Tumor-Experienced Human NK Cells Express High Levels of PD-L1 and Inhibit CD8+ T Cell Proliferation.

Natural Killer (NK) cells play a key role in cancer immunosurveillance. However, NK cells from cancer patients display an altered phenotype and impaired effector functions. In addition, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation, and autoimmunity. Here, we analyzed clear cell renal cell carcinoma (ccRCC) datasets from The Cancer Genome Atlas (TCGA) and observed that a higher expression of NK cell signature genes is associated with reduced survival. Analysis of fresh tumor samples from ccRCC patients unraveled the presence of a high frequency of tumor-infiltrating PD-L1+ NK cells, suggesting that these NK cells might exhibit immunoregulatory functions. In vitro, PD-L1 expression was induced on NK cells from healthy donors (HD) upon direct tumor cell recognition through NKG2D and was further up-regulated by monocyte-derived IL-18. Moreover, in vitro generated PD-L1hi NK cells displayed an activated phenotype and enhanced effector functions compared to PD-L1- NK cells, but simultaneously, they directly inhibited CD8+ T cell proliferation in a PD-L1-dependent manner. Our results suggest that tumors might drive the development of PD-L1-expressing NK cells that acquire immunoregulatory functions in humans. Hence, rational manipulation of these regulatory cells emerges as a possibility that may lead to improved anti-tumor immunity in cancer patients.

Regulatory functions of NK cells during infections and cancer.

After recognition, NK cells can kill susceptible target cells through perforin-dependent mechanisms or by inducing death receptor-mediated apoptosis, and they can also secrete cytokines that are pivotal for immunomodulation. Despite the critical role as effector cells against tumors and virus-infected cells, NK cells have been implicated in the regulation of T cell-mediated responses in different models of autoimmunity, transplantation, and viral infections. Here, we review the mechanisms described for NK cell-mediated inhibition of adaptive immune responses, with spotlight on the emerging evidence of their regulatory role that shapes antitumor immune responses.

Immunomodulation of NK Cell Activity.

Natural killer (NK) cells can kill virus-infected cells and tumor cells without prior sensitization and secrete numerous cytokines and chemokines that modulate the activity of different cells of the immune system. The recognition of target cells is mediated by germ line-encoded receptors, and the activity of NK cells can be further regulated by soluble factors such as cytokines and Toll-like receptor ligands. Thus, NK cells display an exciting potential as a powerful immunotherapeutic tool against malignant diseases, and different strategies are being tested aiming to overcome tumor-induced NK cell suppression and restore NK-cell mediated antitumor activity. This section describes different flow cytometry-based protocols to study NK cell effector functions, which can be used to evaluate the immunomodulatory ability of different therapeutic compounds.

Innate lymphoid cells. New players in tissue homeostasis and inflammatory responses.

In recent times, our understanding of the role of the immune system in different physiopathological situations has increased markedly. A new set of cells, generically known as innate lymphoid cells (ILC), has been discovered in the lymphoid compartment. Five ILC subsets can be recognized according to phenotypic and functional similarities with different subpopulations of T lymphocytes. Unlike T and B lymphocytes, ILC do not express antigen receptors nor undergo selection and clonal expansion upon activation. Instead, they respond rapidly to cytokines and danger signals in infected or inflamed tissues, producing cytokines that direct the immune response toward a type suitable for controlling the initial insult. In addition, ILC establish a crosstalk with other cells of the microenvironment that contributes to the maintenance and restoration of tissue homeostasis. Although many evidences on ILC were obtained from animal models, solid data confirm their existence in humans and their role in various inflammatory disorders. In this article, we address new knowledge on ILC, particularly on their role in the homeostasis of the immune system and in various inflammatory pathologies, in order to present new actors regulating immunity and immunopathology and affecting human health.

