RESUMO
Dendritic cells (DCs), the essential immunoregulatory and APCs, are major producers of the central mediator of inflammation, soluble TNF-α (sTNF). sTNF is generated by TNF-α converting enzyme (TACE) proteolytic release of the transmembrane TNF (tmTNF) ectodomain. The mechanisms of TACE and sTNF regulation in DCs remain elusive. This study newly defines that sTNF regulates TACE in mouse DCs by engaging the AP-2α transcription factor. We found that the expression of AP-2α was higher, whereas the expression and activity of TACE were lower, in wild-type DCs (wtDCs) than in TNF knockout (TNFko) DCs. Exogenous sTNF rapidly and simultaneously induced increases of AP-2α expression and decreases of TACE expression and activity in wtDCs and TNFko DCs, indicating that AP-2α and TACE are inversely dependent on sTNF and are functionally associated. To define this functional association, we identified an AP-2α binding site in TACE promoter and demonstrated, using EMSAs and chromatin immunoprecipitation assays, that AP-2α could bind to TACE promoter in a TNF-dependent manner. Additionally, sTNF simultaneously enhanced AP-2α expression and decreased TACE promoter luciferase activity in DCs. Similarly, transfection of AP-2α cDNA decreased TACE promoter luciferase activity, TACE expression, and TACE enzymatic activity in wtDCs or TNFko DCs. In contrast, transfection of AP-2α small interfering RNA increased TACE promoter luciferase activity, TACE expression, and TACE enzymatic activity in wtDCs. These results show that TACE is a target of, and is downregulated by, sTNF-induced AP-2α transcription factor in DCs.
Assuntos
Proteína ADAM17/metabolismo , Células Dendríticas/metabolismo , Fator de Transcrição AP-2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM17/imunologia , Animais , Imunoprecipitação da Cromatina , Células Dendríticas/imunologia , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica/imunologia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição AP-2/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs -2, -9, and -13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M-1 s-1 and 4300 M-1 s-1, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (kcat/Km) of 5200 M-1 s-1. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 µM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Ácidos Hidroxâmicos/farmacologia , Cinética , Peptídeos/química , Peptídeos/metabolismoRESUMO
A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.3) × 10(4) M(-1) s(-1) and 2.4 (±0.3) × 10(3) M(-1) s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities.
Assuntos
Proteínas ADAM/análise , Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Proteínas ADAM/química , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise , Análise de Regressão , SolubilidadeRESUMO
Dendritic cells (DCs) are the major sentinel, antigen-presenting and regulatory components of the immune system. One of the central DC functions is to rapidly sense and alert host immune system of a pathogen invasion. In the present study, we investigated the role of DC exosomes (DCex) in this sentinel function. We demonstrated that DCex could bind bacterial Toll-like-receptor ligands (TLR-Ls), and acquire their ability to strongly activate bystander DCs. Consequently, bystander DCs enhance the expression of transmembrane tumor necrosis factor, secretion of proinflammatory cytokines and cross-talk with natural killer cells leading to the elevated secretion of IFNγ. These findings newly show that DCex can bind and cross-present TLR-Ls to innate-immunity effector cells, and indicate a potent mechanism to systemically alert the host immune system of pathogen invasion. They also suggest a potential novel strategy to generate effective vaccines by binding TLR-L-immune adjuvants to DCex.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Efeito Espectador/imunologia , Células Dendríticas/imunologia , Exossomos/imunologia , Células Matadoras Naturais/imunologia , Animais , Apresentação de Antígeno/imunologia , Feminino , Interferon gama/metabolismo , Lipopeptídeos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Camundongos SCID , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Recombinant adenovirus-engineered dendritic cells (Ad.DCs) are potent immunologic adjuvants of antiviral and anticancer vaccines. The effectiveness of Ad.DC-based vaccines may depend on the ability of Ad.DCs to crosstalk with natural killer (NK) cells and to activate, polarize, and bridge innate and adaptive immunity. We investigated, for the first time, whether and how human Ad.DCs activate NK cells, and compared the Ad.DC function with that of immature DCs and matured DCs (mDCs). We found that adenovirus transduction and lipopolysaccharide/interferon-gamma-induced maturation increased expression of transmembrane tumor necrosis factor (TNF) and trans-presented (trans) interleukin-15 (IL-15) on DCs, leading to enhanced NK cell activation without enhancing DC susceptibility to NK cell-mediated killing. This crosstalk enhanced NK cell CD69 expression, interferon-gamma secretion, proliferation, and antitumor activities, with Ad.DCs being significantly more effective than immature DCs, but less effective than mDCs. The Ad.DC and mDC crosstalk with NK cells was largely prevented by physical separation of DCs and NK cells, and neutralization of total TNF and IL-15, but not by selective sequestration of soluble TNF. These findings demonstrate that both Ad.DCs and mDCs can efficiently promote innate immune functions by activation of NK cells through the cooperative activities of tmTNF and trans-IL-15 mediated by cell-to-cell contact.
