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Glioblastoma stands as the most frequent primary brain tumor. Despite the multimodal therapy for glioblastoma patients, the survival rate is very low, highlighting the need for novel therapies that improve patient outcomes. Immune checkpoint blockade strategies are achieving promising results in a myriad of tumors and several studies have reported its efficacy in glioblastoma at a preclinical level. ILT2 is a novel immune checkpoint that exerts an inhibitory effect via the interaction with classical and non‐classical HLA class‐I molecules. Herein, we report that ILT2 blockade promotes antitumor responses against glioblastoma. In silico and immunohistochemical analyses revealed that the expression of ILT2 and its ligands HLA‐A, ‐B, ‐C, and ‐E are highly expressed in patients with glioblastoma. Disruption of ILT2 with blocking monoclonal antibodies increased natural killer cell‐mediated IFN‐γ production and cytotoxicity against glioblastoma, partially reverting the immunosuppression linked to this malignancy. In addition, co‐treatment with temozolomide strengthened the antitumor capacity of anti‐ILT2‐treated immune cells. Collectively, our results establish the basis for future studies regarding the clinical potential of ILT2 blockade alone or in combination regimens in glioblastoma. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells have the innate ability to kill cancer cells, however, tumor cells may acquire the capability of evading the immune response, thereby leading to malignancies. Restoring or potentiation of this natural antitumor activity of NK cells has become a relevant therapeutic approach in cancer and, particularly, in hematological cancers. The use of tumor-specific antibodies that promote antibody-dependent cell-mediated cytotoxicity (ADCC) through the ligation of CD16 receptor on NK cells has become standard for many hematologic malignancies. Hematopoietic stem cell transplantation is another key therapeutic strategy that harnesses the alloreactivity of NK cells against cancer cells. This strategy may be refined by adoptive transfer of NK cells that may be previously expanded, activated, or redirected (chimeric antigen receptor (CAR)-NK cells) against cancer cells. The antitumor activity of NK cells can also be boosted by cytokines or immunostimulatory drugs such as lenalidomide or pomalidomide. Finally, targeting immunosubversive mechanisms developed by hematological cancers and, in particular, using antibodies that block NK cell inhibitory receptors and checkpoint proteins are novel promising therapeutic approaches in these malignant diseases. [ABSTRACT FROM AUTHOR]

Metastasis represents the leading cause of cancer-related death mainly owing to the limited efficacy of current anticancer therapies on advanced malignancies. Although immunotherapy is rendering promising results in the treatment of cancer, many adverse events and factors hampering therapeutic efficacy, especially in solid tumors and metastases, still need to be solved. Moreover, immunotherapeutic strategies have mainly focused on modulating the activity of T cells, while Natural Killer (NK) cells have only recently been taken into consideration. NK cells represent an attractive target for cancer immunotherapy owing to their innate capacity to eliminate malignant tumors in a non-Major Histocompatibility Complex (MHC) and non-tumor antigen-restricted manner. In this review, we analyze the mechanisms and efficacy of NK cells in the control of metastasis and we detail the immunosubversive strategies developed by metastatic cells to evade NK cell-mediated immunosurveillance. We also share current and cutting-edge clinical approaches aimed at unleashing the full anti-metastatic potential of NK cells, including the adoptive transfer of NK cells, boosting of NK cell activity, redirecting NK cell activity against metastatic cells and the release of evasion mechanisms dampening NK cell immunosurveillance. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are innate lymphoid cells that, together with CD8 + T lymphocytes, are tightly correlated with immunesurveillance and the elimination of transforming and malignant cells both during early steps of tumorigenesis and metastasis. This capacity is due, but not limited to, their great cytotoxic capacity upon recognition of stress-related activating ligands of NK cells on the surface of tumor cells. Due to the emerging role of NK cells in the response to treatment with immune checkpoint blockers (ICBs) and other immunotherapies, it has become essential to deeply study this immune subset. As mentioned above, NK cell antitumor responses not only rely on their high cytotoxic capacity, cytokine production by this immune subset, such as interferon gamma (IFN-γ) or tumor necrosis factor (TNF-α), also plays a key role on NK cell function. In this chapter, we provide a detailed protocol to measure the intracellular expression of cytokines produced by NK cells upon stimulation. A step-by-step guide to isolate peripheral blood mononuclear cells, purify NK cells from whole blood, co-culture with tumor cell lines and evaluate their cytokine production capacity by flow cytometry is here provided., (© 2020 Elsevier Inc. All rights reserved.)

The immune system exerts a tight cancer surveillance to eradicate nascent and developing tumors. Natural killer (NK) cells rely on their cytotoxic activity to eliminate abnormal or stressed cells, including those undergoing malignant transformation. Thereupon, NK cells, along with CD8 + T cells, are effector immune cells with a pivotal role in cancer immunosurveillance. Due to the outstanding effectivity of immunotherapy in cancer management, strategies aimed at boosting NK cell-mediated tumor recognition and elimination are being thoroughly studied, such as approaches to induce tumor surface expression of NK cell activating ligands. Additionally, the development of methods to evaluate NK cell cytotoxic responses towards malignant cells with distinct immunogenic profiles is of paramount relevance. In this chapter, we detail a simple but highly sensitive and reproducible methodological approach to quantify in vitro NK cell-mediated lysis of a given target cell, e.g. a tumor cell, based on calcein-AM staining and fluorescence detection. Further, this method can be employed to determine the impact of chemical modulators or antibodies blocking cell surface proteins on the antitumor capacity of NK cells, with the aim to improve anticancer immune responses and elucidate the underlying mechanisms regulating NK cell activity., (© 2020 Elsevier Inc. All rights reserved.)

The immune system is able to detect and eliminate nascent and developing tumors. Thus, T lymphocytes of the adaptive immunity recognize cancer cells by detecting tumor-associated antigens, whereas certain innate immune cells scan for molecules that are mainly overexpressed on malignant and infected cells. The best example of the latter is natural killer (NK) cells. The activity of these immune cells is strictly governed by a balance between positive and negative signals provided by stress-regulated molecules that bind NK cell membrane receptors, such as the activatory receptor NKG2D. Given the key role of NK cells in eradicating cancer cells, the development of assays to study the cellular and molecular determinants of NK cell antitumor activity are of great relevance. In this chapter, we describe a flow cytometric in vitro assay to evaluate the cytotoxic activity of NK cells against a given target cell, including tumor cells. Moreover, this method is highly versatile, as it can be complemented with the use of antibodies blocking cell surface proteins (receptors or ligands) or a variety of chemical modulators, which allows the elucidation of molecules and signaling pathways that regulate NK cell anticancer function.

Cancer development is under surveillance by the immune system of the host. Tumor cells can be recognized and killed by cytotoxic lymphocytes- such as CD8+ T lymphocytes and natural killer (NK) cells-mainly through the immune secretion of lytic granules that kill target cells. This process involves the fusion of the granule membrane with the cytoplasmic membrane of the immune effector cell, resulting in surface exposure of lysosomal-associated proteins that are typically present on the lipid bilayer surrounding lytic granules, such as CD107a. Therefore, membrane expression of CD107a constitutes a marker of immune cell activation and cytotoxic degranulation. In this chapter, we detail the steps required to isolate peripheral blood mononuclear cells (PBMCs), coculture them with target tumor cell lines, and evaluate the cytotoxic immune function by means of flow cytometry evaluation of CD107a expression on the surface of NK cells.