B, evaluation of the amount of IFN- secreting tumor-reactive cells in the spleens of mice following DC-lysate vaccinations. of choice for preparing whole tumor lysate vaccine. We then designed and conducted a pilot clinical study to test the biological effects of HOCl-oxidized autologous whole tumor lysate-pulsed DCs (i.e. OCDC vaccine) in five subjects with recurrent ovarian cancer. We reported here the promising immunological and clinical results. Materials and Methods Human DC generation and maturation DC generation was conducted as previously described (19). Normal donors monocytes were cultured in CellGenix DC media (CellGenix, Freiburg, Germany), 2% human AB serum (Valley Biomedical Inc., Winchester, VA), 2 mM L-glutamine, 100units/ml penicillin, 100 g/ml streptomycin (Cellgro, Manassas, VA), 500 IU/ml human granulocyte-macrophage colony stimulating factor (GM-CSF) and 250 IU/ml interleukin (IL)-4 (PeproTech, Rocky Hill, NJ) after obtaining a written informed consent to a tissue and blood procurement study approved by UPenns IRB. Subjects elutriated monocytes were cultured with clinical grade GM-CSF (Leukine?, Bayer Healthcare Pharmaceuticals, Wayne, NJ) and animal-free research grade IL-4 (R&D Systems, Inc., Minneapolis, MN). After 4 days, CD11c, CD14 and HLA-DR were determined on DCs and were >70% pure. After lysate-loading, DCs were matured with lipopolysaccharides (LPS) [60 EU/ml; O:113; gift from Dr. Suffredini at National Institute of Health] and IFN- (2000 IU/ml; Intermune, San Francisco, CA). Human tumor lysate preparations and DC uptake Methods for preparing autologous and allogeneic tumor lysates by by UVB-irradiation (UVB-L) and freeze-thaw cycles (FTL) have been previously described (20). The detailed methodology of preparing whole tumor lysates by HOCl-oxidation was previously published (13) and is described in supplementary materials and methods. After HOCl-oxidation or UVB-irradiation, tumor cells were subjected to 6 freeze-thaw cycles. For uptake experiment, HOCl-oxidized and UVB-irradiated SKOV-3 cells were not frozen and thawed before coculturing with DCs. Human DCs uptake of tumor cells was performed as previously described (13). SKOV-3 cells labeled with PKH26 (Sigma-Aldrich Corp., CA) were cocultured with normal donor DCs (1:1 ratio) for 4 hours at 37C for active uptake, or 4C for passive transfer to DCs. Percentage uptake by DCs was determined by gating on HLA-DR+PKH26+ cells in flow cytometry analysis performed on BD Canto (Becton Dickinson, Franklin Lakes, NJ). Data was analyzed with Pro CellQuest software. Human DC and T-cell cytokine and chemokine analysis Normal donor DCs were pulsed with HOCl-L of 3 ovarian tumor linesCSKOV-3, OVCAR5 and A1847 (ratio of 1 1:1:1 in the lysate mixture), whilst subject DCs were pulsed with HOCl-oxidized autologous tumor lysate in the presence of GM-CSF (500 IU/ml). After 8 hours stimulation with LPS and IFN-, supernatants were analysis. For T-cells, subject PBMCs were cocultured with OCDC, unpulsed autologous DCs or media only and the supernatant analyzed Erythromycin Cyclocarbonate after 24 hours. Mouse bone marrow-derived DC preparation Bone marrow cells were isolated from hind leg femurs and tibias of mice, and plated at 1106 cells/ml in complete IMDM media containing 10% FBS, 50 M 2-mercaptoethanol, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin and 1000 IU/ml GM-CSF. On day 3, floating cells representing granulocytes were removed and replenished with fresh complete IMDM media and 1000 IU/ml Rabbit Polyclonal to MED27 GM-CSF. Day 5, IL-4 was added at 100 IU/ml. Day 6, ID8-ova tumor lysate prepared by HOCl-oxidation, UVB-irradiation or 6 cycles of freeze-thawed were cocultured with DCs at 1:1 ratio for 20 to 24 hours. Then DCs were stimulated with LPS (120 Erythromycin Cyclocarbonate EU/ml) and IFN- (4000 IU/ml) for 16 hours and used. Vaccination of mice and Erythromycin Cyclocarbonate Erythromycin Cyclocarbonate tumor challenge Mouse ID8-ova tumor line was generated to express surface SIINFEKL-H-2Kb complex by lentiviral transduction. Ovalbumin (OVA) plasmid vector pAC-Neo-OVA (21) [plasmid 22533; deposited by Dr. Michael Bevan] was obtained from Addgene (Cambridge, MA), and the full length OVA amplified with polymerase chain reaction and inserted into the pELNS lentiviral vector (22) [kind gift of Dr. Carl June from University of Pennsylvania]. MOI 5 was used, and cell surface SIINFEKL-H-2Kb complexes on transduced ID8 cells were detected by anti-mouse H-2Kb bound to SIINFEKL (Biolegend) after 24 hours of IFN- treatment. Positive cells were sorted by flow cytometery, and.