Assuntos
Infecções por Adenoviridae/imunologia , Comunicação Celular , Células Dendríticas/fisiologia , Interleucina-15/fisiologia , Células Matadoras Naturais/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Feminino , Humanos , Imunidade Inata/imunologia , Interleucina-15/metabolismo , Células K562 , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/fisiologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Ligação Proteica/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The essential innate immunity effector cells, natural killer and dendritic cells, express multiple plasma membrane-associated tumor necrosis factor (TNF) superfamily (TNFSF) ligands that, through simultaneous and synergistic engagement, mediate anti-cancer cytotoxicity. Here, we report that circulating B cells, mediators of adaptive humoral immunity, also mediate this innate anti-cancer immune mechanism. We show that resting human B cells isolated from peripheral blood induce apoptosis of, and efficiently kill a large variety of leukemia and solid tumor cell types. Single-cell RNA sequencing analyses indicate, and flow cytometry data confirm that B cells from circulation express transmembrane TNF, Fas ligand (FasL), lymphotoxin (LT) α1ß2 and TNF-related apoptosis-inducing ligand (TRAIL). The cytotoxic activity can be inhibited by individual and, especially, combined blockade of the four transmembrane TNFSF ligands. B cells from tumor-bearing head and neck squamous cell carcinoma patients express lower levels of TNFSF ligands and are less cytotoxic than those isolated from healthy individuals. In conclusion, we demonstrate that B cells have the innate capacity to mediate anti-cancer cytotoxicity through concurrent activity of multiple plasma membrane-associated TNFSF ligands, that this mechanism is deficient in cancer patients and that it may be part of a general cancer immunosurveillance mechanism.
Assuntos
Linfócitos B , Neoplasias , Ligante Indutor de Apoptose Relacionado a TNF , Proteínas Reguladoras de Apoptose , Humanos , Ligantes , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This article presents the hypothesis that acupuncture enhances anticancer immune functions by stimulating natural killer (NK) cells. It provides background information on acupuncture, summarizes the current scientific understanding of the mechanisms through which NK cells act to eliminate cancer cells, and reviews evidence that acupuncture is associated with increases in NK cell quantity and function in both animals and humans. The key contribution of this article involves the use of cellular immunology and molecular biological theory to interpret and synthesize evidence from disparate animal and human studies in formulating the 'acupuncture immuno-enhancement hypothesis': clinicians may use acupuncture to promote the induction and secretion of NK-cell activating cytokines that engage specific NK cell receptors that endogenously enhance anticancer immune function.
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Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable. Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes. Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells. Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.
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Background: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Methods: Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts. Results: NSCLC cell lines and patient tumors and exosomes consistently showed significant increases of ADAM10sa relative to their normal, inflammatory and/or benign-tumor controls. Additionally, stage IA-IIB NSCLC primary tumors of patients who died of the disease exhibited greater increases of ADAM10sa than those of patients who survived 5 years following diagnosis and surgery. In contrast, NSCLC cell lines and patient tumors and exosomes did not display increases of ADAM17sa. Conclusions: This study is the first to investigate enzyme-specific proteolytic activities as potential cancer biomarkers. It provides a proof-of-concept that ADAM10sa could be a biomarker for NSCLC early detection and outcome prediction. To ascertain that ADAM10sa is a useful cancer biomarker, further robust clinical validation studies are needed.
