555. Langerhans Cells Modified by Gene Transfer of Full Length CMV pp65 Are More Potent Inducers of Cytolytic T Lymphocytes in a CD4-Dependent Manner Than Either CMV p495 Gene-Modified or p495 Peptide-Pulsed Langerhans Cells

Abstract

The use of dendritic cells (DCs) for immunotherapy is often limited to defined Ags with known MHC restrictions. Investigators are pursuing a number of approaches to introduce whole Ag, however. These would increase the range of MHC alleles presenting immunogenic peptide Ags by allowing DCs to process and present epitopes tailored to their own MHC alleles. We addressed this issue by generating recall immune responses in vitro against human cytomegalovirus (HCMV) by seropositive donors. CD34+ HPCs were transduced twice on RetroNectin-coated plates by GalV-pseudotyped oncoretroviral vectors encoding either the full-length HCMV pp65 or the HLA-A*0201-restricted CMV pp65495-503 peptide (p495). Differentiation and maturation of CD34+ HPC-derived Langerhans-type dendritic cells (LCs) followed over the next 14 days. These pp65-transduced and p495-transduced LCs were compared with nontransduced but p495-pulsed LCs, for the generation of CMV-specific CTLs after two rounds of autologous T cell stimulation without additional cytokines. Stimulation by pp65-transduced LCs, p495-transduced LCs, and p495-pulsed LCs respectively increased p495/HLA-A*0201 tetramer positive T cells by 51.3±24.1, 33.8±20.9, and 10.4±10.7-fold (n=4 different donors, P<0.05). CMV specific CD8+ CTLs induced by pp65-transduced LCs, p495-transduced LCs, and p495-pulsed LCs achieved respective frequencies of 714.4±280.8, 582.6±218.6, and 231.0±102.5 spot-forming cells (SFC) per 105 input cells (n=4, P<0.01) against p495-pulsed A*0201 targets in ELISpot assays for IFN-g secretion. pp65-transduced LCs that could present both class I and II MHC-restricted viral epitopes also expanded IFN-g -secreting CD4+ T cells, whereas p495-transduced LCs did not. Stimulation by p495-pulsed-LCs was even associated with a decline in CD4+ T cell numbers. pp65-transduced LCs also stimulated greater lytic activity than did either p495-transduced LCs or p495-pulsed LCs, based on 51Cr release from p495-pulsed A*0201 targets (n=3, P<0.05). CD4+ T cells boosted the overall CTL response, insofar as CD4+ T cell depletion before each round of stimulation decreased the number of effector CD8+ T cells by 70% at d7 and 89% at d14. p495-transduced LCs and p495-pulsed LCs gave similar results. Finally, CMV tetramer positive cells express low levels of CD62L, CD45RA, CD27, and CCR7, consistent with an effector memory phenotype. DCs transduced with a retroviral vector encoding the full-length pp65 protein can thus expand potent CTL presumably directed against multiple epitopes in a CD4+ helper T cell-dependent manner. DCs transduced with retroviral vectors expressing full-length antigens in serum free media should therefore provide effective cell-based vaccines against viral pathogens and cancer.