Dendritic Cells Progenitors Research Articles

専門スタディー2-2 マクロファージ・樹状細胞 マクロファージ・樹状細胞の起源・分化と機能の多様性

単球，マクロファージ及び樹状細胞は単核貪食細胞に属し，病原性微生物への免疫反応のみならず組織発生や代謝など生体恒常性維持に中心的な役割を担っている．これらの単核貪食細胞は免疫表現型や機能が重複するため，その分化起源や生体反応における役割，重要性は長い間議論されてきた．近年，分化誘導システムの樹立，各前駆細胞の同定，多重染色による細胞表面マーカー解析，遺伝子改変マウスを用いた解析，さらに細胞系譜解析から単核貪食細胞の分化起源及び維持機構が明らかになってきた．従来の説では，組織内マクロファージは生体骨髄の血液単球由来であると提唱されていたが，最新の研究成果では定常状態における組織内マクロファージの多くは胎生期幹細胞・前駆細胞由来であり，単球の寄与は少ないことが明らかになった．こうした中，我々のグループは世界に先駆けて樹状細胞のみに分化する共通樹状細胞前駆細胞（common dendritic cell progenitor: CDP）を同定した．この発見は樹状細胞と単球では分化起源が異なることを示唆している．また，ヒト単核貪食細胞の前駆細胞も同定されている．そこで今回の専門スタディではマウス及びヒトの樹状細胞，単球，マクロファージの起源とその分化維持機構について概説する．さらに分化起源，機能，局在，免疫表現型をもとにした新しい統一名称について紹介する．

A Common Progenitor for Macrophages, Osteoclasts and Dendritic Cells Newly Discovered in Human Bone Marrow and Cord Blood Yields Dendritic Cells with T-Cell Priming Ability

Dendritic cells (DC) have potent antigen-presentation and T-cell priming ability and therefore hold great promise in cancer immunotherapy. However, DC vaccination has not yet delivered a reliable clinical response rate, despite great efforts. Since primary DCs are rare (up 0.2-1.5% of circulating leukocytes), therapeutic DCs are generally derived from peripheral blood monocytes by culture with GM-CSF (moDC). Vaccines composed of moDC loaded with tumor antigens can induce potent and long-lasting tumour-specific immune responses in patients, but such positive results are infrequent and unpredictable. To improve success rate, research has focused on moDC culture regimens, antigen loading and activation strategies and methods of DC injection. Nevertheless, to date clinical trials using moDC have not yielded statistically significant treatment benefits over conventional strategies. Current attention has therefore shifted to the rare primary DCs that circulate in the blood under homeostatic conditions.Knowing the identity of the precursors of these DCs may facilitate the ex-vivo or in-vivo generation of DCs via the homeostatic pathway, potentially yielding DCs with optimal T cell priming ability. We (Xiao et al. Stem Cell Rep. 2015) and others (Lee et al. J. Exp. Med. 2015) have recently identified a population with DC progenitor potential in human bone marrow and cord blood, respectively. This population can be isolated on basis of a CD34+ c-KIT+ FLT3+ IL3Rαhigh phenotype and is furthermore Lin- CD10- CD11b- CD45RA+ CD38+. We have shown that this population is highly enriched for or identical to a common progenitor (P) of macrophages (M), osteoclasts (O) and DCs (D) and termed it MODP. We also identified the progenitor directly upstream from the MODP that still has granulocyte (G) differentiation potential and termed it GMODP.We hypothesized that DCs generated from GMODP or MODP under homeostatic conditions would have superb T-cell priming capacity. To examine this, the progenitors were sorted by flow cytometry from human bone marrow or cord blood and cultured with Flt3 ligand, M-CSF and IL-3 to generate DCs. We also tested the effect of a mensenchymal stem cell (MSC) feeder layer. Within 2-3 weeks of culture, 1000 DC progenitors generated approximately 150,000-250,000 DCs. Co-culture with MSC increased DC output significantly, at least 2 fold. The progenitor-derived DCs could be discerned into CD141+ conventional (c)DC, CD1c+ cDC and CD303+ plasmacytoid (p)DC. To study T-cell priming capacity of progenitor-derived DCs, we set up an in vitro DC-T co-culture assay. CD141+ cDC, CD1c+ cDC and CD303+ pDC were generated from GMODP or MODP of HLA-A2+ donors, flow cytometrically purified, activated with lipopolysaccharide and loaded with MART-126-35 peptide that represents a melanoma-derived tumor antigen. Primary T cells from peripheral blood of unrelated donors were retrovirally transduced to express a T cell antigen receptor (TCR) ab specific for the HLA-A2/MART-126-35 peptide complex. The ability of the DCs to prime a T-cell response was read out by antigen-specific CD8+ T cell proliferation. All DC subsets were able to induce MART-1 specific T cell proliferation, with the CD1c+ cDCs being most potent and the CD303+ pDC being least potent.In conclusion: We have established a culture method to derive DCs with T-cell priming ability from a newly identified DC progenitor. These results are of value for improvement of DC-based immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Potent Stem Cell Mobilization with the Novel CXCR4 Antagonist POL6326 - Results of a Phase IIa Dose Escalation Study in Comparison to G-CSF

