Inhibitory receptors of plasmacytoid dendritic cells as possible targets for checkpoint blockade in cancer.

Plasmacytoid dendritic cells (pDCs) are the major producers of type I interferons (IFNs), which are essential to mount antiviral and antitumoral immune responses. To avoid exaggerated levels of type I IFNs, which pave the way to immune dysregulation and autoimmunity, pDC activation is strictly regulated by a variety of inhibitory receptors (IRs). In tumors, pDCs display an exhausted phenotype and correlate with an unfavorable prognosis, which largely depends on the accumulation of immunosuppressive cytokines and oncometabolites. This review explores the hypothesis that tumor microenvironment may reduce the release of type I IFNs also by a more pDC-specific mechanism, namely the engagement of IRs. Literature shows that many cancer types express de novo, or overexpress, IR ligands (such as BST2, PCNA, CAECAM-1 and modified surface carbohydrates) which often represent a strong predictor of poor outcome and metastasis. In line with this, tumor cells expressing ligands engaging IRs such as BDCA-2, ILT7, TIM3 and CD44 block pDC activation, while this blocking is prevented when IR engagement or signaling is inhibited. Based on this evidence, we propose that the regulation of IFN secretion by IRs may be regarded as an "innate checkpoint", reminiscent of the function of "classical" adaptive immune checkpoints, like PD1 expressed in CD8+ T cells, which restrain autoimmunity and immunopathology but favor chronic infections and tumors. However, we also point out that further work is needed to fully unravel the biology of tumor-associated pDCs, the neat contribution of pDC exhaustion in tumor growth following the engagement of IRs, especially those expressed also by other leukocytes, and their therapeutic potential as targets of combined immune checkpoint blockade in cancer immunotherapy.

Extracellular microRNAs induce dendritic cell-dependent joint inflammation and potentiate osteoclast differentiation via TLR7/8 engagement.

OBJECTIVES: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation. METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors. RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts. CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.

CCRL2 Expression by Specialized Lung Capillary Endothelial Cells Controls NK-cell Homing in Lung Cancer.

Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C-C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27- CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2'-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell-mediated lung immune surveillance.

The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity.

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies.

Inhibition of Class I Histone Deacetylase Activity Blocks the Induction of TNFAIP3 Both Directly and Indirectly via the Suppression of Endogenous TNF-α.

Histone deacetylase inhibitors (HDIs) are promising drugs for the treatment of inflammatory diseases. However, their therapeutical exploitation is slowed down by severe adverse manifestations that can hardly be foreseen, mainly due to incomplete knowledge of how HDIs impact the delicate balance of inflammatory mediators. In this work, we characterized the effects of the HDI trichostatin A (TSA) on the expression of TNFAIP3, which is a crucial inhibitor of the classical NF-kB pathway and an LPS-induced negative feedback regulator. The accumulation of TNFAIP3 mRNA after LPS stimulation showed biphasic behavior, with one wave within the first hour of stimulation and a second wave several hours later, which were both reduced by TSA. By using inhibition and knockdown approaches, we identified two temporally and mechanistically distinct modes of action. The first wave of TNAIP3 accumulation was directly blunted by the histone deacetylase (HDAC) blockade. By contrast, the second wave was decreased mainly because of the lack of endogenous TNF-α induction, which, in turn, depended on the intact HDAC activity. In both cases, class I HDACs appeared to play a nonredundant role, with HDAC3 required, but not sufficient, for TNF-α and TNFAIP3 induction. In addition to TNFAIP3, TNF-α is known to induce many response genes that orchestrate the inflammatory cascade. Thus, suppression of TNF-α may represent a general mechanism through which HDIs regulate a selected set of target genes.

The PDE4 inhibitor tanimilast shows distinct immunomodulatory properties associated with a type 2 endotype and CD141 upregulation.

BACKGROUND: Tanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanced clinical development for chronic obstructive pulmonary disease (COPD). Tanimilast is known to exert prominent anti-inflammatory activity when tested in preclinical experimental models as well as in human clinical studies. Recently, we have demonstrated that it also finely tunes, rather than suppressing, the cytokine network secreted by activated dendritic cells (DCs). This study was designed to characterize the effects of tanimilast on T-cell polarizing properties of DCs and to investigate additional functional and phenotypical features induced by tanimilast. METHODS: DCs at day 6 of culture were stimulated with LPS in the presence or absence of tanimilast or the control drug budesonide. After 24 h, DCs were analyzed for the expression of surface markers of maturation and activation by flow cytometry and cocultured with T cells to investigate cell proliferation and activation/polarization. The regulation of type 2-skewing mediators was investigated by real-time PCR in DCs and compared to results obtained in vivo in a randomized placebo-controlled trial on COPD patients treated with tanimilast. RESULTS: Our results show that both tanimilast and budesonide reduced the production of the immunostimulatory cytokine IFN-γ by CD4+ T cells. However, the two drugs acted at different levels since budesonide mainly blocked T cell proliferation, while tanimilast skewed T cells towards a Th2 phenotype without affecting cell proliferation. In addition, only DCs matured in the presence of tanimilast displayed increased CD86/CD80 ratio and CD141 expression, which correlated with Th2 T cell induction and dead cell uptake respectively. These cells also upregulated cAMP-dependent immunosuppressive molecules such as IDO1, TSP1, VEGF-A and Amphiregulin. Notably, the translational value of these data was confirmed by the finding that these same genes were upregulated also in sputum cells of COPD patients treated with tanimilast as add-on to inhaled glucocorticoids and bronchodilators. CONCLUSION: Taken together, these findings demonstrate distinct immunomodulatory properties of tanimilast associated with a type 2 endotype and CD141 upregulation in DCs and provide a mechanistic rationale for the administration of tanimilast on top of inhaled corticosteroids.

