Veronica Huber

Identification of a New Subset of Myeloid Suppressor Cells in Peripheral Blood of Melanoma Patients With Modulation by a Granulocyte-Macrophage Colony-Stimulation Factor–Based Antitumor Vaccine

PURPOSE Phenotypic and functional features of myeloid suppressor cells (MSC), which are known to serve as critical regulators of antitumor T-cell responses in tumor-bearing mice, are still poorly defined in human cancers. Here, we analyzed myeloid subsets with suppressive activity present in peripheral blood of metastatic melanoma patients and evaluated their modulation by a granulocyte-macrophage colony-stimulating factor (GM-CSF)--based antitumor vaccine. PATIENTS AND METHODS Stage IV metastatic melanoma patients (n = 16) vaccinated with autologous tumor-derived heat shock protein peptide complex gp96 (HSPPC-96) and low-dose GM-CSF provided pre- and post-treatment whole blood specimens. Peripheral-blood mononuclear cells (PBMCs) were analyzed by flow cytometry, separated into cellular subsets, and used for in vitro proliferation assays. PBMCs from stage-matched metastatic melanoma patients (n = 12) treated with non-GM-CSF-based vaccines (ie, HSPPC-96 alone or interferon alfa/melanoma-derived peptides) or sex- and age-matched healthy donors (n = 16) were also analyzed for comparison. RESULTS The lack of or low HLA-DR expression was found to identify a CD14+ cell subset highly suppressive of lymphocyte functions. CD14+HLA-DR-/lo cells were significantly expanded in all metastatic melanoma patients, whereas they were undetectable in healthy donors. Suppressive activity was mediated by transforming growth factor beta (TGF-beta), whereas no involvement of the arginase and inducible nitric oxide synthase pathways could be detected. CD14+HLA-DR-/lo cells, as well as spontaneous ex vivo release and plasma levels of TGF-beta, were augmented after administration of the HSPPC-96/GM-CSF vaccine. No enhancement of the CD14+-mediated suppressive activity was found in patients receiving non-GM-CSF-based vaccines. CONCLUSION CD14+HLA-DR-/lo cells exerting TGF-beta-mediated immune suppression represent a new subset of MSC potentially expandable by the administration of GM-CSF-based vaccines in metastatic melanoma patients.

Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

Background Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. Methodology/Principal Findings We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504±315) or caveolin-1 (619±310) were significantly increased in melanoma patients as compared to healthy donors (223±125 and 228±102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. Conclusions/Significance We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.

Human Tumor-Released Microvesicles Promote the Differentiation of Myeloid Cells with Transforming Growth Factor-β–Mediated Suppressive Activity on T Lymphocytes

Human tumors constitutively release endosome-derived microvesicles, transporting a broad array of biologically active molecules with potential modulatory effects on different immune cells. Here, we report the first evidence that tumor-released microvesicles alter myeloid cell function by impairing monocyte differentiation into dendritic cells and promoting the generation of a myeloid immunosuppressive cell subset. CD14+ monocytes isolated from healthy donors and differentiated with interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor in the presence of tumor-derived microvesicles turned into HLA-DR(-/low) cells, retaining CD14 expression and failing to up-regulate costimulatory molecules, such as CD80 and CD86. These phenotypic changes were paralleled by a significant release of different cytokines, including IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and a dose-dependent suppressive activity on activated T-cell-proliferation and cytolytic functions, which could be reversed by anti-TGF-beta-neutralizing antibodies. Microvesicles isolated from plasma of advanced melanoma patients, but not from healthy donors, mediated comparable effects on CD14+ monocytes, skewing their differentiation toward CD14+HLA-DR-/low cells with TGF-beta-mediated suppressive activity on T-cell-functions. Interestingly, a subset of TGF-beta-secreting CD14+HLA-DR- cells mediating suppressive activity on T lymphocytes was found to be significantly expanded in peripheral blood of melanoma patients compared with healthy donors. These data suggest the development in cancer patients of an immunosuppressive circuit by which tumors promote the generation of suppressive myeloid cells through the release of circulating microvesicles and without the need for cell-to-cell contact. Therapeutic interventions on the crucial steps of this pathway may contribute to restore tumor/immune system interactions favoring T-cell-mediated control of tumor growth in cancer patients.

Immunity to cancer: attack and escape in T lymphocyte–tumor cell interaction

Summary: Tumor cells may express antigens which are recognized in a form of HLA/peptide complexes by T cells. The frequency at which different antigens are seen by T cells of melanoma patients and healthy donors was evaluated by human leukocyte antigen (HLA)/peptide tetramer technology which stains T cells bearing the specific receptor for a given epitope. By this technique, it was found that the majority of metastatic melanoma patients can recognize differentiation antigens (particularly Melan‐A/MART‐1), whereas such a recognition is scanty in the early phase of the disease and in healthy subjects. Despite the presence of melanoma‐specific T cells infiltrating tumor lesions, tumor rejection rarely occurs. Among the different mechanisms of such inefficient antitumor response, this review discusses the possible anti‐T‐cell counterattack mediated by FasL‐positive tumor cells, and shows that FasL is located in the cytoplasm of melanoma cells and is transported in the tumor microenvironment through the release of melanosomes. Additionally, mechanisms of suboptimal T cell activation through tumor cell expression of peptide analogs with antagonist activity are described, together with the possibility of overcoming such anergy induction by the usage of optimized tumor epitopes. Down‐modulation of HLA expression by target tumor cells and its multiple mechanisms is also considered. Finally, we discuss the role of inducible nitric oxide synthases in determining the inhibition of apoptosis in melanoma cells, which can make such tumor cells resistant to the T‐cell attack.

