Common DC Progenitors Research Articles

Energy restriction blocks bone marrow dendritic cell development

Dendritic cells (DC) are antigen presenting cells and sensitive to nutritional modulation. Energy restriction (ER) is a nutritional intervention shown to extend lifespan in many experimental organisms, but recent data in non‐human primates is contradictory. Immunological outcomes of ER are mixed with several reports demonstrating ER amelioration of autoimmune disease, but ER mice are also more susceptible to some primary infections such as influenza. Due to glucocorticoid‐associated increased myelopoiesis, we hypothesized bone marrow DC development would be increased. The objective of this study was to determine the effects of ER on bone marrow DC progenitor numbers. Multicolor flow cytometry was used to characterize bone marrow DC progenitors. We observed increased myeloid progenitors and monocytes, while common DC progenitors and precursor DC were decreased in bone marrow of ER mice. Altered progenitor populations had no effect on in vitro DC development. Therefore we conclude ER bone marrow microenvironment is altered favoring myelopoiesis over lymphopoiesis, but myelopoiesis is also altered. The altered developmental pathway of DC subsets may be responsible for contradictory immunological outcomes of ER populations and provide potential targets for improving vaccination, autoimmune conditions, and infectious disease.Grant Funding Source: NIA R01AG034949 to EMG

P123 Type I IFN augments FLT3 ligand-dependent plasmacytoid dendritic cell development from common lymphoid progenitor

Dendritic cells (DC), belonging to the innate immune system, are specialized for antigen processing and presentation and orchestrate the adaptive immune response to infection. Among different DC populations, plasmacytoid DCs (pDCs) are highly potent type I IFN (IFN-I) producing cells during infection relative to cDCs. Aberrant secretion of IFN-I by pDCs in the absence of infection has been implicated in autoimmune diseases, including systemic lupus erythematosus (SLE) and psoriasis. However, signals dictating pDC generation and activity remain incompletely understood. DC development was done using coculture of common lymphoid progenitors (CLPs) or common dendritic cell progenitors (CDPs) with AC6 stromal cell line in the presence of Flt3 ligand (FL). We were able to derive pDC and cDC in vitro. Here, we report a synergistic role for FL and IFN-I in pDC development from CLPs. Both cDCs and pDC were generated from CLP in response to FL, but pDC generation required higher signal strength of Flt3 receptor and depended on concurrent IFN-I signaling. While CLPs predominantly generated cDCs at low dose of FL, pDCs preferentially developed at higher cytokine doses. Absence of IFN-I receptor 1 (IFNAR1) or mutation of its effector transcription factors STAT1 or STAT2 reduced pDC development in vivo and impaired FL-dependent pDC formation from CLPs in vitro and in vivo. Moreover, exogenous IFN-I enhanced FL-induced pDC genesis, and neutralizing antibody to IFNAR1 abrogated this process. Interestingly, IFN-I was induced in CLPs in response to FL stimulation, facilitating up-regulation of Flt3 expression on CLPs and their differentiation into pDCs. Collectively, these data define a cooperative effect of FL and IFN-I in pDC differentiation from CLPs through an autocrine positive feedback loop that could provide a therapeutic target for treatment of autoimmune disease.

IRF-8 extinguishes neutrophil production and promotes dendritic cell lineage commitment in both myeloid and lymphoid mouse progenitors

AbstractWhile most blood lineages are assumed to mature through a single cellular and developmental route downstream of HSCs, dendritic cells (DCs) can be derived from both myeloid and lymphoid progenitors in vivo. To determine how distinct progenitors can generate similar downstream lineages, we examined the transcriptional changes that accompany loss of in vivo myeloid potential as common myeloid progenitors differentiate into common DC progenitors (CDPs), and as lymphoid-primed multipotent progenitors (LMPPs) differentiate into all lymphoid progenitors (ALPs). Microarray studies revealed that IFN regulatory factor 8 (IRF-8) expression increased during each of these transitions. Competitive reconstitutions using Irf8−/− BM demonstrated cell-intrinsic defects in the formation of CDPs and all splenic DC subsets. Irf8−/− common myeloid progenitors and, unexpectedly, Irf8−/− ALPs produced more neutrophils in vivo than their wild-type counterparts at the expense of DCs. Retroviral expression of IRF-8 in multiple progenitors led to reduced neutrophil production and increased numbers of DCs, even in the granulocyte-macrophage progenitor (GMP), which does not normally possess conventional DC potential. These data suggest that IRF-8 represses a neutrophil module of development and promotes convergent DC development from multiple lymphoid and myeloid progenitors autonomously of cellular context.