En tiempos recientes, nuestra comprensión del rol del sistema inmune en diferentes situaciones fisiopatológicas ha aumentado notablemente. En el compartimiento linfoide se ha descubierto un conjunto de células denominadas células linfoides innatas o innate lymphoid cells (ILC). Las ILC incluyen cinco grupos, clasificados según su similitud fenotípica y funcional con diferentes subpoblaciones de linfocitos T. A diferencia de los linfocitos T y B, las ILC no expresan receptores de antígeno ni sufren selección y expansión clonal cuando se activan. En cambio, responden rápidamente frente a citoquinas y señales de peligro en tejidos infectados o inflamados produciendo citoquinas que dirigen la respuesta inmune hacia un tipo adecuado para controlar la noxa original. Además, las ILC establecen un diálogo cruzado con otras células del microambiente que contribuye al mantenimiento y la restauración de la homeostasis tisular. Si bien muchas evidencias acerca de las ILC fueron obtenidas en modelos animales, existen datos sólidos que confirman su existencia en seres humanos y su papel en diversos trastornos inflamatorios. En este artículo, abordamos los nuevos conocimientos acerca de las ILC, y su rol en la homeostasis del sistema inmune y en diversas patologías inflamatorias, con el fin de presentar nuevos actores que regulan la inmunidad y la inmunopatología, lo que repercute en la salud humana.

Human M2 Macrophages Limit NK Cell Effector Functions through Secretion of TGF-ß and Engagement of CD85j.

NK cells play important roles during immunosurveillance against tumors and viruses as they trigger cytotoxicity against susceptible cells and secrete proinflammatory cytokines such as IFN-γ. In addition, upon activation, macrophages can become proinflammatory (M1) or anti-inflammatory (M2) cells. Although the consequences of the cross-talk between M1 and NK cells are known, the outcome of the cross-talk between M2 and NK cells remains ill-defined. Therefore, in the current work, we investigated the outcome and the underlying mechanisms of the interaction between resting or stimulated human NK cells with M1 or M2. We observed a lower percentage of activated NK cells that produced less IFN-γ upon coculture with M2. Also, CD56dim NK cells cocultured with M2 displayed lower degranulation and cytotoxic activity than NK cells cocultured with M1. Soluble TGF-ß and M2-driven upregulation of CD85j (ILT-2) on NK cells accounted for the diminished IFN-γ production by CD56bright NK cells, whereas M2-driven upregulation of CD85j on NK cells accounted for the generation of hyporesponsive CD56dim NK cells with limited degranulation and cytotoxic capacity. Accordingly, M2 expressed higher amounts of HLA-G, the main ligand for CD85j, than M1. Hyporesponsiveness to degranulation in NK cells was not restored at least for several hours upon removal of M2. Therefore, alternatively activated macrophages restrain NK cell activation and effector functions through different mechanisms, leading to NK cells that display diminished IFN-γ production and at least a transiently impaired degranulation ability. These results unravel an inhibitory circuit of possible relevance in pathological situations.

Interleukin (IL)-23 Stimulates IFN-γ Secretion by CD56bright Natural Killer Cells and Enhances IL-18-Driven Dendritic Cells Activation.

Interleukin (IL)-23 is a member of the IL-12 family of cytokines that, as the other members of this family, is secreted by monocytes, macrophages, and dendritic cells (DC) upon recognition of bacterial, viral, and fungal components. IL-23 is critical during immunity against acute infections, and it is also involved in the development of autoimmune diseases. Although immunoregulatory effects of IL-23 on mouse natural killer (NK) cells have been described, the effect of IL-23 on human NK cells remains ill-defined. In this study, we observed that monocytes stimulated with LPS secreted IL-23 and that blockade of this cytokine during monocyte and NK cell coculture led to a diminished production of IFN-γ by NK cells. Accordingly, rIL-23-induced NK cell activation and stimulated IFN-γ production by CD56bright NK cells. This effect involved MEK1/MEK2, JNK, PI3K, mammalian target of rapamycin, and NF-κB, but not STAT-1, STAT-3, nor p38 MAPK pathways. Moreover, while NK cell-mediated cytotoxicity remained unaltered, antibody-dependent cellular cytotoxicity (ADCC) was enhanced after IL-23 stimulation. In addition, IL-23 displayed a synergistic effect with IL-18 for IFN-γ production by both CD56bright and CD56dim NK cells, and this effect was due to a priming effect of IL-23 for IL-18 responsiveness. Furthermore, NK cells pre-stimulated with IL-18 promoted an increase in CD86 expression and IL-12 secretion by DC treated with LPS, and IL-23 potentiated these effects. Moreover, IL-23-driven enhancement of NK cell "helper" function was dependent on NK cell-derived IFN-γ. Therefore, our results suggest that IL-23 may trigger NK cell-mediated "helper" effects on adaptive immunity, shaping T cell responses during different pathological situations through the regulation of DC maturation.

