RESUMO
Osteoprotegerin (OPG), the soluble decoy receptor of RANKL is released by bone marrow osteoblasts and plays an important role in physiological osteoblastogenesis and pathological bone disease. In earlier studies, we have shown that generated stromal cell lines from the aorta-gonad-mesonephros (AGM)-region serving as good supporters of murine and human hematopoietic progenitor cell (HPC) expansion highly express OPG detected by microarray analysis. Here, we investigated the role of OPG to HPC expansion in vitro. Addition of OPG leads to an enhanced expansion of HPC in liquid culture. In addition, progenitor cell function, measured by colony and cobblestone formation, was increased. The observed effects were partially antagonized by addition of RANKL. In conclusion, these findings suggest an important role of OPG maintaining progenitor cell function in the osteoblastic niche.
Assuntos
Doenças Ósseas/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Osteoblastos/metabolismo , Osteoprotegerina/metabolismo , Antígenos CD34/metabolismo , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Análise em Microsséries , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteoprotegerina/genética , Osteoprotegerina/farmacologia , Ligante RANK/antagonistas & inibidores , Ligante RANK/farmacologiaRESUMO
NK cells play an important role in tumor immunosurveillance and largely contribute to the therapeutic success of anti-tumor antibodies like Rituximab. Here, we studied the role of the TNF family member 4-1BB ligand (4-1BBL) during the interaction of NK cells with chronic lymphocytic leukemia (CLL) cells. 4-1BBL was highly expressed on patient B-CLL cells in all 56 investigated cases. Signaling via 4-1BBL following interaction with 4-1BB, which was detected on NK cells of CLL patients but not healthy individuals, led to the release of immunoregulatory cytokines including TNF by CLL cells. CLL patient sera contained elevated levels of TNF and induced 4-1BB upregulation on NK cells, which in turn impaired direct and Rituximab-induced NK-cell reactivity against 4-1BBL-expressing targets. NK-cell reactivity was not only enhanced by blocking the interaction of NK cell-expressed 4-1BB with 4-1BBL expressed by CLL cells, but also by preventing 4-1BB upregulation on NK cells via neutralization of TNF in patient serum with Infliximab. Our data indicate that 4-1BBL mediates NK-cell immunosubversion in CLL, and thus might contribute to the reportedly compromised efficacy of Rituximab to induce NK-cell reactivity in the disease, and that TNF neutralization may serve to enhance the efficacy of Rituximab treatment in CLL.
Assuntos
Ligante 4-1BB/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Antineoplásicos/farmacologia , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Ligante 4-1BB/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Testes Imunológicos de Citotoxicidade , Feminino , Citometria de Fluxo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rituximab , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Natural killer (NK) cells play an important role in the immunosurveillance of leukemia. Their reactivity is governed by a balance of activating and inhibitory receptors including various members of the tumor necrosis factor receptor (TNFR) family. Here we report that human NK cells acquire expression of the TNFR family member CD137 upon activation, and NK cells of acute myeloid leukemia (AML) patients display an activated phenotype with substantial CD137 expression. CD137 ligand (CD137L) was detectable on leukemic cells in 35% of 65 investigated AML patients, but not on healthy CD34(+) cells, and expression was associated with monocytic differentiation. Bidirectional signaling following CD137-CD137L interaction induced the release of the immunomodulatory cytokines interleukin-10 and TNF by AML cells and directly diminished granule mobilization, cytotoxicity, and interferon-gamma production of human NK cells, which was restored by blocking CD137. Cocultures of NK cells with CD137L transfectants confirmed that human CD137 inhibits NK-cell reactivity, while activating signals were transduced by its counterpart on NK cells in mice. Our data underline the necessity to study the function of seemingly analog immunoregulatory molecules in mice compared with men and demonstrate that CD137-CD137L interaction enables immune evasion of AML cells by impairing NK-cell tumor surveillance in humans.
Assuntos
Ligante 4-1BB/imunologia , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Ligante 4-1BB/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Regulação Leucêmica da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Tolerância Imunológica/imunologia , Células Matadoras Naturais/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Ligação Proteica , Transdução de Sinais/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Regulação para Cima/genéticaRESUMO
Sunitinib and Sorafenib are protein kinase inhibitors (PKI) approved for treatment of patients with advanced renal cell cancer (RCC). However, long-term remissions of advanced RCC have only been observed after IL-2 treatment, which underlines the importance of antitumor immune responses in RCC patients. Because PKI, besides affecting tumor cells, also may inhibit signaling in immune effector cells, we determined how Sunitinib and Sorafenib influence antitumor immunity. We found that cytotoxicity and cytokine production of resting and IL-2-activated PBMC are inhibited by pharmacological concentrations of Sorafenib but not Sunitinib. Analysis of granule-mobilization within PBMC revealed that this was due to impaired reactivity of NK cells, which substantially contribute to antitumor immunity by directly killing target cells and shaping adaptive immune responses by secreting cytokines like IFN-gamma. Analyses with resting and IL-2-activated NK cells revealed that both PKI concentration dependently inhibit cytotoxicity and IFN-gamma production of NK cells in response to tumor targets. This was due to impaired PI3K and ERK phosphorylation which directly controls NK cell reactivity. However, while Sorafenib inhibited NK cell effector functions and signaling at levels achieved upon recommended dosing, pharmacological concentrations of Sunitinib had no effect, and this was observed upon stimulation of NK cell reactivity by tumor target cells and upon IL-2 treatment. In light of the important role of NK cells in antitumor immunity, and because multiple approaches presently aim to combine PKI treatment with immunotherapeutic strategies, our data demonstrate that choice and dosing of the most suitable PKI in cancer treatment requires careful consideration.
Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Indóis/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica/imunologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Células K562 , Camundongos , Niacinamida/análogos & derivados , Compostos de Fenilureia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sorafenibe , SunitinibeRESUMO
The reciprocal interaction of tumor cells with the immune system is influenced by various members of the tumor necrosis factor (TNF)/TNF receptor (TNFR) family, and recently, glucocorticoid-induced TNFR-related protein (GITR) was shown to stimulate antitumor immunity in mice. However, GITR may mediate different effects in mice and men and impairs the reactivity of human natural killer (NK) cells. Here, we studied the role of GITR and its ligand (GITRL) in human acute myeloid leukemia (AML). Surface expression of GITRL was observed on AML cells in six of seven investigated cell lines, and 34 of 60 investigated AML patients whereas healthy CD34(+) cells did not express GITRL. Furthermore, soluble GITRL (sGITRL) was detectable in AML patient sera in 18 of 55 investigated cases. While the presence of GITRL was not restricted to a specific AML subtype, surface expression was significantly associated with monocytic differentiation. Signaling via GITRL into patient AML cells induced the release of TNF and interleukin-10 (IL-10), and this was blocked by the inhibition of mitogen-activated protein kinases extracellular signal-regulated kinase 1/2. Furthermore, triggering GITR by surface-expressed and sGITRL impaired NK cell cytotoxicity and IFN-gamma production in cocultures with leukemia cells, and NK cell reactivity could be restored by blocking GITR and neutralization of sGITRL and IL-10. Thus, whereas a stimulatory role of the GITR-GITRL system in mouse antitumor immunity has been reported, our data show that in humans GITRL expression subverts NK cell immunosurveillance of AML. Our results provide useful information for therapeutic approaches in AML, which, like haploidentical stem cell transplantation, rely on a sufficient NK cell response.
Assuntos
Leucemia Mieloide/imunologia , Fatores de Necrose Tumoral/imunologia , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Feminino , Humanos , Vigilância Imunológica/imunologia , Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , Leucemia Mieloide/sangue , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Fatores de Necrose Tumoral/biossíntese , Adulto JovemRESUMO
Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Monócitos/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptor Cross-Talk/imunologia , Receptores Toll-Like/imunologia , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Ligantes , Monócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Toll-Like/metabolismoRESUMO
NK cell anti-tumor reactivity is governed by a balance of activating and inhibitory receptors including various TNF receptor (TNFR) family members. Here we report that human tumor cells release a soluble form of the TNF family member Glucocorticoid-Induced TNFR-Related Protein (GITR) ligand (sGITRL), which can be detected in cell culture supernatants. Tumor-derived sGITRL concentration-dependently reduced NK cell cytotoxicity and IFN-gamma production, which could be overcome by neutralization of sGITRL using a GITR-Ig fusion protein. Although sGITRL did not induce apoptosis in NK cells, it diminished nuclear localized RelB, indicating that sGITRL negatively modulates NK cell NF-kappaB activity. Furthermore, we detected substantial levels of sGITRL in sera of patients with various malignancies, but not in healthy controls. Presence of sGITRL-containing patient serum in cocultures with tumor cells significantly reduced NK cell cytotoxicity and IFN-gamma production, which could again be restored by neutralization of sGITRL. The strong correlation of tumor incidence and elevated sGITRL levels indicates that sGITRL is released from cancers in vivo, leading to impaired NK cell immunosurveillance of human tumors. Our data suggest that determination of sGITRL levels might be implemented as a tumor marker in patients, and GITRL neutralization may be used to improve immunotherapeutic strategies relying on NK cell reactivity.
Assuntos
Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Inibidores do Fator de Necrose Tumoral , Fatores de Necrose Tumoral/metabolismo , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/imunologia , Humanos , Interferon gama/biossíntese , Células Matadoras Naturais/metabolismo , NF-kappa B/metabolismo , Neoplasias/metabolismo , Testes de Neutralização , Solubilidade , Fatores de Necrose Tumoral/sangueRESUMO
Glucocorticoid-induced TNF-related protein (GITR) has been shown to stimulate T cell-mediated antitumor immunity in mice. However, the functional relevance of GITR and its ligand (GITRL) for non-T cells has yet to be fully explored. In addition, recent evidence suggests that GITR plays different roles in mice and humans. We studied the role of GITR-GITRL interaction in human tumor immunology and report for the first time that primary gastrointestinal cancers and tumor cell lines of different histological origin express substantial levels of GITRL. Signaling through GITRL down-regulated the expression of the immunostimulatory molecules CD40 and CD54 and the adhesion molecule EpCAM, and induced production of the immunosuppressive cytokine TGF-beta by tumor cells. On NK cells, GITR is constitutively expressed and up-regulated following activation. Blocking GITR-GITRL interaction in cocultures of tumor cells and NK cells substantially increased cytotoxicity and IFN-gamma production of NK cells demonstrating that constitutive expression of GITRL by tumor cells diminishes NK cell antitumor immunity. GITRL-Ig fusion protein or cell surface-expressed GITRL did not induce apoptosis in NK cells, but diminished nuclear localized c-Rel and RelB, indicating that GITR might negatively modulate NK cell NF-kappaB activity. Taken together, our data indicate that tumor-expressed GITRL mediates immunosubversion in humans.