Monocyte, Macrophage, and Dendritic Cell

Monocyte, Macrophage, and Dendritic Cell Development: the Human Perspective MATTHEW COLLIN1 and VENETIA BIGLEY1 1

Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom

ABSTRACT The maintenance of monocytes, macrophages, and dendritic cells (DCs) involves manifold pathways of ontogeny and homeostasis that have been the subject of intense study in recent years. The concept of a peripheral mononuclear phagocyte system continually renewed by blood-borne monocytes has been modified to include specialized DC pathways of development that do not involve monocytes, and longevity through self-renewal of tissue macrophages. The study of development remains difficult owing to the plasticity of phenotypes and misconceptions about the fundamental structure of hematopoiesis. However, greater clarity has been achieved in distinguishing inflammatory monocyte-derived DCs from DCs arising in the steady state, and new concepts of conjoined lymphomyeloid hematopoiesis more easily accommodate the shared lymphoid and myeloid phenotypes of some DCs. Cross-species comparisons have also yielded coherent systems of nomenclature for all mammalian monocytes, macrophages, and DCs. Finally, the clear relationships between ontogeny and functional specialization offer information about the regulation of immune responses and provide new tools for the therapeutic manipulation of myeloid mononuclear cells in medicine.

INTRODUCTION Monocytes, macrophages, and dendritic cells (DCs) are populations of myeloid mononuclear cells (MMCs) that provide critical sensing functions in innate immunity and a bridge to adaptive immunity through antigen presentation. They also perform important effector functions and contribute to chronic inflammation and healing. Collectively they have been described as the “mononuclear phagocyte system” (MPS) (1). As originally conceived, the MPS had a single blood-borne precursor, the monocyte. It is now appreciated that the development

and homeostasis of MMCs is considerably more complex. In this chapter, we discuss the ontogeny of these diverse cells and reflect on ways in which ontogeny is linked to functional specialization, plasticity, and immune regulation.

MYTHS, CONTROVERSIES, AND OTHER DIFFICULTIES Resolving the developmental relationships between monocytes, macrophages, and DCs and their respective roles in immunity are challenges that continue to attract strong opinion. For many years, the MPS model was widely accepted and both resident histiocytes and myeloid inflammatory infiltrates were presumed to be populations derived from monocytes (Fig. 1A). The ability of human monocytes to take on a wide range of phenotypes in vitro appeared to support this notion (2, 3).

DCs: a Hematopoietic Lineage The discovery of DCs posed a problem for the classic MPS model (4): if they were included as members of the MPS, this implied monocyte origin, promulgating the Received: 16 July 2015, Accepted: 19 April 2016, Published: 16 September 2016 Editor: Siamon Gordon, Oxford University, Oxford, United Kingdom Citation: Collin M, Bigley V. 2016. Monocyte, macrophage, and dendritic cell development: the human perspective. Microbiol Spectrum 4(5):MCHD-0015-2015. doi:10.1128/microbiolspec .MCHD-0015-2015. Correspondence: Matthew Collin, [email protected] © 2016 American Society for Microbiology. All rights reserved.

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FIGURE 1 Steady-state MMC homeostasis. (A) Classical mononuclear phagocyte model showing the development of LCs, DCs, and macrophages from a common monocyte precursor. (B) Current model of DC, monocyte, and macrophage development in which DCs develop through a discrete hematopoietic lineage arising from a bone marrowresident restricted DC precursor, the CDP. CDPs give rise to pDCs and a myeloid pre-DC population that differentiates into two myeloid DCs (cDC1 and cDC2). It is not known if the pre-DC or cDC1 and cDC2 circulating in the blood give rise to tissue populations of cDC1 and cDC2. Monocytes also contribute to steady-state populations of monocyte-derived macrophages and DCs especially in the skin, gut, and lung. These can be clearly distinguished from the CDP-derived DCs and resident macrophages. Resident macrophages are originally derived from prenatal hematopoiesis. It is unknown whether monocytederived cells can contribute to long-term resident macrophages. cMoP, common monocyte progenitor; MAC, macrophage; mo, monocyte.

