Mirna Balsamo

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.

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.

Targeting the Heat Shock Factor 1 by RNA Interference: A Potent Tool to Enhance Hyperthermochemotherapy Efficacy in Cervical Cancer

Carcinoma of the uterine cervix is one of the highest causes of mortality in female cancer patients worldwide, and improved treatment options for this type of malignancy are highly needed. Local hyperthermia has been successfully used in combination with systemic administration of cisplatin-based chemotherapy in phase I/II clinical studies. Heat-induced expression of cytoprotective and antiapoptotic heat shock proteins (HSP) is a known complication of hyperthermia, resulting in thermotolerance and chemoresistance and hindering the efficacy of the combination therapy. Heat shock transcription factor 1 (HSF1) is the master regulator of heat-induced HSP expression. In the present report, we used small interfering RNA (siRNA) to silence HSF1 and to examine the effect of HSF1 loss of function on the response to hyperthermia and cisplatin-based chemotherapy in HeLa cervical carcinoma. We have identified the 322-nucleotide to 340-nucleotide HSF1 sequence as an ideal target for siRNA-mediated HSF1 silencing, have created a pSUPER-HSF1 vector able to potently suppress the HSF1 gene, and have generated for the first time human cancer cell lines with stable loss of HSF1 function. We report that, although it surprisingly does not affect cancer cell sensitivity to cisplatin or elevated temperatures up to 43 degrees C when administered separately, loss of HSF1 function causes a dramatic increase in sensitivity to hyperthermochemotherapy, leading to massive (>95%) apoptosis of cancer cells. These findings indicate that disruption of HSF1-induced cytoprotection during hyperthermochemotherapy may represent a powerful strategy to selectively amplify the damage in cancer cells and identify HSF1 as a promising therapeutic target in cervical carcinoma.

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.

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.

Evidence that the KIR2DS5 gene codes for a surface receptor triggering natural killer cell function

In this study, after immunization with NK cells from a KIR2DS5+ donor and screening on cell transfectants expressing different members of the killer immunoglobulin‐like receptor (KIR) family, we generated a mAb, DF200, reacting with several KIR2D receptors including KIR2DL1/L2/L3, KIR2DS1/S2 and KIR2DS5. By the analysis of peripheral blood NK cells and in vitro derived NK cell clones, we have demonstrated for the first time that KIR2DS5 is expressed at the cell surface in discrete subsets of NK cells and, after DF200 mAb‐mediated engagement, can induce both cytotoxicity and cytokine release. Using co‐transfection and co‐immunoprecipitation, we found that KIR2DS5 associates with the DAP12 signaling polypeptide. Finally, soluble KIR2DS5‐Fc fusion protein does not bind to cell transfectants expressing different HLA‐C alleles, suggesting that, if KIR2DS5 does recognize HLA‐C molecules, this may only occur in the presence of certain peptides.

Melanoma cells become resistant to NK-cell-mediated killing when exposed to NK-cell numbers compatible with NK-cell infiltration in the tumor

During the past few years, a number of studies reported that different melanoma cell lines could be extensively lysed in vitro by IL‐2‐activated NK cells at appropriate effector/target ratios. Here, we show, by histological evaluation of different melanoma lesions, that NK/target‐cell ratios compatible with those allowing efficient melanoma cell killing in vitro are hardly reached at the tumor site. We then investigated the outcome of cocultures established at low NK/melanoma cell ratios. After initial NK‐mediated lysis, residual melanoma cells acquired resistance to IL‐2‐activated NK cells. This reflected primarily an increased expression, on melanoma cells, of classical and nonclassical HLA class I molecules, accompanied by a partial downregulation of NKG2D‐ligands, and was dependent on NK‐mediated IFN‐γ release. Consistently, melanoma lesions showed a higher HLA class I expression on tumor cells that were proximal to infiltrating NK cells. In long‐term cocultures, the “protective phenotype” acquired by melanoma cells was lost over time. However, this phenomenon was counteracted by downregulation of relevant activating receptors in cocultured NK cells. Analysis of different NK‐cell‐activating cytokines indicated that IL‐15 can partially overcome this novel tumor escape mechanism suggesting that IL‐15, rather than IL‐2, may be eligible for NK‐cell‐based immunotherapy.

GPR56 as a novel marker identifying the CD56dull CD16+ NK cell subset both in blood stream and in inflamed peripheral tissues.

To define novel human NK cell markers, we generated two mAbs specific for G-protein-coupled receptor 56 (GPR56), a surface glycoprotein that appears to be involved in cell-to-cell and cell-to-matrix interactions. GPR56 has been described in selected normal tissues, and in certain tumors, while, as yet, its expression on leukocytes is unknown. In this study, we show that anti-GPR56 mAbs, among leukocytes, prevalently recognize NK cells. In particular, these mAbs brightly stain CD56(dull) CD16(+) NK cells while react poorly with CD56(bright) CD16(+/-) NK cells. Consistently, we found that GPR56 was expressed on NK cells populating inflamed peripheral tissues while it was absent in lymph node-derived NK cells. We also show that activating stimuli, such as cytokines or exposure to monocyte-derived dendritic cell, down-regulate NK cell expression of GPR56 both at the protein and at the transcriptional level. Interestingly, IL-18, known to induce de novo expression of CCR7 on CD56(dull) CD16(+) NK cells, displayed the highest capability of modulating GPR56. Thus, together with the identification of GPR56 as a novel marker capable of discriminating different NK cells subsets, our data suggest that GPR56 may take part to the mechanisms regulating NK cell migration through the blood stream, peripheral tissues and lymph nodes.

Lack of expression of inhibitory KIR3DL1 receptor in patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes

Background Natural killer cell-type lymphoproliferative disease of granular lymphocytes is a disorder characterized by chronic proliferation of CD3−CD16+ granular lymphocytes. By flow cytometry analysis, we previously demonstrated a dysregulation in killer immunoglobulin-like receptor (KIR) expression in natural killer cells from patients with this lymphoproliferative disease, the activating KIR receptors being mostly expressed. We also found that patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes usually had KIR genotypes characterized by multiple activating KIR genes. Design and Methods We investigated the mRNA levels of the KIR3DL1 inhibitory and the related KIR3DS1 activating receptors in 15 patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes and in ten controls. These genes are usually expressed when present in the genome of the Caucasian population. Results We demonstrated the complete lack of KIR3DL1 expression in most of the patients analyzed, with the receptor being expressed in 13% of patients compared to in 90% of controls (P<0.01). Interestingly, studies of the methylation patterns of KIR3DL1 promoter showed a significantly higher methylation status (0.76 ± 0.12 SD) in patients than in healthy subjects (0.49±0.10 SD, P<0.01). The levels of expression of DNA methyl transferases, which are the enzymes responsible for DNA methylation, did not differ between patients and controls. Conclusions In this study we showed, for the first time, a consistent down-regulation of the inhibitory KIR3DL1 signal due to marked methylation of its promoter, thus suggesting that together with the increased expression of activating receptors, the lack of the inhibitory signal could also play a role in the pathogenesis of natural killer cell-type lymphoproliferative disease of granular lymphocytes.

Melanoma immunoediting by NK cells.

The immune system can control the early steps of tumor growth, but it may also induce phenotypic/functional changes of malignant cells during tumor progression, favoring immunoescape mechanisms. In a recent study, we revealed how natural killer (NK) cells can participate in such an immunoediting process, by rendering melanoma cells resistant to NK-mediated killing.