Drug-Inducible, Dendritic Cell-Based Genetic Immunization

Abstract

Determining the mechanism of Ag loading of Langerhans cells (LC) for genetic immunization (GI) is complicated by the inability to distinguish between the response generated by direct transfection of LC from that due to exogenous uptake. To unravel this mechanism, we examined the impact of gene gun treatment on LC with respect to their activation and migration from skin, transgene expression, and ability to initiate humoral and cellular immune responses upon transfer to naive mice. To assess responses generated by direct LC transfection, an RU486-inducible expression system was used as a GI vector. In vitro skin organ cultures were developed from gene gun immunized mouse ear specimens to obtain LC. Gene gun treatment markedly augmented (3-fold) LC migration from ear skin, and these LC expressed the transgene at RNA and protein levels. Transfer of 2 × 105 migratory cells resulted in identical cellular responses to, but 10-fold lower humoral responses than, standard GI. Using an RU486-inducible system, we were able to measure responses generated by directly transfected LC. Our results indicate that direct transfection is a predominant pathway for LC Ag loading. The ability to regulate transgene expression with inducible DC-based vaccines demonstrates a new level of immunological control.

CD4 T Cell-Induced, Bid-Dependent Apoptosis of Cutaneous Dendritic Cells Regulates T Cell Expansion and Immune Responses

“…The cells that migrated from the skin specimens into the culture medium were harvested, passed through a screen to remove large skin debris, and examined for cell counts, viability by trypan blue exclusion, and phenotype. Migratory cells routinely contained Ͼ50% I-A and CD11c double-positive cells, as determined by flow cytometry (32). Additionally, the I-A-positive fraction was 70 -90% double positive for the LC markers, CD205 (DEC-205 clone NLDC145 from Cedarlane Laboratories) and Langerin/CD207 (clones 205C1 and 929F3) (AbCys) (data not shown).…”

The role of particle-mediated DNA vaccines in biodefense preparedness

“…Insofar as the efficacy of PMED DNA vaccines is influenced by the use of a direct intracellular delivery system, the epidermis as a target site also contributes to DNA vaccine efficacy because of its resident population of epidermal dendritic cells, the Langerhans cells (LC), and their ability to process foreign antigens and present them in the draining lymph node (DLN). Dendritic cells containing gold particles and expressing the gene of interest have been identified in the draining lymph nodes of animals following PMED DNA vaccination of the skin [38][39][40][41][42][43]. Moreover, in adoptive transfer experiments, the skin-derived migratory antigen-presenting cells that contribute to the induction of cellular responses were shown to be of bone marrow origin [44], consistent with their identification as epidermal LCs.…”

“…In this study, migratory skin cells isolated from donor mice immunized with an inducible promoter plasmid were collected and transferred into recipient mice, after which gene expression was induced de novo in the DCs in recipient animals by treatment with the drug RU486. The newly induced antigen expression in the transfected donor DCs in recipient animals stimulated strong cellular responses, but humoral responses that were weaker than those observed in directly DNA-vaccinated animals [42]. Because antigen expression was not induced until the migratory skin cells from donor mice were transferred into recipients, a cross-priming mechanism of antigen transfer from non-antigen-presenting cells such as keratinocytes could not have come into play in this case.…”

“…In contrast, the non-migratory cells (keratinocytes) that produce antigen at the skin target site contribute predominantly to the magnitude of the antibody response [45,46]. There is strong evidence for the role of antigen expression in both types of cells in that studies investigating the exclusive expression in either antigen-presenting or non-antigen-presenting cells demonstrate responses that are generally reduced as compared to animals receiving DNA vaccines that are not tissue specific [42,47].…”

Approaches to improved targeting of DNA vaccines

“…DNA vaccine delivered directly and selectively to defined types of DCs was identified as a key mechanism for efficient antigen presentation and induction of CTL responses. [2][3][4][5][6][7][8] Intradermal or intramuscular delivery of DNA vaccines is most commonly used. To date neither route gives unambiguously better results although the skin as a target site for DNA immunizations seems to be more promising, at least theoretically, because the dermis and the epidermis are densely populated by different subsets of DCs.…”