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Monocyte, Macrophage, and Dendritic Cell Development

view that DCs were just a type of specialized macrophage (5). If one accepted their uniqueness, then this suggested the existence of a distinct DC lineage that was not consistent with a unified MPS. The heritage of DCs took some years to emerge following their discovery, but it is now accepted by most that DCs emerge from the bone marrow as a defined hematopoietic lineage that is distinct from monocytes (6) (Fig. 1B). While monocyte function overlaps the remit of DCs in inflammation, it is clear that in the steady state easily distinguishable populations of DCs and monocytes, or their derivatives, exist in the blood and tissues of humans and other mammals (7–11). The significance of a distinct DC lineage in the induction of immunity is still incompletely understood, as although DCs are potent at stimulating naive T cells, they are relatively rare, especially during inflammation, when monocytederived, highly proficient antigen-presenting cells are rapidly formed (12). While monocyte function may be sufficient to resist infection at some level, DCs presumably improve the fitness of the immune system to respond rapidly, to generate anamnestic responses, or to combat a small number of highly virulent pathogens exerting strong selective pressure. The nonredundant role of DCs in infection and evidence of their tolerogenic functions are established in a number of mouse models (13, 14).

Primitive Macrophages A second, major challenge to the MPS model was the more recent discovery that the majority of histiocytes, or tissue-resident macrophages, are extremely stable populations that do not require replenishment by monocytes or any other precursor (15, 16). In the absence of inflammatory challenge, microglia, Langerhans cells (LCs), Kupffer cells, and alveolar, peritoneal, and splenic macrophages persist from fetal or even embryonic hematopoiesis (12, 17, 18). Notable exceptions are the contributions of monocyte-derived cells to skin, gut, and interstitial lung macrophages (10, 19, 20). Significant import has been attached to the primitive origin of tissue macrophages, and recent studies in the heart suggest that they have a much more prominent role in repair than recently recruited monocyte-derived cells (21). It is not clear whether monocyte-independent routes of macrophage differentiation can also persist in some form after birth. Given that hematopoietic stem cells (HSCs) are detectable in the peripheral tissues of mice (22), it is hard to exclude the occurrence of a direct route of macrophage differentiation from an early hematopoietic precursor, residing in adult tissues.

PITFALLS IN THE STUDY OF MMCs This narrative illustrates a number of generic challenges in the study of monocytes, macrophages, and DCs. First, it is important to distinguish between the steady state and inflammation. There is no a priori reason to suppose that inflammatory precursors and their progeny are related to steady-state populations of MMCs. Indeed, temporal and special separation of quiescent and inflammatory pathways of differentiation may provide critical aspects of immune regulation. Major questions remain unsolved, particularly in humans, concerning the functional differences between resident and recruited cells and their respective fates after inflammation has resolved. Second, overreliance on a small number of surface antigens to identify a particular cell type can be misleading. The use of CD11c and major histocompatibility complex class II (MHC-II) to define murine DCs has been criticized (5). In humans, it was already well known that CD11c and MHC-II are highly expressed by both monocytes and DCs and cannot be used to separate them in the blood, although CD11c defines different populations of dermal DCs and macrophages quite clearly in tissues (8). This issue has been partly circumvented by the discovery of more-restricted antigens such as the suite of “blood DC antigens,” or BDCA-1 to -4, in humans (23, 24) and, more recently, new DC and macrophage signposts such as CLEC9A (C-type lectin domain family 9 member A), XCR1 (chemokine XC receptor 1), SIRPα (signal regulatory protein α), and MerTK (MER tyrosine kinase) identified by unbiased computational approaches to classification (25, 26). Third, the plasticity of MMCs to respond to environmental cues means that different anatomical sites are likely to induce specific phenotypes (27). Furthermore, this plasticity inflates the problems already alluded to that inflammation and the steady state are not equivalent and that individual surface antigens may be fickle. A cogent argument has been made to mitigate these problems by using ontogeny as the basis of MMC classification (28). This approach has many attractions, and although definitive ontological experiments are difficult to perform in humans, the concepts developed in mice can often translate to humans. As recent data show, the prenatal development of MMCs is an important dimension to consider, in addition to the lineages that arise later from definitive HSCs (12, 17).

