Analysis of the experiments was performed using the Kaluza, Summit, or FlowJo softwares. Degranulation assays Natural killer cells were co-cultured with target cells for 2?h at an E:T ratio of 1 1:2. effects and the ability to identify nonresponder patients at an early stage would allow clinicians to switch to alternate therapies earlier in the disease. A considerable body of experimental evidence indicates that natural killer (NK) and NKT cells play an important role on the anti-tumor responses induced by BCG in the treatment of superficial bladder carcinoma (4C9). For this reason, it is important to study in detail the characteristics of the immune response mediated by these cells. Natural killer cells usually represent 5C15% of peripheral blood lymphocytes and respond to their targets without prior antigen sensitization; in particular, their activity is important against virus-infected cells and tumor cells [for review, see Ref. (10)]. NK cell cytotoxicity is exerted by release of lytic granules toward the target cell after formation of a cytotoxic immunological synapse, but, instead of depending on a single receptor, like the TCR in T cells, NK cells require the integration of signals produced by a large number of activating and inhibitory receptors [for review, see Ref. (11, 12)]. In this context, while recognition of MHC-I can be a very strong inhibitory signal, activation mediated by certain activating receptors and cytokines can override the recognition of MHC. The presence of different receptors at the NK cell surface defines several NK subsets with different functional properties. Therefore, to understand how NK cells respond against bladder tumors, in the ML349 context of BCG, it is necessary to dissect the contribution of different receptors to NK cell recognition of these tumor cells. Previous data show that NK cells could kill one urothelial tumor cell line, T24 (6, 7) and that effector cells could be recognizing NKG2D ligands on this cell line (9); however, the presence of these ligands in bladder cancer cells has not been previously explored. The purpose of the work presented here was to systematically evaluate the contribution of immune activating and inhibitory ligands to bladder cancer recognition by NK cells in the context of BCG immunotherapy. We report a comprehensive analysis of the immune phenotype of a panel of urothelial tumor cells and their differential ability to be lysed by primary NK cell lines from healthy donors. We identify NKG2D as a key receptor involved in the recognition of bladder cancer cells by activated NK cells, while NKp46 only contributes partially to the response against certain bladder cell lines. The exposure of purified NK cells to BCG does not affect NK function; however, activation of NK cells can be achieved by exposing PBMCs to BCG. Initial analysis of peripheral blood obtained from BCG-treated bladder cancer patients included in a pilot study show more differences in the percentage of NK cells among individuals than in response to treatment. We have also analyzed the amount of NKG2D receptor in the surface of different subpopulations of NK cells. Materials and Methods Reagents Antibodies Monoclonal antibodies specific for ULBP1, 2, 3, MICA, and MICB were purchased from R&D Systems (Abingdon, UK); ICAM-1/CD54 (Immunotech, Clone 84H10); Nectin 2/CD112 (Santa Cruz, Clone B-C12); CD155/PVR (Abcam, Clone D171); E-Cadherin (Immunotech, Clone 67A4); CD58/LFA-3 (Immunotech, Clone AICD58); CD106/VCAM-1 (Pharmingen, Clone 51-10C9); CD48 (Diaclone, Clone MEM102). MHC class I specific antibody [HP-1F7 (13)] and L31 anti-HLA-C antibody (14) were previously described. Conjugated ML349 antibodies for blood lymphocyte subpopulations were from Biolegend and Immunotools. Secondary antibodies, such as FITC- and PE-conjugated anti-mouse Ig, were purchased from DakoCytomation. Blocking antibodies, specific for anti-human NKG2D (clone 149810), NKp46/NCR1 (clone 195314), NKp30/NCR3 (clone 210845), and DNAM-I/CD226 (clone 102511), were purchased from R&D. KLRG1 antibody was kindly provided by Prof. H. Pircher (University Medical Center Freiburg). Fusion proteins of Mouse monoclonal to CD10.COCL reacts with CD10, 100 kDa common acute lymphoblastic leukemia antigen (CALLA), which is expressed on lymphoid precursors, germinal center B cells, and peripheral blood granulocytes. CD10 is a regulator of B cell growth and proliferation. CD10 is used in conjunction with other reagents in the phenotyping of leukemia NKp46 and NKp30 were prepared by exchanging the human Fc portion of the constructs previously described (15, 16) with a murine IgG1 Fc, to minimize possible Fc/Fc receptor interactions during flow cytometry of human cells. Stable transfectants secreting recombinant proteins were produced in HEK293FT cells (Life Technologies) maintained in DMEM 4.5?mg/l glucose medium supplemented with 10% FBS, l-Glutamine, non-essential amino acids, sodium pyruvate, and antibiotics (penicillin/streptomycin), and 1?g/ml puromycin. NKG2D-Fc constructs, prepared by transient transfection in 293T cells, have been previously described (17). Bacillus CalmetteCGurin Tice strain (from Merck ML349 Canada ML349 Inc.) was used for the majority of the experiments. For some experiments, the strains Connaught (from Sanofi Pasteur Limited) and Danish 1331 (kindly provided by Pfizer) were used. Aliquots of reconstituted BCG were prepared and stored at ?20C. Cell lines and peripheral blood subpopulations The bladder cancer cell lines T24, UM-UC-3, J82, RT-112, RT4, and SW780 are available from the ATCC [cell lines were kindly provided by Dr. FX Real (CNIO,.