Ennio Carbone

NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.

Recognition of autologous dendritic cells by human NK cells.

NK cells can recognize and kill tumor as well as certain normal cells. The outcome of the NK‐target interaction is determined by a balance of positive and negative signals initiated by different target cell ligands. We have previously shown that human NK cells kill CD40‐transfected tumor targets efficiently, but the physiological significance of this is unclear. We now demonstrate that human NK cells can kill dendritic cells (DC), known to express CD40 and other co‐stimulatory molecules. The killing was observed with polyclonal NK cells cultured short term in IL‐2 as well as with NK cell clones as effectors, and with allogeneic as well as autologous DC as targets. NK cell recognition could be inhibited, but only partially, by preincubation of target cells with monoclonal antibodies against CD40, suggesting that this molecule may be one of several ligands involved. Addition of TNF‐α of the cultures stimulated the development of a more mature DC phenotype, while addition of IL‐10 resulted in a less mature phenotype, with lower expression of CD40 and other co‐stimulatory molecules. Nevertheless, such DC were more NK susceptible than the differentiated DC. This may be partly explained by a reduced MHC class I expression observed on such cells, since blocking of MHC class I molecules on differentiated DC or CD94 receptors of NK cells led to increased NK susceptibility. The results show that NK cells may interact with DC, and suggest that the outcome of such interactions depend on the cytokine milieu.

Human NK Cells Selective Targeting of Colon Cancer–Initiating Cells: A Role for Natural Cytotoxicity Receptors and MHC Class I Molecules

Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the “differentiated” cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma–derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the “differentiated” tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.

Human Cytomegalovirus Strain-Dependent Changes in NK Cell Recognition of Infected Fibroblasts

NK cells play a key role in the control of CMV infection in mice, but the mechanism by which NK cells can recognize and kill CMV-infected cells is unclear. In this study, the modulation of NK cell susceptibility of human CMV (hCMV)-infected cells was examined. We used a human lung and a human foreskin fibroblast cell line infected with clinical isolates (4636, 13B, or 109B) or with laboratory strains (AD169, Towne). The results indicate that all three hCMV clinical isolates confer a strong NK resistance, whereas only marginal or variable effects in the NK recognition were found when the laboratory strains were used. The same results were obtained regardless of the conditions of infection, effector cell activation status, cell culture conditions, and/or donor-target cell combinations. The NK cell inhibition did not correlate with HLA class I expression levels on the surface of the target cell and was independent of the leukocyte Ig-like receptor-1, as evaluated in Ab blocking experiments. No relevant changes were detected in the adhesion molecules ICAM-I and LFA-3 expressed on the cell surface of cells infected with hCMV clinical and laboratory strains. We conclude that hCMV possesses other mechanisms, related neither to target cell expression of HLA-I or adhesion molecules nor to NK cell expression of leukocyte Ig-like receptor-1, that confer resistance to NK cell recognition. Such mechanisms may be lost during in vitro passage of the virus. These results emphasize the differences between clinical hCMV isolates compared with laboratory strains.

Synergistic effect of IFN-γ and human cytomegalovirus protein UL40 in the HLA-E-dependent protection from NK cell-mediated cytotoxicity

Human CMV (HCMV) has evolved several strategies to evade the immune system of the infected host. Here, we investigated the role of the HCMV‐encoded protein UL40 in the modulation of NK cell lysis. UL40 carries in its leader sequence a nonameric peptide similar to that found in many HLA class I molecules leader sequences. This peptide up‐regulates the expression of HLA‐E, the ligand for the NK cell inhibitory receptor CD94 / NKG2A. The UL40‐encoded HLA‐E‐binding peptide was present in all HCMV clinical (4636, 13B, 109B, 3C) and laboratory (AD169) strains analyzed. However, transfection of UL40 in different cell lines (293T, 721.221, K562) did not consistently confer protection from NK lysis (as measured using NKL and the newly generated NK line Nishi), despite a moderate up‐regulation of HLA‐E. Interestingly, combined transfection and treatment with IFN‐γ increased the inhibitory effect, via an HLA‐E‐ and CD94 / NKG2A‐dependent mechanism. Although cells transfected with UL40 derived from either AD169 or 3C showed protection from NK cell lysis, infection of fibroblasts with the viruses resulted in a strong inhibition only with the clinical strain 3C. Our results suggest that UL40 and IFN‐γ‐dependent up‐regulation of HLA‐E is only one possible mechanism to avoid NK cell recognition of HCMV infected cells.

