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Blood, 1 August 2006, Vol. 108, No. 3, pp. 878-885. Prepublished online as a Blood First Edition Paper on February 28, 2006; DOI 10.1182/blood-2005-11-4545.
HEMATOPOIESIS Mouse plasmacytoid dendritic cells derive exclusively from estrogen-resistant myeloid progenitorsFrom the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA; and Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA.
Current models predict that mouse plasmacytoid dendritic cells (PDCs) derive from lymphoid progenitors. However, we show PDCs arise exclusively from common myeloid progenitors (CMPs) characterized by low-level expression of several lymphoid-associated genes, including a RAG2/GFP reporter transgene. This conclusion is supported by both adoptive transfer experiments and an estrogen treatment strategy that led to marked depletion of very early lymphoid progenitors without affecting RAG2/GFP+ CMPs or the developmental kinetics, RAG-mediated recombinase activity, and cytokine production of PDCs. These data suggest that PDCs arise exclusively from early myeloid progenitors and that promiscuous low-level expression of lymphoid-associated genes is a general feature of PDC progenitors among CMPs.
Mouse plasmacytoid dendritic cells (PDCs) were first described in 2001.1-3 Similar to human PDCs, mouse PDCs are relatively inefficient at presenting pathogenic peptides to immune effector cells,4 but are potent producers of type I interferons, which can induce TH1 polarization in CD8+ T cells and stimulate natural killer (NK) cell-mediated cytotoxicity.5,6 These functions are mediated through the Toll-like receptors (TLRs) specific for viral single-stranded RNA and CpG-containing motifs, TLR-7 and TRL-9, respectively.7
Little is known about the mechanisms underlying PDC differentiation from hematopoietic stem cells (HSCs); however, several lines of evidence suggest significant overlap between the molecular circuitry required for PDC and lymphocyte development. First, PDCs express an array of genes associated with lymphocyte development or function,8-10 and a small fraction of PDCs contain DH-JH rearrangements on the Ig heavy-chain (IgH) locus.11 Second, progenitor pools enriched for lymphoid precursors, including cells designated common lymphoid progenitors (CLPs) or more HSC-proximal early lymphoid progenitors (ELPs) described by Kincade and colleagues, yield PDCs following transfer into adoptive hosts.10,12-15 In addition, several reports demonstrate that myeloid progenitors can also generate PDCs.16 Moreover, recent data suggest that common myeloid progenitor (CMP)–derived PDCs express the lymphoid-associated genes pre-T Currently there are 2 opposing models describing precursor-product relationships for early lymphoid and myeloid progenitors. The first predicts that myeloid- and lymphoid-restricted progenitors can be cleanly identified and separated based on distinct cell surface phenotypes. Data supporting this viewpoint include descriptions of CMP and CLP populations defined by their restricted differentiation potentials for myeloid and lymphoid lineages, respectively.17,18 However, certain observations appear to conflict with this model. For instance, CMPs generate a small but persistent number of B cells,12 and a recently described progenitor thought to be downstream of the CLP retains macrophage potential in vitro.19 The requirement that all lymphoid cells must pass through a CLP intermediate during steady-state lymphopoiesis has also not been determined conclusively. Indeed, early T-lineage progenitors (ETPs) in the thymus can arise independent of CLPs,20 and initiation of recombinase activity among CLPs requires the B lineage–restricted enhancer element eRAG.21 These observations suggest an alternative model in which the CLP population consists primarily of early B-lineage progenitors (EBPs) characterized by residual T-lineage potential. Thus, the ability of EBPs/CLPs to generate PDCs may also reflect the natural differentiation plasticity of early hematopoietic progenitors. We used a combination of experimental strategies to determine whether PDCs arise from early lymphoid versus early myeloid progenitors. These strategies included single and competitive bone marrow (BM) chimeras established by transfer of well-defined multipotent lymphoid and myeloid progenitor populations, evaluation of the impact of the age-associated loss and the estrogen-mediated depletion of ELP pools on PDC development, and an assessment of whether lymphoid-associated genes expressed by BM PDCs are also expressed by PDC progenitors within the CMP pool. Our data indicate that PDCs arise predominantly from a subset of estrogen-resistant Flt3/Flk2+ CMPs that are further characterized by low-level expression of a RAG2/GFP reporter transgene and the additional lymphoid-associated genes terminal deoxynucleotidyl transferase (TdT) and sterile IgH (µ0) transcripts.