Background:Stem cell mobilization (SCM) with G-CSF is efficient but - although overall safe - inconvenient because of the five-day injection regime and certain contraindications. Side effects, sometimes severe, are frequent. These disadvantages fuel the quest for alternative mobilizing agents. Mobilization with the CXCR4-inhibitor plerixafor is rapid, albeit insufficiently efficacious on its own. POL6326, a potent 2nd generation macrocycle CXCR4 antagonist, has demonstrated rapid mobilization kinetics and efficacy in mice. We herein report the results of a Phase IIa dose escalation trial where SCM in response to POL6326 was compared with G-CSF in healthy volunteer stem cell donors.Methods:In this Phase IIa open label trial, healthy volunteer stem cell donors with average mobilization (121±7 CD34+ cells/μL, MW±SEM)after a five-day course of G-CSF, and a wash-out period of at least 6 weeks, received POL6326 at 500-2500 µg/kg as a single 2-hour i.v. infusion. Safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) were assessed in 3-10 subjects/dose group. Subgroups received two doses of POL6326, 1000 and 2500 µg/kg or 1500 and 2500 µg/kg, at least 2 weeks apart (paired intra-individual analysis). For PK and PD blood samples were collected before (0) and at 2, 3, 4, 6, 8 and 24 hrs after infusion start. Complete blood count, CD34+, CFU-C count and PK were assessed at all time points. At 0, 4, 8 and 24 hrs extensive phenotyping of mobilized mature and immature leukocyte subsets was performed. Eight to 14 days after treatment volunteers underwent extensive clinical and laboratory follow-up.Results:POL6326 was very well tolerated. Several volunteers experienced a mild urticarial or itchy macular rash which responded well to H1/H2 blockade. Rating of tolerability/adverse events by volunteers (questionnaire) compared favourably with G-CSF administration. Exposure (Cmax, AUC) was dose-linear. At all doses tested POL6326 mobilized CD34+ progenitor cells and colony-forming cells (CFU-C, Figure 1) exceeding reported peak mobilization with plerixafor in donors at all except the lowest dose levels. In this dataset mobilization after doses of 2000 or 2500 µg/kg did not appear meaningfully stronger than after 1500 µg/kg. The SCM response for CD34+ cells to doses ≥1500 µg/kg was 36.9±2.4/µL (mean±SEM), or 1/3 that of G-CSF (y=0.324x). Good SCM with G-CSF was predictive of good SCM with POL6326 (r=0.63). One/5.7 POL6326-mobilized CD34+ cells was clonogenic (G-CSF: 1 CFU-C/3.4 CD34+ cells) possibly indicating a more immature phenotype of CD34+ cells mobilized by POL6326. POL6326 caused mixed leucocytosis with peak values in the mid-20K/µL. B-lymphocytosis was more and neutrophilia and monocytosis were less pronounced after POL6326 than G-CSF. Compared to G-CSF the subset of plasmocytoid dendritic cell progenitors (pDC) was enriched to a distinct population within the CD34+ cells following SCM with POL6326 as previously described for plerixafor. At the 24 h time point, blood values were well on their way towards normal, and at follow-up all laboratory values had normalized.Summary/Conclusions:The novel CXCR4-antagonist POL6326 is safe, well tolerated, and provides efficient mobilization of HSPCs. Based on the number of mobilized CD34+ cells at higher doses in this study, we conclude that a standard dose of 4x10E6 CD34+ cells/kg can be extracted with a single apheresis for most recipients unless their body weight significantly exceeds the donor weight. However, exploration of alternative dosing regimens may provide even higher mobilization responses. POL6326 can be an effective mobilizing agent for allogeneic donors, including subjects with contra-indications to G-CSF. [Display omitted] [Display omitted] DisclosuresEscot:Polyphor Ltd.: Employment. Douglas:Polyphor Ltd.: Employment. Romagnoli:Polyphor Ltd.: Employment. Chevalier:Polyphor Ltd.: Employment. Dembowsky:Polyphor Ltd.: Consultancy. Hooftman:Polyphor Ltd.: Employment. Bonig:Polyphor Ltd.: Research Funding.