Predictive Outcomes for HER2-enriched Cancer Using Growth and Metastasis Signatures Driven By SPARC.

Understanding the mechanism of metastatic dissemination is crucial for the rational design of novel therapeutics. The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein which has been extensively associated with human breast cancer aggressiveness although the underlying mechanisms are still unclear. Here, shRNA-mediated SPARC knockdown greatly reduced primary tumor growth and completely abolished lung colonization of murine 4T1 and LM3 breast malignant cells implanted in syngeneic BALB/c mice. A comprehensive study including global transcriptomic analysis followed by biological validations confirmed that SPARC induces primary tumor growth by enhancing cell cycle and by promoting a COX-2-mediated expansion of myeloid-derived suppressor cells (MDSC). The role of SPARC in metastasis involved a COX-2-independent enhancement of cell disengagement from the primary tumor and adherence to the lungs that fostered metastasis implantation. Interestingly, SPARC-driven gene expression signatures obtained from these murine models predicted the clinical outcome of patients with HER2-enriched breast cancer subtypes. In total, the results reveal that SPARC and its downstream effectors are attractive targets for antimetastatic therapies in breast cancer.Implications: These findings shed light on the prometastatic role of SPARC, a key protein expressed by breast cancer cells and surrounding stroma, with important consequences for disease outcome. Mol Cancer Res; 15(3); 304-16. ©2016 AACR.

Expression and Functional Role of α7 Nicotinic Receptor in Human Cytokine-stimulated Natural Killer (NK) Cells.

The homomeric α7 nicotinic receptor (nAChR) is one of the most abundant nAChRs in the central nervous system where it contributes to cognition, attention, and working memory. α7 nAChR is also present in lymphocytes, dendritic cells (DCs), and macrophages and it is emerging as an important drug target for intervention in inflammation and sepsis. Natural killer (NK) cells display cytotoxic activity against susceptible target cells and modulate innate and adaptive immune responses through their interaction with DCs. We here show that human NK cells also express α7 nAChR. α7 nAChR mRNA is detected by RT-PCR and cell surface expression of α7 nAChR is detected by confocal microscopy and flow cytometry using α-bungarotoxin, a specific antagonist. Both mRNA and protein levels increase during NK stimulation with cytokines (IL-12, IL-18, and IL-15). Exposure of cytokine-stimulated NK cells to PNU-282987, a specific α7 nAChR agonist, increases intracellular calcium concentration ([Ca(2+)]i) mainly released from intracellular stores, indicating that α7 nAChR is functional. Moreover, its activation by PNU-282987 plus a specific positive allosteric modulator greatly enhances the Ca(2+) responses in NK cells. Stimulation of NK cells with cytokines and PNU-282987 decreases NF-κB levels and nuclear mobilization, down-regulates NKG2D receptors, and decreases NKG2D-dependent cell-mediated cytotoxicity and IFN-γ production. Also, such NK cells are less efficient to trigger DC maturation. Thus, our results demonstrate the anti-inflammatory role of α7 nAChR in NK cells and suggest that modulation of its activity in these cells may constitute a novel target for regulation of the immune response.

NK Cells Restrain Spontaneous Antitumor CD8+ T Cell Priming through PD-1/PD-L1 Interactions with Dendritic Cells.

