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.

Effects of cyclophosphamide and IL-2 on regulatory CD4+ T cell frequency and function in melanoma patients vaccinated with HLA-class I peptides: impact on the antigen-specific T cell response.

The frequency and function of regulatory T cells (Tregs) were studied in stage II-III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4(+) T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8(+) T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8(+) T cell effectors when used in the inflammatory environment of vaccination.

Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy.

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.

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.

Modified peptides in anti-cancer vaccines: are we eventually improving anti-tumour immunity?

The discovery of tumour antigens recognized by T cells and the features of immune responses directed against them has paved the way to a multitude of clinical studies aimed at boosting anti-tumour T cell immunity as a therapeutic tool for cancer patients. One of the different strategies explored to ameliorate the immunogenicity of tumour antigens in vaccine protocols is represented by the use of optimized peptides or altered peptide ligands, whose amino acid sequence has been modified for improving HLA binding or TCR interaction with respect to native epitopes. However, despite the promising results achieved with preclinical studies, the clinical efficacy of this approach has not yet met the expectations. Although multiple reasons could explain the relative failure of altered peptide ligands as more effective cancer vaccines, the possibility that T cells primed by modified tumour peptides might may be unable to effectively cross-recognize tumour cells has not been sufficiently addressed. Indeed, the introduction of conservative amino acid substitutions may still produce diverse and unpredictable changes in the HLA/peptide interface, with consequent modifications of the TCR repertoire that can interact with the complex. This could lead to the expansion of a broad array of T cells whose TCRs may not necessarily react with equivalent affinity with the original antigenic epitope. Considering the results presently achieved with this vaccine approach, and the emerging availability of alternative strategies for boosting anti-tumour immunity, the use of modified tumour peptides could be reconsidered.

Tumor-released microvesicles as vehicles of immunosuppression.

Tumor-released microvesicles, or exosomes, which are abundant in the body fluids of patients with cancer, are likely to be involved in tumor progression. We recently showed that microvesicles released by human melanoma and colorectal carcinoma cells can promote the differentiation of monocytes to myeloid-derived suppressor cells which support tumoral growth and immune escape. These findings underscore an important role for these extracellular organelles in remodeling tumor-stromal interactions to promote malignancy.

Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-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.

Tumor-derived microRNAs induce myeloid suppressor cells and predict immunotherapy resistance in melanoma.

The accrual of myeloid-derived suppressor cells (MDSCs) represents a major obstacle to effective immunotherapy in cancer patients, but the mechanisms underlying this process in the human setting remain elusive. Here, we describe a set of microRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e, miR-125a, miR-146b, miR-99b) that are associated with MDSCs and resistance to treatment with immune checkpoint inhibitors in melanoma patients. The miRs were identified by transcriptional analyses as being responsible for the conversion of monocytes into MDSCs (CD14+HLA-DRneg cells) mediated by melanoma extracellular vesicles (EVs) and were shown to recreate MDSC features upon transfection. In melanoma patients, these miRs were increased in circulating CD14+ monocytes, plasma, and tumor samples, where they correlated with the myeloid cell infiltrate. In plasma, their baseline levels clustered with the clinical efficacy of CTLA-4 or programmed cell death protein 1 (PD-1) blockade. Hence, MDSC-related miRs represent an indicator of MDSC activity in cancer patients and a potential blood marker of a poor immunotherapy outcome.

The acidity of the tumor microenvironment is a mechanism of immune escape that can be overcome by proton pump inhibitors.

We have recently reported that lowering the pH to values that are frequently detected in tumors causes reversible anergy in both human and mouse CD8+ T lymphocytes in vitro. The same occurs in vivo, in the tumor microenvironment and the administration of proton pump inhibitors, which buffer tumor acidity, can revert T-cell anergy and increase the efficacy of immunotherapy.

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.

Limited induction of tumor cross-reactive T cells without a measurable clinical benefit in early melanoma patients vaccinated with human leukocyte antigen class I-modified peptides.

PURPOSE: The progressive immune dysfunctions that occur in patients with advanced melanoma make them unlikely to efficiently respond to cancer vaccines. A multicenter randomized phase II trial was conducted to test whether immunization with modified HLA class I tumor peptides in the context of adjuvant therapy results in better immunologic responses and improved clinical outcomes in patients with early melanoma (stages IIB/C-III). EXPERIMENTAL DESIGN: Forty-three patients were enrolled to undergo vaccination (n = 22) or observation (n = 21). The vaccine included four HLA-A*0201-restricted modified peptides (Melan-A/MART-1([27L]), gp100([210M]), NY-ESO-1([165V]), and Survivin([97M])) emulsified in Montanide ISA51 and injected subcutaneously in combination with cyclophosphamide (300 mg/m(2)) and low-dose IL-2 (3 × 10(6) IU). The immune responses were monitored using ex vivo IFN-γ-ELISpot, HLA/multimer staining, and in vitro short-term peptide sensitization assays. RESULTS: Vaccination induced a rapid and persistent increase in specific effector memory CD8(+) T cells in 75% of the patients. However, this immunization was not associated with any significant increase in disease-free or overall survival as compared with the observation group. An extensive immunologic analysis revealed a significantly reduced cross-recognition of the corresponding native peptides and, most importantly, a limited ability to react to melanoma cells. CONCLUSIONS: Adjuvant setting is an appealing approach for testing cancer vaccines because specific CD8(+) T cells can be efficiently induced in most vaccinated patients. However, the marginal antitumor activity of the T cells induced by modified peptides in this study largely accounts for the observed lack of benefit of vaccination. These findings suggest reconsidering this immunization strategy, particularly in early disease.

Cell-cycle inhibition by Helicobacter pylori L-asparaginase.

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.

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.