The skin contains readily accessible dendritic cells (DCs) with potent antigen presentation function and functional plasticity enabling the integration of antigen specificity with environmentally responsive immune control. Recent studies challenge the established paradigm of cutaneous immune function by suggesting that lymph node-resident DCs, rather than skin-derived DCs (sDCs), are responsible for eliciting T cell immunity against cutaneous pathogens including viral vectors. We show that cutaneous delivery of lentivirus results in direct transfection of sDCs and potent and prolonged antigen presentation. Further, sDCs are the predominant antigen-presenting cells for the induction of potent and durable CD8(+) T cell immunity. These results support the classical paradigm of cutaneous immune function and suggest that antigen presentation by sDCs contributes to the high potency of lentivector-mediated genetic immunization.

Purpose Design and evaluate a new micro-machining based approach for fabricating dissolvable microneedle arrays (MNAs) with diverse geometries and from different materials for dry delivery to skin microenvironments. The aims are to describe the new fabrication method, to evaluate geometric and material capability as well as reproducibility of the method, and to demonstrate the effectiveness of fabricated MNAs in delivering bioactive molecules. Methods Precise master molds were created using micromilling. Micromolding was used to create elastomer production molds from master molds. The dissolvable MNAs were then fabricated using the spin-casting method. Fabricated MNAs with different geometries were evaluated for reproducibility. MNAs from different materials were fabricated to show material capability. MNAs with embedded bioactive components were tested for functionality on human and mice skin. Results MNAs with different geometries and from carboxymethyl cellulose, polyvinyl pyrrolidone and maltodextrin were created reproducibly using our method. MNAs successfully pierce the skin, precisely deliver their bioactive cargo to skin and induce specific immunity in mice. Conclusions We demonstrated that the new fabrication approach enables creating dissolvable MNAs with diverse geometries and from different materials reproducibly. We also demonstrated the application of MNAs for precise and specific delivery of biomolecules to skin microenvironments in vitro and in vivo.

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Dendritic cells (DCs) play a key role in the induction and control of immunity. Genetic engineering of DCs is a promising approach for the development of a broad range of immunomodulatory strategies, for purposes ranging from genetic immunization to tolerance induction. The development of DC-based immunotherapies is limited by the inability to efficiently transfect DCs using naked DNA. Here we demonstrate that after plasmid DNA delivery, the transgene expression level controlled by the human immediate-early cytomegalovirus promoter (hIE-CMVp) is higher in mature DCs than in immature DCs and is further increased after terminal differentiation of DCs by agonist anti-CD40 monoclonal antibody ( IntroductionDendritic cells (DCs) have a key role in initiating and controlling immune responses. In addition to being the most potent antigenpresenting cells (APCs), DCs determine the nature and magnitude of immune responses and provide a link between innate and acquired immunity. 1,2 Genetic engineering of DCs offers potential for the development of immune-regulatory strategies for purposes ranging from immunization to tolerance induction. The capacity of DCs engineered to express transgenic antigens, cytokines, or T-cell costimulatory molecules to induce or bias T helpers 1 and 2 (T H 1/T H 2)-skewed immune responses, or to promote tolerance, is the subject of current investigation. [3][4][5] DNA-based immunization has potential advantages over proteinbased vaccines. [5][6][7][8] These include the simultaneous delivery of transgenic antigens and immunoregulatory genes to DCs. Despite advances in the understanding of DC biology, the development of genetic immunization strategies using DCs transfected with plasmid DNA has been limited by low transfection efficiencies. 9 Currently, the most efficient method for DC transduction is infection by recombinant adenovirus (rAd) at a relatively high multiplicity of infection (MOI). However, the applicability of viral vectors is limited by their potential to interfere with DC function and by the coadministration of viral antigens that elicit strong Band T-cell responses in the host that may limit the readministration of viral vectors. [10][11][12] Indirect evidence suggests that the signaling pathways involved in maturation/terminal differentiation of DCs may enhance the level of transgene expression driven by the human immediate-early cytomegalovirus promoter (hIE-CMVp). We and others have shown that rAd induces the maturation of mouse bone marrow (BM)-derived DCs (BMDCs) mainly by nuclear translocation of nuclear factor-B (NF-B). 11 NF-B is one of the factors necessary to initiate mRNA transcription by hIE-CMVp, and nuclear translocation of NF-B occurs immediately after CMV infection. 13,14 Furthermore, low doses of rAds, engineered to be internalized by cell surface CD40, resulted in enhanced hIE-CMVpdriven transgene expression by DCs. 15 CD40 ligand (L) (CD154) is expressed transiently on the surfaces of activated T cells and mediates the terminal differentiation of DCs. This phe...

In vivo dendritic cells (DC) targeting is an attractive approach with potential advantages in vaccine efficacy, cost, and availability. Identification of molecular adjuvants to in vivo "modulate " DC to coordinately render improved Th1 and CD8 T cell immunity, and attenuated deleterious Treg effects, is a critical challenge. Here, we report that in vivo genetic targeting of the active transcription factor XBP1s to DC (XBP1s/DC) potentiated vaccine-induced prophylactic and therapeutic antitumor immunity in multiple tumor models. This immunization strategy is based on a genetic vaccine encoding both cytomegalovirus (CMV)-driven vaccine Aghsp70 and DC-specific CD11c-driven XBP1s. The novel targeted vaccine induced durable Th1 and CD8 T cell responses to poorly immunogenic self/tumor antigen (Ag) and attenuated tumor-associated Treg suppressive function. Bone marrow (BM)-derived DC genetically modified to simultaneously overexpress XBP1s and express Aghsp70 upregulated CD40, CD70, CD86, interleukin (IL)-15, IL-15Rα, and CCR7 expression, and increased IL-6, IL-12, and tumor necrosis factor (TNF)-α production in vitro. XBP1s/DC elevated functional DEC205(+)CD8α(+)DC in the draining lymph nodes (DLN). The data suggest a novel role for XBP1s in modulating DC to potentiate tumor vaccine efficacy via overcoming two major obstacles to tumor vaccines (i.e., T cell hyporesponsiveness against poorly immunologic self/tumor Ag and tumor-associated Treg-mediated suppression) and improving DEC205(+)CD8α(+)DC.