Selective Disruption of PU.1 in Mature Dendritic Cells Affects Their Tissue Distribution and T Cell Homeostasis

Abstract 518PU.1, a hematopoietic transcription factor, is indispensable for development of conventional dendritic cells (cDCs) from hematopoietic stem cells. However, the function of PU.1 in mature cDC remains unclear. To test the possible role of PU.1 in mature cDCs, we developed mice lacking PU.1 selectively in mature cDCs (DC-PU.1D/D mice) by crossing a PU.1flox mouse line with a transgenic Itgax (CD11c)-Cre strain. In these mice, cDCs were dramatically reduced in spleen, thymus, lymph node, and skin, down to <40%, <25%, <10% and <5% of DCs in control mice respectively, whereas bone marrow cDCs and common dendritic cells progenitors (CDPs) were not affected. Surprisingly, T cell numbers were significantly decreased in DC-PU.1D/D mice, whereas thymic T cell development was normal, suggesting that maintenance of mature T cell pool might be impaired, presumably by dysfunction of PU.1D/D cDCs. In fact, PU.1D/D cDCs failed to efficiently induce ovalbumin-specific T cell response and to produce inflammatory cytokines in response to Toll like receptor (TLR) stimulation both in vitro and in vivo. The intravenous transfer of spleen PU.1D/D cDCs failed to repopulate the spleen of recipient mice, suggesting their poor survival in vivo. Furthermore, the expression of critical molecules for inflammatory responses was downregulated in PU.1D/D cDCs as compared to normal cDCs. These molecules included Myd88 and NFkB that are downstream molecules of TLR signaling, CD86 that is required for T cell stimulation, and CCR7 that is required for cDC migration. These results clearly show that PU.1 is required for development of the functional cDC pool, and the cDC pool plays a critical role in T cell homeostasis. Disclosures:No relevant conflicts of interest to declare.

Modulation of Myeloid-Derived Dendritic Cell Maturity: Unmasking a Novel Role for the Tumor Suppressor p15Ink4b in Immunity

Abstract 2469p15Ink4b, an inhibitor of cyclin-dependent kinases, is a tumor suppressor frequently associated with hematological malignancies. Its inactivation through DNA methylation is one of the most prevalent epigenetic alterations reported in up to 80% of all acute myeloid leukemia (AML) patients. p15Ink4b is also silenced in 50% of patients diagnosed with myelodysplastic syndromes and its silencing correlates with frequent disease progression into AML. During the leukemogenesis process, escape of pre-leukemic cells from immune clearance represents an important step in the establishment of leukemic disease. Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the regulation of immune responses. In immune surveillance, their primary function is to stimulate naïve T cells against pathogens and cancerous cells leading to their effective clearance. However, whether p15Ink4b plays a role in DC development has never been addressed.In this study, we found that expression of p15Ink4b is strongly induced in mouse splenic DCs and during the development of bone marrow-derived DCs (BM-DCs). Increased expression levels were also found during the development of human CD34-derived DCs suggesting an important role for p15Ink4b in DC maturation. To investigate the function of p15Ink4b during the differentiation and maturation of DCs we used the previously generated p15Ink4bfl/fl-LysMcre conditional knockout mice, where a myeloid-specific deletion of p15Ink4b closely mimics inactivation of the gene in AML. The knockout mice developed nonreactive monocytosis and were predisposed to retrovirus-induced AML. These results provided strong experimental evidence for a role of the gene as a tumor suppressor for myeloid leukemia.Myeloid-specific deletion of p15Ink4b in mice resulted in a reduction in the common DC progenitor pool as compared to wild type mice. p15Ink4bfl/fl-LysMcre mice had significantly fewer and less mature myeloid DCs (mDCs) than the wild type mice whereas other DC subtypes were not affected. Consistent with this data, BM cells from the p15Ink4bfl/fl-LysMcre mice cultured in vitro, generated BM-DCs that express lower levels of the antigen presenting (MHCII) and the co-stimulatory (CD80, CD86) molecules when activated with LPS. Re-expression of p15Ink4b in knockout BM-DCs resulted in an increase in the expression of both co-stimulatory molecules confirming a role for p15Ink4b in the regulation of the maturation process of DCs. The incomplete maturation of BM-DCs correlated with a reduced ability to activate T cells in a MHCII-mismatched mixed leukocyte reaction, and to uptake antigen suggesting that loss of p15Ink4b affects the function of BM-DCs.Taken together, our results indicate a novel role for p15Ink4b in mDC development, and suggest that frequent inactivation of p15Ink4b in myeloid malignancies could lead to an inefficient anti-leukemic immune response during leukemogenesis. Our data also have an important translational significance. AML blasts isolated from patients and differentiated ex-vivo into DCs represent a powerful immunotherapy tool. However, AML-DCs have reportedly a partially impaired maturation process as compared to DCs from healthy donors. We propose that re-expression of p15 in AML-DCs may overcome some of the limitations of a DC-based immunotherapy for AML patients. Disclosures:No relevant conflicts of interest to declare.

