Common Dendritic Progenitors Research Articles

In Utero Hematopoietic Cell Transplantation of Lineage- c-Kit- Sca-1- Cells Is Responsible for Tolerance Induction

IntroductionIn utero hematopoietic cell transplantation (IUHCT) of allogenic bone marrow mononuclear cells (BM MNC) results in mixed hematopoietic chimerism in the murine model. In contrast, highly enriched HSCs (LSKs: Lin- c-Kit+ Sca-1+ cells) do not engraft after allogeneic IUHCT. Tracking studies in the first 72 hours after IUHCT demonstrate relatively large numbers of donor cells in the fetal thymus after IUHCT of BM MNCs, but not after IUHCT of LSK cells. This suggests that a sub-population of BM MNCs are capable of homing to the thymus within a window of opportunity for tolerance induction and that this population is lost, with enrichment of HSC.The aim of this study is to identify the specific cell population within BM MNCs that is responsible for tolerance induction. To achieve this goal, we developed a practical model for determination of prenatal tolerance induction and tested subpopulations of BM MNC for their ability to induce tolerance.MethodsDonor BM MNCs and subpopulations were isolated from 6-week old C57BL/6TgN(act-EGFP)OsbY01) (B6GFP) mice. Lineage (CD3, B220, CD11b, Gr-1) or CD3 depletion of donor cells was performed using magnetic sorting (Auto-MACS Pro Separator) and anti-Biotin microbeads.To isolate the Lin- subpopulations, anti-biotin antibody was used to exclude Lin+ cells, and then the remaining cells were stained for c-Kit and Sca-1 markers.Donor subpopulations were generated by sorting using the FACSAria.IUHCT was performed at E14 in Balb/c fetuses via vitelline vein injection. Post-natal injections were performed at P0 through the facial vein. Donor cell engraftment was assessed in peripheral blood at 4 and 12 weeks of age by flow cytometry (% GFP+ cells within CD45+ population).Thymi and livers of E16 fetuses were harvested and GFP cells were detected using a fluorescence stereomicroscope.Statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Results are expressed as mean±SEM. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children's Hospital of Philadelphia.ResultsTo develop the model for tolerance assessment we determined that injection of 10^7 CD3 depleted BM MNCs into Balb/c pups at P0 resulted in no engraftment. In contrast, IUHCT at E14 of 10^7 BM MNCs resulted in chimerism (13.2±2.01% chimerism) at 4 weeks with associated donor specific tolerance. Finally, P0 injection of 10^7 CD3 depleted BM MNCs into in mice that had received E14 injections of 10^7 BM MNCs resulted in engraftment with enhancement of chimerism at 4 weeks (38.02±3.67% in E14 plus P0 group vs 0.008±0.005 in P0 only group, p=0.0001).After establishing the model to test tolerance, we performed IUHCT of subpopulations of cells derived from BM MNCs, using the dose of each subpopulation found in 10^7 BM MNCs, to test their potential for tolerance induction. Mice that underwent IUHCT of 10^5 Lin- cells prior to the P0 injection of 10^7 CD3depleted BM MNCs had higher chimerism at 4 weeks than mice that received E14 injections of 10^5 Lin+ cells (9.843±0.79% vs 0.088±0.056%, p=0.0001).Thymuses were harvested from E16 fetuses that were injected at E14 with different subsets of the Lin- population (c-Kit+ Sca-1+, c-Kit- Sca-1+, c-Kit+ Sca-1-, c-Kit- Sca-1int, and c-Kit- Sca-1- cells). The only thymuses to show GFP cells at E16 were those that had received E14 Lin- c-Kit- Sca-1- cells. Mice that received Lin- c-Kit- Sca-1- cells at E14 prior to P0 injections showed chimerism at 4 weeks when compared to those that had received Lin- c-Kit+ Sca-1-, Lin- c-Kit+ Sca-1+, Lin- c-Kit- Sca-1int, or Lin- c-Kit- Sca-1hi cells at E14, in which chimerism was absent (6.755±0.95% vs 0.0%; p=0.02.)ConclusionWe have demonstrated that it is the Lin- c-Kit- Sca-1- subpopulation of BM MNCs that induces tolerance and prevents rejection of subsequent injections from the same donor. We have also shown that only the Lin- c-Kit- Sca-1- population migrates to the thymus in time for the induction of central tolerance. Furthermore, mouse fetuses that were given Lin- c-Kit- Sca-1- intravascularly at E14 were able to become chimeric with a postnatal CD3 depleted MNCs transplantation without immunosuppression or myeloablation. These findings suggest that a c-Kit- Sca-1- progenitor is responsible for tolerance induction, likely via common dendritic progenitors that home to the thymus. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TNFα Rescues Dendritic Cell Development in Hematopoietic Stem and Progenitor Cells Lacking C/EBPα.

