Gabriella Pietra

Melanoma Cells Inhibit Natural Killer Cell Function by Modulating the Expression of Activating Receptors and Cytolytic Activity

Natural killer (NK) cells play a key role in tumor immune surveillance. However, adoptive immunotherapy protocols using NK cells have shown limited clinical efficacy to date, possibly due to tumor escape mechanisms that inhibit NK cell function. In this study, we analyzed the effect of coculturing melanoma cells and NK cells on their phenotype and function. We found that melanoma cells inhibited the expression of major NK receptors that trigger their immune function, including NKp30, NKp44, and NKG2D, with consequent impairment of NK cell-mediated cytolytic activity against various melanoma cell lines. This inhibitory effect was primarily mediated by indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). Together, our findings suggest that immunosuppressive barriers erected by tumors greatly hamper the antitumor activity of human NK cells, thereby favoring tumor outgrowth and progression.

HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes

HLA-E-restricted T cell receptor αβ+ CD8+ cytolytic T lymphocytes (CTLs) exist as monoclonal expansions in the peripheral blood of some individuals. Here, we show that they recognize, with high avidity, peptides derived from the UL40 protein of different human cytomegalovirus (CMV) strains. Recognition results in the induction of cytotoxicity, IFN-γ production and cell proliferation. Autologous cells pulsed with CMV-derived peptides become susceptible to lysis by HLA-E-restricted CTLs and induce their proliferation. The high avidity for CMV-derived peptides may explain how these cells are generated in vivo and suggest their possible role in the host defenses against CMV, a virus that evolved various mechanisms to down-regulate classical HLA class I molecules, thus escaping detection by conventional CTLs.

Melanoma-associated fibroblasts modulate NK cell phenotype and antitumor cytotoxicity

Although the role of the tumor microenvironment in the process of cancer progression has been extensively investigated, the contribution of different stromal components to tumor growth and/or evasion from immune surveillance is still only partially defined. In this study we analyzed fibroblasts derived from metastatic melanomas and provide evidence for their strong immunosuppressive activity. In coculture experiments, melanoma-derived fibroblasts sharply interfered with NK cell functions including cytotoxicity and cytokine production. Thus, both the IL-2-induced up-regulation of the surface expression of NKp44, NKp30, and DNAM-1 triggering receptors and the acquisition of cytolytic granules were inhibited in NK cells. This resulted in an impairment of the NK cell-mediated killing of melanoma target cells. Transwell cocultures and the use of specific inhibitors suggested that cell-to-cell contact was required for inducing DNAM-1 modulation. In contrast, modulation of NKp44 and NKp30 was due to PGE2 released by fibroblasts during coculture. Normal skin fibroblasts could also partially affect NK cell phenotype and function. However, the inhibitory effect of tumor-derived fibroblasts was far stronger and directly correlated with their ability to produce PGE2 either constitutively or upon induction by NK cells.

Effect of tumor cells and tumor microenvironment on NK-cell function.

The ability of tumors to manage an immune‐mediated attack has been recently included in the “next generation” of cancer hallmarks. In solid tumors, the microenvironment that is generated during the first steps of tumor development has a pivotal role in immune regulation. An intricate net of cross‐interactions occurring between tumor components, stromal cells, and resident or recruited immune cells skews the possible acute inflammatory response toward an aberrant ineffective chronic inflammatory status that favors the evasion from the hosts defenses. Natural killer (NK) cells have powerful cytotoxic activity, but their activity may be eluded by the tumor microenvironment. Immunosubversion, immunoediting or immunoselection of poorly immunogenic tumor cells and interference with tumor infiltration play a major role in evading NK‐cell responses to tumors. Tumor cells, tumor‐associated fibroblasts and tumor‐induced aberrant immune cells (i.e. tolerogenic or suppressive macrophages, dendritic cells (DCs) and T cells) can interfere with NK‐cell activation pathways or the complex receptor array that regulate NK‐cell activation and antitumor activity. Thus, the definition of tumor microenvironment‐related immunosuppressive factors, along with the identification of new classes of tissue‐residing NK‐like innate lymphoid cells, represent key issues to design effective NK‐cell‐based therapies of solid tumors.

Natural killer cells kill human melanoma cells with characteristics of cancer stem cells

Experimental and clinical data suggest that tumours harbour a cell population retaining stem cell characteristics that can drive tumorigenesis. CD133 is considered an important cancer stem cells (CSC)-associated marker. In a large variety of human malignancies, including melanoma, CD133(+) cells have been reported to comprise CSC. In this study, we show that melanoma cell lines are highly heterogeneous for the expression of several stem cell-associated markers including CD133, c-kit/CD117 and p75 neurotrophin receptor/CD271. Since no information is available on the ability of NK cells to recognize and lyse melanoma stem cells, we assessed whether melanoma cell lines, characterized by stem cell-like features, were susceptible to lysis by IL-2-activated NK cells. We show that activated NK cells efficiently kill malignant melanoma cell lines that were enriched in putative CSC by the use of different selection methods (i.e. CD133 expression, radioresistance or the ability to form melanospheres in stem cell-supportive medium). NK cell-mediated recognition and lysis of melanoma cells involved different combinations of activating NK receptors. Since CSC have been reported to be both drug resistant and radioresistant, our present data suggest that NK-based adoptive immunotherapy could represent a novel therapeutic approach to possibly eradicate metastatic melanoma.

