Silvia Zanellato

CD56brightCD16− NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation

Recent studies suggested that human CD56brightCD16− NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56dimCD16+ and CD56brightCD16− NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56brightCD16− than in CD56dimCD16+ NK cells. CD57 was mostly expressed by CD56dimCD16+ NK cells. CD203a/PC-1 expression was restricted to CD56brightCD16− NK cells. CD56brightCD16− NK cells produce ADO and inhibit autologous CD4+ T cell proliferation. Such inhibition was 1) reverted pretreating CD56brightCD16− NK cells with a CD38 inhibitor and 2) increased pretreating CD56brightCD16− NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56brightCD16− NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4+ T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56brightCD16− NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56brightCD16− NK cells act as “regulatory cells” through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases.

Polarization of Tumor Infiltrating Leukocytes from Innate Immunity and their role in the Pro-angiogenic Phenotype in NSCLC

Non-small cell lung cancer (NSCLC), the most frequent lung cancer (80%), can be phenotypically classified into two main subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (ADC). While SCC has relatively rapid doubling times from the onset, ADC has longer doubling times initially that become reduced during tumor progression, suggesting a key role for the microenvironment. During lung tumor progression, a complex and dynamic interplay occurs between proliferating tumor cells and stromal, endothelial and immune tumor-conditioned host cells within the tumor microenvironment (TUMIC). Several factors within the TUMIC, such as hypoxia, cytokines and soluble factors, appear to blunt the anti-tumor immune response and polarize immune cells towards a pro-tumor phenotype. Phenotypically and functionally altered immune cells found in cancer patients include macrophages, neutrophils, myeloid, dendritic, and even NK cells. We studied tumor infiltrating (TINK) and tumor associated (TANK) NK cells in NSCLC. NSCLC TINKs and TANKs show similarities to decidual NK cells, being polarized toward tissue builders, rather than killers, and producing pro-angiogenic cytokines. The functionally polarized immune cells in NSCLC provide the stromal support and neovascularization required for NSCLC tumor expansion and progression in a feed-forward mechanism, leading to tumor progression. Further, systemic alterations of immune cells are also present in NSCLC patients. The precise knowledge of these immune cell alterations within the TUMIC has become crucial for diagnosis, targeted therapeutic intervention, as well as prevention, of NSCLC cancer.

Natural Killer Cells from Malignant Pleural Effusion Are Endowed with a Decidual-Like Proangiogenic Polarization

Natural killer (NK) cells are crucial in tumor recognition and eradication, but their activity is impaired in cancer patients, becoming poorly cytotoxic. A particular type of NK cells, from the decidua, has low cytotoxicity and shows proangiogenic functions. We investigated whether NK cells from peripheral blood (PB) and pleural effusions of patients develop decidual-like NK phenotype and whether exposure to IL-2 can restore their killing ability in the presence of pleural fluids. NK cells from pleural effusion of patients with inflammatory conditions (iPE, n = 18), primary tumor (ptPE, n = 18), and metastatic tumor (tmPE, n = 27) acquired the CD56brightCD16− phenotype. NK cells from both ptPE and tmPE showed increased expression for the CD49a and CD69 decidual-like (dNK) markers and decreased levels of the CD57 maturation marker. NK from all the PE analyzed showed impaired degranulation capability and reduced perforin release. PE-NK cells efficiently responded to IL-2 stimulation in vitro. Addition of TGFβ or cell-free pleural fluid to IL-2 in the culture medium abrogated NK cell CD107a and IFNγ expression even in healthy donors (n = 14) NK. We found that tmPE-NK cells produce VEGF and support the formation of capillary-like structures in endothelial cells. Our results suggest that the PE tumor microenvironment can shape NK cell polarization towards a low cytotoxic, decidual-like, highly proangiogenic phenotype and that IL-2 treatment is not sufficient to limit this process.

The therapeutic T-cell response induced by tumor delivery of TNF and melphalan is dependent on early triggering of natural killer and dendritic cells.

The fusion protein L19mTNF (mouse TNF and human antibody fragment L19 directed to fibronectin extra domain B) selectively targets the tumor vasculature, and in combination with melphalan induces a long‐lasting T‐cell therapeutic response and immune memory in murine models. Increasing evidence suggests that natural killer (NK) cells act to promote effective T‐cell‐based antitumor responses. We have analyzed the role of NK cells and dendritic cells (DCs) on two different murine tumor models: WEHI‐164 fibrosarcoma and C51 colon carcinoma, in which the combined treatment induces high and low rejection rates, respectively. In vivo NK‐cell depletion strongly reduced the rejection of WEHI‐164 fibrosarcoma and correlated with a decrease in mature DCs, CD4+, and CD8+ T cells in the tumor‐draining LNs and mature DCs and CD4+ T cells in the tumor 40 h after initiation of the therapy. NK‐cell depletion also resulted in the impairment of the stimulatory capability of DCs derived from tumor‐draining LNs of WEHI‐164‐treated mice. Moreover, a significant reduction of M2‐type infiltrating macrophages was detected in both tumors undergoing therapy. These results suggest that the efficacy of L19mTNF/melphalan therapy is strongly related to the early activation of NK cells and DCs, which are necessary for an effective T‐cell response.

