RESUMEN
Tumor-associated macrophages (TAM) are abundant in several tumor types and usually correlate with poor prognosis. Previously, we demonstrated that anti-inflammatory macrophages (M2) inhibit NK cell effector functions. Here, we explored the impact of TAM on NK cells in the context of clear-cell renal cell carcinoma (ccRCC). Bioinformatics analysis revealed that an exhausted NK cell signature strongly correlated with an M2 signature. Analysis of TAM from human ccRCC samples confirmed that they exhibited an M2-skewed phenotype and inhibited IFN-γ production by NK cells. Moreover, human M0 macrophages cultured with conditioned media from ccRCC cell lines generated macrophages with an M2-skewed phenotype (TAM-like), which alike TAM, displayed suppressive activity on NK cells. Moreover, TAM depletion in the mouse Renca ccRCC model resulted in delayed tumor growth and reduced volume, accompanied by an increased frequency of IFN-γ-producing tumor-infiltrating NK cells that displayed heightened expression of T-bet and NKG2D and reduced expression of the exhaustion-associated co-inhibitory molecules PD-1 and TIM-3. Therefore, in ccRCC, the tumor microenvironment polarizes TAM toward an immunosuppressive profile that promotes tumor-infiltrating NK cell dysfunction, contributing to tumor progression. In addition, immunotherapy strategies targeting TAM may result in NK cell reinvigoration, thereby counteracting tumor progression.
Asunto(s)
Carcinoma de Células Renales , Interferón gamma , Neoplasias Renales , Células Asesinas Naturales , Macrófagos Asociados a Tumores , Células Asesinas Naturales/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Interferón gamma/metabolismo , Interferón gamma/inmunología , Humanos , Animales , Ratones , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Progresión de la Enfermedad , Línea Celular Tumoral , Microambiente Tumoral/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
NKG2D is a major natural killer (NK) cell-activating receptor that recognizes eight ligands (NKG2DLs), including MICA, and whose engagement triggers NK cell effector functions. As NKG2DLs are upregulated on tumor cells but tumors can subvert the NKG2D-NKG2DL axis, NKG2DLs constitute attractive targets for antibody (Ab)-based immuno-oncology therapies. However, such approaches require a deep characterization of NKG2DLs and NKG2D cell surface expression on primary tumor and immune cells. Here, using a bioinformatic analysis, we observed that MICA is overexpressed in renal cell carcinoma (RCC), and we also detected an association between the NKG2D-MICA axis and a diminished overall survival of RCC patients. Also, by flow cytometry (FC), we observed that MICA was the only NKG2DL over-expressed on clear cell renal cell carcinoma (ccRCC) tumor cells, including cancer stem cells (CSC) that also coexpressed NKG2D. Moreover, tumor-infiltrating leukocytes (TIL), but not peripheral blood lymphoid cells (PBL) from ccRCC patients, over-expressed MICA, ULBP3 and ULBP4. In addition, NKG2D was downregulated on peripheral blood NK cells (PBNK) from ccRCC patients but upregulated on tumor-infiltrating NK cells (TINK). These TINK exhibited impaired degranulation that negatively correlated with NKG2D expression, diminished IFN-γ production, upregulation of TIM-3, and an impaired glucose intake upon stimulation with cytokines, indicating that they are dysfunctional, display features of exhaustion and an altered metabolic fitness. We conclude that ccRCC patients exhibit a distorted MICA-NKG2D axis, and MICA emerges as the forefront NKG2DL for the development of targeted therapies in ccRCC.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Carcinoma de Células Renales/terapia , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Neoplasias Renales/terapia , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Receptores de Células Asesinas NaturalesRESUMEN
Immune checkpoint inhibitors (ICI) revolutionized the field of immuno-oncology and opened new avenues towards the development of novel assets to achieve durable immune control of cancer. Yet, the presence of tumor immune evasion mechanisms represents a challenge for the development of efficient treatment options. Therefore, combination therapies are taking the center of the stage in immuno-oncology. Such combination therapies should boost anti-tumor immune responses and/or target tumor immune escape mechanisms, especially those created by major players in the tumor microenvironment (TME) such as tumor-associated macrophages (TAM). Natural killer (NK) cells were recently positioned at the forefront of many immunotherapy strategies, and several new approaches are being designed to fully exploit NK cell antitumor potential. One of the most relevant NK cell-activating receptors is NKG2D, a receptor that recognizes 8 different NKG2D ligands (NKG2DL), including MICA and MICB. MICA and MICB are poorly expressed on normal cells but become upregulated on the surface of damaged, transformed or infected cells as a result of post-transcriptional or post-translational mechanisms and intracellular pathways. Their engagement of NKG2D triggers NK cell effector functions. Also, MICA/B are polymorphic and such polymorphism affects functional responses through regulation of their cell-surface expression, intracellular trafficking, shedding of soluble immunosuppressive isoforms, or the affinity of NKG2D interaction. Although immunotherapeutic approaches that target the NKG2D-NKG2DL axis are under investigation, several tumor immune escape mechanisms account for reduced cell surface expression of NKG2DL and contribute to tumor immune escape. Also, NKG2DL polymorphism determines functional NKG2D-dependent responses, thus representing an additional challenge for leveraging NKG2DL in immuno-oncology. In this review, we discuss strategies to boost MICA/B expression and/or inhibit their shedding and propose that combination strategies that target MICA/B with antibodies and strategies aimed at promoting their upregulation on tumor cells or at reprograming TAM into pro-inflammatory macrophages and remodeling of the TME, emerge as frontrunners in immuno-oncology because they may unleash the antitumor effector functions of NK cells and cytotoxic CD8 T cells (CTL). Pursuing several of these pipelines might lead to innovative modalities of immunotherapy for the treatment of a wide range of cancer patients.
Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Animales , Terapia Combinada , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Proteínas Ligadas a GPI/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ligandos , Terapia Molecular Dirigida , Neoplasias/patología , Neoplasias/terapia , Receptores de Células Asesinas Naturales/genética , Receptores de Células Asesinas Naturales/metabolismo , Transducción de Señal , Escape del Tumor , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Although natural killer (NK) cells infiltrate clear cell renal cell carcinomas (ccRCC), the most frequent malignancy of the kidney, tumor progression suggests that they become dysfunctional. As ccRCC-driven subversion of NK cell effector functions is usually accompanied by phenotypic changes, analysis of such alterations might lead to the identification of novel biomarkers and/or targets in immuno-oncology. Consequently, we performed a phenotypic analysis of peripheral blood NK cells (PBNK) and tumor-infiltrating NK cells (TINK) from ccRCC patients. Compared to HD, PBNK from ccRCC patients exhibited features of activated cells as shown by CD25, CD69 and CD62L expression. They also displayed increased expression of DNAM-1, CD48, CD45, MHC-I, reduced expression of NKG2D, and higher frequencies of CD85j+ and PD-1+ cells. In addition, compared to PBNK from ccRCC patients, TINK exhibited higher expression of activation markers, tissue residency features and decreased expression of the activating receptors DNAM-1, NKp30, NKp46, NKp80 and CD16, suggesting a more inhibitory phenotype. Analysis of The Cancer Genome Atlas (TCGA) revealed that CD48, CD45, CD85j and PD-1 are significantly overexpressed in ccRCC and that their expression is associated with an NK cell infiltration signature. Calculation of z-scores revealed that their expression on PBNK, alone or combined, distinguished ccRCC patients from HD. Therefore, these molecules emerge as novel potential biomarkers and our results suggest that they might constitute possible targets for immunotherapy in ccRCC patients.
Asunto(s)
Carcinoma de Células Renales/etiología , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/etiología , Neoplasias Renales/metabolismo , Células Asesinas Naturales/inmunología , Recuento de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Anciano , Biomarcadores , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/terapia , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Expresión Génica , Humanos , Inmunofenotipificación , Neoplasias Renales/patología , Neoplasias Renales/terapia , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , NefrectomíaRESUMEN
BACKGROUND: Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. METHODS: We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. RESULTS: Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. CONCLUSIONS: Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Linfoma/inmunología , Proteínas Recombinantes de Fusión/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Animales , Brucella/enzimología , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Linfoma/patología , Linfoma/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/terapiaRESUMEN
The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b(+)Gr-1(+), mostly CD11b(+)Ly6G(+)Ly6C(int) and CD11b(+)Ly6G(-)Ly6C(high) cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b(+)Ly6G(+)Ly6C(int) cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b(+)Ly6G(+)Ly6C(int) cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b(+)Gr-1(+) cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b(+)Gr-1(+) cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b(+)Ly6G(+)Ly6C(int) cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.
Asunto(s)
Antígenos Ly/inmunología , Neoplasias de la Mama/inmunología , Antígeno CD11b/inmunología , Células Asesinas Naturales/inmunología , Acetato de Medroxiprogesterona/farmacología , Células Mieloides/inmunología , Animales , Antígenos Ly/metabolismo , Antineoplásicos Hormonales/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Antígeno CD11b/metabolismo , Diferenciación Celular , Proliferación Celular , Citotoxicidad Inmunológica , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Receptores de Glucocorticoides/metabolismo , Factor de Transcripción STAT3/metabolismo , Células Tumorales CultivadasRESUMEN
Two populations of human natural killer (NK) cells can be identified in peripheral blood. The majority are CD3(-)CD56(dim) cells while the minority exhibits a CD3(-)CD56(bright) phenotype. In vitro evidence indicates that CD56(bright) cells are precursors of CD56(dim) cells, but in vivo evidence is lacking. Here, we studied NK cells from a patient that suffered from a melanoma and opportunistic fungal infection during childhood. The patient exhibited a stable phenotype characterized by a reduction in the frequency of peripheral blood CD3(-)CD56(dim) NK cells, accompanied by an overt increase in the frequency and absolute number of CD3(-)CD56(bright) cells. These NK cells exhibited similar expression of perforin, CD57 and CD158, the major activating receptors CD16, NKp46, NKG2D, DNAM-1, and 2B4, as well as the inhibitory receptor CD94/NKG2A, on both CD56(bright) and CD56(dim) NK cells as healthy controls. Also, both NK cell subpopulations produced IFN-γ upon stimulation with cytokines, and CD3(-)CD56(dim) NK cells degranulated in response to cytokines or K562 cells. However, upon stimulation with cytokines, a substantial fraction of CD56(dim) cells failed to up-regulate CD57 and CD158, showed a reduction in the percentage of CD16(+) cells, and CD56(bright) cells did not down-regulate CD62L, suggesting that CD56(dim) cells could not acquire a terminally differentiated phenotype and that CD56(bright) cells exhibit a maturation defect that might result in a potential altered migration pattern. These observations, support the notion that NK cells of this patient display a maturation/activation defect that precludes the generation of mature NK cells at a normal rate accompanied by CD56(dim) NK cells that cannot completely acquire a terminally differentiated phenotype. Thus, our results provide evidence that support the concept that in vivo CD56(bright) NK cells differentiate into CD56(dim) NK cells, and contribute to further understand human NK cell ontogeny.
Asunto(s)
Antígeno CD56 , Diferenciación Celular , Linaje de la Célula , Células Asesinas Naturales , Antígeno CD56/sangre , Antígeno CD56/genética , Antígeno CD56/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Citometría de Flujo , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células K562 , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunologíaRESUMEN
MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.