We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141+ DC, CD1c+ DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c+ DC and CD141+ DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c+ DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141+ DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c+ DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8+ T cells, CD141+ DC are superior in their capacity to cross-present protein Ag to CD8+ T cells following activation with polyinosinic-polycytidylic acid. CD141+ DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141+ and CD1c+ DC and evaluate adjuvants and DC-targeting strategies in vivo.

+subsets that display unique gene expression profiles, suggesting specialized functions. CD1c+ DCs express TLR4 while CD141 + DCs do not, thus predicting that these two subsets have differential capacities to respond to Escherichia coli. We isolated highly purified CD1c + and CD141 + DCs and compared them to in vitro generated monocyte-derived DCs (MoDCs) following stimulation with whole E. coli. As expected, MoDCs produced high levels of the proinflammatory cytokines TNF, IL-6, and IL-12, were potent inducers of Th1 responses, and processed E. coli-derived Ag. In contrast, CD1c + DCs produced only low levels of TNF, IL-6, and IL-12 and instead produced high levels of the anti-inflammatory cytokine IL-10 and regulatory molecules IDO and soluble CD25. Moreover, E. coli-activated CD1c + DCs suppressed T-cell proliferation in an IL-10-dependent manner. Contrary to their mouse CD8 + DC counterparts, human CD141 + DCs did not phagocytose or process E. coli-derived Ag and failed to secrete cytokines in response to E. coli. These data demonstrate substantial differences in the nature of the response of human blood DC subsets to E. coli. Keywords: E. coli r Human dendritic cells r IL-10 See accompanying Commentary by Qian and CaoSupporting Information available online IntroductionDCs are potent antigen-presenting cells that play a fundamental role in the induction and regulation of innate and adaptiveCorrespondence: Dr. Kristen J. Radford e-mail: kradford@mmri.mater.org.au immune responses against microbial pathogens. DCs detect microbial products using a sophisticated repertoire of PRR that include the toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors, retinoic acid * These authors contributed equally to this work.C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2012. 42: 1512-1522 Immunity to infection 1513 inducible gene 1 (RIG-I)-like receptors and C-type lectins. The nature of the immune response to a given pathogen is tightly regulated by the DC network, which consists of multiple subsets that are equipped with unique PRRs and are endowed with specialized functions. DCs in humans and mice can be categorized into three broad subtypes. These three subtypes are: (i) inflammatory DCs that are mobilized rapidly from monocytes in response to infection and inflammation (monocyte-derived DCs; MoDCs), (ii) migratory DCs that reside in the peripheral tissues and migrate to lymphoid tissues after encounter with Ag, and (iii) the blood and lymphoid tissue resident DCs that are comprised of plasmacytoid and myeloid DCs [1]. Further complexity arises within the steadystate myeloid DC subsets, which in humans are defined as lineage (CD3,14,15,19,20,56 ResultsMoDCs are the main DC subset responsive to E. coli CD1c + and CD141 + DCs were enriched from healthy donor leukapheresis products by immuno-magnetic selection, and flow sorted to high purity (>99%) using our established human DC isolation protocols [8,19]. Autologous MoDCs were differentiated from...

Cross-presentation is the mechanism by which exogenous Ag is processed for recognition by CD8 + T cells. Murine CD8α + DCs are specialized at cross-presenting soluble and cellular Ag, but in humans this process is poorly characterized. In this study, we examined uptake and cross-presentation of soluble and cellular Ag by human blood CD141 + DCs, the human equivalent of mouse CD8α + DCs, and compared them with human monocyte-derived DCs (MoDCs) and blood CD1c + DC subsets. MoDCs were superior in their capacity to internalize and cross-present soluble protein whereas CD141 + DCs were more efficient at ingesting and cross-presenting cellular Ag. Whilst cross-presentation by CD1c + DCs and CD141 + DCs was dependent on the proteasome, and hence cytosolic translocation, cross-presentation by MoDCs was not. Inhibition of endosomal acidification enhanced cross-presentation by CD1c + DCs and MoDCs but not by CD141 + DCs. These data demonstrate that CD1c + DCs, CD141 + DCs, and MoDCs are capable of crosspresentation; however, they do so via different mechanisms. Moreover, they demonstrate that human CD141 + DCs, like their murine CD8α + DC counterparts, are specialized at cross-presenting cellular Ag, most likely mediated by an enhanced capacity to ingest cellular Ag combined with subtle changes in lysosomal pH during Ag processing and use of the cytosolic pathway.Keywords: Antigen processing r Cross-presentation r Human dendritic cells Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionDendritic cells (DCs) are professional APCs that are uniquely able to process and present antigen (Ag) to prime naïve T-cell Correspondence: Dr. Kristen J. Radford e-mail: kristen.radford@mater.uq.edu.au responses. DCs in human and mouse can be classified into a number of subsets that vary in location, phenotype, and specialized function [1]. These include (i) inflammatory monocyte-derived DCs (MoDCs) that develop from monocytes and are rapidly * These authors contributed equally to this work.C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 330 Meng-Chieh Chiang et al. Eur. J. Immunol. 2016. 46: 329-339 recruited to sites of inflammation, (ii) plasmacytoid DCs, which are key producers of type I IFN, and (iii) "classical" or "conventional" DCs (cDCs), which can be further categorized based on location into "lymphoid-resident" and "migratory" DCs [1]. The lymphoidresident DCs capture Ag directly in situ, whereas migratory DCs reside in the peripheral organs (e.g. lung, skin, and gut) where they capture Ag then migrate to lymphoid tissues to share their Ag with other lymphoid-resident DCs, or present Ag directly to T cells. cDCs can be further segregated into two main subsets: (i) the mouse CD11b + cDC subset and human CD1c + DC equivalent; and (ii) the mouse CD8α + lymphoid-resident DC, related mouse CD103 + tissue resident cDCs, and the human equivalent CD141 + DC that can now be collectively defined by coexpression of the C-type lectin-like re...