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Dendritic cells are professional antigen-presenting cells associated with efficient antigen processing and presentation to T cells. However, recent evidence also suggests that dendritic cells may mediate direct tumoricidal functions. In this study, we investigated the mechanism by which murine dendritic cells mediate the apoptotic death of murine lymphoma cell lines, and whether dendritic cell effector function could be enhanced by preconditioning tumor cells with the protein phosphatase inhibitor nitric oxide (NO) by altering the balance of proapoptotic/antiapoptotic proteins in the treated cells. We observed that NO donor compound sensitized lymphomas to dendritic cell-mediated cytotoxicity in vitro. Both immature and spontaneously matured bone marrow-derived dendritic cells (SM-DC) were capable of inducing tumor cell apoptosis, with SM-DCs serving as comparatively better killers. Fas ligand (FasL)-Fas engagement proved important in this activity because elevated expression of membrane-bound FasL was detected on SM-DCs, and dendritic cells derived from FasL-deficient mice were less capable of killing NO-sensitized tumor cells than wild-type dendritic cells. As FasL-deficient dendritic cells were still capable of mediating a residual degree of tumor killing, this suggests that FasL-independent mechanisms of apoptosis are also involved in dendritic cell-mediated tumor killing. Because NO-treated tumor cells displayed a preferential loss of survivin protein expression via a proteasome-dependent pathway, enhanced tumor sensitivity to dendritic cell-mediated killing may be associated with the accelerated turnover of this critical antiapoptotic gene product. Importantly, NO-treated tumor cells were also engulfed more readily than control tumor cells and this resulted in enhanced cross-presentation of tumor-associated antigens to specific T cells in vitro.
Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , Linfoma/imunologia , Óxido Nítrico/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Reações Cruzadas , Células Dendríticas/efeitos dos fármacos , Proteína Ligante Fas , Proteínas Inibidoras de Apoptose , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Linfoma/patologia , Glicoproteínas de Membrana/imunologia , Camundongos , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Óxido Nítrico/farmacocinética , Doadores de Óxido Nítrico/farmacocinética , Doadores de Óxido Nítrico/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras , Survivina , Linfócitos T/imunologia , Receptor fas/imunologiaRESUMO
Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/- and ADAM17-/- mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/- MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms.
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Dendritic cells (DC) and natural killer (NK) cells are essential components of the innate immune system, which rapidly sense and eliminate invading pathogens and transformed cells, mediate inflammation, and initiate adaptive immune responses. During the early immune events, DC and NK cells interact and regulate each other. The cellular "cross talk" and its molecular mediators are believed to be critical to the quality and magnitude of innate and adaptive immune responses. The goal of the present manuscript is to identify and initially assess major molecular mediators of DC-NK cell interaction. We have previously shown that DC and NK cells constitutively express several tumor necrosis factor family ligands (TNFfLs) and corresponding TNF family receptors (TNFfRs). Therefore, DC and NK cells might be able to interact via cognate interplays of TNFfLs and TNFfRs. Here, we provide initial experimental evidence supporting this possibility. We found that combined but not individual ligation of several TNFfRs induced substantial increases in secretion of interleukin-12 and interferon-gamma by DC and NK cells, respectively. In contrast, specific, individual disruptions of the engagements of the corresponding TNfL-TNFfR pairs greatly impaired DC and NK cell abilities to reciprocally mediate the increases in cytokine secretion. These findings indicate that multiple TNFfLs mediate DC-NK cell interaction.
Assuntos
Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Animais , Comunicação Celular/imunologia , Feminino , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Modelos BiológicosRESUMO
Dendritic cells (DCs) were adenovirally engineered to constitutively and durably secrete the potent Th1-biasing cytokines interleukin (IL)-12 (AdIL12DC) and/or IL-18 (AdIL18DC) and evaluated for their ability to promote therapeutic antitumor immunity in murine sarcoma models. Injection of either AdIL12DC or AdIL18DC into day 7 CMS4 or MethA tumors resulted in tumor rejection or slowed tumor growth when compared with control cohorts. Importantly, intratumoral injection with DCs engineered to secrete both IL-12 and IL-18 (AdIL12/IL18DC) resulted in complete and the most acute rejection of any treatment group analyzed. This strategy was also effective in promoting the regression of contralateral, untreated tumors. Both CD4+ and CD8+ T cells were required for tumor rejection. CD8+ splenic T cells from mice treated with AdIL12/IL18DC produced the highest levels of IFN-gamma in response to tumor rechallenge in vitro and displayed the broadest repertoire of Tc1-type reactivity to acid-eluted, tumor-derived peptides among all treatment cohorts. This apparent enhancement in cross-presentation of tumor-associated epitopes in vivo may result from the increased capacity of engineered DCs to kill tumor cells, survive tumor-induced apoptosis, and present immunogenic MHC/tumor peptide complexes to T cells after intratumoral injection. In support of this hypothesis, cytokine gene-engineered DCs expressed higher levels of MHC and costimulatory molecules, as well as Fas ligand and membrane-bound tumor necrosis factor alpha, with the latter markers associated with elevated tumoricidal activity in vitro. Cytokine gene-engineered DCs appeared to have a survival advantage in situ when injected into tumor lesions, to be found in approximation with regions of tumor apoptosis, and to have the capacity to ingest apoptotic tumor bodies. These results support the ability of combined cytokine gene transfer to enhance multiple effector functions mediated by intralesionally injected DCs that may concertedly promote cross-priming and the accelerated immune-mediated rejection of tumors.