Pharmacological activation of p53 triggers anticancer innate immune response through induction of ULBP2

Escape of tumor cells from cell-intrinsic barrier mediated by tumor suppressors and cell-extrinsic barrier mediated by the immune system is crucial for tumorigenesis. Growing evidence suggests that reactivation of tumor suppressor function or restoration of anticancer immunity is promising strategy for anticancer therapy due to their high potential to combat cancer. p53, a key tumor suppressor, represses tumorigenesis by eliciting growth arrest, apoptosis or senescence in cancer cells. Here, we unravel that, apart from these cell-autonomous effects, p53 activates the innate immune response against cancer cells. Our results show that pharmacological reactivation of p53 can stimulate the expression of ULPB2, a ligand for NK cell activating receptor NKG2D in human tumor cells of different origin, which enhance the susceptibility of tumor cells to NK cell-mediated killing. The molecular mechanism controlling ULPB2 expression by p53 is neither ATM/ATR- nor caspase-dependent. Using several approaches, we identified p53 as a direct transcriptional regulator of ULBP2 and found a p53 response element within ULBP2 gene, which confers the p53 regulation. Furthermore, we demonstrated that demethylation of p53-binding region within ULBP2 gene was required for p53-dependent induction of ULPB2, which can be achieved via repression of DNA methyltransferases (DNMTs) by p53. This molecular evidence for the direct control of immunosurveillance by p53 links tumor suppressor activation to innate immune stimuli and provides a possibility to integrate cell-extrinsic and -intrinsic defenses against tumorigenesis by pharmacological activation of p53, which may increase the probability to achieve a durable therapeutic success.

Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded autologous or allogeneic natural killer (NK) cells mismatched for MHC class I molecules and inhibitory NK receptors. NK cell–based immunoadjuvant therapies are carried out in combination with standard chemotherapeutic protocols. In the presented study, we characterized the effect of 28 frequently used chemotherapeutic agents on the capacity of NK cells to kill target cells. We found that treatment of NK cells with the drugs vinblastine, paclitaxel, docetaxel, cladribine, chlorambucil, bortezomib, and MG-132 effectively inhibited NK cell–mediated killing without affecting the viability of NK cells. On the other hand, the following drugs permitted efficient NK cell–mediated killing even at concentrations comparable with or higher than the maximally achieved therapeutic concentration in vivo in humans: asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine. [Mol Cancer Ther 2007;6(2):644–54]

LIR-1 expression on lymphocytes, and cytomegalovirus disease in lung-transplant recipients

Human cytomegalovirus infection is a major cause of morbidity after lung transplantation. LIR-1 (leucocyte immunoglobulin-like receptor-1) is an inhibitory cell surface receptor that has high affinity for an MHC class I homologue (UL18) encoded by human cytomegalovirus. We aimed to investigate whether reactivation of human cytomegalovirus affects the expression of LIR-1. We measured LIR-1 expression on peripheral blood lymphocytes from 13 lung-transplant recipients and established human cytomegalovirus load using PCR. Eight patients developed cytomegalovirus disease. The percentage of cells expressing LIR-1 increased in the patients who developed cytomegalovirus disease several weeks before viral DNA was detectable by PCR. Measurement of LIR-1 expression might allow early identification of cytomegalovirus disease in lung-transplant patients.

Lipid Droplets: A New Player in Colorectal Cancer Stem Cells Unveiled by Spectroscopic Imaging

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label‐free Raman spectroscopy and it directly correlates with well‐accepted CR‐CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR‐CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR‐CSCs. Stem Cells 2015;33:35–44

New views on natural killer cell-based immunotherapy for melanoma treatment

Natural killer (NK) cell-based immunotherapies treat hematopoietic malignancies, but are less effective against solid tumors. Here, we review recent data on NK cell recognition of melanoma at various stages of the disease and propose a combinatorial strategy to exploit fully the potential of NK cells. Depending on the stage of melanoma progression, NK cell-based therapies could be combined with pharmacological and T cell-based immunotherapies, to: (i) prevent lymph node metastases by redistributing cytotoxic NK cells; (ii) boost NK cell activity using chemotherapy to upregulate activating ligands on tumor cells; and (iii) target visceral metastases by transfer of autologous or allogeneic NK cells.