Phenotype, function and clinical implications of myeloid-derived suppressor cells in cancer patients

The involvement of a smouldering microenvironment is currently considered a cancer hallmark and a required step for tumour cells to disable specific immunity while promoting angiogenesis and stroma remodelling. Nevertheless, the molecular pathways driving such aberrant interactions in human cancer and their actual implication in disease progression are still poorly defined. Here, we will report about the remarkable efforts devoted by our group as well as many other scientists to dissect this process focusing on tumour-mediated activation of myeloid dysfunctional pathways occurring in cancer patients. Indeed, myeloid-derived suppressor cells (MDSC), playing a crucial role as cellular regulators of immune responses, have been extensively shown to restrain tumour immunity through a vast array of molecular mechanisms and to promote tumour progression in different murine models. Although in mice the phenotypic features of these cells were defined initially rather generally by Gr1+ and CD11b+ co-expression, more recent studies have unravelled the actual complexity of this population and the existence of different cell subsets. This complexity is even more remarked in the human setting, where heterogeneous populations of myeloid cells with variable phenotype and immunosuppressive features have been described in patients affected by different types of tumours. The lack of homogeneous properties of human MDSC has made these cells a controversial and still unacknowledged player in cancer-related immune suppression and disease progression. Nevertheless, with the efforts of the scientific community, MDSC will soon reveal their key role thereby becoming novel targets for innovative therapeutic strategies.

Recent advances on the role of tumor exosomes in immunosuppression and disease progression

Exosomes are endosomal-derived nanovesicles released by most cells types, including tumor cells, and principally involved in intercellular communication in physiology and disease. Tumor exosomes are gaining increasing interest in medicine and oncology as efficient tools for the delivery of defined signals. Representing the acellular replicas of tumor cells, they contain a great variety of bioactive molecules, such as proteins, RNA, miRNA and DNA. Their great ability to recirculate in body fluids and their structure allow them to transport their cargo to distant targets. Major studies have shown that tumor exosomes convey information not only between tumor cells but also to other cell types, including different immune cell components. There is increasing evidence that these nanovesicles may contribute to cancer progression by influencing different immune cell types, likely blunting specific T cell immunity and skewing innate immune cells toward a pro-tumorigenic phenotype. Because of this function and the additional property to deliver molecular signals modulating neoangiogenesis and stroma remodeling, tumor exosomes are believed to play a role in tumor progression by favoring metastatic niche onset. This review outlines the recent knowledge on immune suppressive mechanisms mediated by tumor exosomes. We will discuss our view on the role of these nanovesicular structures in cancer progression and how their presence could interfere with cancer therapy.

Modulation of Microenvironment Acidity Reverses Anergy in Human and Murine Tumor-Infiltrating T Lymphocytes

Stimulating the effector functions of tumor-infiltrating T lymphocytes (TIL) in primary and metastatic tumors could improve active and adoptive T-cell therapies for cancer. Abnormal glycolysis, high lactic acid production, proton accumulation, and a reversed intra-extracellular pH gradient are thought to help render tumor microenvironments hostile to roving immune cells. However, there is little knowledge about how acidic microenvironments affect T-cell immunity. Here, we report that lowering the environmental pH to values that characterize tumor masses (pH 6-6.5) was sufficient to establish an anergic state in human and mouse tumor-specific CD8(+) T lymphocytes. This state was characterized by impairment of cytolytic activity and cytokine secretion, reduced expression of IL-2Rα (CD25) and T-cell receptors (TCR), and diminished activation of STAT5 and extracellular signal-regulated kinase (ERK) after TCR activation. In contrast, buffering pH at physiologic values completely restored all these metrics of T-cell function. Systemic treatment of B16-OVA-bearing mice with proton pump inhibitors (PPI) significantly increased the therapeutic efficacy of both active and adoptive immunotherapy. Our findings show that acidification of the tumor microenvironment acts as mechanism of immune escape. Furthermore, they illustrate the potential of PPIs to safely correct T-cell dysfunction and improve the efficacy of T-cell-based cancer treatments.

Human Tumor-Derived Heat Shock Protein 96 Mediates In Vitro Activation and In Vivo Expansion of Melanoma- and Colon Carcinoma-Specific T Cells

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8+ T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8+ T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-γ and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A27–35 in three of five HLA-A*0201 melanoma patients, and of CEA571–579 and EpCAM263–271 in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8+ T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients.

Immune Surveillance Properties of Human NK Cell-Derived Exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by “normal” cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.