Vaccines delivered to the skin by microneedles – with and without adjuvants – have increased immunogenicity with lower doses than standard vaccine delivery techniques such as intramuscular (i.m.) or intradermal (i.d.) injection. However, the mechanisms behind this skin-mediated ‘adjuvant’ effect are not clear. Here, we show that the dynamic application of a microprojection array (the Nanopatch) to skin generates localized transient stresses invoking cell death around each projection. Nanopatch application caused significantly higher levels (~65-fold) of cell death in murine ear skin than i.d. injection using a hypodermic needle. Measured skin cell death is associated with modeled stresses ~1–10 MPa. Nanopatch-immunized groups also yielded consistently higher anti-IgG endpoint titers (up to 50-fold higher) than i.d. groups after delivery of a split virion influenza vaccine. Importantly, co-localization of cell death with nearby live skin cells and delivered antigen was necessary for immunogenicity enhancement. These results suggest a correlation between cell death caused by the Nanopatch with increased immunogenicity. We propose that the localized cell death serves as a ‘physical immune enhancer’ for the adjacent viable skin cells, which also receive antigen from the projections. This natural immune enhancer effect has the potential to mitigate or replace chemical-based adjuvants in vaccines.

ABSTRACTmolecules is ideally suited for the capture of lipid antigens 3 . CD1 clefts derive from deep invaginations into the CD1 core structure and form two or four pockets 5,6,7,8,9. In general, the pockets surround a large portion of the lipidic antigens so that their hydrocarbon moieties are sequestered from solvent and the hydrophilic headgroups protrude for T cell contact. However, each of the four types of human CD1 proteins has a cavity with unique architecture, which endows each CD1 isoform with the ability to present specific types of lipids. Whereas MHC proteins allow broad access to peptides that span the entire platform, CD1 proteins possess an

Human immune cell subsets develop in immunodeficient mice following reconstitution with human CD34+ hematopoietic stem cells. These “humanized” mice are useful models to study human immunology and human-tropic infections, autoimmunity, and cancer. However, some human immune cell subsets are unable to fully develop or acquire full functional capacity due to a lack of cross-reactivity of many growth factors and cytokines between species. Conventional dendritic cells (cDCs) in mice are categorized into cDC1, which mediate T helper (Th)1 and CD8+ T cell responses, and cDC2, which mediate Th2 and Th17 responses. The likely human equivalents are CD141+ DC and CD1c+ DC subsets for mouse cDC1 and cDC2, respectively, but the extent of any interspecies differences is poorly characterized. Here, we exploit the fact that human CD141+ DC and CD1c+ DC develop in humanized mice, to further explore their equivalency in vivo. Global transcriptome analysis of CD141+ DC and CD1c+ DC isolated from humanized mice demonstrated that they closely resemble those in human blood. Activation of DC subsets in vivo, with the TLR3 ligand poly I:C, and the TLR7/8 ligand R848 revealed that a core panel of genes consistent with DC maturation status were upregulated by both subsets. R848 specifically upregulated genes associated with Th17 responses by CD1c+ DC, while poly I:C upregulated IFN-λ genes specifically by CD141+ DC. MYCL expression, known to be essential for CD8+ T cell priming by mouse DC, was specifically induced in CD141+ DC after activation. Concomitantly, CD141+ DC were superior to CD1c+ DC in their ability to prime naïve antigen-specific CD8+ T cells. Thus, CD141+ DC and CD1c+ DC share a similar activation profiles in vivo but also have induce unique signatures that support specialized roles in CD8+ T cell priming and Th17 responses, respectively. In combination, these data demonstrate that humanized mice provide an attractive and tractable model to study human DC in vitro and in vivo.