Assuntos
Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Interleucina-12/imunologia , Interleucina-18/imunologia , Sarcoma Experimental/terapia , Células Th1/imunologia , Adenoviridae/genética , Animais , Proteínas Reguladoras de Apoptose , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Células Dendríticas/virologia , Epitopos de Linfócito T/imunologia , Feminino , Interleucina-12/biossíntese , Interleucina-12/genética , Interleucina-12/metabolismo , Interleucina-18/biossíntese , Interleucina-18/genética , Interleucina-18/metabolismo , Linfotoxina-alfa/biossíntese , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Sarcoma Experimental/imunologia , Ligante Indutor de Apoptose Relacionado a TNF , Células Th1/metabolismo , Transdução Genética , Fator de Necrose Tumoral alfa/biossínteseRESUMO
TNF is a potent promoter of carcinogenesis and potentially important target for cancer prevention. TNF is produced as functionally distinct transmembrane and soluble molecules (tmTNF and sTNF, respectively), but their individual roles in carcinogenesis are unexplored. We investigated the participation of tmTNF and sTNF in chemically induced carcinogenesis in mice. We found that injection of XPro1595, a dominant-negative TNF biologic (DN-TNF) and specific antagonist of sTNF, decreased tumor incidence and growth, and prolonged survival of 3-methylcholanthrene (MCA)-injected mice. Similar results were obtained following the exclusion of both TNF forms by either TNF-receptor 2-Fc fusion protein (TNFR2-Fc) treatment or TNF gene deletion. In addition, gene deletion of TNFR1, which is preferentially triggered by sTNF, was temporarily blocked, whereas gene deletion of TNFR2, which is preferentially triggered by tmTNF, enhanced MCA-induced carcinogenesis. Concomitantly with carcinogenesis induction, MCA increased circulating IL1α, accumulation of myeloid-derived suppressor cells (MDSC), STAT3 phosphorylation, and immunosuppression in the spleen. In sharp contrast, DN-TNF treatment dramatically decreased IL1α and increased the essential immunoregulatory cytokines IL1ß, IL12p70, and IL17 in the peripheral blood of MCA-injected mice. In addition, MDSC accumulation, STAT3 phosphorylation, and immunosuppression in MCA-injected mice were prevented by DN-TNF treatment, TNFR2-Fc treatment, and/or gene deletion of TNF or TNFR1, but not deletion of TNFR2. These findings reveal that sTNF is both an essential promoter of carcinogenesis and a pivotal regulator of MDSCs, and indicate that sTNF could be a significant target for cancer prevention and therapy. Cancer Immunol Res; 4(5); 441-51. ©2016 AACR.
