackgroundMonocyte infiltration is involved in the pathogenesis of many retinal degenerative conditions. This process traditionally depends on local expression of chemokines, though the roles of many of these in the degenerating retina are unclear. Here, we investigate expression and in situ localization of the broad chemokine response in a light-induced model of retinal degeneration.MethodsSprague–Dawley (SD) rats were exposed to 1,000 lux light damage (LD) for up to 24 hrs. At time points during (1 to 24 hrs) and following (3 and 7 days) exposure, animals were euthanized and retinas processed. Microarray analysis assessed differential expression of chemokines. Some genes were further investigated using polymerase chain reaction (PCR) and in situ hybridization and contrasted with photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Recruitment of retinal CD45+ leukocytes was determined via fluorescence activated cell sorting (FACS), and expression of chemokine receptors determined using PCR.ResultsExposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10. Their upregulation correlated strongly with peak photoreceptor death, at 24 hrs exposure. In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE). This preceded large increases in the number of CD45+ cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.ConclusionsOur data indicate that retinal degeneration induces upregulation of a broad chemokine response whose expression is coordinated by Müller cells, microglia, and RPE. The findings inform our understanding of the processes govern the trafficking of leukocytes, which are contributors in the pathology of retinal degenerations.

Chemokine receptors in Atlantic salmon

Teleost sequence data have revealed that many immune genes have evolved differently when compared to other vertebrates. Thus, each gene family needs functional studies to define the biological role of individual members within major species groups. Chemokine receptors, being excellent markers for various leukocyte subpopulations, are one such example where studies are needed to decipher individual gene function. The unique salmonid whole genome duplication that occurred approximately 95 million years ago has provided salmonids with many additional duplicates further adding to the complexity and diversity. Here we have performed a systematic study of these receptors in Atlantic salmon with particular focus on potential inflammatory receptors.Using the preliminary salmon genome data we identified 48 chemokine or chemokine-like receptors including orthologues to the ten receptors previously published in trout. We found expressed support for 40 of the bona fide salmon receptors. Eighteen of the chemokine receptors are duplicated, and when tested against a diploid sister group the majority were shown to be remnants of the 4R whole genome duplication with subsequent high sequence identity. The salmon chemokine receptor repertoire of 40 expressed bona fide genes is comparably larger than that found in humans with 23 receptors. Diversification has been a major driving force for these duplicate genes with the main variability residing in ligand binding and signalling domains.

Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

TLR activation of tumor‐associated macrophages from ovarian cancer patients triggers cytolytic activity of NK cells

We analyzed the functional outcome of the interaction between tumor-associated macrophages (TAMs) and natural killer (NK) cells. TAMs from ascites of ovarian cancer patients displayed an alternatively activated functional phenotype (M2) characterized by a remarkably high frequency and surface density of membrane-bound IL-18. Upon TLR engagement, TAMs acquired a classically activated functional phenotype (M1), released immunostimulatory cytokines (IL-12, soluble IL-18), and efficiently triggered the cytolytic activity of NK cells. TAMs also induced the release of IFN-γ from NK cells, which however was significantly lower compared with that induced by in vitro-polarized M2 cells. Most tumor-associated NK cells displayed a CD56(bright) , CD16(neg) or CD56(bright) , CD16(dim) phenotype, and very poor cytolytic activities, despite an increased expression of the activation marker CD69. They also showed downregulation of DNAM-1, 2B4, and NTB-A activating receptors, and an altered chemokine receptor repertoire. Importantly however, when appropriately stimulated, NK cells from the patients, including those cells isolated from ascites, efficiently killed autologous TAMs that expressed low, "nonprotective" levels of HLA class I molecules. Overall, our data show the existence of a complex tumor microenvironment in which poorly cytolytic/immature NK cells deal with immunosuppressive tumor-educated macrophages.

Natural Killer Cells and Neuroblastoma: Tumor Recognition, Escape Mechanisms, and Possible Novel Immunotherapeutic Approaches

Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and arises from developing sympathetic nervous system. Most primary tumors localize in the abdomen, the adrenal gland, or lumbar sympathetic ganglia. Amplification in tumor cells of MYCN, the major oncogenic driver, patients’ age over 18 months, and the presence at diagnosis of a metastatic disease (stage IV, M) identify NB at high risk of treatment failure. Conventional therapies did not significantly improve the overall survival of these patients. Moreover, the limited landscape of somatic mutations detected in NB is hampering the development of novel pharmacological approaches. Major efforts aim to identify novel NB-associated surface molecules that activate immune responses and/or direct drugs to tumor cells and tumor-associated vessels. PVR (Poliovirus Receptor) and B7-H3 are promising targets, since they are expressed by most high-risk NB, are upregulated in tumor vasculature and are essential for tumor survival/invasiveness. PVR is a ligand of DNAM-1 activating receptor that triggers the cytolytic activity of natural killer (NK) cells against NB. In animal models, targeting of PVR with an attenuated oncolytic poliovirus induced tumor regression and elimination. Also B7-H3 was successfully targeted in preclinical studies and is now being tested in phase I/II clinical trials. B7-H3 down-regulates NK cytotoxicity, providing NB with a mechanism of escape from immune response. The immunosuppressive potential of NB can be enhanced by the release of soluble factors that impair NK cell function and/or recruitment. Among these, TGF-β1 modulates the cytotoxicity receptors and the chemokine receptor repertoire of NK cells. Here, we summarize the current knowledge on the main cell surface molecules and soluble mediators that modulate the function of NK cells in NB, considering the pros and cons that must be taken into account in the design of novel NK cell-based immunotherapeutic approaches.