Despite the classical function of NK cells in the elimination of tumor and of virus-infected cells, evidence for a regulatory role for NK cells has been emerging in different models of autoimmunity, transplantation, and viral infections. However, this role has not been fully explored in the context of a growing tumor. In this article, we show that NK cells can limit spontaneous cross-priming of tumor Ag-specific CD8(+) T cells, leading to reduced memory responses. After challenge with MC57 cells transduced to express the model Ag SIY (MC57.SIY), NK cell-depleted mice exhibited a significantly higher frequency of SIY-specific CD8(+) T cells, with enhanced IFN-γ production and cytotoxic capability. Depletion of NK cells resulted in a CD8(+) T cell population skewed toward an effector memory T phenotype that was associated with enhanced recall responses and delayed tumor growth after a secondary tumor challenge with B16.SIY cells. Dendritic cells (DCs) from NK cell-depleted tumor-bearing mice exhibited a more mature phenotype. Interestingly, tumor-infiltrating and tumor-draining lymph node NK cells displayed an upregulated expression of the inhibitory molecule programmed death ligand 1 that, through interaction with programmed death-1 expressed on DCs, limited DC activation, explaining their reduced ability to induce tumor-specific CD8(+) T cell priming. Our results suggest that NK cells can, in certain contexts, have an inhibitory effect on antitumor immunity, a finding with implications for immunotherapy in the clinic.

Regulatory Dendritic Cells Restrain NK Cell IFN-γ Production through Mechanisms Involving NKp46, IL-10, and MHC Class I-Specific Inhibitory Receptors.

Cross-talk between mature dendritic cells (mDC) and NK cells through the cell surface receptors NKp30 and DNAM-1 leads to their reciprocal activation. However, the impact of regulatory dendritic cells (regDC) on NK cell function remains unknown. As regDC constrain the immune response in different physiological and pathological conditions, the aim of this work was to investigate the functional outcome of the interaction between regDC and NK cells and the associated underlying mechanisms. RegDC generated from monocyte-derived DC treated either with LPS and dexamethasone, vitamin D3, or vitamin D3 and dexamethasone instructed NK cells to secrete lower amounts of IFN-γ than NK cells exposed to mDC. Although regDC triggered upregulation of the activation markers CD69 and CD25 on NK cells, they did not induce upregulation of CD56 as mDC, and silenced IFN-γ secretion through mechanisms involving insufficient secretion of IL-18, but not IL-12 or IL-15 and/or induction of NK cell apoptosis. Blocking experiments demonstrated that regDC curb IFN-γ secretion by NK cells through a dominant suppressive mechanism involving IL-10, NK cell inhibitory receptors, and, unexpectedly, engagement of the activating receptor NKp46. Our findings unveil a previously unrecognized cross-talk through which regDC shape NK cell function toward an alternative activated phenotype unable to secrete IFN-γ, highlighting the plasticity of NK cells in response to tolerogenic stimuli. In addition, our findings contribute to identify a novel inhibitory role for NKp46 in the control of NK cell function, and have broad implications in the resolution of inflammatory responses and evasion of antitumor responses.

IL-27 stimulates human NK-cell effector functions and primes NK cells for IL-18 responsiveness.

IL-27, a member of the IL-12 family of cytokines, is produced by APCs, and displays pro- and anti-inflammatory effects. How IL-27 affects human NK cells still remains unknown. In this study, we observed that mature DCs secreted IL-27 and that blockade of IL-27R (CD130) reduced the amount of IFN-γ produced by NK cells during their coculture, showing the importance of IL-27 during DC-NK-cell crosstalk. Accordingly, human rIL-27 stimulated IFN-γ secretion by NK cells in a STAT1-dependent manner, induced upregulation of CD25 and CD69 on NK cells, and displayed a synergistic effect with IL-18. Preincubation experiments demonstrated that IL-27 primed NK cells for IL-18-induced IFN-γ secretion, which was associated with an IL-27-driven upregulation of T-bet expression. Also, IL-27 triggered NKp46-dependent NK-cell-mediated cytotoxicity against Raji, T-47D, and HCT116 cells, and IL-18 enhanced this cytotoxic response. Such NK-cell-mediated cytotoxicity involved upregulation of perforin, granule exocytosis, and TRAIL-mediated cytotoxicity but not Fas-FasL interaction. Moreover, IL-27 also potentiated Ab-dependent cell-mediated cytotoxicity against mAb-coated target cells. Taken together, IL-27 stimulates NK-cell effector functions, which might be relevant in different physiological and pathological situations.