The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001-is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide-a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

SARS-CoV-2-associated ssRNAs activate inflammation and immunity via TLR7/8.

The inflammatory and IFN pathways of innate immunity play a key role in the resistance and pathogenesis of coronavirus disease 2019 (COVID-19). Innate sensors and SARS-CoV-2-associated molecular patterns (SAMPs) remain to be completely defined. Here, we identified single-stranded RNA (ssRNA) fragments from the SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and function, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream of these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identified TLR7/8 as a crucial cellular sensor of ssRNAs encoded by SARS-CoV-2 involved in host resistance and the disease pathogenesis of COVID-19.

Bartonella henselae Persistence within Mesenchymal Stromal Cells Enhances Endothelial Cell Activation and Infectibility That Amplifies the Angiogenic Process.

Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, Bartonella henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of B. henselae, the mechanisms by which B. henselae induces EC activation are not completely clear, as well as the possible contributions of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections, exerting antimicrobial properties but also acting as a shelter for pathogens. Here, we delved into the role of MSCs as a reservoir of B. henselae and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclearly bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of epidermal growth factor receptor (EGFR), Toll-like receptor 2 (TLR2), and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF-7), matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), interleukin 6 (IL-6), platelet-derived growth factor D (PDGF-D), chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 8 (CXCL8). Supernatants from B. henselae-infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion, and reorganization in tube-like structures. Altogether, these results indicate MSCs as a still underestimated niche for persistent B. henselae infection and reveal MSC-EC cross talk that may contribute to exacerbate bacterium-induced angiogenesis and granuloma formation.

TIM4 expression by dendritic cells mediates uptake of tumor-associated antigens and anti-tumor responses.

Acquisition of cell-associated tumor antigens by type 1 dendritic cells (cDC1) is essential to induce and sustain tumor specific CD8+ T cells via cross-presentation. Here we show that capture and engulfment of cell associated antigens by tissue resident lung cDC1 is inhibited during progression of mouse lung tumors. Mechanistically, loss of phagocytosis is linked to tumor-mediated downregulation of the phosphatidylserine receptor TIM4, that is highly expressed in normal lung resident cDC1. TIM4 receptor blockade and conditional cDC1 deletion impair activation of tumor specific CD8+ T cells and promote tumor progression. In human lung adenocarcinomas, TIM4 transcripts increase the prognostic value of a cDC1 signature and predict responses to PD-1 treatment. Thus, TIM4 on lung resident cDC1 contributes to immune surveillance and its expression is suppressed in advanced tumors.

Urethane-induced lung carcinogenesis.

Chemical induced carcinogenesis together with genetically engineered mouse models represent important approaches for the study of the complex mechanisms involving genotype and environmental factors in cancer development, including lung cancer. The induction of lung tumor in mice with urethane (ethyl carbamate) is considered a valuable model of Kras-driven lung cancer. However, inbred mouse strains show variable susceptibility to lung tumor formation, with C57BL/6 background, widely used to study many transgenic and null mutations, highly resistant to lung carcinogenesis. Here is described a protocol of urethane-induced lung cancer effective in lung tumor induction in C57BL/6J strain. Multiple urethane injections are needed to overcome genetic resistance and induce in a reproducible manner lung carcinogenesis in C57BL/6J background mice.

The PDE4 Inhibitor Tanimilast Blunts Proinflammatory Dendritic Cell Activation by SARS-CoV-2 ssRNAs.

Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases associated with chronic inflammatory conditions, such as COPD, psoriasis and atopic dermatitis. Tanimilast (international non-proprietary name of CHF6001) is a novel, potent and selective inhaled PDE4 inhibitor in advanced clinical development for the treatment of COPD. To begin testing its potential in limiting hyperinflammation and immune dysregulation associated to SARS-CoV-2 infection, we took advantage of an in vitro model of dendritic cell (DC) activation by SARS-CoV-2 genomic ssRNA (SCV2-RNA). In this context, Tanimilast decreased the release of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (CCL3, CXCL9, and CXCL10) and of Th1-polarizing cytokines (IL-12, type I IFNs). In contrast to ß-methasone, a reference steroid anti-inflammatory drug, Tanimilast did not impair the acquisition of the maturation markers CD83, CD86 and MHC-II, nor that of the lymph node homing receptor CCR7. Consistent with this, Tanimilast did not reduce the capability of SCV2-RNA-stimulated DCs to activate CD4+ T cells but skewed their polarization towards a Th2 phenotype. Both Tanimilast and ß-methasone blocked the increase of MHC-I molecules in SCV2-RNA-activated DCs and restrained the proliferation and activation of cytotoxic CD8+ T cells. Our results indicate that Tanimilast can modulate the SCV2-RNA-induced pro-inflammatory and Th1-polarizing potential of DCs, crucial regulators of both the inflammatory and immune response. Given also the remarkable safety demonstrated by Tanimilast, up to now, in clinical studies, we propose this inhaled PDE4 inhibitor as a promising immunomodulatory drug in the scenario of COVID-19.

Molecular Basis for CCRL2 Regulation of Leukocyte Migration.

CCRL2 is a seven-transmembrane domain receptor that belongs to the chemokine receptor family. At difference from other members of this family, CCRL2 does not promote chemotaxis and shares structural features with atypical chemokine receptors (ACKRs). However, CCRL2 also differs from ACKRs since it does not bind chemokines and is devoid of scavenging functions. The only commonly recognized CCRL2 ligand is chemerin, a non-chemokine chemotactic protein. CCRL2 is expressed both by leukocytes and non-hematopoietic cells. The genetic ablation of CCRL2 has been instrumental to elucidate the role of this receptor as positive or negative regulator of inflammation. CCRL2 modulates leukocyte migration by two main mechanisms. First, when CCRL2 is expressed by barrier cells, such endothelial, and epithelial cells, it acts as a presenting molecule, contributing to the formation of a non-soluble chemotactic gradient for leukocytes expressing CMKLR1, the functional chemerin receptor. This mechanism was shown to be crucial in the induction of NK cell-dependent immune surveillance in lung cancer progression and metastasis. Second, by forming heterocomplexes with other chemokine receptors. For instance, CCRL2/CXCR2 heterodimers were shown to regulate the activation of ß2-integrins in mouse neutrophils. This mini-review summarizes the current understanding of CCRL2 biology, based on experimental evidence obtained by the genetic deletion of this receptor in in vivo experimental models. Further studies are required to highlight the complex functional role of CCRL2 in different organs and pathological conditions.

Functional Role of Dendritic Cell Subsets in Cancer Progression and Clinical Implications.

Dendritic cells (DCs) constitute a complex network of cell subsets with common functions but also with many divergent aspects. All dendritic cell subsets share the ability to prime T cell response and to undergo a complex trafficking program related to their stage of maturation and function. For these reasons, dendritic cells are implicated in a large variety of both protective and detrimental immune responses, including a crucial role in promoting anti-tumor responses. Although cDC1s are the most potent subset in tumor antigen cross-presentation, they are not sufficient to induce full-strength anti-tumor cytotoxic T cell response and need close interaction and cooperativity with the other dendritic cell subsets, namely cDC2s and pDCs. This review will take into consideration different aspects of DC biology, including the functional role of dendritic cell subsets in both fostering and suppressing tumor growth, the mechanisms underlying their recruitment into the tumor microenvironment, as well as the prognostic value and the potentiality of dendritic cell therapeutic targeting. Understanding the specificity of dendritic cell subsets will allow to gain insights on role of these cells in pathological conditions and to design new selective promising therapeutic approaches.

Endogenous murine microbiota member Faecalibaculum rodentium and its human homologue protect from intestinal tumour growth.

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigenesis. We identified two anti-tumourigenic strains of the microbiota-Faecalibaculum rodentium and its human homologue, Holdemanella biformis-that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymethane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endogenous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

The Atypical Receptor CCRL2 Is Essential for Lung Cancer Immune Surveillance.

