RESUMEN
Single and combination cytokines offer promise in some patients with advanced cancer. Many spontaneous and experimental cancers naturally express ligands for the lectin-like type-2 transmembrane stimulatory NKG2D immunoreceptor; however, the role this tumor recognition pathway plays in immunotherapy has not been explored to date. Here, we show that natural expression of NKG2D ligands on tumors provides an effective target for some cytokine-stimulated NK cells to recognize and suppress tumor metastases. In particular, interleukin (IL)-2 or IL-12 suppressed tumor metastases largely via NKG2D ligand recognition and perforin-mediated cytotoxicity. By contrast, IL-18 required tumor sensitivity to Fas ligand (FasL) and surprisingly did not depend on the NKG2D-NKG2D ligand pathway. A combination of IL-2 and IL-18 stimulated both perforin and FasL effector mechanisms with very potent effects. Cytokines that stimulated perforin-mediated cytotoxicity appeared relatively more effective against tumor metastases expressing NKG2D ligands. These findings indicate that a rational choice of cytokines can be made given the known sensitivity of tumor cells to perforin, FasL, and tumor necrosis factor-related apoptosis-inducing ligand and the NKG2D ligand status of tumor metastases.
Asunto(s)
Citocinas/uso terapéutico , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Metástasis de la Neoplasia/terapia , Receptores Inmunológicos/metabolismo , Animales , Apoptosis/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Proteína Ligando Fas , Citometría de Flujo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-18/inmunología , Interleucina-18/metabolismo , Interleucina-2/inmunología , Interleucina-2/metabolismo , Células Asesinas Naturales/metabolismo , Ligandos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK , Proteínas Citotóxicas Formadoras de Poros , Receptores de Células Asesinas Naturales , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Células Tumorales CultivadasRESUMEN
Natural killer (NK) cells are innate effector lymphocytes necessary for defence against stressed, microbe-infected, or malignant cells. NK cells kill target cells by either of two major mechanisms that require direct contact between NK cells and target cells. In the first pathway, cytoplasmic granule toxins, predominantly a membrane-disrupting protein known as perforin, and a family of structurally related serine proteases (granzymes) with various substrate specificities, are secreted by exocytosis and together induce apoptosis of the target cell. The granule-exocytosis pathway potently activates cell-death mechanisms that operate through the activation of apoptotic cysteine proteases (caspases), but can also cause cell death in the absence of activated caspases. The second pathway involves the engagement of death receptors (e.g. Fas/CD95) on target cells by their cognate ligands (e.g. FasL) on NK cells, resulting in classical caspase-dependent apoptosis. The comparative role of these pathways in the pathophysiology of many diseases is being dissected by analyses of gene-targeted mice that lack these molecules, and humans who have genetic mutations affecting these pathways. We are also now learning that the effector function of NK cells is controlled by interactions involving specific NK cell receptors and their cognate ligands, either on target cells, or other cells of the immune system. This review will discuss the functional importance of NK cell cytotoxicity and the receptor/ligand interactions that control these processes.
Asunto(s)
Citotoxicidad Inmunológica/fisiología , Células Asesinas Naturales/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/fisiología , Apoptosis , Citocinas/inmunología , Gránulos Citoplasmáticos/fisiología , Exocitosis/fisiología , Humanos , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/fisiología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores del Factor de Necrosis Tumoral/metabolismo , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/fisiologíaRESUMEN
The NKG2D receptor on NK cells can recognize a variety of ligands on the tumor cell surface. Using a mouse renal cancer (Renca), we show that NKG2D recognition by NK cells was crucial for their ability to limit tumor metastases in vivo in both liver and lungs using perforin-dependent effector mechanisms. However, for the R331 cell line established from Renca, NKG2D recognition and perforin-dependent lysis played no role in controlling liver metastases. R331 cells were also more resistant to perforin-dependent lysis by NK cells in vitro. We therefore used these phenotypic differences between Renca and R331 to further investigate the crucial receptor:ligand interactions required for triggering lytic effector functions of NK cells. Reconstitution of R331 cells with ICAM-1, but not Rae-1gamma, restored NKG2D-mediated, perforin-dependent lysis. Interestingly, R331 cells were efficiently lysed by NK cells using death ligand-mediated apoptosis. This death ligand-mediated killing did not depend on NKG2D recognition of its ligands on tumor cells. This result suggests that the intracellular signaling in NK cells required for perforin and death ligand-mediated lysis of tumor target cell are quite distinct, and activation of both of these antitumor lytic effector functions of NK cells could improve therapeutic benefits for certain tumors.