Dendritic cell lineage commitment is instructed by distinct cytokine signals

Dendritic cells (DC) develop from hematopoietic stem cells, which is guided by instructive signals through cytokines. DC development progresses from multipotent progenitors (MPP) via common DC progenitors (CDP) into DC. Flt3 ligand (Flt3L) signaling via the Flt3/Stat3 pathway is of pivotal importance for DC development under steady state conditions. Additional factors produced during steady state or inflammation, such as TGF-β1 or GM-CSF, also influence the differentiation potential of MPP and CDP. Here, we studied how gp130, GM-CSF and TGF-β1 signaling influence DC lineage commitment from MPP to CDP and further into DC. We observed that activation of gp130 signaling promotes expansion of MPP. Additionally, gp130 signaling inhibited Flt3L-driven DC differentiation, but had little effect on GM-CSF-driven DC development. The inflammatory cytokine GM-CSF induces differentiation of MPP into inflammatory DC and blocks steady state DC development. Global transcriptome analysis revealed a GM-CSF-driven gene expression repertoire that primes MPP for differentiation into inflammatory DC. Finally, TGF-β1 induces expression of DC-lineage affiliated genes in MPP, including Flt3, Irf-4 and Irf-8. Under inflammatory conditions, however, the effect of TGF-β1 is altered: Flt3 is not upregulated, indicating that an inflammatory environment inhibits steady state DC development. Altogether, our data indicate that distinct cytokine signals produced during steady state or inflammation have a different outcome on DC lineage commitment and differentiation.

Cell-intrinsic role for IFN-α–STAT1 signals in regulating murine Peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response

AbstractPlasmacytoid dendritic cells (pDCs) reside in bone marrrow and lymphoid organs in homeostatic conditions and typically secrete abundant quantities of type I interferons (IFNs) on Toll-like receptor triggering. Recently, a pDC population was identified within Peyer patches (PPs) of the gut that is distinguished by its lack of IFN production; however, the relationship of PP pDCs to pDCs in other organs has been unclear. We report that PP pDCs are derived from common DC progenitors and accumulate in response to Fms-like tyrosine kinase 3 ligand, yet appear divergent in transcription factor profile and surface marker phenotype, including reduced E2-2 and CCR9 expression. Type I IFN signaling via STAT1 has a cell-autonomous role in accrual of PP pDCs in vivo. Moreover, IFN-α enhances pDC generation from DC progenitors by a STAT1-dependent mechanism. pDCs that have been developed in the presence of IFN-α resemble PP pDCs, produce inflammatory cytokines, stimulate Th17 cell generation, and fail to secrete IFN-α on Toll-like receptor engagement. These results indicate that IFN-α influences the development and function of pDCs by inducing emergence of an inflammatory (Th17-inducing) antigen-presenting subset, and simultaneously regulating accumulation of pDCs in the intestinal microenvironment.

