news & views HEMATOPOIESIS Plasmacytoid dendritic cells: origin matters Plasmacytoid dendritic cells (pDCs) are type I interferon–producing cells with antigen-presenting potential. pDC populations are composed of transcriptionally and functionally heterogeneous cellular subsets with distinct hematopoietic precursor origin. Markus G. Manz D endritic cells (DCs) were characterized about 45 years ago by the late Nobel laureate Ralf Steinman and colleagues. Since then, multiple distinct subsets of the DC family have been identified, all equipped with the hallmarks of extracellular antigen uptake, migration, antigen-presenting ability and induction of activation or tolerance of the adaptive immune system1. Plasmacytoid DCs (pDCs), initially also called ‘plasmacytoid T cells’ or ‘plasmacytoid monocytes’, are a sub-population of DCs with large capacity for producing type I interferon (and are thus also called ‘natural interferon-producing cells’)2. In this issue of Nature Immunology, Fernandes et al. identify proliferating immediate progenitors of pDCs in the bone marrow of mice3. Moreover, they developmentally and transcriptionally delineate the heterogeneity of tissue pDC populations and relate this heterogeneity to differentiation pathways derived from common DC progenitors (CDPs) and cytokine receptor IL-7Rα–expressing pre-pDC progenitors that are newly identified here. On the basis of the discovery that after in vitro stimulation with selected cytokines, monocytes can differentiate into either macrophages or DCs (including Langerhans cell–like cells), it was assumed that this would reflect the main differentiation pathways in vivo. Subsequent in vivo research has revealed that these differentiation pathways are possible but instead reflect inflammation- and repairdriven cellular differentiation. Somewhat surprisingly, homeostasis-relevant tissue macrophages and cells, such as microglia and Langerhans cells, are embryonically seeded, are long-lived, undergo self-renewal and are replaced by adult hematopoietic stem cell (HSC)-derived cells only after tissue damage4. In contrast, lymphoid-tissue classical DC (cDC) and pDC populations do not undergo self-renewal and live only few days (cDCs) or 1–2 weeks (pDCs)5. Thus, both cDCs and pDCs require continuous progenitor cell–derived input to maintain the size of the population in vivo at steady state. During the past two decades, DC subpopulations and their respective differentiation pathways have been defined in hematopoietic reconstitution settings at steady state and, less frequently, in situations of inflammation and infection. This has been achieved through the use of population-based and clonal in vitro and in vivo transfer assays, as well as genetic gain- and loss-of-function approaches5–7. It is clear that Flt3L is the key non-redundant cytokine for the sustained quantitative development of both cDCs and pDCs. The main cDC populations at steady state in vivo are derived from myeloid-restricted early hematopoietic progenitors (MPs). MPs generate CDPs8,9 via differentiation though macrophage-DC progenitors10 or generate CDPs directly11. CDPs subsequently differentiate into pre-cDCs12 that then produce cDCs. While on a clonal level CDPs are able to produce both cDCs and pDCs, their overall pDC production is low relative to the quantitative occurrence of pDCs versus cDCs in vivo. Thus, alternative pDC developmental pathways are possible. Indeed, major pDC- and minor cDC-differentiation potential has been identified in a population tightly related to and possibly in part derived from CDPs (and myelo-lymphiod progenitors) but lacking the surface marker M-CSFR while expressing large amounts of the transcription factor E2-2 (TCF4), which is essential for pDC development13. Fernandes et al. now revise this further3. They first show in elegant classical in vitro differentiation and in vivo transfer assays that major pDC-differentiation potential lies in Lin–c-Kitint–loFlt3+M-CSFR–IL-7Rα+ cells, suggestive of lymphoid progenitor derivation due to the expression of IL-7Rα. They further subdivide this population into three subpopulations on the basis of the expression of Ly6D, a marker for early B cell differentiation, and of SiglecH, a member of the sialic acid–binding immunoglobulin-like lectin family, which is expressed on pDCs. Ly6D+SiglecH+ cell populations contain purely pDC-committed cells with low proliferation potential, which the authors call ‘pre-pDCs’ in analogy to previously defined pre-cDCs12. While CDPs and the IL-7Rα+ precursors exist at about the same frequency in steady-state bone marrow, they seem not to give rise to each other. The in vitro and in vivo pDC output from the newly identified IL-7Rα+ population is five- to tenfold higher than that from CDPs or M-CSFR–IL-7Ra– pDC progenitors13. By transcriptional analysis, Fernandes et al. then demonstrate pDClineage specification at the Ly6D+SiglecH+ progenitor stage3. Moreover, by bulk and single-cell RNA-based next-generation sequencing, focused protein-expression analysis and computational modeling, they reveal pDC heterogeneity in the bone marrow and spleen: a smaller ‘pDC-like’ population that produces the cytokine IFN-αonly after stimulation with the oligodeoxynucleotide CpG-A, not after stimulation with CpG-B, has higher expression of major histocompatibility complex class II after stimulation with CpG-A, and has more-efficient antigen processing and greater T cell–stimulating ability (which resembles the characteristics of cDCs); and a larger population of ‘regular’ pDCs, with a greater capacity to produce IFN-αand less T cell–stimulating ability. On the basis of their transcriptional profiles and function, ‘pDC-like’ cells are derived from CDPs, while pDCs are derived from IL-7Rα+Ly6D+SiglecH+ progenitor cells, which suggests that the different tissue pDC clusters might be ‘developmentally encoded’ (Fig. 1). Such findings are