The analysis of the natural killer-like activity of human cytolytic T lymphocytes revealed HLA-E as a novel target for TCR α/β-mediated recognition

Cytolytic T lymphocytes (CTL) are known to recognize antigen peptides in association with major histocompatibility complex (MHC) class I molecules expressed on target cells. However, a fraction of human CD8+ CTL has been shown to lyse certain natural killer (NK)‐susceptible target cells via still undefined mechanism(s). These CD8+ T cells, hereafter referred to as NK‐CTL, are frequently composed of cells expressing one single TCR Vβ expansion (different in different individuals), display a memory phenotype and express HLA class I‐specific inhibitory NK receptors. Here we show that cell populations or clones of NK‐CTL isolated from three healthy donors homogeneously expressed Vβ16, Vβ9 and Vβ3 TCR, respectively. Various clones isolated under limiting dilution conditions from Vβ16+ cells of donor 1 displayed identical TCR Vβ and Vα rearrangements, thus suggesting a substantial monoclonality of the NK‐CTL subset analyzed. NK‐CTL lysed a number of NK‐susceptible tumor target cells with the exception of those characterized by β2‐microglobulin (β2m) deficiency. However, the latter targets became susceptible to lysis upon β2m transfection. Using monoclonal antibodies specific for the relevant TCR Vβ or β2m we provide evidence suggesting that target cell lysis by NK‐CTL is mediated by the TCR itself upon recognition of β2m‐associated proteins. The cellular distribution of the potential β2m‐associated proteins in susceptible target cells suggested, as a likely candidate for TCR‐mediated recognition, the non‐classical HLA‐E molecule. The use, as target cells, of the murine TAP2‐deficient RMA‐S cells, either untransfected or transfected with HLA‐E, and loaded with an appropriate HLA‐E‐binding peptide, provided the direct demonstration that HLA‐E represents a ligand recognized by the TCR expressed by NK‐CTL. This is the first evidence that human TCR α/β can recognizeHLA‐E molecules, thus revealing a novel type of TCR‐mediated recognition, which may offer new insight in immune responses in both normal and disease conditions.

Identification of HLA-E-specific alloreactive T lymphocytes: A cell subset that undergoes preferential expansion in mixed lymphocyte culture and displays a broad cytolytic activity against allogeneic cells

Previous studies showed that a subset of CD8+ cytolytic T lymphocytes expressing HLA class I-specific natural killer inhibitory receptors (iNKR) is characterized by the ability to recognize HLA-E and to mediate T cell receptor-dependent killing of different NK cell-susceptible tumor target cells. In this study, we show that this CD8+ T cell subset can also undergo extensive proliferation in mixed lymphocyte cultures in response to allogeneic cells. Analysis of their cytolytic activity revealed a broad specificity against a panel of allogeneic phytohemagglutinin-induced blasts derived from HLA-unmatched donors. On the other hand, autologous and certain allogeneic phytohemagglutinin blasts were resistant to lysis. We used as target cells the transporter associated with antigen processing (TAP)-2−/− murine RMA-S cell line cotransfected with β2-microglobulin and HLA-E*01033. On loading these cells with different HLA-E-binding peptides derived either from HLA class I leader sequences or viral proteins, we could show that HLA-E-specific cytolytic T lymphocytes recognized many, but not all, peptides analyzed. These data suggest that these cells may recognize, on allogeneic cells, HLA-E with peptides that are not present in the host of origin. In addition to their ability to proliferate in response to allogeneic stimulation and to lyse, most allogeneic cells may have important implications in transplantation and in antitumor immune responses.

The emerging role of HLA-E-restricted CD8+ T lymphocytes in the adaptive immune response to pathogens and tumors

Human leukocyte antigen (HLA)-E is a nonclassical major histocompatibility complex (MHC) class I molecule of limited sequence variability that is expressed by most tissues albeit at low levels. HLA-E has been first described as the ligand of CD94/NKG2 receptors expressed mainly by natural killer (NK) cells, thus confining its role to the regulation of NK-cell function. However, recent evidences obtained by our and other groups indicate that HLA-E complexed with peptides can interact with αβ T-cell receptor (TCR) expressed on CD8+ T cells. Although, HLA-E displays a selective preference for nonameric peptides, derived from the leader sequence of various HLA class I alleles, several reports indicate that it can present also “noncanonical” peptides derived from both stress-related and pathogen-associated proteins. Because HLA-E displays binding specificity for innate CD94/NKG2 receptors, as well as all the features of an antigen-presenting molecule, its role in both natural and acquired immune responses has recently been re-evaluated.

The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.

BackgroundCTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma.MethodsCTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student’s t-test.ResultsAll melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts.ConclusionsOur studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.

Hypoxia downregulates the expression of activating receptors involved in NK-cell-mediated target cell killing without affecting ADCC.

In certain infection sites or tumor tissues, the disruption of homeostasis can give rise to a hypoxic microenvironment, which, in turn, can alter the function of different immune cell types and favor the progression of the disease. Natural killer (NK) cells are directly involved in the elimination of virus‐infected or transformed cells, however it is unknown whether their function is affected by hypoxia or not. In this study, we show that NK cells adapt to a hypoxic environment by upregulating the hypoxia‐inducible factor 1α. However, NK cells lose their ability to upregulate the surface expression of the major activating NK‐cell receptors (NKp46, NKp30, NKp44, and NKG2D) in response to IL‐2 (or other activating cytokines, including IL‐15, IL‐12, and IL‐21). These altered phenotypic features correlate with reduced responses to triggering signals resulting in impaired capability of killing infected or tumor target cells. Remarkably, hypoxia does not significantly alter the surface density and the triggering function of the Fc‐γ receptor CD16, thus allowing NK cells to maintain their capability of killing target cells via antibody‐dependent cellular cytotoxicity. This finding offers an important clue for exploitation of NK cell in antibody‐based immunotherapy of cancer.