Identification of CCR9− murine plasmacytoid DC precursors with plasticity to differentiate into conventional DCs

AbstractWhereas the final differentiation of conventional dendritic cells (CDCs) from committed precursors occurs locally in secondary lymphoid or peripheral tissues, plasmacytoid dendritic cells (PDCs) are thought to fully develop in the bone marrow from common DC progenitors before migrating to the periphery. In our study, we define, for the first time, a subpopulation of CCR9− major histocompatibility complex class IIlow PDCs in murine bone marrow, which express E2-2 and are immediate precursors of CCR9+ fully differentiated PDCs. However, CCR9− PDCs have the plasticity to acquire the phenotype and function of CD11b+ CD8α− major histocompatibility complex class IIhigh CDC-like cells under the influence of soluble factors produced by intestinal epithelial cells or recombinant GM-CSF. This deviation from the PDC lineage commitment is regulated on the level of transcription factors reflected by down-regulation of E2-2 and up-regulation of ID2, PU.1, and BATF3. Thus, CCR9− PDCs are immediate PDC precursors that can be reprogrammed to differentiate into CDC-like cells with higher antigen-presenting and cytokine-producing capacity under the influence of the local tissue microenvironment.

Complexity of dendritic cell subsets and their function in the host immune system

Dendritic cells (DCs) are professional antigen-presenting cells that are critical for induction of adaptive immunity and tolerance. Traditionally DCs have been divided into two discrete subtypes, which comprise conventional and non-conventional DCs. They are distributed across various organs in the body and comprise a heterogeneous population, which has been shown to display differences in terms of surface marker expression, function and origins. Recent studies have shed new light on the process of DC differentiation and distribution of DC subtypes in various organs. Although monocytes, macrophages and DCs share a common macrophage-DC progenitor, a common DC progenitor population has been identified that exclusively gives rise to DCs and not monocytes or macrophages. In this review, we discuss the recent advances in our understanding of DC differentiation and subtypes and provide a comprehensive overview of various DC subtypes with emphasis on their function and origins. Furthermore, in light of recent developments in the field of DC biology, we classify DCs based on the precursor populations from which the various DC subsets originate. We classify DCs derived from common DC progenitor and pre-DC populations as conventional DCs, which includes both migratory and lymphoid-resident DC subsets and classify monocyte-derived DCs and plasmacytoid DCs as non-conventional DCs.

Asynchronous and modular repression of neutrophil production by IRF8 leads to the development of progenitors with partially overlapping lineage potentials. (153.24)

Abstract Most previous studies have assumed that the maturation of a particular blood cell type downstream of hematopoietic stem cells follows a singular developmental route; therefore the identification of distinct hematopoietic progenitors with partially overlapping lineage potentials has led to considerable controversy. For example, common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) both generate dendritic cells upon adoptive transfer in vivo. To determine the molecular mechanisms by which progenitors with partially overlapping lineage potentials arise, we examined the transcriptional changes that occur as lymphoid-primed multipotent progenitors (LMPPs) differentiate into CLPs, and as CMPs differentiate into common dendritic cell progenitors (CDPs). In both of these independent pathways, we determined that the expression of the transcription factor IRF8 was strongly induced. IRF8-/- mice showed reduced numbers of both CLPs and CDPs. Competitive bone marrow reconstitutions also demonstrated cell-intrinsic defects in the generation of CD8a- dendritic cells, previously reported to be normal in IRF8-/- mice. Microarray comparisons between dendritic cells derived from either wild type CMPs or CLPs demonstrated that these cells are identical. Thus, the asynchronous expression of IRF8 leads to the formation of progenitors with partially overlapping developmental potentials, which can resolve themselves to ultimately generate the same mature cell type.

