Edited by: Lutz Walter, Leibniz-Institute for Primate Research, Germany
Reviewed by: Evelyn Ullrich, Goethe University Frankfurt, Germany; Ralf Dressel, University Medical Center Göttingen, Germany
Specialty section: This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology
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Natural killer (NK) cells participate in the early immune response against melanoma and also contribute to the development of an adequate adaptive immune response by their crosstalk with dendritic cells and cytokine secretion. Melanoma resistance to conventional therapies together with its high immunogenicity justifies the development of novel therapies aimed to stimulate effective immune responses against melanoma. However, melanoma cells frequently escape to CD8 T cell recognition by the down-regulation of major histocompatibility complex (MHC) class I molecules. In this scenario, NK cells emerge as potential candidates for melanoma immunotherapy due to their capacity to recognize and destroy melanoma cells expressing low levels of MHC class I molecules. In addition, the possibility to combine immune checkpoint blockade with other NK cell potentiating strategies (e.g., cytokine induction of activating receptors) has opened new perspectives in the potential use of adoptive NK cell-based immunotherapy in melanoma.
Melanoma is largely resistant to current therapies as chemotherapy and radiotherapy (
It has been postulated that melanoma ability of inducing an immune response contributes to patient survival. Thus, melanoma is usually highly immunogenic and induces cytotoxic T cell (CTL)-mediated immune responses. Tumor infiltrating lymphocytes (TILs) have been identified in melanoma lesions usually associated with spontaneous tumor regression and favorable prognostic in primary melanoma (
Innate immune responses against melanoma have also been described. Natural killer (NK) cells constitute the first line of defense against transformed cells as tumors or virus-infected cells.
It is well known that age affects both adaptive and innate immune responses against tumors (
Altogether, these characteristics of melanoma reinforce the previous consideration of melanoma as a suitable model for studying tumor immunity. Here, we review the current state of knowledge on NK cell-mediated recognition and lysis of melanoma cells and the up to date immunotherapeutic strategies against melanoma based on NK cells.
The key role played by NK cells as a first line of defense against tumors has been established in hematological malignancies based on the graft-versus-leukemia effect (
Natural killer cells express several activating receptors that after cross-linking with their respective ligands trigger NK cell degranulation releasing their cytotoxic granule content leading to target cell apoptosis (Figure
A characteristic that makes melanoma a prototype for the study of NK cell-mediated tumor destruction is the fact that melanoma cells frequently show altered expression of MHC class I molecules (
Mature NK cells express CD16 (FcγR-III) that mediates antibody-dependent cell cytotoxicity (ADCC) representing an effective mechanism of lysis of antibody-coated target cells. However, it has been described that NK cell activation is associated with metalloproteinase-mediated cleavage of CD16 molecules. The treatment with metalloproteinase inhibitors prevented CD16 down-regulation and increased NK cell polyfunctionality (cytokine production and degranulation). The use of metalloproteinase inhibitors in monoclonal antibody (mAb)-based immunotherapy is proposed to benefit cancer patients (
We have previously analyzed a large panel of melanoma cell lines from the “European Searchable Tumor Cell Line and Data Bank” (ESTDAB,
The identification of cellular ligands for the natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46 has remained elusive until recently. The use of chimera proteins constructed using the extracellular domain of NKp30, NKp44, or NKp46 fused to the Fc immunoglobulin domain (NCR-Fc) or to an amino-terminal isoleucine zipper (NCR-ILZ) allowed to analyze the expression of NCR ligands on tumor cells. A high variability in the binding of NCR chimeras to melanoma cells was observed with melanoma cell lines expressing ligands for NKp30 and NKp44 but not for NKp46 (
Recently, several cellular ligands for NCRs have been identified. NKp30 recognizes B7-H6 that has been found expressed on melanoma cell lines (
Natural killer cell recognition and lysis of melanoma cells involve different receptor–ligand interactions including NKG2D-, DNAM-1-, and NCRs-activating receptors. The expression pattern of ligands for activating receptors on melanoma and the expression of MHC class I molecules recognized by inhibitory receptors will determine the activation of NK cells (Figures
The role of NKG2D in NK cell recognition and lysis of melanoma cells has been extensively discussed. Whereas, NKG2D is clearly involved in the lysis of melanoma cells expressing high levels of NKG2D ligands, and NCRs and DNAM-1 are the receptors involved in the elimination of melanoma cells with low expression of ligands for NKG2D. Thus, it has been described that NCRs and DNAM-1 cooperation is frequently involved in the lysis of melanoma cells both in humans and in mice (
The majority of studies analyzing effector–target interactions in melanoma are performed using cell lines cultured as monolayer or in suspension testing ligand expression correlation with CTL- or NK cell-susceptibility to lysis. Recently, the use of three-dimensional (3D) cell culture systems has been proposed for the analysis of melanoma interaction with lymphocytes. Thus, melanoma cells grown in 3D architecture showed lower recognition by melanoma-specific CTLs compared to those melanoma cells growing in 2D monolayers. It has been proposed that culture in 3D affects the expression of molecules involved in melanoma recognition by CTLs (