Dendritic cells (DCs) are crucial effectors of the immune system, which are formed from hematopoietic stem and progenitor cells (HSPCs) by a multistep process regulated by cytokines and distinct transcriptional mechanisms. C/EBPα is an important myeloid transcription factor, but its role in DC formation is not well defined. Using a CebpaCre-EYFP reporter mouse model, we show that the majority of splenic conventional DCs are derived from Cebpa-expressing HSPCs. Furthermore, HSPCs isolated from Cebpa knockout (KO) mice exhibited a marked reduced ability to form mature DCs after in vitro culture with FLT3L. Differentiation analysis revealed that C/EBPα was needed for the formation of monocytic dendritic progenitors and their transition to common dendritic progenitors. Gene expression analysis and cytokine profiling of culture supernatants showed significant downregulation of inflammatory cytokines, including TNFα and IL-1β as well as distinct chemokines in KO HSPCs. In addition, TNFα-induced genes were among the most dysregulated genes in KO HSPCs. Intriguingly, supplementation of in vitro cultures with TNFα at least partially rescued DC formation of KO HSPCs, resulting in fully functional, mature DCs. In conclusion, these results reveal an important role of C/EBPα in early DC development, which in part can be substituted by the inflammatory cytokine TNFα.

Spatial Mapping of Myelopoiesis Reveals the Bone Marrow Niche for Monocyte Dendritic Cell Progenitors

Monocytes, macrophages and dendritic cells are indispensable for innate immunity. Myelopoiesis takes place in the bone marrow (BM) through differentiation of a common myeloid progenitor into monocyte dendritic cell progenitors (MDP) or granulocyte monocyte progenitors (GMP). MDP differentiate into myeloid progenitors (MoP) or common dendritic progenitors (cDP) that give rise to monocytes or dendritic cells (DC) repectively. GMP can also generate neutrophil-like monocytes via an intermediate monocyte progenitor [MP; Immunity. 2017 47(5):890] and neutrophils via a granulocyte progenitor (GP). CSF1 (M-CSF) is the key cytokine that regulates monopoiesis, CSF1 loss causes profound defects in monocyte, dendritic cell, macrophage and osteoclast generation. Classical studies using Csf1-/- chimeric mice demonstrated that CSF1 is produced by BM stromal cells. However the identity of these CSF1-producing cells is unknown. A major limitation is the lack of immunofluorescence protocols to map progenitor interaction with candidate niches. We have found that CD11b-Ly6C-CD117+CD115+ cells are MDP; Lin-Ly6C-CD117+CD16/32+CD115- cells are GMP; CD11b-Ly6C+CD117+CD115+ are MP/MoP; CD11b-Ly6C+CD117+CD115- are GP; CD11b+CD115+Ly6Chi and CD11b+CD115+Ly6Clo cells are classical and non-classical monocytes; and MHCIIhi reticular cells are DC. We used the markers above to map the 3D position of every myeloid cell in the sternum and assessed the relationships between myeloid progenitors, their offspring and candidate niches in situ with single cell resolution. To test whether the interactions observed were specific we obtained the X, Y and Z coordinates for every hematopoietic cell in the sternum (detected using αCD45 and αTer119). We then used these coordinates to randomly place each type of myeloid cell, at the same frequencies found in vivo, through the BM to generate a random distribution for each myeloid cell type. HSC localize to sinusoidal, arteriolar and endosteal niches. However, myeloid progenitors are exclusively perisinusoidal (mean MDP distance to sinusoids, arterioles, and endosteum observed 5, 134, and 105μm vs 9, 86, and 69µm in the random simulation). Myeloid progenitors rarely localize with HSC indicating that progenitors abandon the HSC niche upon differentiation. Strikingly, we found that granulopoiesis, monopoiesis and DCpoiesis occur in distinct sinusoidal locations and that MDP are tightly associated with sinusoids, dendritic cells and Ly6Clo monocytes (2.0 DC and 4.4 Ly6Clo monocytes observed within 50µm of an MDP vs 0.9 DC and 1.8 Ly6Clo monocytes in the random simulation p=0.02 and p<0.0001) but not with MoP/MP or Ly6Chi classical monocytes. The results above suggest that the stromal cells that provide the signals that regulate MDP will localize to the sinusoids. Analyses of Csf1 expression in two recently published scRNAseq studies of BM stroma showed that perivascular stromal cells and osteoblastic cells are the major CSF1 sources with sinusoidal endothelial cells expressing much lower levels. Csf1 deletion in perivascular cells using LepR-cre mice and in osteoblastic cells using Ocn-cre mice did not impact Ly6Chi classical or Ly6Clo non-classical monocytes in peripheral blood. We also did not find any defects in BM MDP, GMP, MoP numbers or colony forming activity or in monocyte or dendritic cell numbers. In sharp contrast we found that conditional Csf1 deletion in endothelial cells using Cdh5-cre mice led to a 3.9-fold defect in Ly6Clo non-classical monocytes in the blood (1.89 vs 0.47 x105/ml in the +/- controls vs Cdh5-cre:Csf1-/Δ mice; p=0.03). In the BM these mice showed a 1.4 reduction in MDP numbers (0.72 vs 0.5x104/femur; p=0.04) further compounded by a 2.7-fold loss in MDP-derived CFU-M (22 vs 8 colonies/100 cells; p=0.009) indicating a dramatic reduction in MDP function. This in turn led to a 2.3-fold reduction in Ly6Clo non classical monocytes (9.5 vs 4.1x104/femur; p=0.01) and a 1.2-fold reduction in cDC (2.7 vs 2.1 x104cDC/femur p=0.005). In summary we have imaged for the first time myeloid progenitors; mapped their differentiation into mature myeloid cells; quantified their interaction with candidate niche cells; showed that sinusoids are the exclusive site of monocyte and dendritic cell production; and demonstrated that endothelial cells are a niche that regulates MDP numbers and function via CSF1. Disclosures No relevant conflicts of interest to declare.