TGF-β1 Accelerates Dendritic Cell Differentiation from Common Dendritic Cell Progenitors and Directs Subset Specification toward Conventional Dendritic Cells

Dendritic cells (DCs) in lymphoid tissue comprise conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that develop from common DC progenitors (CDPs). CDPs are Flt3(+)c-kit(int)M-CSFR(+) and reside in bone marrow. In this study, we describe a two-step culture system that recapitulates DC development from c-kit(hi)Flt3(-/lo) multipotent progenitors (MPPs) into CDPs and further into cDC and pDC subsets. MPPs and CDPs are amplified in vitro with Flt3 ligand, stem cell factor, hyper-IL-6, and insulin-like growth factor-1. The four-factor mixture readily induces self-renewal of MPPs and their progression into CDPs and has no self-renewal activity on CDPs. The amplified CDPs respond to all known DC poietins and generate all lymphoid tissue DCs in vivo and in vitro. Additionally, in vitro CDPs recapitulate the cell surface marker and gene expression profile of in vivo CDPs and possess a DC-primed transcription profile. TGF-β1 impacts on CDPs and directs their differentiation toward cDCs. Genome-wide gene expression profiling of TGF-β1-induced genes identified instructive transcription factors for cDC subset specification, such as IFN regulatory factor-4 and RelB. TGF-β1 also induced the transcription factor inhibitor of differentiation/DNA binding 2 that suppresses pDC development. Thus, TGF-β1 directs CDP differentiation into cDCs by inducing both cDC instructive factors and pDC inhibitory factors.

The Transcription Factor PU.1 Controls Dendritic Cell Development and Flt3 Cytokine Receptor Expression in a Dose-Dependent Manner

The transcription factor PU.1 plays multiple context and concentration dependent roles in lymphoid and myeloid cell development. Here we showed that PU.1 (encoded by Sfpi1) was essential for dendritic cell (DC) development in vivo and that conditional ablation of PU.1 in defined precursors, including the common DC progenitor, blocked Flt3 ligand-induced DC generation in vitro. PU.1 was also required for the parallel granulocyte-macrophage colony stimulating factor-induced DC pathway from early hematopoietic progenitors. Molecular studies demonstrated that PU.1 directly regulated Flt3 in a concentration-dependent manner, as Sfpi1(+/-) cells displayed reduced expression of Flt3 and impaired DC formation. These studies identify PU.1 as a critical regulator of both conventional and plasmacytoid DC development and provide one mechanism how altered PU.1 concentration can have profound functional consequences for hematopoietic cell development.

Origin of the Lamina Propria Dendritic Cell Network

CX(3)CR1(+) and CD103(+) dendritic cells (DCs) in intestinal lamina propria play a key role in mucosal immunity. However, the origin and the developmental pathways that regulate their differentiation in the lamina propria remain unclear. We showed that monocytes gave rise exclusively to CD103(-)CX(3)CR1(+) lamina propria DCs under the control of macrophage-colony-stimulating factor receptor (M-CSFR) and Fms-like thyrosine kinase 3 (Flt3) ligands. In contrast, common DC progenitors (CDP) and pre-DCs, which give rise to lymphoid organ DCs but not to monocytes, differentiated exclusively into CD103(+)CX(3)CR1(-) lamina propria DCs under the control of Flt3 and granulocyte-macrophage-colony-stimulating factor receptor (GM-CSFR) ligands. CD103(+)CX(3)CR1(-) DCs but not CD103(-)CX(3)CR1(+) DCs in the lamina propria constitutively expressed CCR7 and were the first DCs to transport pathogenic Salmonella from the intestinal tract to the mesenteric lymph nodes. Altogether, these results underline the diverse origin of the lamina propria DC network and identify mucosal DCs that arise from pre-DCs as key sentinels of the gut immune system.

Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow

Lymphoid tissue plasmacytoid and conventional dendritic cells (DCs) are continuously regenerated from hematopoietic stem cells. The cytokine dependence and biology of plasmacytoid and conventional DCs suggest that regeneration might proceed through common DC-restricted developmental intermediates. By selecting for cytokine receptor expression relevant to DC development, we identify here highly cycling Lin(-)c-Kit(int)Flt3(+)M-CSFR(+) cells with a distinct gene-expression profile in mouse bone marrow that, on a clonal level in vitro and as a population both in vitro and in vivo, efficiently generated plasmacytoid and conventional DCs but no other lineages, which increased in number after in vivo injection of the cytokine Flt3 ligand. These clonogenic common DC progenitors thus define a cytokine-regulated DC developmental pathway that ensures the supply of various DC populations.

