In order to assess pp65-reactivity, human CD3+CD8+ T cells detectable in the PBL were analyzed by tetramer staining. The frequencies of pp65-specific circulating CD3+CD8+ T cells were approximately eight-fold higher in mice preconditioned with SmyleDC/pp65 or SmartDC/pp65 as compared to control mice (Fig. 8b). Human CD3+CD8+ T cells were isolated from the spleen by FACS sorting and used in IFN-γ ELISPOT

assay. The human T cells were pulsed with pp65 peptide pool or with a mixture of recall antigenic peptides. Using this approach, we were able to confirm the engraftment and expansion of functional human CD3+CD8+ T cells in the spleen (Fig. 8c). Mice injected with SmyleDC/pp65 showed higher frequencies of CD8+ T

cells producing IFN-γ than mice pre-conditioned with SmartDC/pp65. XL184 solubility dmso Cytomegalovirus is a relevant issue AZD6244 in stem cell transplantation, particularly because immune suppressed transplanted patients do not respond well to vaccinations, underscoring the need for novel cell-based therapies. With respect to existing DC vaccination therapies, they are very cost intensive, poorly viable in vivo, scarcely bio-distribute to lymphatic tissues and are far away from a standardized cellular product for larger clinical trials [29]. We have previously demonstrated in homologous and humanized mouse models that SmartDCs generated with IC-LV are significantly more viable in vivo (several weeks) than conventional DCs (1–2 days) and immunization with SmartDCs result into massive recruitment and expansion of antigen-reactive T cells in lymph nodes or in the vaccination site [5], [6] and [10].

Spilucel-T, the only FDA approved and marketed cell therapy product, is not a highly stable product and therefore has to be prepared fresh for three rounds of infusion. We have recently demonstrated the feasibility of up-scaling SmartDC production using GMP-compatible methods, which was achieved in 28 h of ex vivo cell manipulation and the cell product could be conveniently cryopreserved without precluding its potency [7]. Although novel in the field of immunotherapy, lentiviral vectors have now lined up for several clinical Calpain trials of human gene therapy (for hematopoieitic, metabolic and neurologic disorders), and large scale GMP production is developing in Europe and in the United States [30] and [31]. Thus, innovative genetically modified iDCs may become a practical and valuable option for immunotherapy of immune-compromised transplanted patients at risk of CMV infection, since besides its reduced time of ex vivo manipulation, high viability in vivo and antigenic properties, its 2–3 weeks production of cytokine stimuli may improve the immunization milieu and accelerate the homeostatic immune reconstitution of human T cells.