Abstract 3244: Tumor infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells (TINKs): A new paradigm in colorectal cancer

Tumor infiltrating immune cells often show a skewed phenotype that reflects attenuation of anti-tumor activity and enhancement of pro-tumor activities, including angiogenesis. Natural Killer (NK) cells are effectors lymphocytes of innate immunity, primarily involved in immunosurvelliance against tumors through their cytotoxic activity. We previously reported that NKs from Non Small Cell Lung Cancer (NSCLC) show the decidual-like CD56brightCD16-VEGFhighPlGFhighIL-8+IFNγlow phenotype [Bruno et al, Neoplasia, 2014; Bruno et al, JNCI, 2014]. Here we investigated whether tumor associated (TANKs) and tumor infiltrating (TINKs) NK cells undergo an angiogenic-switch in colorectal cancer (CRC). NK subset distribution and cytokine profiling were performed by multicolor flow cytometry, using peripheral blood and tissue samples from CRC patients. Conditioned media (CM) from FACS-sorted NKs were used either for secretomic profiling, by antibody membrane array or angiogenesis functional assay on human umbilical endothelial vein cells (HUVECs) We found that CD56brightCD16− NK cells predominate in CRC adjacent and tumor tissues, produce VEGF, PlGF, IL-8 and show impaired cytotoxicity. Further, TINK/TANKs from CRC patients express the decidual NK markers CD9 and CD49a. Secretomic analysis on CRC peripheral blood NKs revealed up regulation for several pro angiogenic factors, including Angiogenin, Angiopoietin-1/2, TIMP-1/2, Tie-2, MMP1, MMP9. Media conditioned by FACS sorted NK cells from peripheral blood and tumor tissue of CRC patients were able to induce HUVEC proliferation, migration and the formation of capillary like structures. Our data demonstrate that TINK/TANKS from CRC patients are switched toward a pro-angiogenic/pro-tumor phenotype and function. We propose that TINK/TANKs could represent the hallmark for a new paradigm in CRC inflammation. 1. Bruno A, Focaccetti C, Pagani A, Imperatori AS, Spagnoletti M, Rotolo N, Cantelmo AR, Franzi F, Capella C, Ferlazzo G, Mortara L, Albini A, Noonan DM. The proangiogenic phenotype of natural killer cells in patients with non-small cell lung cancer. Neopla sia. 2013 Feb;15(2):133-42. 2. Bruno A, Ferlazzo G, Albini A, Noonan DM. A think tank of TINK/TANKs: tumor infiltrating/tumor-associated natural killer cells in tumor progression and angiogenesis. J Natl Cancer Inst. 2014 Aug;106(8):dju200 Citation Format: Antonino Bruno, Barbara Bassani, Davide Giuseppe D’Urso, Silvia Zanellato, Elisabetta Gini, Elisa Cassinotti, Luigi Boni, Lorenzo Mortara, Adriana Albini, Douglas M. Noonan. Tumor infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells (TINKs): A new paradigm in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3244.

Tumor-infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells: a new player in the inflammatory orchestration of tumor angiogenesis in colon cancer

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide and a prevailing cause of cancer-related mortality. Several factors increase the risk of developing the tumor, including age, inflammatory bowel disease, personal and/or family history of colorectal tumors (adenoma or adenocarcinoma), and environmental factors. In spite of the extensive evidence indicating a role for inflammation in both colon cancer insurgence and progression, there is relatively little information concerning the role of inflammatory cells in CRC progression. Natural Killer (NK) cells are effectors lymphocytes of innate immunity that can potentially control tumors by their cytotoxic activity. Several immune cells within the tumors, like macrophages, neutrophils, myeloid-derived suppressor (MSDCs) cells and dendritic cells (DCs) have been reported to acquire an altered phenotype that reflects attenuation of anti-tumor activity and enhancement of pro-tumor activities, including angiogenesis. We recently identified a peculiar NKs subset infiltrating Non Small Cell Lung Cancer (NSCLC), that we termed TINKs, described as CD56brightCD16- NKs, able to produce large amount of VEGF, PlGF and IL-8 and induce angiogenesis in vitro. We therefore extended our studies on CRC TINKs and TANKs. NK cell were isolated from blood and tissue (adjacent-normal/tumour) samples from patients with CRC then phenotipically and functionally characterized for surface antigen expression and cytokine profiling by multiparametric flow cytometry. Finally, functional studies were performed on human umbelical vein cells (HUVECs), using conditioned media (CM) derived from isolated NKs. We found that the CD56brightCD16- NK cells predominate in both the tumors and adjacent tissues derived from colo-rectal carcinoma (CRC) samples and that are able to release substantial amounts of pro-angiogenic factors, including VEGF, PlGF and IL-8, exert low cytotoxic activities and induce migration of and capillary-like structure formation of endothelial cells in vitro. When we look at the molecular mechanisms involved in TINK and TANK angigoenic switch, we fould out that TGFβ1, an abundant cytokine within the tumor microenvironent, strongly up-regulates VEGF and PlGF release by peripheral blood NK cells from healthy age-matched donors. Taken together, the “switched” phenotype and function of tumor infiltrating NK cells acquire a broad implications in the role of immune response against tumors, ranging from a deficient control of cancer to an altered crosstalk with other relevant players of both innate ad acquired immune response. This places NK cells as a new player in the inflammatory orchestration of tumor angiogenesis. Citation Format: Antonino Bruno, Barbara Bassani, Silvia Zanellato, Sara Canali, Lorenzo Dominioni, Luigi Boni, Cassinotti Elisa, Giulia David, Katiuscia Dallaglio, Lorenzo Mortara, Douglas M. Noonan, Adriana Albini. Tumor-infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells: a new player in the inflammatory orchestration of tumor angiogenesis in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2015-2367