Using pp65 soluble peptides loaded externally on iDCs, both SmyleDCs and SmartDCs potently stimulated T cells to proliferate and upregulated the production of several inflammatory cytokines (IFN-γ, IL-3, GM-CSF, TNF-α, IL-8, IL-5), resulting into “licensed antigen presentation” of different pp65 antigenic determinants in vitro (20–30% of the cells stimulated in vitro for 7 days were reactive against pp65 epitopes). The pp65-reactive T cells stimulated in vitro with peptides were in the majority characterized as T central memory (TCM, 43–58%) and T effector memory (TEM, 40–47%) phenotype. Interestingly, SmyleDCs bypassed the requirement of additional in vitro maturation with recombinant

cytokines for optimal antigen-specific T cell stimulation ( Fig. S7), indicating that SmyleDCs are more endogenously activated than SmartDCs. When the pp65 antigen was provided internally, in the form of a full-length pp65 antigen expressed stably for 3 weeks mTOR inhibitor by a co-transduced ID-LV, we observed potent stimulation of pp65-reactive multivalent T cells in vitro ( Fig. 5 and Fig. 6). Notably, in this setting the majority of the T cells displayed a TEM phenotype (although 10–40% of TCM were also observed); possibly indicating that pp65 internal processing by the iDCs per se provided higher immune stimulation. Moreover, these iDCs were endowed with potent functional activities in vivo, as

they were able to directly stimulate the generation of effector CD8+ T cell responses in NRG mice reconstituted with human lymphocytes. Since NRG mice lack a functional lymphatic system and lymph nodes to where iDCs could migrate to, it is therefore likely that find more iDCs

stimulated T cells directly on the immunization sites. too Based on these results, the use of SmyleDCs or SmartDCs co-expressing pp65 for the development of prophylactic vaccines to boost the immune response in lymphopenic hosts at high risk of HCMV infection should be considered. It has been demonstrated that the transfer of adoptive immunity against HCMV after HSCT depends on the specificity and memory phenotype of specific T cells in the donor [44]. Thus, a potential population target for vaccination in order to avoid HCMV recurrent reactivations would be immunosuppressed HCMV seropositive recipients of grafts from seropositive or seronegative donors. After transplantation, recipients would be vaccinated with iDCs produced from donor’s monocytes in order to minimize graft-versus-host disease. Regarding the choice between the two types of iDCs for an antiviral vaccine, it is tempting to speculate that SmyleDCs would be the first choice, based on several proposed superior attributes conferred to DCs produced in the presence of IFN-α instead of IL-4 which led to a recent clinical trials using ex vivo generated DCs as vaccines for chronically infected HIV-1 patients (http://clinicaltrials.gov/ct2/show/NCT00796770) [21].