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Neoplasias Experimentais/prevenção & controle , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Citocinas/biossíntese , Células Dendríticas/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Deleção de Genes , Tolerância Imunológica , Células Matadoras Naturais/imunologia , Metilcolantreno , Camundongos Endogâmicos C57BL , Camundongos SCID , Terapia de Alvo Molecular/métodos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Transcrição STAT3/metabolismo , Solubilidade , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/uso terapêuticoRESUMO
Activated, adherent natural killer (A-NK) cells represent a distinct subpopulation of interleukin (IL)-2-stimulated NK cells, which are selectively endowed with the increased expression of integrins and ability to adhere to solid surfaces, migrate into, infiltrate, and destroy cancerous tissues. The present study defines the phenotype and functions of precursors of A-NK (pre-A-NK) cells in humans. Peripheral blood pre-A-NK cells, in contrast to the rest of NK cells, express a novel epitope of CD56 neuronal cell adhesion molecule, termed ANK-1, and increased cell-surface levels of integrins. Pre-A-NK cells also express low levels of CD56 and CD161, and some express CD162 receptor, do not express CD25 or activation markers, and are effective mediators of NK cytotoxicity. Thus, pre-A-NK cells are generally similar to CD56(dim) NK cells. However, pre-A-NK cells differ from the main NK cell subpopulation by having a lower expression level of CD16 and a lower ability to mediate redirected antibody-dependent, cell-mediated cytotoxicity. More importantly, pre-A-NK cells are preferentially endowed with the ability to rapidly respond to IL-2 by integrin-mediated adherence to endothelial cells, extracellular matrix, and plastic. This early, specific response of pre-A-NK cells to IL-2 is followed by their activation, vigorous proliferation, and differentiation into phenotypically and functionally similar A-NK cells. Pre-A-NK cells represent only approximately 26% of peripheral blood NK cells but encompass the majority of NK cells in normal and cancerous, solid tissues. We conclude that pre-A-NK cells represent a distinct subset of resting, mature NK cells with the characteristics indicative of their ability to migrate and reside in solid tissues.
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Antígeno CD56/imunologia , Epitopos/imunologia , Células Matadoras Naturais/metabolismo , Células-Tronco/metabolismo , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Antígeno CD56/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Citotoxicidade Imunológica/imunologia , Humanos , Imunofenotipagem , Integrinas/imunologia , Integrinas/metabolismo , Interleucina-2/imunologia , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologiaRESUMO
BACKGROUND: Natural killer (NK) cells and dendritic cells (DCs) mediate tumor cell apoptosis using tumor necrosis factor superfamily ligands (TNFSFLs). This cytotoxicity is an important anticancer immune defense mechanism. METHODS: We examined TNFSFL expression and apoptotic tumoricidal activity (ATA) of purified NK cells and DCs, and peripheral blood mononuclear leukocytes (PBMLs) of healthy individuals and patients with head and neck cancer (HNC) before and after cancer ablation. RESULTS: PBMLs, NK cells and DCs, but not NK-cell/DC-depleted PBMLs, expressed multiple TNFSFLs and mediated ATA. Both TNFSFL expression and ATA were suppressed in tumor-bearing, and restored in tumor-ablated patients with (HNC) Soluble TNF superfamily receptors (solTNFSFRs) were increasingly bound by PBNLs of tumor-bearing HNC patients. Dissociation of solTNFSFR led to more pronounced increases in TNFSFL expression and ATA of PBMLs of patients with HNC than healthy individuals. CONCLUSION: NK-cell and DC TNFSFL expression and ATA are suppressed in patients with HNC. This suppression is tumor-dependent and possibly mediated by solTNFSFRs.
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Apoptose/imunologia , Células Dendríticas/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Tolerância Imunológica , Células Matadoras Naturais/imunologia , Fatores de Necrose Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Autocrine and paracrine cell communication can be conveyed by multiple mediators, including membrane-associate proteins, secreted proteins and exosomes. Exosomes are 30-100 nm endosome-derived vesicles consisting in cytosolic material surrounded by a lipid bilayer containing transmembrane proteins. We have previously shown that dendritic cells (DCs) express on their surface multiple TNF superfamily ligands (TNFSFLs), by which they can induce the apoptotic demise of tumor cells as well as the activation of natural killer (NK) cells. In the present study, we demonstrate that, similar to DCs, DC-derived exosomes (DCex) express on their surface TNF, FasL and TRAIL, by which they can trigger caspase activation and apoptosis in tumor cells. We also show that DCex activate NK cells and stimulate them to secrete interferonγ (IFNγ) upon the interaction of DCex TNF with NK-cell TNF receptors. These data demonstrate that DCex can mediate essential innate immune functions that were previously ascribed to DCs.