Mice Six- to 10-week-old C57BL/6 mice and B6.Ly5.2 (referred to herein as B6.Ly5SJL) mice were purchased from the National Cancer Institute animal facility (Frederick, MD) or the Jackson Laboratory (Bar Harbor, ME). (C57BL/6 x B6.Ly5SJL) F1s and aged C57BL/6 mice were generated and maintained in our animal colony. NG-BAC (RAG2/GFP) transgenic reporter mice22 were kindly provided by Dr Michel Nussenzweig (Rockefeller University, New York, NY). H2-SVEX21 transgenic mice were maintained in our animal colonies. Antibodies and analytical flow cytometry
For flow cytometric analyses BM, spleen, and thymus suspensions were prepared and stained with optimal dilutions of directly conjugated fluorescent antibodies as previously described,23 then analyzed on a 10-color LSR II flow cytometer (Becton Dickinson, San Jose, CA) equipped with 4 lasers for excitation of UV-, violet-, blue-, and red-excited dyes. Antibodies used included fluorescein (FL), phycoerythrin (PE), PE-Cy5.5, PE-Cy7, allophycocyanin (APC), APC-Cy7, or biotin (BI)–conjugated versions of purified antibodies to the following cell surface antigens: B220 (RA3-6B2), CD11b (M1/70), Gr-1 (8C5), Ter-119, CD3 (2C11), CD127/IL-7R Cell sorting Cell suspensions were applied to an 11-parameter MoFlo (DakoCytomation, Fort Collins, CO) or one of two 12-parameter FACSDiva high-speed cell sorters. The MoFlo and one of the two FACSDivas are equipped with 2 Coherent Innova argon lasers tuned for blue (488 nm) or UV (351/363) excitation and a Coherent Spectrum argon/krypton laser tuned to 647 nm for excitation of APC and its derivatives. The alternative FACSDiva is equipped with argon and argon/krypton lasers for excitation in the blue and red wavelengths as described and a Coherent Innova 302C krypton laser tuned to 407 nm for violet excitation. For the MoFlo, stained cell suspensions were applied at a sheath pressure of 60 psi with a drop delay of approximately 98 000 drops/s. This resulted in sorting rates of 28 000 to 30 000 cells/s with abort rates of 10% to 12%. For the FACSDiva, cells were applied at a sheath pressure of 40 psi and a drop delay frequency of approximately 70 000 drops/s resulting in sort trigger rates of 20 000 to 25 000 cells/s. Intravenous transfers Hosts were maintained on water containing a Bactrim suspension (400 mg sulfamethoxazole and 80 mg trimethoprim/500 mL water) for 1 week prior to, and at least 3 weeks following, lethal (900 R) irradiation. Hosts were irradiated 1 day before intravenous transfer of 500 to 1000 sorted progenitor populations mixed with either additional sorted precursors or 1 to 2 x 105 unfractionated host-type BM cells as indicated. In mice receiving CMPs subdivided based on Flt3/Flk2 expression, 2000 donor cells were given per recipient. Intrathymic transfers Intrathymic transfers were performed as previously described.23 Briefly, 500 freshly sorted BM progenitors from female C57BL/6 (Ly5B6) adults were injected into thymi of anesthetized female B6.Ly5SJL mice given 500 R 6 hours previously. Estrogen administration
17 In vivo BrdU labeling Continuous in vivo BrdU labeling was performed as previously described27 with the addition of the appropriately conjugated antibodies. Briefly, adult estrogen-treated and control C57BL/6 mice were inoculated with 0.5 mg BrdU (Sigma, St Louis, MO) in PBS every 12 hours for 0.5 to 7 days. BM and spleen cells were stained with PE-CD19, PE-CD3, PE-NK1.1, APC-Cy7 B220, APC-CD11c, and biotinylated Ly6C followed by streptavidin-Pacific blue in standard fluorescence-activated cell sorting (FACS) buffer, washed twice with protein-free PBS, then permeabilized using "Fix and Perm" (Caltag). Subsequently, cells were washed, incubated with DNaseI, washed, and then stained with FL-anti-BrdU antibodies (Becton Dickinson) before analysis. RT-PCR