CCRL2 is a nonsignaling seven-transmembrane domain receptor. CCRL2 binds chemerin, a protein that promotes chemotaxis of leukocytes, including macrophages and natural killer (NK) cells. In addition, CCRL2 controls the inflammatory response in different pathologic settings, such as hypersensitivity, inflammatory arthritis, and experimental autoimmune encephalitis. Here, we investigated the role of CCRL2 in the regulation of lung cancer-related inflammation. The genetic deletion of Ccrl2 promoted tumor progression in urethane-induced and in Kras G12D/+/p53 LoxP lung tumor mouse models. Similarly, a Kras-mutant lung tumor displayed enhanced growth in Ccrl2-deficient mice. This phenotype was associated with a reduced inflammatory infiltrate characterized by the impaired recruitment of several leukocyte populations including NK cells. Bone marrow chimeras showed that CCRL2 expression by the nonhematopoietic cell compartment was responsible for the increased tumor formation observed in Kras-mutant Ccrl2-deficient mice. In human and mouse lungs, CCRL2 was expressed by a fraction of CD31+ endothelial cells, where it could control NK infiltration. Elevated CCRL2 expression in biopsies from human lung adenocarcinoma positively correlated with clinical outcome. These results provide evidence for a crucial role of CCRL2 in shaping an anti-lung tumor immune response.

Chemokine and chemotactic signals in dendritic cell migration.

Dendritic cells (DCs) are professional antigen-presenting cells responsible for the activation of specific T-cell responses and for the development of immune tolerance. Immature DCs reside in peripheral tissues and specialize in antigen capture, whereas mature DCs reside mostly in the secondary lymphoid organs where they act as antigen-presenting cells. The correct localization of DCs is strictly regulated by a large variety of chemotactic and nonchemotactic signals that include bacterial products, DAMPs (danger-associated molecular patterns), complement proteins, lipids, and chemokines. These signals function both individually and in concert, generating a complex regulatory network. This network is regulated at multiple levels through different strategies, such as synergistic interactions, proteolytic processing, and the actions of atypical chemokine receptors. Understanding this complex scenario will help to clarify the role of DCs in different pathological conditions, such as autoimmune diseases and cancers and will uncover new molecular targets for therapeutic interventions.

Leukocyte trafficking in tumor microenvironment.

The tumor microenvironment consists of both malignant and non-malignant cells and a plethora of soluble mediators. Different types of tumors have specific tumor microenvironments characterized by distinct chemokines and chemotactic factors that influence leukocyte recruitment. The immune cell infiltrate continuously interacts with stroma cells and influence tumor growth. Emerging evidence suggests that the regulation of the composition and the metabolic state of tumor-associated leukocytes may represent a new promising intervention strategy. Here we summarize the current knowledge on the role of tumor-associated immune cells in tumor growth and dissemination, with a specific focus on the nature of the chemotactic factors responsible for their accumulation and activation in tumors.

A CCR4 antagonist reverses the tumor-promoting microenvironment of renal cancer.

Elevated expression of the chemokine receptor CCR4 in tumors is associated with poor prognosis in several cancers. Here, we have determined that CCR4 was highly expressed in human renal cell carcinoma (RCC) biopsies and observed abnormal levels of CCR4 ligands in RCC patient plasma. An antagonistic anti-CCR4 antibody had antitumor activity in the RENCA mouse model of RCC. CCR4 inhibition did not reduce the proportion of infiltrating leukocytes in the tumor microenvironment but altered the phenotype of myeloid cells, increased NK cell and Th1 cytokine levels, and reduced immature myeloid cell infiltrate and blood chemokine levels. In spite of prominent changes in the myeloid compartment, the anti-CCR4 antibody did not affect RENCA tumors in T cell-deficient mice, and treatment with an anti-class II MHC antibody abrogated its antitumor activity. We concluded that the effects of the anti-CCR4 antibody required the adaptive immune system and CD4+ T cells. Moreover, CCL17-induced IFN-γ production was reduced when Th1-polarized normal CD4+ T cells were exposed to the CCR4 ligand, evidencing the involvement of CCR4 in Th1/Th2 regulation. The anti-CCR4 antibody, alone or in combination with other immune modulators, is a potential treatment approach to human solid cancers with high levels of CCR4-expressing tumor-infiltrating leukocytes and abnormal plasma CCR4 ligand levels.

A dynamic inflammatory cytokine network in the human ovarian cancer microenvironment.

Constitutive production of inflammatory cytokines is a characteristic of many human malignant cell lines; however, the in vitro and in vivo interdependence of these cytokines, and their significance to the human cancer microenvironment, are both poorly understood. Here, we describe for the first time how three key cytokine/chemokine mediators of cancer-related inflammation, TNF, CXCL12, and interleukin 6, are involved in an autocrine cytokine network, the "TNF network," in human ovarian cancer. We show that this network has paracrine actions on angiogenesis, infiltration of myeloid cells, and NOTCH signaling in both murine xenografts and human ovarian tumor biopsies. Neutralizing antibodies or siRNA to individual members of this TNF network reduced angiogenesis, myeloid cell infiltration, and experimental peritoneal ovarian tumor growth. The dependency of network genes on TNF was shown by their downregulation in tumor cells from patients with advanced ovarian cancer following the infusion of anti-TNF antibodies. Together, the findings define a network of inflammatory cytokine interactions that are crucial to tumor growth and validate this network as a key therapeutic target in ovarian cancer.