Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8α+ dendritic cells

Abstract

In mouse, a subset of dendritic cells (DCs) known as CD8α+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8α+ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8α+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8α+ DCs, human DNGR-1+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell–derived signals. Notably, DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy.

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

“…They expressed IRF8 and TLR3, two hallmarks of this subset in blood and spleen, 22,23 albeit at lower levels than their in vivo DC counterparts. Furthermore, similar to blood CLEC9A 1 cDCs, they stimulated CD4 1 T cells to a lesser extent than the CD1b/c 1 DCs in an MLR, cross-presented antigen and produced IL-6, TNF-a and Mip-1a.…”

“…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. It may be that the cultured DCs in general, similar to mouse cultured DCs, represent an immature DC subset that requires a number of different stimuli to be fully activated.…”

“…5,7 However, it has been demonstrated recently that both CD1b/c 1 and CD141 1 CLEC9A 1 cDCs as well as CD304 1 pDCs in both human blood and spleen can cross-present antigen, albeit with different efficiencies. [20][21][22][23][24] We therefore tested the ability of Flt3L-derived human DCs to cross-present influenza virus protein to a CD8 1 Tcell clone specific for an epitope in the matrix of influenza virus. CD8 1 T-cell activation was measured as IFN-c secretion by ELISpot assay.…”

“…14 The CD141 1 DCs have been suggested to be equivalent to the mouse CD8 1 DCs sharing a similar transcriptional signature 15 and expression of Necl2, CLEC9A and XCR1. [16][17][18][19][20][21] Furthermore, the CD141 1 DCs have been shown to produce IL-12p70 and cross-present antigen, [20][21][22][23] both characteristics of the mouse CD8 1 DC subset. The CD1b/c 1 DC is thought to be the equivalent of the mouse CD8 2 DC, based on a similar transcriptional profile.…”

“…25 Culturing HSCs enriched from granulocyte colonystimulating factor (G-CSF)-mobilized blood in the presence of Flt3L and thrombopoietin (TPO) has been shown to generate in good yield, functional pDC that are equivalent to their in vivo counterparts in their capacity to produce interferon (IFN)-a in response to TLR stimulation. 26,27 Most recently, Poulin et al 23 have developed a culture system that supports the generation of CD141 1 CLEC9A 1 DCs from cord blood precursors. However, despite many different culture conditions used to generate functional DC in vitro, a culture system capable of generating the pDC and the cDC subtypes including the CD1b/c 1 and CD141 1 CLEC9A 1 DC found within human lymphoid tissue and blood has not been described.…”