The C-type lectin receptor CLEC9A mediates antigen uptake and (cross-)presentation by human blood BDCA3+ myeloid dendritic cells

Abstract

CLEC9A is a recently discovered C-type lectin receptor involved in sensing necrotic cells. In humans, this receptor is selectively expressed by BDCA3 ؉ myeloid dendritic cells (mDCs), which have been proposed to be the main human cross-presenting mDCs and may represent the human homologue of murine CD8 ؉ DCs. In mice, it was demonstrated that antigens delivered with antibodies to CLEC9A are presented by CD8 ؉ DCs to both CD4 ؉ and CD8 ؉ T cells and induce antitumor immunity in a melanoma model. Here we assessed the ability of CLEC9A to mediate antigen presentation by human BDCA3 ؉ mDCs, which represent < 0.05% of peripheral blood leukocytes. We demonstrate that CLEC9A is only expressed on immature BDCA3 ؉ mDCs and that cell surface expression is lost after TLR-mediated maturation. CLEC9A triggering via antibody binding rapidly induces receptor internalization but does not affect TLR-induced cytokine production or expression of costimulatory molecules. More importantly, antigens delivered via CLEC9A antibodies to BDCA3 ؉ mDCs are presented by both MHC class I (cross-presentation) and MHC class II to antigen-specific T cells. We conclude that CLEC9A is a promising target for in vivo antigen delivery in humans to increase the efficiency of vaccines against infectious or malignant diseases. (Blood. 2012; 119(10):2284-2292) IntroductionDendritic cells (DCs) are central players in the induction of adaptive immune responses. 1 They reside in peripheral tissues, where they are positioned to capture antigens. In the immature state, DCs continuously sample their environment by receptormediated endocytosis, pinocytosis, and phagocytosis. Once DCs also encounter danger signals, such as those present in pathogens, endogenous danger molecules, inflammatory cytokines, or immune complexes, they become activated and migrate to the lymph nodes and differentiate into mature DCs, which is accompanied by stabilization of peptide-MHC complexes on the cell surface, up-regulation of costimulatory molecules, and cytokine release. These alterations contribute to optimal antigen presentation to T lymphocytes. DCs have the unique capacity to process extracellular antigens for cross-presentation via MHC class I. This feature allows DCs to induce CD8 T-cell responses against dying cells, tumor cells, and viruses that do not replicate in DCs.In human peripheral blood, 2 main populations of DCs can be distinguished: CD11c-positive myeloid DCs (mDCs) and CD11c-negative plasmacytoid DCs (pDCs). 2,3 Human mDCs can be subdivided further on the basis of differential surface expression of BDCA1 (CD1c), CD16, and BDCA3 (CD141). 4,5 Because the frequency of circulating mDCs in human blood is very low (Ͻ 2% of the peripheral blood leukocytes), many studies exploit in vitro-generated monocyte-derived DCs (moDCs) as "surrogate mDCs" because of the relative ease of obtaining large quantities of these cells. DC subsets are heterogeneous in the expression of cell surface markers and pathogen-recognition receptors, cytokine production after stimulation, as w...

The equivalents of human blood and spleen dendritic cell subtypes can be generated in vitro from human CD34+ stem cells in the presence of fms-like tyrosine kinase 3 ligand and thrombopoietin

“…This result is in keeping with a recent study demonstrating that human blood BDCA3 1 DCs stimulated with Poly(I : C) produce only low levels of IL-12p70. 54 Furthermore, a recent study on in vitro derived CD141 1 CLEC9A 1 DCs differentiated from HSCs in the presence of a cocktail of cytokines including SCF, Flt3L, IL-3, IL-6, GM-SCF and IL-4 has shown a similar poor response of this population to TLR stimulation with respect to IL-12p70 production. 23 They demonstrated that a further stimulus of antigenspecific T cells was required to produce IL-12p70.…”

Fcγ receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3+ dendritic cells

“…[10][11][12] Human BDCA-3 ϩ DCs internalize dead cell material and crosspresent exogenous soluble or cell-associated proteins to CD8 ϩ T cells. [11][12][13] Recombinant soluble HCMV pp65 antigen was crosspresented with increased efficiency by BDCA-3 ϩ DCs to antigenspecific CD8 ϩ T cells. 12 For vaccination strategies, full grasp on the uptake and processing mechanisms and preferentially a further increase in CD8 ϩ T-cell stimulation potency is desired, which was the aim of our study.…”

Differential uptake and cross‐presentation of soluble and necrotic cell antigen by human DC subsets

“…In mice, uptake and cross-presentation of soluble Ag is mediated by the mannose receptor (MR) in in vitro BM-derived DCs, whilst this process is independent of MR in splenic CD8α + and CD11b + DCs [20]. In humans, MoDCs express high levels of MR whereas CD1c + DCs and CD141 + DCs express little if any MR [21,22], which may account for the lower uptake of soluble protein by CD1c + DCs and CD141 + DCs and suggests that similar uptake mechanisms occur between species. Consistent with their known higher levels of lysosomal proteases [22,23] the proteasome for processing of the pp65 NLV epitope, suggesting preferential use of the vacuolar pathway by MoDCs.…”