4146). NK cell number, subset distribution and function, as measured by cluster Ebastine of differentiation 107a degranulation, did not exhibit any significant alterations as a result of sunitinib treatment. These results indicate that sunitinib Rabbit Polyclonal to RAB18 does not negatively affect NK cell function, which supports the pursuit of therapeutic modalities that combine immunomodulation and NK cell-stimulating approaches. analyses were performed to investigate whether NK cells may be affected by sunitinib treatment. Materials and methods Patients and sample preparation The current immunomonitoring study was approved by the ethics committee of the Friedrich-Alexander University Erlangen-Nuremberg (Erlangen, Germany), according to the Declaration of Helsinki (approval no. 4146). All patients provided written informed consent. Samples (10C20 ml heparinized peripheral blood) were collected from 3 patients between June 2010 and June 2011. The inclusion criteria for patients included a minimum age of 18 years and a confirmed mRCC diagnosis. The samples were collected prior to TKI administration (day 0) and then at 3C5, 8C10, 17C23 and 48 (1 patient only) weeks after the start of TKI treatment. Of the 3 patients, 2 were followed-up subsequent to the termination of TKI therapy. Peripheral blood mononuclear cells (PBMCs) were isolated from the samples by density gradient centrifugation (Pancoll human; Pan-Biotech GmbH, Aidenbach, Germany). Control group characteristics The control group consisted of four healthy individuals with an age range of 44C60 years. These individuals were monitored over 91 days. Blood samples were obtained on days 0, 60 and 91. Flow cytometry The following mouse anti-human monoclonal antibodies and antibody conjugates were used in different panels: Anti-T-cell receptor /-allophycocyanin (APC; dilution, 1:100; Ebastine cat. no., 130-091-237) and anti-cluster of differentiation (CD) 335-APC (dilution, 1:30; cat. no., 130-092-609; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany); anti-CD4-phycoerythrin (PE) -Cy7 (dilution, 1:100; cat. no., 557852), streptavidin-APC-Cy7 (dilution, 1:500; cat. no., 554063), anti-CD314-biotin (dilution, 1:50;cat. no., 552866) andanti-CD127-PE (dilution, 1:300;cat. no., 557938; BD Pharmingen, San Diego, CA, USA);anti-CD25-PE (dilution, 1:60; cat. no., 341,011), anti-CD336-PE (dilution, 1:50; cat. no., 558563), anti-CD8-peridinin chlorophyll (PerCP; dilution, 1:100; cat. no., 345774), anti-CD117-PerCP-Cy5.5 (dilution, 1:50; cat. no., 333950), anti-CD19-V450 (dilution, 1:300; cat. no., 560353) and anti-CD3-V450 (dilution, 1:200;cat. no., 560366;BD Biosciences, Franklin Lakes, NJ, USA), anti-CD3-APC-Cy7 (dilution, 1:300; cat. no., 300318), anti-CD16-PE-Cy7 (dilution, 1:300; cat. no., 302016), anti-CD19-PerCP (dilution, 1:50; cat. no., 302228), anti-CD107a-PE (dilution, 1:10; cat. no., 328608), anti-CD56-fluorescein isothiocyanate (FITC; dilution, 1:50; cat. Ebastine no., 318304) andanti-CD19-PE-Cy7 (dilution, 1:100; cat. no., 302216; Biolegend, Inc., San Diego, CA, USA); anti-CD27-FITC (dilution, 1:70; cat. no. MHCD2701) and anti-CD45-PE (dilution, 1:200; cat no., MHCD4504; Invitrogen; Thermo Ebastine Fisher Scientific, Inc., Waltham, MA, USA) andanti-CD159a-biotin (dilution, 1:30; cat. no., PNIM2750; Beckman Coulter, Inc., Brea, CA, USA). A total of 2106 PBMCs with antibodies were incubated for 10 min at 4C in the dark for each staining procedure. The PBMCs were then washed with phosphate-buffered saline (PBS) for 4 min and then suspended in PBS. Staining with DAPI was performed directly prior to cytometric analysis (dilution, 1:6,000, 4C, 2 min) to identify dead cells and isotype controls (IgG1-PE, IgG2a-FITC, IgG1-PerCP) were performed prior to analyzing the samples with fluorescence-activated cell sorting (FACS). The samples were examined using a BDFACSCanto II? (BD Biosciences) with three lasers (488, 633 and 405 nm). The experiment was repeated at least five times before the start of analyzing patients’ data, and then performed once with each patient sample. Data were analyzed by FlowJo 7.6.5 software (FlowJo, LLC, Ashland, OR, USA). Functional in vitro assays To determine the activity of NK cells, the extent of degranulation Ebastine was evaluated by CD107a staining following the incubation of whole blood samples with target cells. Cells from the human MHC-I deficient erythroleukaemic cell line K562 (American Type Culture Collection, Manassas, VA, USA) were used as the target cells. The cells were maintained in Iscove’s modified Dulbecco’s medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FCS (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin. A total of 200 l from the heparinized whole blood samples was incubated with 2105 K562 target cells for 3 h at 37C, as previously described by Claus (21). Erythrocyte lysis was then performed in an ammonium-chloride-potassium buffer for 10 min at 4C; the lysis procedure was repeated 2C3 times for each sample. In order to stain the cells for FACS, PBMC were incubated with anti-CD107a-PE, anti-CD3-APC-Cy7, anti-CD19-PerCP, anti-CD56-FITC, anti-CD16-PE-Cy7 or anti-CD335-APC.