Asunto(s)
Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Molécula 1 de Adhesión Intercelular/biosíntesis , Neoplasias Renales/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Proteínas de la Membrana/fisiología , Receptores Inmunológicos/fisiología , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/secundario , Línea Celular Tumoral , Células Cultivadas , Citotoxicidad Inmunológica/genética , Molécula 1 de Adhesión Intercelular/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Hepáticas Experimentales/inmunología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/secundario , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Subfamilia K de Receptores Similares a Lectina de Células NK , Proteínas Citotóxicas Formadoras de Poros , Receptores de Células Asesinas NaturalesRESUMEN
Cytotoxic lymphocytes express a large family of granule serine proteases, including one member, granzyme (Grz)M, with a unique protease activity, restricted expression, and distinct gene locus. Although a number of Grzs, including GrzM, have been shown to mediate target cell apoptosis in the presence of perforin, the biological activity of Grz has been restricted to control of a number of viral pathogens, including two natural mouse pathogens, ectromelia, and murine CMV (MCMV). In this article, we describe the first reported gene targeting of GrzM in mice. GrzM-deficient mice display normal NK cell/T cell development and homeostasis and intact NK cell-mediated cytotoxicity of tumor targets as measured by membrane damage and DNA fragmentation. GrzM-deficient mice demonstrated increased susceptibility to MCMV infection typified by the presence of more viral inclusions and transiently higher viral burden in the visceral organs of GrzM-deficient mice compared with wild-type (WT) mice. The cytotoxicity of NK cells from MCMV-infected GrzM-deficient mice remained unchanged and, like WT control mice, GrzM-deficient mice eventually effectively cleared MCMV infection from the visceral organs. In contrast, GrzM-deficient mice were as resistant as WT control mice to mouse pox ectromelia infection, as well as challenge with a number of NK cell-sensitive tumors. These data confirm a role for GrzM in the host response to MCMV infection, but suggest that GrzM is not critical for NK cell-mediated cytotoxicity.
Asunto(s)
Ectromelia Infecciosa/inmunología , Infecciones por Herpesviridae/inmunología , Muromegalovirus , Serina Endopeptidasas/fisiología , Animales , Citotoxicidad Inmunológica , Granzimas , Infecciones por Herpesviridae/patología , Homeostasis , Células Asesinas Naturales/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Muromegalovirus/inmunología , Neoplasias Experimentales/inmunología , Linfocitos T/fisiologíaRESUMEN
We have investigated the primary and secondary immunity generated in vivo by a MHC class I-deficient tumor cell line that expressed CD80 (B7-1). CD80 expression enhanced primary NK cell-mediated tumor rejection in vivo and T cell immunity against secondary tumor challenge. CD80 expression enhanced primary NK cell-mediated tumor rejection, and both NK cell perforin and IFN-gamma activity were critical for the rejection of MHC class I-deficient RMA-S-CD80 tumor cells. This primary rejection process stimulated the subsequent development of specific CTL and Th1 responses against Ags expressed by the MHC class I-deficient RMA-S tumor cells. The development of effective secondary T cell immunity could be elicited by irradiated RMA-S-CD80 tumor cells and was dependent upon NK cells and IFN-gamma in the priming response. Our findings demonstrate a key role for IFN-gamma in innate and adaptive immunity triggered by CD80 expression on tumor cells.
Asunto(s)
Antígeno B7-1/metabolismo , Memoria Inmunológica , Interferón gamma/fisiología , Células Asesinas Naturales/inmunología , Neoplasias Experimentales/inmunología , Animales , División Celular , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Cinética , Activación de Linfocitos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/patología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Células Tumorales CultivadasRESUMEN
We have investigated the primary immunity generated in vivo by MHC class I-deficient and -competent tumor cell lines that expressed the NKG2D ligand retinoic acid early inducible-1 (Rae-1) beta. Rae-1beta expression on class I-deficient RMA-S lymphoma cells enhanced primary NK cell-mediated tumor rejection in vivo, whereas RMA-Rae-1beta tumor cells were rejected by a combination of NK cells and CD8(+) T cells. Rae-1beta expression stimulated NK cell cytotoxicity and IFN-gamma secretion in vitro, but not proliferation. Surprisingly, only NK cell perforin-mediated cytotoxicity, but not production of IFN-gamma, was critical for the rejection of Rae-1beta-expressing tumor cells in vivo. This distinct requirement for perforin activity contrasts with the NK cell-mediated rejection of MHC class I-deficient RMA-S tumor cells expressing other activating ligands such as CD70 and CD80. Thus, these results indicated that NKG2D acted as a natural cytotoxicity receptor to stimulate perforin-mediated elimination of ligand-expressing tumor cells.