RESUMO
Recombinant adenovirus-engineered dendritic cells (Ad.DC) are potent vaccines for induction of anti-viral and anti-cancer T cell immunity. The effectiveness of Ad.DC vaccines may depend on the newly described ability of Ad.DC to crosstalk with natural killer (NK) cells via cell-to-cell contact, and to mediate activation, polarization and bridging of innate and adaptive immunity. For this interaction to occur in vivo, Ad.DC must be able to attract NK cells from surrounding tissues or peripheral blood. We developed a novel live mouse imaging system-based NK-cell migration test, and demonstrated for the first time that human Ad.DC induced directional migration of human NK cells across subcutaneous tissues, indicating that Ad.DC-NK cell contact and interaction could occur in vivo. We examined the mechanism of Ad.DC-induced migration of NK cells in vitro and in vivo. Ad.DC produced multiple chemokines previously reported to recruit NK cells, including immunoregulatory CXCL10/IP-10 and proinflammatory CXCL8/IL-8. In vitro chemotaxis experiments utilizing neutralizing antibodies and recombinant human chemokines showed that CXCL10/IP-10 and CXCL8/IL-8 were critical for Ad.DC-mediated recruitment of CD56(hi)CD16(-) and CD56(lo)CD16(+) NK cells, respectively. The importance of CXCL8/IL-8 was further demonstrated in vivo. Pretreatment of mice with the neutralizing anti-CXCL8/IL-8 antibody led to significant inhibition of Ad.DC-induced migration of NK cells in vivo. These data show that Ad.DC can recruit spatially distant NK cells toward a vaccine site via specific chemokines. Therefore, an Ad.DC vaccine can likely induce interaction with endogenous NK cells via transmembrane mediators, and consequently mediate Th1 polarization and amplification of immune functions in vivo.
RESUMO
Natural killer (NK) cells and dendritic cells (DCs) are essential effector cells of the innate immune system that rapidly recognize and eliminate microbial pathogens and abnormal cells, and induce and regulate adaptive immune functions. While NK cells express perforin and granzymes in the lysosomal granules and transmembrane tumor necrosis factor superfamily ligands (tmTNFSFL) on the plasma membrane, DCs express only tmTNFSFL on the plasma membrane. Perforin and granzymes are cytolytic molecules, which NK cells use to mediate a secretory/necrotic killing mechanism against rare leukemia cell targets. TNFSFL are pleiotropic transmembrane molecules, which can mediate a variety of important functions such as apoptosis, development of peripheral lymphoid tissues, inflammation and regulation of immune functions. Using tmTNFSFL, NK cells and DCs mediate a cell contact-dependent non-secretory apoptotic cytotoxic mechanism against virtually all types of cancer cells, and cross talk that leads to polarization and reciprocal stimulation and amplification of Th1 type cytokines secreted by NK cells and DCs. In this paper, we review and discuss the supporting evidence of the non-secretory, tmTNFSFL-mediated innate mechanisms of NK cells and DCs, their roles in anticancer immune defense and potential of their modulation and use in prevention and treatment of cancer.
Assuntos
Imunidade Inata/imunologia , Fatores de Necrose Tumoral/imunologia , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Interleucina-15/metabolismo , Interleucina-15/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias/genética , Neoplasias/imunologia , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Necrose Tumoral/genéticaRESUMO
Tumor necrosis factor alpha converting enzyme (TACE) is a sheddase overexpressed in cancers that generates cancer cell growth and survival factors, and is implicated in carcinogenesis and tumor growth. This indicates that TACE could be a potentially important cancer biomarker. Unexpectedly, TACE expression in cancer tissues does not correlate with cancer stage or invasiveness. Although TACE sheddase activity is a more direct and potentially better indicator of TACE biology and might be a better cancer biomarker than TACE expression, it has not been studied in cancer tissues. In the present study, we developed a reliable specific assay for quantification of TACE sheddase activity, investigated TACE activity and TACE protein expression in head and neck cancer (HNC) tissues, and examined the correlation of the results with HNC clinical stages and likelihood to recur. We found that HNC cell lines and tissues contained remarkably higher quantities of TACE activity and TACE protein than normal keratinocytes or oral mucosa. siRNA silencing of TACE resulted in the inhibition of release of the tumorogenic factors amphiregulin and transforming growth factor alpha, and tumor protective factors tumor necrosis factor receptors from HNC cells. Importantly, TACE activity, but not TACE protein expression, was significantly higher in large, T3/T4, primary tumors relative to small, T1/T2, primary tumors, and especially in primary tumors likely to recur relative to those unlikely to recur. These data show that increased TACE activity in cancer is biologically and clinically relevant, and indicate that TACE activity could be a significant biomarker of cancer prognosis.