Isolation of a Common Natural Type-I Interferon Producing Cell and Dendritic Cell Progenitor Population in Mouse Bone Marrow.

Most type-I interferon producing cells (IPCs) and dendritic cells (DCs) are non-dividing cells with a short in vivo half-live of several days, and thus need to be continuously replaced. A common differentiation pathway for IPCs and DCs, and accordingly, the existence of common IPC and DC progenitors remains controversial. Flt3-ligand (Flt3L) is a non-redundant cytokine for in vivo IPC and DC development: IPC and DC differentiation potential is confined to Flt3+-hematopoietic progenitors; Flt3L KO mice show massively reduced IPCs and DCs. In contrast to Flt3, the “myeloid” cytokines GM-CSF and M-CSF seem to be less relevant in steady-state IPC and DC differentiation, however, they might be critically important in inflammatory conditions.To identify a candidate common IPC and DC progenitor population, we evaluated Flt3 and “myeloid” cytokine receptor expression in mouse bone marrow. We found that c-kitintlin− cells contained a Flt3+M-CSFR+ fraction that in Flt3L supplemented cultures gave rise to about 95% pure CD11c+MHC class II+ cells, consisting of both CD11c+B220+ IPCs and CD11c+B220− DCs, at a efficiency comparable to that of hematopoietic stem cells. In the presence of GM-CSF, Flt3+M-CSFR+c-kitintlin− cells gave rise to CD11c+CD11b+ DCs but not CD11c−CD11b+ macrophages/monocytes. Furthermore, Flt3+M-CSFR+c-kitintlin− cells possessed very poor, if any activity in myeloid colony forming assays, and lacked pre-B cell colony forming activity.In both, lethally and sub-lethally irradiated mice, transferred Flt3+M-CSFR+c-kitintlin− cells differentiated into CD11c+B220+ IPCs, CD11c+CD8α+, and CD11c+CD8α− conventional DC subsets, while no other hematopoietic cells were detectable. In vivo reconstitution and CFSE-labeling experiments showed that Flt3+M-CSFR+c-kitintlin− cells extensively proliferate in the lethally irradiated mice, reaching peak progeny levels of IPC and DC at day 10 after transplantation, indicating high proliferative, but limited self-renewal capacity of these cells. Quantitative RT-PCR analysis revealed high expression of DC and IPC-development affiliated genes (such as PU.1, STAT3, GM-CSFR, and CX3CR1), but no lymphoid- and erythroid-development affiliated gene transcription. These data suggest the existence of common developmental intermediates for both IPCs and DCs in mouse bone marrow, and thus might provide new insights into the regulation of IPC and DC differentiation in steady-state and inflammation.

Essential role for ICSBP in the in vivo development of murine CD8α+dendritic cells

AbstractInterferon (IFN) consensus sequence-binding protein (ICSBP) is an important transcription factor regulating proinflammatory cytokine production and the development of mononuclear phagocytes in vitro. Here we analyzed the role of ICSBP in the in vivo differentiation of 3 major subsets of murine dendritic cells (DCs). We found that ICSBP is predominantly expressed by the CD8α+subset, and more important, that ICSBP−/−mice have a profound and selective deficiency in CD8α+DEC205+DCs in lymphoid tissues. Studies using wild-type/ICSBP−/−chimeras revealed that this defect in CD8α+DC development is intrinsic to bone marrow–derived progenitors and not dependent on ICSBP expression in the nonhemopoietic compartment. Because DC precursor frequencies are unaltered in the bone marrow of ICSBP−/−mice, ICSBP appears to function by regulating CD8α+DC differentiation downstream from the generation of common DC progenitors. Although CD8α−DCs are present in normal numbers in ICSBP−/−animals, up-regulation of CD40, CD80, and major histocompatibility complex (MHC) class II expression was found to be impaired in this subset after in vivo microbial stimulation. Together these results demonstrate that ICSBP is critically required for the in vivo differentiation of CD8α+DCs and may also influence the functional maturation of the CD8α−subsets.