Expression of IFN- was slightly (albeit significantly) higher in peripheral blood CD56+CD16- NK cells from AdC compared to healthy controls and even higher in SCC with significant differences between AdC and healthy controls (Figure 3). production. Peripheral blood CD56+CD16- NK cells from patients with the squamous cell carcinoma (SCC) subtype showed higher VEGF and PlGF production compared to those from patients with adenocarcinoma (AdC) and controls. Higher IL-8 production was found for both SCC and AdC compared to controls. Supernatants derived from NSCLC CD56+CD16- NK cells induced endothelial cell chemotaxis and formation of capillary-like structures is known to be effected by TGF and has been previously suggested to induce a polarization of peripheral blood NK cells toward a dNK-like CD56superbrightCD16- phenotype [23,24]. exposure of peripheral blood NK cells from healthy donors to TGF1 upregulated production of angiogenic cytokines, suggesting a role for this cytokine in inducing a proangiogenic NK phenotype. Patients, Materials, and Methods Patient Selection and Samples Samples (tumor tissue and macroscopically normal adjacent tissues) from 31 patients with NSCLC were obtained during surgical resections after obtaining informed consent in an institutional ethics committee-approved study. The patient population characteristics are shown in Table W1. Tissue samples were placed in phosphate-buffered saline (PBS; LONZA, Basel, Switzerland) with 1% Pen/Strep (Sigma- Aldrich, St Louis, MO) at 4C for no more than 18 hours before processing. Peripheral blood samples were drawn from the same patients before surgical intervention into blood collection heparinized tubes, stored at 4C, and processed within 18 hours. Patients with diabetes, human immunodeficiency virus (HIV)/hepatitis C virus (HCV)/hepatitis B virus (HBV) infection, overt chronic inflammatory conditions, previously treated with chemotherapy or radiotherapy, or those iatrogenically immunosuppressed or having undergone myeloablative therapies were excluded. As controls, adjacent normal lung samples were obtained from patients who underwent minimal lung resection for bullectomy to treat pneumothorax following informed consent and processed as above (Table W1). Peripheral blood samples were obtained from healthy donors. Patient Characteristics Lys05 NK cells were isolated from blood, lung tumor, and adjacent healthy tissues from 31 NSCLC patients having undergone tumor resection (median age, 71; range, 44C79), as well as blood and macroscopically normal lung tissue from 10 patients having undergone minimal lung resection for bullectomy (median age, 27; range, 16C69), whose characteristics are shown in Tables 1 and W1. Consistent with the population at risk, the majority of the cancer patients were males (90%) and either former or current smokers (90%). The most frequent subtype was AdC (17; 55%), followed by SCC (9; 29%) and tumors of other subtypes. Lung tissue controls were predominantly male (90%) and current or former smokers (70%; Table W1). Table 1 Characteristics of All Patients with Resected NSCLCs Analyzed. for 30 minutes at room temperature with no brake. The lymphocyte-containing ring at Lys05 the interface was collected in a new tube and washed twice in PBS by centrifugation. Solid Tissue Enzymatic Digestion The solid tissues obtained (tumor, adjacent normal, and non-oncologic lung tissues) were extensively washed in PBS to remove cell debris and eventual red blood cell aggregates and mechanically minced by scissors to obtain small fragments that were enzymatically digested with a cocktail containing DNAse (100 g/ml; Roche, Mannhein, Germany) and Collagenase (1 mg/ml; Sigma-Aldrich) in RPMI 1640 supplemented with Pen/Strep for 1 hour at 37C. The suspension was then filtered on cell strainers [Becton Dickinson (BD), San Jose, CA], while the remaining tissue fragments were processed in a tissue dissociator (gentleMACS; Miltenyi Biotec, Auburn, CA) and subsequently filtered as above. The total single cell suspension was washed by centrifugation Mouse monoclonal to EhpB1 in PBS to remove residual enzymes. Phenotypic Characterization of Tumor Infiltrating NK Cells Cells from blood, tumor, and normal adjacent tissue (3 x 105 cells per sample) were stained with the following monoclonal antibodies (mAbs) in a direct immunofluorescence assay and assessed by flow cytometry (FACS Canto I; BD): Leucogate [BD; fluorescein isothiocyanate (FITC)-conjugated anti-human CD45 and phycoerythrin-conjugated (PE) anti-human CD14] was used to gate on lymphocytes. FITC-conjugated anti-human CD16 (BD, clone 3G8), peridinin-chlorophyll-protein complex (PerCP)-conjugated anti-human CD3 (Miltenyi Biotec, clone BW264/56), and allophycocyanin (APC)-conjugated anti-human CD56 (Miltenyi Biotec, clone AF12-7H3) were used to detect NK cells. Negative controls included directly labeled FITC-conjugated, PerCP-conjugated, and APC-conjugated isotype-matched irrelevant mAbs (BD). Lys05 Briefly, after physical parameter setting (Forward and Side Scatter), lymphocyte populations were identified by gating on CD45-positive cells, and then the NK cell subpopulations were distinguished by gating on CD3-negative cells/CD56-positive cells using the isotypic controls. The CD3-CD56+ NK population was evaluated for CD16 expression. Cytokine and Angiogenic Growth Factor Expression by NK Cells The total cell suspensions were incubated overnight in RPMI 1640 supplemented with heat-inactivated FBS (Euroclone, Milan, Italy), Pen/Strep, and IL-2 (100 U/ml; R&D Systems, Minneapolis, MN) at 37C and 5% CO2. Cells (3 x.