Cells were sorted directly into RNA-lysis buffer consisting of 4.23 mM guanidium isothiocyanate, 0.67% Sarcosyl, 33.3 µM citrate buffer, and 134 mM 2-ME and extracted and RNA precipitated as described.28 cDNA for each sample was synthesized using the First Strand cDNA synthesis kit (Roche Applied Science, Indianapolis, IN) before loading normalization by polymerase chain reaction (PCR) using Methylcellulose cultures Methylcellulose culture conditions were as previously described.17 Triplicate cultures were established by plating sorted cells at 500 cells/plate according to the manufacturer's instructions. On day 12, discrete colonies were counted and typed by morphology: M, colony-forming unit (CFU) macrophage; G, CFU granulocyte; GM, CFU granulocyte/macrophage, E, burst-forming unit (BFU) erythroid; GEM, granulocytes, erythrocytes, and macrophages; Mix, GEM plus megakaryocytes. ELISA
BM PDCs (CD19–CD3–NK1.1–CD11c+PDCA+CD11b–B220+Ly6c+) were sorted as described (see "Cell sorting"). Triplicate cultures, each containing 3 x 104 sorted cells, were stimulated in vitro with influenza virus at 300 U/mL overnight in triplicate. Supernatant was collected and analyzed using an enzyme-linked immunosorbent assay (ELISA) kit specific for IFN- Statistical analyses Statistical analyses were performed by calculating the mean and the standard error of the mean (SEM) of data generated from a minimum of 3 mice per group or an unpaired Student t test.
Age-associated loss of ELPs but not PDCs
Current data suggest that potential PDC precursors are enriched among Flt3/Flk2+ early lymphoid and myeloid precursors.10,12,13 Candidate Flt3/Flk2+ lymphoid progenitors include IL-7R These data raise questions about whether Flt-3+ ELPs are obligate precursors for BM PDCs. Alternatively, the source of BM PDCs and the resulting cellular dynamics associated with BM PDC development, including their cellular half-life, may be altered in the aged environment. Accordingly, we initiated a series of experiments to test the degree to which both early lymphoid and early myeloid progenitors generate PDCs. These experiments included the use of single and double BM chimeras to test the PDC differentiation potential of well-defined Flt3/Flk2+ lymphoid progenitors, and estrogen-depletion studies designed to test the impact of selective depletion of early lymphoid progenitors on PDC differentiation. EBPs/CLPs do not yield PDCs in vivo
Our first set of adoptive transfer experiments was designed to compare the relative capacity of Flt3/Flk2+ cells within the EBP/CLP and LSK-Flt3+ progenitor pools to generate splenic PDCs in vivo. Five hundred sorted LSK-Flt-3+ cells or EBPs/CLPs (Lin–IL-7R
Lymphoid-specified ELPs are inefficient PDC progenitors Given that c-kithigh LSK-Flt3+ precursors were more efficient than EBPs/CLPs at generating PDCs (Figure 2) and BM PDCs exhibit evidence of ongoing or past RAG2 expression (see Figure 6D), we hypothesized that PDCs arise primarily from the c-kithighFlt3/Flk2+RAG1/2+ ELP population described by Igarashi et al.31 However, because CMPs also generate PDCs following adoptive transfer, we established a competitive scenario to test the relative efficiency with which each population contributes to the PDC pool. Accordingly, we established mixed BM chimeras in which 1000 sorted CMPs and ELPs (or EBPs/CLPs) were coinjected into the same recipients. For these experiments, c-kithigh ELPs and EBPs/CLPs were identified and sorted from C57BL/6-backcrossed NG-BAC (Ly5B6+) transgenics (Figure 3A), CMPs were sorted from B6.Ly5SJL congenics, and 1000 cells from each population were mixed together with 250 host-type HSCs before transfer into (C57BL/6 x B6.Ly5SJL) F1s. With this strategy, EBP/CLP- and ELP-derived cells were identified as Ly5B6+Ly5SJL–, CMP-derived cells were identified as Ly5B6–Ly5SJL+, and host-type HSC-derived cells were Ly5B6+Ly5SJL+. Surprisingly, as shown in Figure 3B-C neither EBPs/CLPs nor ELPs generated significant numbers of PDCs in mixed BM chimeras. Indeed, analyses of multiple recipients within each group showed that whereas on average CMPs produced 40% of the total PDC pool in adoptive hosts, equal numbers of ELPs and EBPs/CLPs generated 3% and less than 1% of the PDC pool, respectively. These data suggest that PDCs arise primarily from within the CMP progenitor pool.