GATA-2 Regulates Dendritic Cell Differentiation

(Background) Dendritic cells (DCs) are critical regulators of the immune response, but their differentiation mechanism remains unclear. Heterozygous germline GATA-2 mutations in humans cause MonoMAC syndrome, characterized by monocytopenia and predisposition to myelodysplasia/acute myeloid leukemia. In this syndrome, DC count decreases profoundly, with an increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. In the present study, we analyzed the role of GATA-2 in DC differentiation and the underlying molecular mechanisms.(Method)Gata2 haploinsufficient mice (Gata2+/−: Tsai et al. Nature 1994) and tamoxifen-inducible Gata2-knockout mice (Gata2flox/flox/ER-Cre: Charles et al. Molecular Endocrinology 2006) were used. To generate conditional Gata2 knockouts in vivo, Gata2flox/flox/ER-Cre mice were intraperitoneally injected with 1-μg tamoxifen on days 1-3 and 8-10 and evaluated on days 20-22. Isolation of splenic DCs and bone marrow (BM) precursors, including LSK (Lin- Sca1+ Kit+ cell), CMP (common myeloid-restricted progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid-restricted progenitor), and CDP (common dendritic cell precursor), were separated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For the in vitro analysis of Gata2-knockout, BM cells were cultured with CD45.1+ BM feeder cells from SJL mice (The Jackson Laboratory) with FLT3L (200 ng/mL) and 4-hydroxytamoxifen (Sigma). For transcription profiling, SurePrint G3 mouse GE microarray (Agilent) was used, and the data was subsequently analyzed with ImmGen database (http://www.immgen.org). Promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using CMP fraction and erythroid-myeloid-lymphoid (EML) hematopoietic precursor cell line (ATCC) with antibodies to GATA-2 (sc-9008, Santa Cruz Biotechnology).(Results) Quantitative RT-PCR analysis showed abundant Gata2 expression in LSK and CMP fractions, with detectable expression in GMP, CLP, and CDP fractions and in vitro differentiated DCs. Although the DC count did not change in Gata2 haploinsufficient mice, it significantly and profoundly decreased in Gata2 conditional knockout mice. To examine the role of GATA-2 during DC differentiation, we knocked out Gata2 during in vitro DC differentiation, starting from LSK, CMP, GMP, CLP, and CDP fractions obtained from Gata2flox/flox/ER-Cre mice. Gata2 knockout significantly decreased CD11c+ DC counts from LSK, CMP, and CDP fractions, while those from CLP and GMP were unaffected, implying the importance of GATA-2 during DC differentiation in the pathway from LSK to CDP via CMP, not via CLP nor GMP.To elucidate the underlying molecular mechanisms, we performed expression profiling with control and Gata2 -knockout DC progenitors from CMP of Gata2flox/flox/ER-Cre mice. Gata2 knockout caused >5-fold upregulation and downregulation of 67 and 63 genes, respectively. Although genes critical for the DC differentiation, e.g., Spi1, Ikzf1, and Gfi1, were not detected among the GATA-2-regulated gene ensemble, we found significant enrichment of myeloid-related and T lymphocyte-related genes among the downregulated and upregulated gene ensembles, respectively. We focused on Gata3 upregulation (7.33-fold) as a potential key mechanism contributing to Gata2 knockout-related impaired DC differentiation. Quantitative ChIP analysis with both CMP fraction and EML cell line demonstrated obvious GATA-2 chromatin occupancy at the consensus GATA-binding motif within Gata3+190 kb, which was conserved with human. Furthermore, addition of Gata3 +190 kb region to the Gata3 promoter (~0.5 kb) significantly decreased luciferase activity, which was significantly recovered by the deletion of GATA sequence within Gata3 +190 kb, in EML cells.(Conclusion) GATA-2 seems to play an important role for cell fate specification toward myeloid versus T lymphocytes, and thus contributing to the DC differentiation. Our data offer a better understanding of the pathophysiology of MonoMAC syndrome. DisclosuresFujiwara:Chugai Pharmaceuticals. Co., Ltd.: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Ishizawa:GSK: Research Funding; Takeda: Research Funding; Celgin: Speakers Bureau; Kyowa Kirin: Research Funding; Celgin: Research Funding; Janssen: Research Funding; Takeda: Speakers Bureau; Kyowa Kirin: Speakers Bureau; Pfizer: Speakers Bureau.

