All corresponding isotypes were purchased from BD Bioscience (Heidelberg, Germany). For intracellular staining, the BD Cytofix/Cytoperm Kit (BD Bioscience) was used. For stimulation, we used anti-CD3

mAb from Beckman Coulter, rh-IL-2 from Stratmann (Hamburg, Germany), rh-GM-CSF and rh-IL-4 from R&D Systems, rh-IL-10, rh-IL-15, and rh-TGF-β from Peprotech-Tebu (Frankfurt, Germany). For cell PLX-4720 research buy culture assays, complete medium (Rxx10) consisting of RPMI 1640 supplemented with 10% v/v ΔFCS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and L-glutamine (2 mmol/L) was used. All cells were cultured in this medium and incubated in a humidified atmosphere at 37°C with 5% CO2. With the permission and supervision of the Local Ethical Committee, human peripheral blood mononuclear cells (PBMCs) were purified from heparinized venous whole blood from healthy donors by density gradient separation using Biocoll according to manufacturer’s guidelines (Biochrom AG). NK cells were purified from PBMCs using NK Cell Isolation Kit from Miltenyi Biotec (Bergisch Gladbach, Germany)

according BIBW2992 mouse to the manufacturer’s instructions to deplete non-NK cells. The purity of NK cells was confirmed by flow cytometry, and contamination with T cells and B cells was always below 1%. CD4+CD25− T cells were isolated from PBMCs using Regulatory T Cell Separation Kit from Miltenyi Biotec according to the manufacturer’s instructions. CD4+CD25− T cells were used for generation of autologous responder T cells. CD4+CD25+ nTreg Tenofovir cells were separated from PBMCs using the CD4+CD25+ regulatory T cell Isolation Kit from Miltenyi Biotec according to manufacturer’s instruction. To this end, lymphocytes were depleted of non-CD4+ T cells and positively selected for CD4+CD25+ T cells. Monocytes within PBMCs were separated from lymphocytes by plastic adherence. Monocytes were differentiated into immature DCs (iDCs) within 7 days in the presence of IL-4 and GM-CSF (500 IU/mL each with

medium change on days 3 and 5). PCI-13 cells, a HLA-A2+ human squamous cell carcinoma of the head and neck (HNSCC), were used to generate tumor iTreg cells. PCI-13 was a kind gift from the Whiteside Laboratory at the University of Pittsburgh Cancer Institute 43. Colo699 (human lung adenocarcinoma cell line) cells were used as target cells in cytotoxicity assays. Transduction of cells with an adenovirus encoding the human NKG2D-ligand MICA (Ad-MICA) was performed earlier in our laboratory 44. The human erythroleukemia line K562 was obtained from DSMZ (Braunschweig). All tumor cell lines were routinely tested and confirmed to be mycoplasma free. CD4+CD25− T cells were co-cultured with autologous iDCs and mitomycin C treated (0.5  mg/mL, for 30 min) PCI-13 cells at a ratio of 10:1:1 with 106 T cells/mL in Rxx10 medium for 10 days.