Lymphoid-related gene expression among Flk2+CMP Because our adoptive transfer studies identified CMPs as the most efficient PDC progenitors, we next sought to better understand the cellular pathway underlying the activation of lymphoid-associated genes in BM PDCs. We began by assessing RAG2/GFP expression in CMPs and related myelo-erythroid BM progenitor fractions from NG-BAC mice using the flow cytometric strategy described by Akashi et al.17 Although evidence for RAG-1/2 expression has not been obtained through the analysis of RAG1/GFP knock-in mice,10 we reasoned that NG-BAC transgenic mice might provide a more sensitive assessment of low levels of RAG2 transcription due to the presence of multiple copies of the BAC transgene. Indeed, as shown in Figure 4A, 20% to 30% of CMPs from NG-BAC transgenic mice were RAG2/GFPlow, and GFP expression in these cells correlated with surface expression of Flt3/Flk2 and AA4. Further, though we did not reproducibly observe evidence for RAG1 or RAG2 transcripts by RT-PCR, we routinely were able to amplify TdT and µ0 transcripts from sorted RAG2/GFPlow CMPs (Figure 4B). Compared to their RAG2/GFP–Flt3/Flk2– counterparts, RAG2/GFPlowFlt3/Flk2+ CMPs also generated relatively few myeloid colonies in methylcellulose cultures optimized for myeloid lineage differentiation (Figure 4C), and consistent with previous data12,13 PDC differentiation potential among CMP subsets as determined by adoptive transfer of CMP subsets from NG-BAC transgenics (Ly5B6+) into B6.Ly5SJL congenics was restricted to RAG2/GFPlowFlt3/Flk2+ CMPs (Figure 5). Thus, PDC differentiation potential is enriched among a subset of CMPs further characterized by diminished myeloid differentiation potential and low but readily detectable µ0 and TdT expression. Depletion of ELPs does not perturb PDC development
The data described, together with the previous observation that Flt3/Flk2+ CMPs are more efficient PDC progenitors than Flt3/Flk2– CMPs (Figure 5),12,13 suggests that RAG2/GFPlowFlt3/Flk2+ CMPs may serve as a primary PDC progenitor pool. To further test this notion and directly test whether early lymphoid progenitors including c-kithighFlt3/Flk2+ ELPs and EBP/CLPs are requisite for PDC differentiation, we treated normal adult NG-BAC transgenics with 17
We also scrutinized the impact of 17  -estradiol treatment on PDC development and function. First we considered the possibility that despite our observations, at steady state a small fraction of BM PDCs nonetheless derive from lymphoid progenitors. In this regard, it should be noted that low frequencies of PDCs contain DH-JH rearrangements on the IgH locus.11 Consistent with this observation, we found that 10% to 15% of BM PDCs express the VEX-based RAG1/2-dependent V(D)J recombinase transgenic reporter substrate carried by H2-SVEX transgenic mice21 (Figure 7A). To test whether frequencies of recombinase-positive BM PDCs were diminished by in vivo 17-estradiol treatment, H2-SVEX transgenics were exposed to 17-estradiol for 1 to 3 weeks, then frequencies of VEX+ PDCs and EBPs/CLPs determined. As shown (Figure 7A-B), whereas frequencies of VEX+ PDCs were unaffected by 17-estradiol treatment after even 3 weeks, VEX+ EBPs/CLPs were clearly and rapidly depleted via this strategy. Again, absolute numbers of BM PDCs were not affected by 17-estradiol treatment (not shown).