Enhancer Landscape Dynamics and the Role of IRF8 in Mononuclear Phagocyte Development

Monocytes and dendritic cells (DCs) are critical mononuclear phagocytes that regulate innate and adaptive immune responses. Hematopoietic stem cells give rise to monocytes and DCs via intermediate myeloid progenitor populations, such as granulocyte-monocyte progenitors (GMPs), monocyte-DC progenitors (MDPs), and common monocyte progenitors (cMoPs) or common DC progenitors (CDPs). However, the molecular mechanism underlying their lineage determination is poorly understood. Recently, promoter-distal enhancers have been found to be key for cell fate decision. In this study, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) analysis of three histone modifications (H3K4me1, H3K27ac, and H3K4me3, representing primed enhancers, active enhancers, and transcriptional intiation, respectively) in seven myeloid populations (GMPs, MDPs, cMoPs, CDPs, monocytes, DCs, and neutrophils), and identified approximately sixty thousand putative enhancer regions. We found that a majority of monocyte- and DC-specific active enhancers were gradually established at progenitor stages prior to gene expression and terminal differentiation. Computational DNA motif analysis implicated that these enhancers were regulated by combinations of lineage-determining transcription factors such as PU.1, RUNX, C/EBP, and IRF. Indeed, ChIP-seq of PU.1 confirmed that the myeloid master regulator PU.1 was the common transcription factors bound to enhancers at all the stages examined. IRF binding motifs were enriched at the enhancer regions in MDPs, cMoPs and CDPs, but not GMPs. Among IRFs, IRF8, a partner of PU.1, has been shown to be highly expressed in MDPs and their descendants, and to be required for MDP-to-CDP and cMoP-to-monocyte transitions. Indeed, ChIP-seq analysis in Irf8-/- progenitors demonstrated that the enhancer landscapes of Irf8-/- GMPs, MDPs, and cMoPs all remained similar to that of wild-type GMPs. Moreover, ChIP-seq for IRF8 binding revealed that IRF8 directly promoted the priming and activation of many enhancers in MDPs and cMoPs. These results contribute to a comprehensive understanding of how transcription factors govern the enhancer landscape dynamics during mononuclear phagocyte development. DisclosuresNo relevant conflicts of interest to declare.

Systemic Inflammation Recruits Ccr7+ Dendritric-Biased Granulocyte-Macrophage Progenitors to Lymphatic Circulation in a Non-Canonical Traf6-Dependent Manner

A small fraction of hematopoietic stem and progenitor cells (HSPCs) is cyclically released into the bloodstream from bone marrow (BM). The existence of HSCP-lymphatic-blood circulation with the potential to give rise to extramedullary tissue-resident myeloid cells has been described. However, the functional role and underlying mechanisms of this circulation are not known. We hypothesized that inflammation may be a mediator of HSPC mobilization to lymph. In order to determine if inflammation was related to increased circulation of HSPCs through lymph, we analyzed the HSPC content in lymph nodes (LN) of patients with lymphadenitis. Analysis of CFU and side population (SP+) cell content confirmed that LNs from patients with reactive lymphadenitis and patients with inflammatory Hodgkin's disease contained ~2-fold higher HSPCs than in patients diagnosed of non-Hodgkin's lymphoma. To identify the pattern and mechanisms that regulate HSPCs during inflammation. We analyzed the content of HSPCs in blood (PB), BM, LN, thoracic duct (TD) and other organs after systemic administration of lipopolysaccharide (LPS) and compared with the normal circadian migratory rhythms in control animals (PBS). Mobilization of BM myeloid progenitors (mostly granulo-macrophage progenitors-GMPs, but not stem cells), to LN and TD peaked as early as three hours after LPS administration, and followed a very different pattern than the mobilization kinetics in PB. By using 3D reconstruction of confocal microscopy imaging of complete LN from lymphatic endothelium reporter mice (Lyve1Cre-eGFP), we localized all myeloid progenitors in the mantle zone of secondary follicles. We note that they did not enter the follicle germ centers. To determine the molecular mechanisms at play to induce HSPC mobilization to the LN upon LPS administration, we analyzed whether the recruitment of HSPCs in LN required Traf6, a mediator downstream of the LPS/TLR4 signaling pathway. Inducible deficiency of Traf6 driven by Mx1-Cre recombinase completely abolished the migration of myeloid progenitors to LN, but not to PB, induced by LPS as compared with their Mx1-Cre;Wt littermates indicating that the mechanism controlling HSPC recruitment to the LN was distinct from the one controlling HSPC migration to PB. Using a combination of in vivo and in vitro assays, we found that Traf6 activity regulates HSPC migration in a non-cell autonomous manner that depends on its expression in a small (~1%) fraction of non-progenitor LN myeloid cells expressing high levels of Ccl19. In vivo neutralization of Ccr7, the receptor for Ccl19, results in abrogation of myeloid progenitor mobilization to LN. GMPs migrating to LN were biased to differentiate into dendritic cells within 7 days post-migration. Using Cx3cr1-GFP transgenic mice as macrophage-dendritic cell progenitor (MDP) reporters, we found that the vast majority of GMPs recruited to the LN were MDPs, and thus already poised to DC differentiation. Finally, a combination of genetic and pharmacological approaches revealed that Traf6 signaling effects are NFkB independent but dependent upon Irak1/4, Ucb13 and IKKβ resulting in SNAP23 phosphorylation and exocytosis of pre-formed cytokines from LN myeloid cells. This study identifies the cellular and molecular basis of inflammation dependent migration of DC progenitors and suggests that the mobilization of HSPCs from BM to LN results in homeostatic replenishment of highly specialized antigen presenting cells. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic program and transcription factor circuitry of dendritic cell development.