Broad MICA/B expression in the small bowel mucosa: a link between cellular stress and celiac disease.

The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B(+) T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B(+) B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role.

Histone deacetylase inhibitors impair NK cell viability and effector functions through inhibition of activation and receptor expression.

HDACi are being used as a novel, therapeutic approach for leukemias and other hematological malignancies. However, their effect on immune cells remains ill-defined, as HDACi may impair immune surveillance. In this work, we demonstrate that TSA, VPA, and NaB inhibited IFN-γ production by CD56(dim) and CD56(bright) NK cells and NK cell-mediated cytotoxicity against K562 target cells. HDACi promoted minor NK cell apoptosis but inhibited nuclear mobilization of NF-κB p50, which was accompanied by a robust down-regulation of NKG2D and NKp46 on resting NK cells and of NKG2D, NKp44, NKp46, and CD25 on cytokine-activated NK cells. Decreased CD25 expression promoted a weakened IFN-γ secretion upon restimulation of NK cells with IL-2, whereas reduced expression of NKG2D and NKp46 was accompanied by an impaired NKG2D- and NKp46-dependent cytotoxicity. Moreover, NK cells from normal mice treated in vivo with TSA displayed a diminished expression of NK1.1, NKG2D, and NKp46 and secreted reduced amounts of IFN-γ upon ex vivo stimulation with cytokines. Thus, our preclinical results indicate that HDACi exert deleterious effects on NK cell function, which may weaken immune surveillance and facilitate relapse of the malignant disease in HDACi-treated patients.

Premalignant quiescent melanocytic nevi do not express the MHC class I chain-related protein A.

The MHC class I chain-related protein A (MICA) is an inducible molecule almost not expressed by normal cells but strongly up-regulated in tumor cells. MICA-expressing cells are recognized by natural killer (NK) cells, CD8+ abTCR and gdTCR T lymphocytes through the NKG2D receptor. Engagement of NKG2D by MICA triggers IFN-g secretion and cytotoxicity against malignant cells. Although most solid tumors express MICA and this molecule is a target during immune surveillance against tumors, it has been observed that high grade tumors from different histotypes express low amounts of cell surface MICA due to a metalloprotease-induced shedding. Also, melanomas develop after a complex process of neotransformation of normal melanocytes. However, the expression of MICA in premalignant stages (primary human quiescent melanocytic nevi) remains unknown. Here, we assessed expression of MICA by flow cytometry using cell suspensions from 15 primary nevi isolated from 11 patients. When collected material was abundant, cell lysates were prepared and MICA expression was also analyzed by Western blot. We observed that MICA was undetectable in the 15 primary nevi (intradermic, junction, mixed, lentigo and congenital samples) as well as in normal skin, benign lesions (seborrheic keratosis), premalignant lesions (actinic keratosis) and benign basocellular cancer. Conversely, a primary recently diagnosed melanoma showed intense cell surface MICA. We conclude that the onset of MICA expression is a tightly regulated process that occurs after melanocytes trespass the stage of malignant transformation. Thus, analysis of MICA expression in tissue sections of skin samples may constitute a useful marker to differentiate between benign and malignant nevi.

Nuclear factor (NF)-κB controls expression of the immunoregulatory glycan-binding protein galectin-1.