Finally, we assessed the impact of 17
Several studies suggest that PDCs develop from both lymphoid and myeloid sources.10,12,13,16,34 However, our data indicate that during steady-state hematopoiesis, PDCs arise mainly from a subset of PDC-biased CMPs further defined via low-level expression of genes associated with early lymphocyte development. Several lines of evidence support this conclusion. First, whereas aging leads to dramatic reductions in numbers of EBPs/CLPs and upstream LSK-Flk2+ progenitors, we did not observe a corresponding loss of BM PDCs. Second, CMPs were more effective than EBPs/CLPs and RAG2+ ELPs at generating PDCs in competitive BM chimeras and, consistent with previous observations, among CMPs differentiation into PDCs was restricted to Flt3/Flk2+RAG2/GFPlow cells. Third, depletion of EBPs/CLPs and RAG2+ ELPs with 17 -estradiol failed to affect the cellular dynamics, function, and absolute number of BM PDCs including numbers of recombinase-positive cells within the BM PDC pool. Finally, only Flt3/Flk2+RAG2/GFPlow cells within the CMP pool expressed low but detectable levels of TdT and µ0, and transcripts for each of these genes were also detected in BM PDCs.
The expression of µ0 and RAG-1/2 transcripts by BM PDCs and PDC-biased Flt3/Flk2+AA4low CMPs appears to represent an example of lineage promiscuity in gene expression. Previous studies have shown that myeloid and erythroid lineage genes are expressed in HSCs and, at lower levels, in early lymphoid and myeloid progenitors, respectively.35 Our data extend these observations by showing that certain lymphoid-associated genes are activated within the CMP pool. This observation suggests that transcription factors responsible for activation of early lymphoid genetic programs are transiently operative in CMPs. Although the identity of these transcriptional regulators will require further investigation, likely candidates include the Ikaros family of zinc-finger proteins, a possibility supported by the correlated expression of µ0 and RAG-1/2 with surface Flt3/Flk2 levels and previous data suggesting that Ikaros promotes Flt3/Flk2 expression.36 Other potentially relevant transcriptional regulators include the Ets family transcription factor Spi-B and the E2a-encoded E-box factors E12 and E47.37,38 Indeed, PDC differentiation from human progenitors is inhibited through overexpression of negative regulators of E-box proteins,37 and expression of RAG-1/2 is promoted in B-lineage precursors through the binding of E2a encoded proteins to the eRAG enhancer element within the RAG-1/2 locus.39
In contrast to other reports,10,12-15 we found that EBPs/CLPs are devoid of PDC potential as revealed by adoptive transfer into irradiated recipients. Moreover, more HSC-proximal lymphoid progenitors such as ELPs are also relatively inefficient PDC progenitors. Discrepancies between our adoptive transfer experiments and data reported by others may by due to one or more of several factors. First, one significant difference between our adoptive transfer protocol from other published experiments relates to the number of sorted cells transferred into each irradiated host. In our experiments, we routinely transfer no more than 2000 sorted progenitor cells per recipient, compared with 2 x 104 used in other studies.10,12,13,16 Thus, we are less likely to observe complications due to the cotransfer of small number of contaminating HSCs or CMPs. Second, we suggest that the simultaneous use of Flt3/Flk2, AA4, and RAG2 expression to define Lin–IL-7R
Our experiments with estrogen-treated and aged mice immediately suggest that ELPs are dispensable for PDC production. Whereas the lack of lymphoid progenitors in aged mice suggests that PDC development may not require lymphoid progenitors, these data do not rule out any changes in PDC longevity that may be associated with aging. In fact, it should be emphasized that aging leads to increased self-renewal activity for HSCs,42 and age-related changes in HSC function may result in alterations in the cellular dynamics of downstream populations such as BM PDCs.