Dendritic cells (DC) are professional antigen presenting cells that develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Multipotent progenitors (MPP) are committed to DC restricted common DC progenitors (CDP), which differentiate into specific DC subsets, classical DC (cDC) and plasmacytoid DC (pDC). To determine epigenetic states and regulatory circuitries during DC differentiation, we measured consecutive changes of genome-wide gene expression, histone modification and transcription factor occupancy during the sequel MPP-CDP-cDC/pDC. Specific histone marks in CDP reveal a DC-primed epigenetic signature, which is maintained and reinforced during DC differentiation. Epigenetic marks and transcription factor PU.1 occupancy increasingly coincide upon DC differentiation. By integrating PU.1 occupancy and gene expression we devised a transcription factor regulatory circuitry for DC commitment and subset specification. The circuitry provides the transcription factor hierarchy that drives the sequel MPP-CDP-cDC/pDC, including Irf4, Irf8, Tcf4, Spib and Stat factors. The circuitry also includes feedback loops inferred for individual or multiple factors, which stabilize distinct stages of DC development and DC subsets. In summary, here we describe the basic regulatory circuitry of transcription factors that drives DC development.

Defining human dendritic cell progenitors by multiparametric flow cytometry.

Human dendritic cells (DCs) develop from progressively restricted bone marrow (BM) progenitors: these progenitor cells include granulocyte, monocyte and DC progenitor (GMDP) cells; monocyte and DC progenitor (MDP) cells; and common DC progenitor (CDP) and DC precursor (pre-DC) cells. These four DC progenitors can be defined on the basis of the expression of surface markers such as CD34 and hematopoietin receptors. In this protocol, we describe five multiparametric flow cytometry panels that can be used as a tool (i) to simultaneously detect or phenotype the four DC progenitors, (ii) to isolate DC progenitors to enable in vitro differentiation or (iii) to assess the in vitro differentiation and proliferation of DC progenitors. The entire procedure from isolation of cells to flow cytometry can be completed in 3-7 h. This protocol provides optimized antibody panels, as well as gating strategies, for immunostaining of BM and cord blood specimens to study human DC hematopoiesis in health, disease and vaccine settings.

Rapamycin-conditioned dendritic cells induced immune tolerance through the regulation of Treg/Th17 cells in mice

To investigate the effect of tolerogenic dendritic cells (Tol-DC) generated by Rapamycin (Rapa) on the differentiation of Treg/Th17 cells and explore the possible mechanism of tolerance induction. DC progenitors from mouse bone marrow were propagated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin (IL)-4 stimulation for 6 days in the presence or absence of Rapa (20 ng/ml). During DC culture, morphology of cell was observed under electron microscope. Cell surface expression of CD11c, CD40 and CD80 was analyzed by flow cytometry. The antigen-presenting function of DC was determined by one-way mixed leukocyte reactions. In vivo, the recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control, Rapa, immature DC (imDC) and Tol-DC group. The survival time of the skin allograft was observed and Treg/Th17 cells were analyzed by flow cytometry in each group. The immunephenotypic analysis showed that in comparison with those in the control group and the LPS group the expression of the co-stimulatory molecules CD40 and CD80 were significantly lower in the Rapa-group and Rapa+LPS group. The ability to stimulate proliferation of T cells of the same genotype in the Rapa-group was significantly inhibited (P<0.01). In the in vivo experiment, the mice's survival time remarkably prolonged, the percentage of Treg cells was enhanced and Th17 cells was reduced in the mice's spleen in Tol-DC group. Tol-DC generated by Rapamycin can significantly induce immune tolerance through up-regulate Tregs and down-regulate Th17 cells. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs, which can be loaded with donor antigen, and potentially used to promote organ transplant tolerance.

Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand.

Dendritic cells (DCs), including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) are critical for initiating and controlling the immune response. However, study of DC, particularly pDC, function is hampered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation preferentially favor the production of cDCs over pDCs. Here, we demonstrated that pDCs could be efficiently generated in vitro from common lymphoid progenitors (CLPs) using Flt3 ligand (FL) in three different culture systems, namely feeder-free, BM-feeder and AC-6-feeder. This was in stark contrast to common DC progenitors (CDPs), in which cDCs were prominently generated under the same conditions. Moreover, the efficiency and function of pDCs generated from these three systems varied. While AC-6 system showed the greatest ability to support pDC development from CLPs, BM-feeder system was able to develop pDCs with better functionality. pDCs could also be expanded in vivo using hydrodynamic gene transfer of FL, which was further enhanced by the combined treatment of FL and IFN-α. Interestingly, IFN-α selectively promoted the proliferation of CLPs and not CDPs, which might contribute to enhanced pDC development. Together, we have defined conditions for in vitro and in vivo generation of pDCs, which may be useful for investigating the biology of pDCs.

Multicomponent mannose-containing liposomes efficiently deliver RNA in murine immature dendritic cells and provide productive anti-tumour response in murine melanoma model

Here we demonstrate the ability of mannosylated liposomes (ML) targeted to mannose receptors (MR) to perform the targeted delivery of model plasmid DNA encoding EGFP and total tumour RNA into murine bone-marrow-derived dendritic cells (DCs) and enhance the efficiency of anti-tumour response triggered by these DCs in murine melanoma model. ML consist of cationic lipid 2X3 (1,26-Bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and 2.5, 5 or 10% mol. of novel mannosylated lipoconjugates. In the structure of lipoconjugates d-mannose was attached to ditetradecylglycerol residue via succinyl (lipoconjugate 1) or diethylsquarate (lipoconjugate 2) linker groups. ML spontaneously form complexes with plasmid DNA and RNA due to electrostatic interaction between positively charged lipid amino group and negatively charged phosphate of nucleic acids.ML demonstrated the benefit in transfection efficiency (TE) of pDNA into DC progenitors and immature DCs in comparison with the control liposomes at low N/P (nitrogen to phosphate) ratios (1/1 and 2/1) but not at high N/P ratios where the TE was comparable with control liposomes. Moreover, ML at low N/P were more effective in RNA delivery into immature DCs in comparison with DC progenitors. At high N/P ratios liposomal formulations containing 5 and 10% mol. of mannosylated lipoconjugate 2 with diethylsquarate linker were the most effective (up to 50% of transfected cells). DCs transfected ex vivo with ML/melanoma B16 RNA complexes after i.v. injection into mice caused five- to six-fold inhibition of melanoma lung metastasis number. Moreover, the i.v. injection of ML/melanoma B16 RNA complexes into mice induced generation of the melanoma B16-specific cytotoxic T-lymphocytes, which were two-fold more efficient in B16 cell killing than those from control liposome group.

DCs are ready to commit.

Dendritic cell progenitors commit to a specific conventional dendritic cell fate earlier than previously thought, by initiating transcription-factor regulatory circuits unique to their subtype.

Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow.

Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α(+) (CD103(+)) cDC1 lineage, and the CD11b(+) cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H(-)Ly6C(-) pre-DCs) or cDC2 lineage (Siglec-H(-)Ly6C(+) pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.

Batf3 maintains autoactivation of Irf8 for commitment of a CD8α(+) conventional DC clonogenic progenitor.