An expansion of highly cytotoxic CD57+ NK cells has been found in tumor-infiltrating lymph nodes in melanoma patients. Their potential use as a source of cytotoxic NK cells for adoptive immunotherapy is discussed (
Natural killer cell activation depends on a tune balance mediated by inhibitory and activating signals transmitted through surface receptors upon contact with their respective ligands. In this process, the interaction between MHC class I molecules on target cells and MHC class I-specific inhibitory receptors on NK cells represents a major checkpoint regulating NK cell functions (
The discovery of inhibitory receptor-recognizing ligands other than MHC class I molecules such as TIGIT or the programed cell death-1 (PD-1) molecules constitute novel checkpoints in NK cell activation that requires further consideration (
Together with the expression level of MHC class I molecules on melanoma cells and the expression of MHC class I-specific inhibitory receptors on autologous NK cells, the expression of activating receptors on NK cells, and their ligands on melanoma are key actors in the final balance leading to an effective NK cell activation (
Immune evasion by tumor cells through the down-regulation of MHC class I molecules to avoid CD8 T cell recognition constitutes a well-known mechanism used by melanoma (
It has been also proposed that NK cell-mediated immunosurveillance against melanoma can generate immunoselection of melanoma cell variants with low expression of ligands for activating receptors that are resistant to NK cells (
The down-regulation of NK cell-activating receptors has been described as an additional mechanism that contributes to tumor escape in cancer patients (
T cell immunoreceptor with immunoglobulin and ITIM domains signaling after interaction with its ligands suppresses NK cell production of IFN-γ (
Finally, suppression of NK cells by factors or cytokines secreted either by tumor cells or other cells in the tumor microenvironment such as myeloid derived suppressor cells (MDSCs) or macrophages can also contribute to immunoescape of cytotoxic cells (
All these mechanism together may contribute to the alterations of NK cell phenotype and function described in cancer patients.
Different strategies of melanoma immunotherapy developed during the last decade focused on the use of checkpoints inhibitors or immune modulators, oncolytic virus therapy, cancer vaccines, adoptive T cell, and NK cell therapies and the use of cytokines (
Category | Strategy | Start date–completion date | Melanoma patients | Phase/status | Identifier/reference |
---|---|---|---|---|---|
Autologous NK cells | LAK cells in combination with.IL-2 (i.v.) | 1985 | Seven metastatic melanoma | Phase I completed | Rosenberg et al. ( |
Autologous NK cells combined with IL-2 (i.v.) and chemotherapy | 2006–2009 | Seven metastatic melanoma | Phase II completed | NCT00328861 Parkhurst et al. ( |
|
Autologous NK cells and bortezomib (proteasome inhibitor) | 2015 recruiting participants | Hematological and solid tumors including metastatic melanoma | Phase I | NCT00720785 | |
Allogeneic NK cells | Allogeneic haploidentical NK cells | 2004 | 10 metastatic melanoma | Phase I completed | Miller et al. ( |
Allogeneic haploidentical NK cells (from PBMC) combined with chemotherapy | 2009–2012 | Refractory or relapsed melanoma | Phase I/II completed | NCT00846833 | |
Mismatched LAK followed by IL-2 (i.v.) | 2009–2014 | Malignant melanoma | Phase II completed | NCT00855452 | |
NK cell line | NK92 cells | One metastatic melanoma | Phase I completed | Arai et al. ( |
|
Checkpoints/immune modulators | anti-KIR and anti-CTLA-4 |
2012–2015 |
Advanced solid tumors |
Safety study |
NCT01750580 |
There are different strategies to exploit the possibility to modulate NK cells in melanoma immunotherapy. The use of new forms of cytokine therapies or mAbs against tumor antigens can directly contribute to enhance NK cytotoxicity whereas immune checkpoints regulators constitute a novel immunotherapy strategy to modulate immune responses through their interaction with inhibitory receptors on immune cells.
Different cytokines have demonstrated a role in tumor immunity. Two cytokines have been approved by the Food and Drug Administration (FDA) for melanoma treatment as single agent: high doses of IL-2 for metastatic melanoma and IFN-α for the adjuvant therapy of Stage III melanoma based on the results obtained in clinical trials using high doses of IL-2 in metastatic melanoma patients (
As indicated before, one of the major checkpoints in NK cell activation is mediated by MHC class I-specific inhibitory receptors interacting with their ligands on target cells. Thus, blockade of this checkpoint constitutes an emerging area of research. Two NK cell checkpoint inhibitors lirilumab (anti-KIR mAb) and IPH2201 (anti-NKG2A mAb) are currently under revision. A safety study to analyze anti-KIR mAb in combination with ipilimumab (anti-CTLA4) (NCT01750580) is completed and a Phase I clinical trial of anti-KIR mAb in combination with anti-PD-1 is still recruiting patients (NCT01714739). IL-18 secretion by tumor cells upregulates PD-1 on NK cells (
The clinical efficacy and pharmacological activity of anti-NKG2A mAb IPH2201 are going to be analyzed in clinical trials currently recruiting patients with squamous cell carcinoma of the oral cavity for an efficacy study of pre-operative use of IPH2201 (NCT02331875) or for a dose-ranging study of patients with high grade serious carcinoma of ovarian, fallopian tubes, or peritoneal origin (NCT02459301). The results of these trials may open new perspectives for melanoma treatment.