The negative impact of sex steroids on early lymphoid progenitors has been reported33,43; however, little is known about their effect on DC subsets. It has been demonstrated that estrogen promotes the differentiation of CD11c+CD11bint cells from BM precursors and that this capacity was abrogated when such cells were grown ex vivo in hormone-deficient medium.44 In addition, 17
Recent work suggests that initiation of TdT and RAG1 expression occur in nonoverlapping subsets of estrogen-sensitive Flt3/Flk2+ ELPs.31 This observation suggests that establishment of lymphoid-associated patterns of gene expression is asynchronous and likely reflective of the stochastic activation and implementation of discrete transcriptional regulatory circuits within HSC-proximal lymphoid progenitors. Our finding that PDC-biased CMPs are resistant to 17 The notion that PDC production is primarily directed via myeloid progenitors is congruent with data reported by Chicha et al34 who investigated PDC differentiation from human lymphoid and myeloid subsets in vitro. Interestingly, our data also suggest that the genotypic characteristics associated with PDCs are established in Flt3/Flk2+ CMPs. We provide evidence that expression of a number of lymphoid-associated genes, such as TdT, µ0, and Flt3/Flk2, is also detectable in these cells, suggestive of heterogeneity within the CMP pool. This phenomenon has been addressed previously. D'Amico and Wu identified the Flt3+ CMP and EBP/CLP fractions as being efficient progenitors for DCs and PDCs as compared with their Flt3/Flk2– cohorts.13 Flt3/Flk2 expression was also investigated in the CMP pool of PU.1 reporter mice,47 where these authors found that the Flt3/Flk2+ CMPs also expressed PU.1 at high levels, consistent with a myeloid phenotype, and generated granulocyte myeloid precursors (GMPs) after in vitro culture and myeloid progeny in vivo. Importantly, in agreement with previous data13 the Flt3/Flk2+PU.1hi CMPs were more efficient DC progenitors than Flt3/Flk2– subsets. Moreover, these data suggest that Flt3/Flk2 expression may be an important determinant for DC generation as Flt3/Flk2 PU.1hi CMPs demonstrate a lower capacity for DC generation.12 Why TdT and µ0 expression correlate with Flt3/Flk2 levels in CMPs is unclear. Nonetheless, our data illustrate that the CMP pool should be viewed as a heterogeneous mixture of progenitors undergoing specification toward the myeloid, erythroid, and PDC lineages. Further investigation into the mechanisms underpinning lineage specification and commitment for PDCs will greatly enhance our understanding of hematopoiesis as well as PDC differentiation and function.
We thank Drs Avinash Bhandoola and Michael Cancro for helpful discussions and critically reviewing this manuscript. We also gratefully acknowledge the expert technical support in flow cytometry provided by the Abramson Cancer Center Flow Cytometry and Cell Sorting Shared Resource and in particular the efforts of Richard Schretzenmair, William Murphy, and William DeMuth.
Submitted November 16, 2005; accepted February 9, 2006.
Prepublished online as Blood First Edition Paper, February 28, 2006; DOI 10.1182/blood-2005-11-4545.
Supported by National Institutes of Health grants AI052861 and AI058066. D.A. is the recipient of a Career Development Award from the Leukemia and Lymphoma Society.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David Allman, University of Pennsylvania School of Medicine, 36th and Hamilton Walk, 230 John Morgan Bldg, Philadelphia, PA 19104; e-mail: dallman{at}mail.med.upenn.edu.
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Abstract
Introduction 
and RAG1.
, TER-119, Gr-1, and CD11b. Initial gating of Lin–IL-7R
indicates E2 Rx mice; 
, placebo controls). (C) Placebo and 
indicates E2 Rx mice; 
, placebo controls. (D) ELISA analysis of IFN-