The transcription factors Batf3 and IRF8 are required for development of CD8α+ conventional dendritic cells (cDCs), but the basis for their actions was unclear. Here, we identify two novel Zbtb46+ progenitors that separately generate CD8α+ and CD4+ cDCs and arise directly from the common DC progenitor (CDP). Irf8 expression in the CDP depends on prior PU.1-dependent autoactivation, and specification of pre-CD8 DC progenitors requires IRF8 but not Batf3. However, upon pre-CD8 DC specification, Irf8 autoactivation becomes Batf3-dependent at a CD8α+ cDC-specific enhancer containing multiple AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3−/− mice that specify toward pre-CD8 DCs fail to complete CD8α+ cDC development due to decay of Irf8 autoactivation, and divert to the CD4+ cDC lineage.

Chronic alcohol consumption induces peripheral skewed dentritic cell sets without affecting dendritic cell progenitors. (HEM5P.238)

Abstract Alcoholism impairs innate and adaptive immune responses and increases susceptibility to infectious diseases, caused among others by alcohol induced defects in dendritic cell (DC) function and frequency. This study aims at understanding how DC turnover is affected by chronic alcohol exposure, characterizing its influence on DC development at the progenitor stages and the underlying mechanisms. Alcohol (EtOH) is delivered in the drinking water to C57BL/6 mice at 20% v/v (Meadows-Cook diet). Lymphoid tissue (LT) DCs from thymus and spleen, non-lymphoid tissue (NLT) DCs from liver and lung and bone marrow (BM) progenitors are analyzed by flow cytometry. After 12 weeks of EtOH exposure BM populations at the Macrophage-DC Progenitor (MDP) stage decrease by 60% (P&amp;lt;0,05) but there is no difference in the numbers of Common DC Progenitors (CDP). In the liver we observe a 70% increase (P&amp;lt;0,05) of plasmacytoid DCs (pDC) while CD103+ classic DCs (cDC) decrease by 50% (P&amp;lt;0,01). Concordantly hepatic T cell numbers decrease: CD8+ CTL by 40% (P&amp;lt;0,01), CD4+ TH by 30% (P&amp;lt;0,005) and CD4+ TREG by 45% (P&amp;lt;0,05). In summary, alcohol in a chronic model does not change CDP numbers, the main progenitor of pDC and cDC, suggesting little effect on DC development. There is however a major imbalance between pDC and cDC, reflected by the impact on steady-state T cell subsets. Going forward we will elucidate the underlying mechanisms responsible for this peripheral effect of alcohol on DC populations.

Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow.

In mice, two restricted dendritic cell (DC) progenitors, macrophage/dendritic progenitors (MDPs) and common dendritic progenitors (CDPs), demonstrate increasing commitment to the DC lineage, as they sequentially lose granulocyte and monocyte potential, respectively. Identifying these progenitors has enabled us to understand the role of DCs and monocytes in immunity and tolerance in mice. In humans, however, restricted monocyte and DC progenitors remain unknown. Progress in studying human DC development has been hampered by lack of an in vitro culture system that recapitulates in vivo DC hematopoiesis. Here we report a culture system that supports development of CD34(+) hematopoietic stem cell progenitors into the three major human DC subsets, monocytes, granulocytes, and NK and B cells. Using this culture system, we defined the pathway for human DC development and revealed the sequential origin of human DCs from increasingly restricted progenitors: a human granulocyte-monocyte-DC progenitor (hGMDP) that develops into a human monocyte-dendritic progenitor (hMDP), which in turn develops into monocytes, and a human CDP (hCDP) that is restricted to produce the three major DC subsets. The phenotype of the DC progenitors partially overlaps with granulocyte-macrophage progenitors (GMPs). These progenitors reside in human cord blood and bone marrow but not in the blood or lymphoid tissues.

Limited niche availability suppresses murine intrathymic dendritic-cell development from noncommitted progenitors

AbstractThe origins of dendritic cells (DCs) and other myeloid cells in the thymus have remained controversial. In this study, we assessed developmental relationships between thymic dendritic cells and thymocytes, employing retrovirus-based cellular barcoding and reporter mice, as well as intrathymic transfers coupled with DC depletion. We demonstrated that a subset of early T-lineage progenitors expressed CX3CR1, a bona fide marker for DC progenitors. However, intrathymic transfers into nonmanipulated mice, as well as retroviral barcoding, indicated that thymic dendritic cells and thymocytes were largely of distinct developmental origin. In contrast, intrathymic transfers after in vivo depletion of DCs resulted in intrathymic development of non–T-lineage cells. In conclusion, our data support a model in which the adoption of T-lineage fate by noncommitted progenitors at steady state is enforced by signals from the thymic microenvironment unless niches promoting alternative lineage fates become available.