The inflammatory response is a self-limiting process which involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Galectin-1 (Gal-1), an endogenous lectin found in peripheral lymphoid organs and inflammatory sites, elicits a broad spectrum of biological functions predominantly by acting as a potent anti-inflammatory factor and as a suppressive agent for T-cell responses. However, the molecular pathways underlying Gal-1 expression and function remain poorly understood. Here we identified a regulatory loop linking Gal-1 expression and function to NF-κB activation. NF-κB-activating stimuli increased Gal-1 expression on T cells, an effect which could be selectively prevented by inhibitors of NF-κB signaling. Accordingly, transient transfection of the p65 subunit of NF-κB was sufficient to induce high Gal-1 expression. Using in silico studies and chromatin immunoprecipitation analysis we have identified a functional NF-κB binding site within the first intron of the LGALS1 gene. In addition, our results show that exogenous Gal-1 can attenuate NF-κB activation, as shown by inhibition of IκB-α degradation induced by pro-inflammatory stimuli, higher cytoplasmic retention of p65, lower NF-κB DNA binding activity and impaired transcriptional activation of target genes. The present study suggest a novel regulatory loop by which NF-κB induces expression of Gal-1, which in turn may lead to negative control of NF-κB signaling.

Cytokine regulation of natural killer cell effector functions.

Initially described as effectors of natural cytotoxicity and critical players for the control of viral infections and tumor growth, recent investigations unraveled more widespread functions for the natural killer (NK) cells. Through the establishment of a crosstalk with dendritic cells, NK cells promote T helper-1- and cytotoxic T lymphocyte-mediated immunity, whereas through the establishment of a crosstalk with macrophages, NK cells contribute to the activation of their microbicidal functions. Recent evidence has shown that NK cells also display memory, a characteristic thought to be privative of T and B cells, and that NK cells acquire their mature phenotype during a complex ontogeny program which tunes their activation threshold. Cytokines play critical roles in regulating all aspects of immune responses, including lymphoid development, homeostasis, differentiation, tolerance, and memory. Cytokines such as interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, and type I interferons constitute pivotal factors involved in the maturation, activation, and survival of NK cells. In addition, the discovery of novel cytokines is increasing the spectrum of soluble mediators that regulate NK cell immunobiology. In this review, we summarize and integrate novel concepts about the role of different cytokines in the regulation of NK cell function. We believe that a full understanding of how NK cells become activated and develop their effector functions in response to cytokines and other stimuli may lead to the development of novel immunotherapeutic strategies for the treatment of different types of cancer, viral infections, and chronic autoimmune diseases.

Overcoming the hurdles of tumor immunity by targeting regulatory pathways in innate and adaptive immune cells.

The improved understanding of the biochemical nature of tumor antigens and the identification of cellular and molecular mechanisms leading to activation of innate and adaptive immune cells have been of paramount importance in the progress of tumor immunology. Studies on the intricate network of interactions between tumor and immune cells have revealed novel regulatory signals, including cell surface inhibitory receptors and costimulatory molecules, intracellular regulatory pathways, immunosuppressive cytokines and proapoptotic mediators, which may operate in concert to orchestrate tumor-immune escape. This emerging portfolio of inhibitory checkpoints can influence the physiology of innate immune cells including dendritic cells, macrophages and natural killer (NK) cells, as well as different subsets of T cells to fine tune their effector function. The synergistic combination of strategies aimed at overcoming regulatory signals and/or stimulating effector pathways, may offer therapeutic advantage as adjuvants of conventional anticancer therapies. Based on this premise, we will discuss here how the control of the effector functions of innate and adaptive immune cells and the manipulation of regulatory pathways, either alone or in combination, could be exploited for therapeutic purposes in cancer patients.

Tumour-experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands.

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-gamma secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here, we investigated the early events occurring during T cell-tumour cell interactions, and their impact on NK cell functions. We observed that on co-culture with some melanomas, activated CD4(+) T cells promoted degranulation, and NKG2D- and NKp46-dependent IFN-gamma secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4(+), CD8(+) and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity.

Immunology south of the equator in the Americas.

Despite a troubled economic and political past, a tradition of fundamental research in immunology and infectious diseases has been fostered in Argentina, Brazil and Chile, as well as in other South American countries.

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity.

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.