at days-2 and 5. active immunization to block NKp46, through immunizing with recombinant NKp46, inhibited the development of T1D in murine models [10]. These findings motivated us to explore new therapeutic approaches for T1D based on manipulation of NKp46 function. In order to accomplish this, one tactic would be to block the NKp46 ligands. However, the precise nature of NKp46 ligands is not fully revealed. Several reports have shown that NKp46 recognizes cellular ligands expressed on tumor cells, dendritic cells, viral-infected cells, and Langerhans -cells [17,18]. Currently identified NKp46 ligands include viral hemagglutinins [19], ICA-110381 the cellular ligand vimentin [20] and the cellular co-ligands heparan sulfate proteoglycans (HSPG) [21C23]. However, these two cellular ligands hardly present a suitable target for manipulation of NKp46 function through blocking of target cellular ligand. Therefore, in the current study we investigated the methodology of antibody-mediated manipulation of the NKp46 function. We developed and characterized a new anti-murine NKp46 mAb named NCR1.15. Treatment of mice with NCR1.15 did not deplete NK cells, but suppressed their NKp46-mediated function. In accordance, NCR1.15 ICA-110381 treatment of T1D-prone mice significantly prolonged the time to T1D development. Research Design and Methods Cells Cells that were used in this study: YAC-1, murine lymphoma (TIB-160, ATCC); PD1.6, murine thymic virus-induced lymphoma [24]; Ba/F3-Rae1 mouse pro-B lymphocyte ectopically expressing Rae-1 NKG2D ligand [25]; BW-hNKp46 and BW-mNKp46 T-cell lymphoma ectopically expressing the murine or the human NKp46 [19]. Production of NCR1.15 mAb 129/sv/J mice, lacking the expression of endogenous mNKp46, were used for the production of mouse monoclonal antibodies against mNKp46. These mice were immunized twice with 100ug/mice of mNKp46-Ig fusion protein, followed by a boost immunization and a subsequent fusion with the mouse Sp2/0 cell line. First hybridoma screening for specific antibodies was performed using ELISA. Positive hybridomas were selected and cloned several times to ensure monoclonality. Antibodies obtained from these clones were further characterized using different techniques. Antibodies and Fusion proteins The following antibodies were used in this study: BioLegendanti-NKp46 mAb (clone 29A1.4), anti-CD3 (clone145C2C11), anti-NK1.1 (clone PK136), anti-CD49b (clone DX5), anti-CD27 (clone LG.3A10), anti-NKG2D (clone C7), anti-CD107a/LAMP-1 (clone 1D4B); anti-CD11b (eBioscience, clone M1/70); anti-human NKp46 (461-G1,[26]); mouse IgG1, control for injections, rat IgG2a, isotype control for FACS (BioLegend, clone RTK2758). The Production of mNKp46-Ig, LIR1-Ig and mNKG2D-Ig as previously described [25,27,28]. ELISA To determine the specific binding between NCR1.15 and the mNKp46 receptor plates were coated overnight at 4C with 5g/ml of the recombinant proteins. Blocking buffer (PBS supplemented with 10%FBS) was applied for 2 hours at room temperature, after which plates were washed with PBS with 0.05% Tween 20 (PBST) and incubated with 2 g/ml of NCR1.15, 461-G1or PBS for 2 hours at room temperature. Following washing with PBST biotin-conjugated sheep anti-mouse IgG (GE Healthcare, NA931V) was added for 1 hr at 1:750 dilution. Following washing streptavidin-HRP (Jackson, 016-030-084) diluted 1:1000 was added for 30 min. Following washing TMB (DAKO, S1599) was added optical density was read at 650 nm (Thermo Electron Corporation Multiskan Spectum). Bimolecular interaction analysis A BIAcore 3000 device fitted with CM5 sensor chips (BIAcore, Uppsala, Sweden) was used for studying the interactions between NKp46 and NCR1.15 in conjunction with BIAevaluation software (v4.1). To activate the chip, we used the ICA-110381 EDC/NHS amine coupling procedure according to the manufacturers protocol (BIAcore), followed by addition of NKp46, which was immobilized in the different flow cells, followed by blocking the free active groups with 1 M ethanolamine. Rabbit Polyclonal to p14 ARF Different analyte concentrations were injected, each followed by regeneration of the surface using 10 mM NaOH. Data were analyzed using a 1:1 Langmuir binding model. CD107a degranulation assay Spleen lymphocytes were isolated 3.