Increased tumor sensitivity to NK cells has been observed after treatment with proteasome inhibitors, doxorubicin or histone deacetylase inhibitors that upregulates the expression of NKG2D ligands, the secretion of proinflammatory cytokines, or the expression of TNF receptors. However, when combining these therapies with NK cell adoptive transfer, a strict control of NK cell function should be taken into account (
Novel strategies are in progress aimed to redirect NK cell cytotoxicity by CD16-directed bispecific and trispecific killer engagers (BiKEs and TriKEs respectively) constructed using one (BiKEs) or two (TriKEs) variable single-chain fragments against tumor-associated antigens. BiKEs and TriKEs trigger NK cell activation through CD16 (
Optimal adoptive cancer immunotherapy should link both innate and adaptive immune responses. NK cells may contribute to the adaptive immune responses by favoring DC maturation and priming of T cells. The bidirectional crosstalk between NK cells and DC was demonstrated for the first time by Gerosa et al. in 2002 (
Lymphokine-activated killer cells were used for the first time in melanoma patients by Roserberg et al. (
Clinical trials of adoptive NK cell-based immunotherapy against melanoma are very limited. A Phase II trial (NCT00328861) completed in 2009 combined autologous NK cells with intravenous (i.v.) IL-2 and chemotherapy. Although no clinical effect was observed, the transferred NK cells persisted in the peripheral blood from 14 weeks to several months suggesting that combined therapy with antibodies could be beneficial (
Another trial using autologous NK cells combined with the proteasome inhibitor bortezomib is ongoing (NCT00720785). The use of bortezomib has been related to the upregulation of NKG2D ligands on tumor cells that may promote NK cell recognition and lysis of tumor cells (
Because, the expression of activating receptors on NK cells from tumor-bearing patients is frequently found down-regulated, the efficacy of autologous NK cells expanded
Few clinical trials using allogeneic NK cells for melanoma treatment have been reported usually combined with chemotherapy. It has been shown that NK cell activation of activating receptors together with administration of anti-tumor antibodies have substantial anti-cancer effects supporting that the combination of allogeneic NK cells and antibody therapy can be an efficient strategy in clinical trials (
The difficulties of expanding large numbers of clinical grade NK cells (
A strategy to redirect NK cell cytotoxicity against melanoma is the use of chimeric antigen receptor (CAR)-modified NK cells. CARs consist of an external domain that specifically recognizes a given tumor antigen, linked with one or more intracellular signaling domains that trigger cytotoxic cell activation. NK cell lines, peripheral blood NK cells, and NK cells derived from human pluripotent stem cells can be engineered to express CARs. These CAR-transduced NK cells can specifically recognize and kill a variety of tumor targets expressing the surface target antigen [for review in Ref. (
Stimulation of the immune system has been considered a possible therapy for melanoma for many years. Experimental and clinical efforts have focused in exploring possibilities to use different elements of the adaptive and innate immune responses to control and eliminate melanoma cells. However, the heterogeneity of these tumors makes necessary a detailed analysis of the possible interactions between the melanoma and the immune system cells. NK cells are undoubted components within the anti-melanoma immunotherapy arsenal. The potential efficacy of NK cell-based immunotherapy in melanoma patients will rely on melanoma phenotype (expression of ligands for activating receptors and low expression of MHC class I molecules for the use of autologous NK cells), NK cell status (no exhausted, no senescent), NK cell phenotype (high level of NKG2D, NCRs and DNAM-1; CD16 expression for ADCC), microenvironment (proinflammatory versus inhibitory), NK cell crosstalk with other cell types (e.g., DCs, macrophages, MDSCs). The better understanding of the interactions between NK cells and melanoma will open the possibility to use combined strategies of checkpoints blockade and cytokine or activating receptor stimulation to enhance autologous NK cell cytotoxic capacity. These strategies should also be considered to modulate NK cell functionality in protocols of adoptive therapy against melanoma using autologous, allogeneic, or engineered
RT and RS designed the manuscript. RT, ED, and RS contributed to the writting and revised the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We apologize to our colleagues whose work was not cited due to space limitations. This work was supported by grants SAF2009-09711 and SAF2013-46161-R (to RT) from the Ministry of Economy and Competitiveness of Spain, PS09/00723 and PI13/02691 (to RS) from Spanish Ministry of Health and CTS-208 from Junta de Andalucia (to RS) and grants to INPATT research group (GRU10104 and GR15183) and PRI09A029 from Junta de Extremadura and University of Extremadura (to RT and ED) cofinanced by European Regional Development Funds (FEDER). This work was also supported by contracts QLRT-2001-00668 (Outcome and Impact of Specific Treatment in European Research on Melanoma, OISTER) and LABPOLE project from the Ministry of Economy and Competitiveness cofinanced by FEDER.