important, as they enhance understanding of the development and heterogeneity of pDCs. Also, they emphasize some limitations of research approaches currently used, and they stipulate new biological questions. In the definition of hematopoietic stem and Nature Immunology | www.nature.com/natureimmunology © 2018 Nature America Inc., part of Springer Nature. All rights reserved. news & views HSCs MPPs Microglia MLPs Granulocytes NK, B, T cells CLPs MPs MDPs Langerhans cells Pre-pDCs CDPs Monocyte subtypes Pre-cDCs Macrophages Inflammatory macrophages Inflammatory DCs c-Kitint–lo Flt3+ M-CSFR– IL-7Ra+ SiglecH+ Ly6D+ ~0.1% of BM cells c-Kitint Flt3+ M-CSFR+ IL-7Ra– ~0.1% of BM cells pDC-like cells cDC subtypes IRF8 independent ~5–30% of steady-state spleen pDCs Type 1 IFN+ APC function pDCs IRF8 dependent ~70–90% of steady-state spleen pDCs Type 1 IFN+++ Low APC function Clonally or population-based documented major differentiation pathways Differentiation pathways induced by inflammation or tissue damage Immunophenotypic and functional populations or clonally defined cell populations BM BM and/or tissues Embryonic origin and/or self-renewal New findings in Fernades et al. Fig. 1 | Differentiation pathways of antigen-presenting cells (DCs, pDCs and macrophages) in mice. Self-renewing HSCs generate highly proliferative, nonself-renewing progenitor populations that subsequently commit to lineage-restricted progenitor cells and final immature and mature effector populations. These differentiation pathways are highly controlled environmentally and in a cell-intrinsic way. Arrows indicate experimentally documented differentiation pathways (for multipotent progenitors (MPPs), myelo-lymphoid progenitors (MLPs), MPs, macrophage-DC progenitors (MDPs), CDPs, cDCs, common lymphoid progenitors (CLPs) and pDCs). Newly identified pDC sub-populations and their progenitor and differentiation pathways identified by Fernandes et al.3 are highlighted in the outlined oval: the main pDC populations in bone marrow (BM) and spleen are derived from an IL-Rα+ progenitor cell (pre-pDC) population that parallels the previously identified pre-cDC population, while a minor transcriptionally and functionally distinct pDC population, called ‘pDC-like cells’ by authors3, is independently derived from CDPs. pDCs and pDC-like cells are phenotypically similar but transcriptionally and functionally distinct cell populations. NK, natural killer; IRF8, transcription factor; IFN, interferon. Credit: Marina Corral Spence/Springer Nature progenitor cell (HSPC) sub-populations, the in vivo long-term clonal ‘readout’ of HSCs allows relatively clear-cut conclusions to be drawn. However, with a decrease in the proliferative burst size of HSPC populations in subsequent lineage differentiation, robust readouts become more challenging. A positive readout defines a potential but does not necessarily prove a major relevant pathway in steadystate or demand situations in vivo (as, for example, those induced by γ-irradiation in this study), while a negative readout might simply define insufficient assay capacity. In addition, phenotype-based selection of populations by flow cytometry might lead, in some cases, to unavoidably ‘fuzzy’ population definitions. Remarkably, many of the closely related populations discussed here might include some overlap due to varying signal strength, slightly different gating in flow cytometry and, most importantly, a continuum of expression of the respective surface markers, such as M-CSFR and IL-7Rα, used to define cell populations. Similarly, gene expression– guided lineage tracing is challenged by ‘leakiness’ and/or insufficient reporting tools. Finally, this might indeed be ‘fuzzy’ biology, as lineage commitment in vivo might not be as linear as applied assays and subsequent arrows on charts would suggest (Fig. 1). These are issues that have kept the field busy in the context of the definition of many HSPC populations. The integration of classical biological readouts with single-cell RNA and computational analysis, as done in the current study3, is pushing experimental approaches and understanding to the next level. Additional studies are needed to determine the environmental cues that guide the development of pDCs and pDC-like cells and that, in various tissues, imprint their function in infectious disease, autoimmunity, tissue repair and also neoplastic disease. Furthermore, while some hints of this already exist, the existence, differentiation and function of human homologs of pDCs will need to be investigated. ❐ Markus G. Manz Department of Hematology and Oncology, University and University Hospital Zurich, Zurich, Switzerland. e-mail: email@example.com Published: xx xx xxxx https://doi.org/10.1038/s41590-018-0143-x Nature Immunology | www.nature.com/natureimmunology © 2018 Nature America Inc., part of Springer Nature. All rights reserved. news & views References 1. Banchereau, J. & Steinman, R. M. Nature 392, 245–252 (1998). 2. Colonna, M., Trinchieri, G. & Liu, Y. J. Nat. Immunol. 5, 1219–1226 (2004). 3. Fernandes, P.R. et al. Nat. Immunol. https://doi.org/10.1038/ s41590-018-0136-9 (2018). 4. Ginhoux, F. & Jung, S. Nat. Rev. Immunol. 14, 392–404 (2014). 5. Geissmann, F. et al. Science 327, 656–661 (2010). 6. Merad, M., Sathe, P., Helft, J., Miller, J. & Mortha, A. Annu. Rev. Immunol. 31, 563–604 (2013). 7. Murphy, T. L. et al. Annu. Rev. Immunol. 34, 93–119 (2016). 8. Naik, S. H. et al. Nat. Immunol. 8, 1217–1226 (2007). 9. Onai, N. et al. Nat. Immunol. 8, 1207–1216 (2007). 10. Fogg, D. K. et al. Science 311, 83–87 (2006). 11. Sathe, P. et al. Immunity 41, 104–115 (2014). 12. Liu, K. et al. Science 324, 392–397 (2009). 13. Onai, N. et al. Immunity 38, 943–957 (2013). Competing interests The author declares no competing interests. Nature Immunology | www.nature.com/natureimmunology © 2018 Nature America Inc., part of Springer Nature. All rights reserved.