Asunto(s)
Rechazo de Injerto/inmunología , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Receptores Inmunológicos/fisiología , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Células Cultivadas , Citotoxicidad Inmunológica/genética , Rechazo de Injerto/genética , Interferón gamma/metabolismo , Células Asesinas Naturales/metabolismo , Ligandos , Activación de Linfocitos/genética , Linfoma de Células T/inmunología , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subfamilia K de Receptores Similares a Lectina de Células NK , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores Inmunológicos/metabolismo , Receptores de Células Asesinas Naturales , Tretinoina/fisiología , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
NKG2D is an activation receptor on NK cells and has been demonstrated as a primary cytotoxicity receptor for mouse NK cells. Primary rejection of class I-deficient RMA-S lymphoma cells expressing the NKG2D ligand, retinoic acid early inducible-1beta, was critically dependent upon NK cell perforin and occurred independently of T cells. NKG2D-triggered NK cell rejection of RMA-S-retinoic acid early inducible-1beta tumor primed a secondary tumor-specific T cell response mediated by both CD4+ and CD8+ T cells in the effector phase. Surprisingly, during the priming phase, CD4+ T cells, but not CD8+ T cells, were also required to generate this secondary T cell immunity; however, T cell priming was independent of Th1 cytokines, such as IFN-gamma and IL-12. These data imply a novel pathway for priming T cell immunity, that is, stimulated upon NK cell-mediated cytotoxicity of NKG2D ligand-expressing tumor cells, dependent upon CD4+ T cells in the primary phase, and independent of conventional Th1-type immunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica , Rechazo de Injerto/inmunología , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/fisiología , Células TH1/inmunología , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica/genética , Inmunidad Celular/genética , Inmunidad Innata/genética , Inmunización Secundaria , Memoria Inmunológica/genética , Inyecciones Subcutáneas , Células Asesinas Naturales/trasplante , Linfoma de Células T/inmunología , Linfoma de Células T/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subfamilia K de Receptores Similares a Lectina de Células NK , Trasplante de Neoplasias , Receptores de Células Asesinas Naturales , Linfocitos T Reguladores/inmunologíaRESUMEN
Cell death is mediated by cytotoxic lymphocytes through various granule serine proteases released with perforin. The unique protease activity, restricted expression, and distinct gene locus of granzyme M suggested this enzyme might have a novel biological function or trigger a novel form of cell death. Herein, we demonstrate that in the presence of perforin, the protease activity of granzyme M rapidly and effectively induces target cell death. In contrast to granzyme B, cell death induced by granzyme M does not feature obvious DNA fragmentation, occurs independently of caspases, caspase activation, and perturbation of mitochondria and is not inhibited by overexpression of Bcl-2. These data raise the likelihood that granzyme M represents a third major and specialized perforin-dependent cell death pathway that plays a significant role in death mediated by NK cells.
Asunto(s)
Serina Endopeptidasas/fisiología , Animales , Apoptosis , Western Blotting , Caspasas/metabolismo , Muerte Celular , Línea Celular , Cromo/metabolismo , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática , Eritrocitos/metabolismo , Granzimas , Células HeLa , Humanos , Yodo/metabolismo , Células Jurkat , Células K562 , Células Asesinas Naturales/metabolismo , Linfocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocondrias/metabolismo , Perforina , Proteínas Citotóxicas Formadoras de Poros , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/metabolismo , Ovinos , Factores de TiempoRESUMEN
Natural killer (NK) cells may modulate the development of adaptive immune responses, but until now there has been little evidence to support this hypothesis. We investigated the primary and secondary immunity elicited by various tumor cell lines that express CD70 and interact with CD70 ligand (CD27), which is constitutively expressed on NK cells. CD70 expression enhanced primary tumor rejection in vivo as well as T cell immunity against secondary tumor challenge. Primary rejection of major histocompatibility complex (MHC) class I-deficient RMA-S.CD70 tumor cells was mediated by NK cells and perforin- and interferon-gamma-dependent mechanisms. This NK cell-mediated process also efficiently evoked the subsequent development of tumor-specific cytotoxic and T helper type 1 responses to the parental, MHC class I-sufficient, RMA tumor cells. Thus CD27-CD70 interactions provide a key link between innate NK cell responses and adaptive T cell immunity.