Ikaros cooperates with Notch activation and antagonizes TGFβ signaling to promote pDC development.

Plasmacytoid and conventional dendritic cells (pDCs and cDCs) arise from monocyte and dendritic progenitors (MDPs) and common dendritic progenitors (CDPs) through gene expression changes that remain partially understood. Here we show that the Ikaros transcription factor is required for DC development at multiple stages. Ikaros cooperates with Notch pathway activation to maintain the homeostasis of MDPs and CDPs. Ikaros then antagonizes TGFβ function to promote pDC differentiation from CDPs. Strikingly, Ikaros-deficient CDPs and pDCs express a cDC-like transcriptional signature that is correlated with TGFβ activation, suggesting that Ikaros is an upstream negative regulator of the TGFβ pathway and a repressor of cDC-lineage genes in pDCs. Almost all of these phenotypes can be rescued by short-term in vitro treatment with γ-secretase inhibitors, which affects both TGFβ-dependent and -independent pathways, but is Notch-independent. We conclude that Ikaros is a crucial differentiation factor in early dendritic progenitors that is required for pDC identity.

Impact of the c-MybE308G mutation on mouse myelopoiesis and dendritic cell development.

Booreana mice carrying the c-Myb308G point mutation were analyzed to determine changes in early hematopoiesis in the bone marrow and among mature cells in the periphery. This point mutation led to increased numbers of early hematopoietic stem and progenitor cells (HSPCs), with a subsequent reduction in the development of B cells, erythroid cells, and neutrophils, and increased numbers of myeloid cells and granulocytes. Myelopoiesis was further investigated by way of particular subsets affected. A specific question addressed whether booreana mice contained increased numbers of dendritic-like cells (L-DC subset) recently identified in the spleen, since L-DCs arise in vitro by direct differentiation from HSPCs co-cultured over splenic stroma. The non-lethal c-Myb mutation in booreana mice was associated with significantly lower representation of splenic CD8- conventional dendritic cells (cDCs), inflammatory monocytes, and neutrophils compared to wild-type mice. This result confirmed the bone marrow origin of progenitors for these subsets since c-Myb is essential for their development. Production of L-DCs and resident monocytes was not affected by the c-MybE308G mutation. These subsets may derive from different progenitors than those in bone marrow, and are potentially established in the spleen during embryogenesis. An alternative explanation may be needed for why there was no change in CD8+ cDCs in booreana spleen since these cells are known to derive from common dendritic progenitors in bone marrow.

Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase.Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. DisclosuresNo relevant conflicts of interest to declare.

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.

