Cd86 clone it2.2

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy donors (Oklahoma Blood Institute). Monocytes were enriched from PBMCs using magnetic bead separation (Miltenyi Biotech). Purity was >90% by flow cytometry and viability was >99% by trypan blue exclusion post enrichment. Monocytes were stimulated with 10 ng/ml recombinant hIL-10 (Peprotech) or recombinant vIL-10 (R&D systems) for indicated times. STAT3 phosphorylation was detected by flow cytometry using antibodies directed against phospho-STAT3 Y705 (BD Biosciences). To inhibit signaling through IL-10R, monocytes were stimulated with hIL-10 or vIL-10 in the presence or absence of a neutralizing antibody to IL-10R1 (Clone: 37607, R&D systems), and STAT phosphorylation was measured as above. To differentiate monocytes into macrophages, cells were cultured with 50 ng/ml M-CSF (R&D systems). On day 6 cells were additionally stimulated with IFNγ (20 ng/ml, Peprotech), IL4 (20 ng/ml, Peprotech), hIL-10 (10 ng/ml), or vIL-10 (10 ng/ml) for 24 h. Surface markers were stained using following antibodies: CD14 Clone M5E2, CD163 Clone GHI/61, CD32 Clone FLI8.26 (BD Biosciences), CD16 Clone 3G8, CD64 Clone 10.1, HLA-DR Clone LN3, CD86 Clone IT2.2 (BioLegend). Cells were acquired on BD LSR II or BD Celesta (BD Biosciences) and data were analyzed using FlowJo (TreeStar, v10).

Antibodies to the following proteins were purchased from the indicated vendors: mouse IgG1 Kappa (clone MOPC21) Sigma-Aldrich (St. Louis, MO, USA). CD137L (clone 5F4) Biolegend (San Diego, CA, USA). CD3 (clone OKT3), CD40 (clone 5C3) and PD-L1 (clone M1H1) Affymetrix eBioscience (San Diego, CA, USA). CD80 (clone 2D10), CD86 (clone IT2.2) and CD70 (clone 113-16) Biolegend. Phospho-Akt (Ser473) (clone D9E), Pan-Akt (clone 40D4), Phospho-S6 Ribosomal Protein (Ser235/236), S6 Ribosomal Protein (clone 54D2), Phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204), p44/42 MAPK (Erk1/2, clone L34F12), phospho-AMPKα (Thr172, clone 40H9), AMPKα (clone F6), phospho-GSK-3β (Ser9, clone D85E12), GSK-3β (clone 3D10), rabbit IgG-HRP, mouse IgG-HRP, beta-actin (clone 13E5), and PathScan® Intracellular Signaling Array Kit from Cell Signaling Technology (Danvers, MA, USA). GAPDH (clone 6C5) Abcam (Cambridge, UK). LY294002 and Rapamycin from Cell Signaling Technology. DMSO, 2-DG, C75, and TOFA from Sigma-Aldrich.

For experiments with AMG 330, cells were incubated at 37 °C (in 5% CO₂ and air) in 96-well round bottom plates (BD Falcon) at 5–10 × 10³ cells/well in culture medium containing various concentrations of the BiTE antibody construct (kindly provided by Amgen, Amgen Research GmbH, Munich, Germany) as well as T-cells at different E/T cell ratios.^{8 (link)} In some experiments, blocking PD-L1 (clone 29E.2A3),^{14 (link)} PD-L2 (clone MIH18)^{15 (link)} or CD86 (clone IT2.2)^{16 (link)} antibodies, or an activating CD28 (clone CD28.2; all from BioLegend, San Diego, CA, USA) antibody were added at 1 μg/ml. After 48 h, cell numbers and drug-induced cytotoxicity, using 4′,6-diamidino-2-phenylindole (DAPI) to detect nonviable cells, were determined using a LSRII cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo (Tree Star, Ashland, OR, USA). Leukemic cells were identified by forward/side-scatter properties and negativity for CellVue Burgundy dye.^{8 (link)} For experiments with other cytotoxic agents, tumor cell lines were incubated in medium containing various concentrations of cytarabine or mitoxantrone (Sigma-Aldrich, St Louis, MO, USA) for 72 h, after which cell numbers and drug-induced cytotoxicity were quantified by flow cytometry.