Thrombopoietin cooperates with FLT3-ligand in the generation of plasmacytoid dendritic cell precursors from human hematopoietic progenitors

Abstract

IntroductionDendritic cells (DCs) are professional antigen-presenting cells (APCs) that display an extraordinary capacity to stimulate naive T cells and to initiate primary immune responses. 1 Recent studies suggest that DCs also play critical roles in the induction of peripheral immunologic tolerance, regulate the types of T-cell immune responses, and function as effector cells in innate immunity against microbes. 2,3 The diverse functions of DCs in immune regulation depend on the functional plasticity of DCs at the immature stage and the diversity of DC subsets and lineages. 2 Progress in understanding the molecular regulation of DC development from hematopoietic progenitor cells (HPCs) has led to the development of in vitro culture systems for the generation of large numbers of myeloid DCs from CD34 ϩ HPCs with granulocyte macrophage-colony-stimulating factor (GM-CSF) and tumor necrosis factor ␣ (TNF-␣) 4 or peripheral blood monocytes with GM-CSF and interleukin-4 (IL-4). 5,6 These culture systems not only permit detailed studies of DC biology, they allow the development of DC-based cancer vaccines. 7 Type 1 interferon-producing cells (IPCs), or plasmacytoid DC precursors, are distinct from myeloid-derived DC types. IPCs appear to be lymphoid-related because they express antigens or mRNA transcript related to lymphocytes (CD2, CD5, CD7, pre-Tcell receptor-␣ [TCR␣], immunoglobulin germline J chain, immunoglobulin-like 14.2, and Spi-B), and they display neither common myeloid antigens (CD11b, CD13, CD33) nor macrophage function or differentiation potential. IPCs express a set of microbial pattern recognition receptors (Toll-like receptors 7 and 9) different from that expressed by myeloid DCs (Toll-like receptors 2-6). 8 In antimicrobial innate immune responses, though monocytes or immature myeloid DCs play a critical role in phagocytoses of bacteria and parasites, IPCs play a major role in antiviral immunity by rapidly producing large amounts of type 1 interferon after viral infection. The critical role of IPCs in human antiviral immunity is suggested by the observation that the loss of IPCs correlates with disease progression to AIDS in HIV-infected patients. 9 In patients of systemic lupus erythematosus (SLE), constitutive activation of IPCs by dsDNA and anti-dsDNA antibody complexes appears to contribute to the pathogenesis of SLE. 10 In allogeneic bone marrow transplantation, increasing evidence indicates that IPCs may play an important role in immune responses after hematopoietic stem cell transplantation (HSCT) to facilitate engraftment and to prevent graft-versus-host disease (GVHD). [11][12][13] Understanding the molecular mechanisms underlying IPC development from HPCs may provide a novel therapy for viral [16][17][18][19][20] Studies also show that FLT3-L treatment increases the numbers of CD11c ϩ myeloid DCs and CD4 ϩ CD123 high CD11c Ϫ IPCs in the peripheral blood of human donors by 13-fold and 48-fold, respectively. 21,22 These findings suggest that FLT3-L directly induces the differentiation of C...

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

“…Purified CD34 1 HSCs were cultured in vitro with Flt3L and TPO for 14, 21 and 28 days. In agreement with Chen et al, 26 21 days was optimal for the expansion and development of all DC subsets identified 26 and this time point was thus used in all subsequent experiments (data not shown). At day 21, five major populations of cells could be distinguished: CD14 1 monocytes, CD14 2 HLA-DR 1 CD11c 1 CD1b/c 1 and CD14 2 HLA-DR 1 CD11c int CLEC9A 1 cDC, HLA-DR int CD11c 2 CD123 1 pDC and HLA-DR 2 CD11c 2 CD123 2 CD1b/c 2 CLEC9A 2 cells (subsequently referred to as DC marker-negative cells) (Figure 1b and d).…”

“…[48][49][50][51][52] We now show that in addition to pDCs, 26 this culture system also supports the development of both the CD1b/c 1 and CLEC9A 1 DCs and CD14 1 monocytes. This is in keeping with observations that humans injected with Flt3L show an increase in both DCs and monocytes.…”

“…28 It has also been shown that CD11c 1 cDCs and CD123 1 pDCs can be differentiated in vitro from CD34 1 HSC obtained from human foetal liver and adult G-CSFmobilized blood, in the presence of Flt3L and TPO (FL/TPO cultures). 26 We compared these two culture conditions for the ability of CD34 1 HSC isolated from G-CSF-mobilized blood to differentiate into the cDC subsets found in vivo in human blood.…”

“…26 They responded to CpG by producing IFN-a and IL-6 similar to in vivo human DCs, albeit at lower absolute levels, but did not produce Mip-1a. In addition, they were capable of cross-presenting antigen, but at a lower efficiency than their in vivo counterparts.…”

“…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.…”