Evolution and function of chemokine receptors in the immune system of lower vertebrates

Chemokine receptors and their counterpart ligands are one of the evolutionary innovations of vertebrates. They play a guiding role in the coordination of cell trafficking in many biological processes. Comparative syntenic and phylogenetic analyses provide insight into the evolution of chemokine receptors and suggest that the repertoire of chemokine receptors varies in each species, regardless of the evolutionary position of the species. Despite the rapid evolution of chemokine receptors, the expression and function of orthologous chemokine receptors in lower and higher vertebrates are very similar. This is also true for the chemokine ligands that have been examined so far, such as CXCL8, CXCL12, and CCL25. As examples, this review will discuss how the evolution of the chemokine receptor CXCR4 is coincident with the emergence of lymphocytes in jawless vertebrates (lamprey); and that, in jawed vertebrates, CXCR4 and CCR9 are involved in thymus colonization. In myeloid cells, the function of CXCR1 in neutrophils and the expression of CXCR3 in macrophages and DCs are evolutionarily conserved between fish and mammals. In this context, medaka and zebrafish are outstanding models for studying the function of chemokines and their receptors.

Neuroblastoma-Derived TGF-β1 Modulates the Chemokine Receptor Repertoire of Human Resting NK Cells

In this study, we show that neuroblastoma (NB) cell conditioning affects the chemokine receptor repertoire of human resting NK cells. In particular, NB cells upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated CX3CR1 in the CD56(dim) subset. On the contrary, the expression of CXCR1 and CCR7 remained unaltered. The phenomenon was dependent on the release by NB cells of TGF-β1, and rTGF-β1 induced a chemokine receptor repertoire identical to that of NB-conditioned NK cells. The immune modulatory role of TGF-β1 appears to be dose dependent because low amounts of the cytokine were sufficient to modulate CXCR4 and CX3CR1 expression, intermediate amounts modified that of CXCR3, and high amounts were necessary to downregulate the expression of the NKp30 activating receptor. Notably, a similar receptor modulation was observed in rTGF-β2-conditioned NK cells. Finally, the analysis of NK cells from patients with stage 4 NB suggests that NB conditioning could exert in vivo an immune modulatory effect resembling that emerged from in vitro experiments. Altogether our data propose a novel tumor escape-mechanism based on the modulation of chemokine receptors that play pivotal roles in NK cells bone marrow homing, egress, or recruitment into peripheral tissues.

The chemokine receptor CCR3 participates in tissue remodeling during atopic skin inflammation

BackgroundRecent studies provided insights into the recruitment and activation pathways of leukocytes in atopic dermatitis, however, the underlying mechanisms of tissue remodeling in atopic skin inflammation remain elusive. ObjectiveTo identify chemokine-mediated communication pathways regulating tissue remodeling during atopic skin inflammation. MethodsAnalysis of the chemokine receptor repertoire of human dermal fibroblasts using flow cytometry and immunofluorescence. Quantitative real-time polymerase chain reaction and immunohistochemical analyses of chemokine expression in atopic vs. non-atopic skin inflammation. Investigation of the function of chemokine receptor CCR3 on human dermal fibroblasts through determining intracellular Ca2+ mobilization, cell proliferation, migration, and repair capacity. ResultsAnalyses on human dermal fibroblasts showed abundant expression of the chemokine receptor CCR3 in vitro and in vivo. Among its corresponding ligands (CCL5, CCL8, CCL11, CCL24 and CCL26) CCL26 demonstrated a significant and specific up-regulation in atopic when compared to psoriatic skin inflammation. In vivo, epidermal keratinocytes showed most abundant CCL26 protein expression in lesional atopic skin. In structural cells of the skin, TH2-cytokines such as IL-4 and IL-13 were dominant inducers of CCL26 expression. In dermal fibroblasts, CCL26 induced CCR3 signaling resulting in intracellular Ca2+ mobilization, as well as enhanced fibroblast migration and repair capacity, but no proliferation. ConclusionTaken together, findings of the present study suggest that chemokine-driven communication pathways from the epidermis to the dermis may modulate tissue remodeling in atopic skin inflammation.