For in vitro stimulation assay, autologous CD8+ T cells were isolated from PBMCs of CMV-seropositive donors with magnetic beads following the manufacturer’s protocol (Miltenyi Biotec). SmyleDCs or SmartDCs alone or peptide loaded DCs were co-cultured in a 24-well-plate with 3 × 106 T cells/well at ratio of 1:100 (APC: T-cell) in serum-free Cellgro

medium. 1 × 106 autologous feeder cells (CD8−) were gamma-irradiated with 30 Gy and added to the culture. After 3 days, the cells were split and replenished on alternate days with Cellgro medium containing IL-2 (10 IU/ml) (Novartis Pharma GmbH, Germany) and kept at 37 °C. After 7 days of initiation of culture, stimulated T cells were harvested and washed Fludarabine chemical structure http://www.selleckchem.com/products/gw3965.html twice with PBS and analyzed for their pp65-reactivity with tetramer staining. PE-conjugated tetramers (HLA-A*0201-NLVPMVATV, pp65 amino acids (aa) 495–503; HLA-B*0702-TPRVTGGGAM, pp65 aa 417–426; Beckman coulter), ECD-conjugated anti-human CD3 and PCy7-conjugated anti-human CD8 were used. In addition, the expanded pp65-specific T cells were also analyzed for T cell subpopulations using FITC-conjugated anti-CD45RA, PCy5-conjugated anti-CD62L (Beckman Coulter). The cells were acquired and

analyzed by flow cytometry using a FC500 apparatus (Beckman Coulter). In addition, T cells stimulated with iDCs co-expressing full-length pp65 (transduced with ID-LV-pp65) were analyzed for IFN-γ production by Enzyme Linked Immuno Spot Technique

(ELISPOT). Stimulated T cells were seeded at a density of 20,000 cells per well in 96-well ELISPOT plate coated with anti-human IFN-γ (Mabtech AB, Germany). The cells were incubated overnight in the presence of 10 μg/ml of pp65 overlapping peptide pool. After incubation, cells were washed and plates were further incubated with biotin-conjugated anti-human IFN-γ Dipeptidyl peptidase antibody followed by alkaline phosphatase-conjugated streptavidin. Plates were developed using NBT/BCIP liquid substrate (Sigma) and analyzed with an ELISPOT reader (AELVIS GmbH, Germany). Handling of mice for in vivo studies was previously described [10]. Briefly, NOD.Cg-Rag1tm1MomIl2rgtm1Wjl (NOD/Rag1(−/−)/IL-2rγ(−/−), NRG) mice were bred and maintained under pathogen free condition in an IVC system (BioZone, United Kingdom). All procedures involving mice were reviewed and approved by the Lower Saxony and followed the guidelines provided by the Animal Facility at Hannover Medical School. For studies of human T cells engraftment and antigen specific T cell expansion, mice were primed with 5 × 105 SmyleDCs or SmartDCs (in 100 μL of PBS) co-transduced with ID-LV-pp65, by subcutaneous injection at the hind flank using a 27-gauge needle. The iDCs were allowed to self-differentiate in vivo for 7 days. 5 × 106 cells freshly isolated autologous CD8+ T cells (in 100 μL of PBS) were then intravenously infused through the lateral tail vein.