S1PR4 is required for plasmacytoid dendritic cell differentiation.

The sphingolipid sphingosine-1-phosphate (S1P) has various functions in immune cell biology, regulating survival, proliferation, and, most prominently, migration. S1P couples to five G protein-coupled receptors (S1PR1-5) to transduce its effects on immune cell function. Expression of S1PR4 is restricted to immune cells. However, its impact on immune cell biology is largely elusive. In the current study, we intended to answer the question of whether S1P might affect plasmacytoid dendritic cell (pDC) migration, which dominantly express S1PR4. pDC are highly specialized cells producing large amounts of type I interferon in response to TLR7/9 ligands after viral infection or during autoimmunity. Surprisingly, we noticed a reduced abundance of pDC, particularly CD4- pDC, in all organs of S1PR4-deficient vs. wildtype mice. This effect was not caused by altered migration of mature pDC, but rather a reduced potential of pDC progenitors, especially common DC progenitors, to differentiate into pDCs. In vitro studies suggested that reduced S1PR4-deficient pDC progenitor differentiation into mature pDC might be explained by both migration and differentiation of pDC progenitors in the bone marrow. As S1PR4 also affected the differentiation of CD34+ human hematopoietic stem cells into pDC, interfering with S1PR4 might be useful to reduce pDC numbers during autoimmunity.

The Innate Immune System Favors Emergency Monopoiesis at the Expense of DC Differentiation to Control Bacterial Infections

Dendritic cells (DCs) are critical in host defense against infection, bridging the innate and adaptive immune system. Patients with sepsis display reduced circulating and splenic DCs and impaired DC function that may contribute to prolonged immune suppression and exacerbation of infection. However, the mechanisms of pathogen-induced DC depletion remain poorly understood. Here, a mouse model of systemic bacterial infection was employed to analyze the impact of different bacterial pathogens on DC development in vivo. We found that the numbers of bone marrow (BM) hematopoietic progenitors committed to the DC lineages were reduced following systemic infection with different Gram-positive and Gram-negative bacteria. In parallel, a TLR4-dependent increase of committed monocyte progenitors in the BM as well as mature monocytes in the spleen was observed. In line, adoptively transferred FLT3+ myeloid progenitors (MPs) developed preferentially to monocytes at the expense of DCs in infected animals. Analyses performed on mixed BM chimeras suggested that both the reduction of DC progenitors and the induction of monopoiesis following infection were dependent on extrinsic TLR4 signaling driving the secretion of IFN-g regulated chemokines. Consistently, these effects were completely abrogated by suppression of IFN-g signaling.Elevated monocyte numbers in the spleen triggered by infection were due to a CCR2-dependent egress from the BM. In CCR2-deficient mice, in which monocytosis reportedly is abrogated, we observed a significantly increased bacterial load in the spleen and a reduced survival rate, highlighting the importance of monocytes for bacterial clearance. Together, our data provide evidence for a general response of myeloid progenitors upon bacterial infection to enhance monocyte production, thereby increasing the availability of innate immune cells as a first line of defense against invading pathogens. Concomitantly the development of DCs is impaired, which may be responsible for transient immunosuppression in e.g. bacterial sepsis. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic control of Ccr7 expression in distinct lineages of lung dendritic cells.

Adaptive immune responses to inhaled allergens are induced following CCR7-dependent migration of precursor of dendritic cell (pre-DC)-derived conventional DCs (cDCs) from the lung to regional lymph nodes. However, monocyte-derived (moDCs) in the lung express very low levels of Ccr7 and consequently do not migrate efficiently to LN. To investigate the molecular mechanisms that underlie this dichotomy, we studied epigenetic modifications at the Ccr7 locus of murine cDCs and moDCs. When expanded from bone marrow precursors, moDCs were enriched at the Ccr7 locus for trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with transcriptional repression. Similarly, moDCs prepared from the lung also displayed increased levels of H3K27me3 at the Ccr7 promoter compared with migratory cDCs from that organ. Analysis of DC progenitors revealed that epigenetic modification of Ccr7 does not occur early during DC lineage commitment because monocytes and pre-DCs both had low levels of Ccr7-associated H3K27me3. Rather, Ccr7 is gradually silenced during the differentiation of monocytes to moDCs. Thus, epigenetic modifications of the Ccr7 locus control the migration and therefore the function of DCs in vivo. These findings suggest that manipulating epigenetic mechanisms might be a novel approach to control DC migration and thereby improve DC-based vaccines and treat inflammatory diseases of the lung.