Asunto(s)
Antígenos CD , Rechazo de Injerto/inmunología , Memoria Inmunológica , Células Asesinas Naturales/inmunología , Linfoma de Células T/inmunología , Proteínas de la Membrana/inmunología , Subgrupos de Linfocitos T/inmunología , Neoplasias del Timo/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Ligando CD27 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Interferón gamma/fisiología , Linfoma de Células T/patología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Modelos Inmunológicos , Trasplante de Neoplasias/inmunología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Neoplasias del Timo/patología , Células Tumorales Cultivadas/trasplanteRESUMEN
The antimetastatic effect of the CD1d-binding glycolipid, alpha-galactosylceramide (alpha-GalCer), is mediated by NK1.1(+)T (NKT) cells; however, the mechanisms behind this process are poorly defined. Although it has been shown to involve NK cells and interferon-gamma (IFN-gamma) production, the way these factors collaborate to mediate effective tumor rejection and the importance of other factors characteristic of NKT cell and NK cell activation are unknown. Using gene-targeted mice and antibody treatments, the critical need for interleukin 12 (IL-12), IFN-gamma, and NK cells has been shown in the antimetastatic activity of alpha-GalCer in the lungs and the liver. By contrast, in lung and liver metastasis models, cytotoxic molecules expressed by NK cells and NKT cells (perforin, Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand) and an NKT cell-secreted cytokine, IL-4, were not necessary for the antitumor activity of alpha-GalCer. Like IL-12, IL-18 was required for optimal serum IFN-gamma induction and control of lung metastases by alpha-GalCer. IL-18 was unnecessary for alpha-GalCer-related suppression of liver metastases. Most importantly, after adoptive transfer of alpha-GalCer-reactive NKT cells or NK cells into NKT cell-deficient, IFN-gamma-deficient, or RAG-1-deficient mice, it was demonstrated that the sequential production of IFN-gamma by NKT cells and NK cells was absolutely required to reconstitute the antimetastatic activity of alpha-GalCer.
Asunto(s)
Galactosilceramidas/farmacología , Interferón gamma/fisiología , Células Asesinas Naturales/inmunología , Metástasis de la Neoplasia/prevención & control , Traslado Adoptivo/métodos , Animales , Citotoxicidad Inmunológica , Galactosilceramidas/administración & dosificación , Galactosilceramidas/inmunología , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-12/genética , Interleucina-12/fisiología , Interleucina-18/genética , Interleucina-18/fisiología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/trasplante , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Perforina , Proteínas Citotóxicas Formadoras de PorosRESUMEN
Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.
Asunto(s)
Péptidos/química , Péptidos/metabolismo , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Bovinos , Línea Celular , Quimotripsina/metabolismo , Granzimas , Humanos , Hidrólisis , Cinética , Ratones , Péptidos/farmacología , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Serina Endopeptidasas/genética , Serina Endopeptidasas/aislamiento & purificación , Inhibidores de Serina Proteinasa/química , Spodoptera , Especificidad por Sustrato , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Blastocyst MHC is a recently identified mouse MHC class Ib gene, which is selectively expressed in blastocyst and placenta, and may be the mouse homolog of HLA-G gene the products of which have been implicated in protection of fetal trophoblasts from maternal NK cells and evasion of some tumor cells from NK cell attack. In this study, we identified two blastocyst MHC gene transcripts encoding a full-length alpha-chain (bc1) and an alternatively spliced form lacking the alpha2 domain (bc2), which may be homologous to HLA-G1 and HLA-G2, respectively. Both placenta and a teratocarcinoma cell line predominantly expressed the bc2 transcript. When these cDNAs were expressed in TAP-deficient RMA-S or TAP-sufficient RMA cells, only bc1 protein was expressed on the surface of RMA cells, but both bc1 and bc2 proteins were retained in the cytoplasm of RMA-S cells. Significantly, the RMA-S cells expressing either bc1 or bc2 were protected from lysis by NK cells in vitro. This protection was at least partly mediated by up-regulation of Qa-1(b) expression on the surface of RMA-S cells, which engaged the CD94/NKG2A inhibitory receptor on NK cells. More importantly, the bc1- or bc2-expressing RMA-S cells were significantly protected from NK cell-mediated rejection in vivo. These results suggested a role for blastocyst MHC in protecting TAP-deficient trophoblasts and tumor cells from NK cell attack in vivo.