Distinct Progenitor Origin Distinguishes a Lineage of Dendritic-Like Cells in Spleen

The dendritic cell (DC) compartment comprises subsets of cells with distinct phenotypes. Previously this lab reported methodology for hematopoiesis of dendritic-like cells in vitro dependent on a murine splenic stromal cell line (5G3). Co-cultures of lineage-depleted bone marrow (Lin− BM) over 5G3 continuously produced a distinct population of dendritic-like “L-DC” for up to 35 days. Here the progenitor of L-DC is investigated in relation to known BM-derived hematopoietic progenitors. It is shown here that L-DC-like cells also derive from the CD150+Flt3− long-term reconstituting-hematopoietic stem cells (HSC), and also from the Flt3+ multipotential progenitor subset in BM. Lin− BM co-cultures also produce a transient population of cells resembling conventional (c) DC. Production of cDC-like cells is shown here to be transient and M-CSF dependent, and also appears following co-culture of described common dendritic progenitors or monocyte dendritic progenitors over 5G3. BM cells from C57BL/6-flt3Ltm1lmx and C57BL/6-Csf2tm1Ard mice which lack cDC precursors and monocytes, are shown here to contain L-DC progenitors which can seed 5G3 co-cultures. L-DC are functionally distinct cells, in that they arise independently of M-CSF, and by direct differentiation from HSC.

Differential requirement for Hoxa9 in the development and differentiation of B, NK, and DC-lineage cells from Flt3+ multipotential progenitors

Hoxa9 is a homeodomain transcription factor important for the generation of Flt3+hiIL-7R- lymphoid biased-multipotential progenitors, Flt3+IL-7R+ common lymphoid progenitors (CLPs), and B cell precursors (BCP) in bone marrow (BM). In addition to B-cell, Flt3+IL-7R+ CLPs possess NK and DC developmental potentials, although DCs arise from Flt3+IL-7R- myeloid progenitors as well. In this study, we investigated the requirement for Hoxa9, from Flt3+ or Flt3- progenitor subsets, in the development of NK and DC lineage cells in BM. Flt3+IL-7R+Ly6D- CLPs and their Flt3+IL-7R+Ly6D+ B lineage-restricted progeny (BLP) were significantly reduced in hoxa9−/− mice. Interestingly, the reduction in Flt3+IL-7R+ CLPs in hoxa9−/− mice had no impact on the generation of NK precursor (NKP) subsets, the differentiation of NKP into mature NK cells, or NK homeostasis. Similarly, percentages and numbers of common dendritic progenitors (CDP), as well as their plasmacytoid or conventional dendritic cell progeny in hoxa9−/− mice were comparable to wildtype. These findings reveal distinct requirements for Hoxa9 or Hoxa9/Flt3 molecular circuits in regulation of B versus NK and DC development in BM.

Direct Sensing of Toll-Like Receptor Agonists by Dendritic Cell Progenitors Leads to Their Homing to Inflamed Lymph Nodes Via Regulation of CXCR4 and CCR7

AbstractAbstract 3895Dendritic cells (DCs), the key antigen-presenting cell population, continuously need to be regenerated from bone marrow (BM) hematopoietic stem and progenitor cells. Common dendritic progenitors (CDP) were previously shown to efficiently generate DCs in lymphoid and non-lymphoid tissues. How the dissemination of bone marrow (BM) DC-progenitors to peripheral tissues is regulated upon demand remains elusive to date. Acute microbial infections are sensed via Toll-like receptors (TLR). Recent studies showed that stem and progenitor cells express TLRs. We found that CDPs in the BM of mice express relative high levels of Tlr2, Tlr4 and Tlr9, and hypothesized that these might be involved in regulating CDP migration. CDPs in steady-state expressed high levels of Cxcr4, but no, or low Ccr7. Upon direct stimulation with the respective TLR-agonists in vitro, CDPs rapidly down-regulated Cxcr4 and up-regulated Ccr7 mRNA and protein. CDPs that were stimulated with TLR-agonists for only 2 h preferentially homed to the lymph nodes (LN) in expense of BM in steady-state recipients. When TLR-agonists were injected subcutaneously, CDPs gave rise to increased numbers of plasmacytoid DCs, classical DCs, and DCs with a skin-derived migratory phenotype in inflamed LNs on day 4. This was not due to increased proliferative activity. Injecting the CXCR4 antagonist AMD3100 demonstrated that the retention of CDPs in the BM depends on CXCR4. Furthermore, CCR7 was important for the engraftment of CDP-derived DCs into LNs in steady-state and during inflammation. In conclusion, DC progenitors in the bone marrow are capable to directly sense TLR-agonists via their cognate receptors in systemic infections. This results in differential expression of chemokine receptors and consecutive migration of DC-progenitors to inflamed LNs. This mechanism helps to restore DC subsets during ongoing immune responses and to return to DC homeostasis once the inflammation ceases. Disclosures:No relevant conflicts of interest to declare.