Adenoviral (AdV) vectors can be used to transduce a wide range of human cells and tissues. However, pre-existing immunity to AdV, and enhancement of this immunity after repeated administration, limits their clinical application. This may be especially relevant when vectors are loaded into APCs. Helper-dependent AdV (Hd-AdV), in which viral coding regions are replaced by human stuffer DNA, offers a new approach for limiting antiviral responses. To evaluate their immunogenicity, human dendritic cells (DCs) were infected with either an Hd-AdV or a conventional replication-deficient E1-deleted AdV (E1-AdV) and were evaluated for their capacity to stimulate antiviral T cell responses. Hd-AdV proved to be 50- to 275-fold more effective than E1-AdV at expressing the lacZ transgene in human DCs. PCR demonstrated similar transduction efficiencies, but RT-PCR revealed much higher expression of transgene mRNA after transduction with Hd-AdV. Functionally, DCs transduced with Hd-AdV stimulated the proliferation of autologous T cells to the same level as DCs transduced with E1-AdV. Identical viral-specific T cell responder frequencies were observed and T cells stimulated with either type of AdV-transduced DC lysed viral-infected target cells. Disrupting transcription of vector-based genes had no effect on T cell activation, suggesting that responses against both vectors were directed against preformed components of the viral capsid. We conclude that Hd-AdV vectors can be used to obtain higher transgene expression in human DCs but that they still evoke a vector-related immune response similar to that generated by E1-AdV.

One of the limitations of adenovirus vectors is the lack of machinery necessary for their integration into host chromosomes, resulting in short-term gene expression in dividing cells. We analyzed frequencies of integration and persistence of gene expression from integrated adenovirus vectors. Both E1-substituted and helper-dependent adenovirus vectors achieved similar integration efficiencies of ∼10−3 to 10−5 per cell, with the helper-dependent vector showing slightly higher efficiencies. In stable cell pools, gene expression of the integrated vector persisted for at least 50 cell divisions without selection. However, some stable cell clones showed changes in gene expression, which were accompanied by structural changes in the integrated vector DNA.

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Dendritic cells (DCs) are rare antigenpresenting cells that play a central role in stimulating immune responses. The combination of recombinant granulocyte macrophage-colony-stimulating factor (rGM-CSF) and recombinant interleukin-4 (rIL-4) provides an important stimulus for generating DCs from murine bone marrow precursors in vitro. Using miniature osmotic pumps, we now demonstrate that continuous infusion of these cytokines for 7 days had a similar effect in vivo, increasing the number and function of splenic DCs. Administration of rGM-CSF/rIL-4 (10 g/d each) increased the concentration of CD11 ؉ DCs by 2.7-fold and the absolute number of splenic DCs by an average of 5.7-fold. DC number also increased in peripheral blood and lymph nodes. The resultant DCs exhibited a different phenotype and function than those in control mice or mice treated with rGM-CSF alone. rGM-CSF/IL-4 increased both the myeloid (CD11c ؉ /CD11b ؉ ) and the lymphoid (CD11c ؉ /CD8␣ ؉ ) subpopulations, whereas rGM-CSF increased only myeloid DCs. DCs were highly concentrated in the T-cell areas of white pulp after rGM-CSF/IL-4 administration, whereas they were diffusely distributed throughout white pulp, marginal zones, and red pulp in mice treated with rGM-CSF alone. rGM-CSF/rIL-4 also significantly increased the expression of major histocompatibility complex (MHC) class I and MHC class II on CD11c ؉ cells and increased their capacity to take up antigens by macropinocytosis and receptor-mediated endocytosis. Splenic DCs generated in response to rGM-CSF/rIL-4 were functionally immature in terms of allostimulatory activity, but this activity increased after short-term in vitro culture. Systemic treatment with rGM-CSF/rIL-4 enhanced the response to an adenoviral-based vaccine and led to antigen-specific retardation in the growth of established tumor. We conclude that systemic therapy with the combination of rGM-CSF/rIL-4 provides a new approach for generating DCs in vivo.

Humanized mouse models provide a unique opportunity to study human immune cells in vivo, but traditional models have been limited to the evaluation of non-specific T-cell interactions due to the absence of antigen-presenting cells. In this study, immunodeficient NOD/SCID/IL2r-γnull (NSG) mice were engrafted with human peripheral blood lymphocytes alone or in combination with donor-matched monocyte-derived dendritic cells (DC) to determine whether antigen-specific T-cell activation could be reconstituted. Over a period of 3 weeks, transferred peripheral blood lymphocytes reconstituted the spleen and peripheral blood of recipient mice with predominantly human CD45-positive lymphocytes. Animals exhibited a relatively normal CD4/CD8 ratio (average 1.63:1) as well as reconstitution of CD3/CD56 (averaging 17.8%) and CD20 subsets (averaging 4.0%). Animals reconstituted with donor-matched CD11c+ DC also demonstrated a CD11c+ population within their spleen, representing 0.27% to 0.43% of the recovered human cells with concurrent expression of HLA-DR, CD40, and CD86. When immunized with adenovirus, either as free replication-incompetent vector (AdV) or as vector-transduced DC (DC/AdV), there was activation and expansion of AdV-specific T-cells, an increase in Th1 cytokines in serum, and skewing of T-cells toward an effector/memory phenotype. T-cells recovered from animals challenged with AdV in vivo proliferated and secreted a Th1-profile of cytokines in response to DC/AdV challenge in vitro. Our results suggest that engrafting NSG mice with a combination of lymphocytes and donor-matched DC can reconstitute antigen responsiveness and allow the in vivo assessment of human immune response to viruses, vaccines, and other immune challenges.