THE REPERTOIRE OF HUMAN MONOCYTES, MACROPHAGES, AND DCs Monocytes Monocytes are the archetypal MMCs, constituting ∼10% of human peripheral blood mononuclear cells.



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They are blood borne by convention, although recent studies in humans and mice describe steady-state populations of tissue monocytes or monocyte-derived cells (9, 19, 20). Monocytes have a plethora of functions in inflammation and rapidly differentiate into cells with DC-like and macrophage phenotypes that can be difficult to separate from resident populations. Recent transcriptomic profiling experiments highlight the complexity of monocyte differentiation potential (29). Human monocytes are heterogeneous in their expression of CD14 and CD16 (Fc receptor III), and this has been used to divide them into subpopulations (30). CD14

and CD16 are both continuously distributed (Fig. 2), but the majority of monocytes are CD14+ and are defined as classical or inflammatory monocytes. Most of the precursor functions defined in vitro, including the formation of monocyte-derived DCs and macrophages, arise from classical monocytes (29). Some studies ascribe distinct functions to “intermediate” CD14+CD16+ monocytes at the vertex of the two-dimensional plot. These have the highest MHC-II expression and cytokine production and may be derived by activation of classical monocytes. Nonclassical CD16+ monocytes also express high MHC-II and costimulatory antigens, leading some

FIGURE 2 Human blood monocytes and DCs. Gating strategy to identify human monocytes and DCs in peripheral blood. Monocytes and DCs are all found in the HLA-DR+, lineage-negative compartment. CD14 versus CD16 displays monocyte subsets and double-negative DC populations. These may be separated in a variety of ways. Here the markers CD123 and CD11c are used to define pDCs and myeloid DCs. The latter can be separated into cDC1 and cDC2 using CD141 and CD1c, respectively.

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authors to regard them as DCs (24). However, transcriptional profiling and unsupervised hierarchical clustering is unequivocal that all monocytes cluster independently of DCs (9, 31). Within the CD16+ subset a population expressing the antigen 6-sulfo LacNAc (SLAN-DCs) is reported to secrete large amounts of tumor necrosis factor α, interleukin-1β (IL-1β), and IL-12 and to respond rapidly to inflammatory stimuli (32). Other studies suggest that CD16+ monocytes, including SLAN+ cells, have low inflammatory activity and are homologous to murine Gr-1/Ly6Clo “patrolling” monocytes (33). Inconsistencies may have arisen through different gating of the CD14 versus CD16 plot and inclusion or exclusion of the CD14+CD16+ intermediate cells, which have a highly activated phenotype. Anatomically, it appears that nonclassical monocytes are well positioned to infiltrate tissues or to control diapedesis across the endothelium; the importance of both of these potential functions remains to be tested in humans (34). The origin of human nonclassical monocytes remains controversial. Kinetic studies in the mouse suggest that they are derived from classical monocytes (35), although this is recently disputed (36). Human CD16+ monocytes also have higher expression of the nuclear factor Nur77, proposed to control murine nonclassical monocyte differentiation (37). There is a prominent literature in humans describing abnormalities of monocyte development in disease, notably the appearance of “sick” monocytes with lower MHC-II expression and impaired antimicrobial functions (38). Variation in the CD14/CD16 ratio due to expansion or contraction of either subset is also associated with many different conditions. Of note, CD16+ monocytes are highly sensitive to depletion following treatment with corticosteroids (39). The appearance of monocytic “myeloid-derived suppressor cells” that can inhibit T-cell responses is associated with cancer and chronic infection (40). These demographic changes may simply represent a “left shift” in myelopoiesis, but nonetheless this appears to provide immune regulation under conditions of stress or chronic inflammation. Finally, heterogeneous expression of CD68, CD163, and CD202b (Tie2; angiopoietin receptor) has been noted among human monocytes. Tie2+ monocytes are found throughout classical, intermediate, and nonclassical populations and are implicated in tumor angiogenesis (41).

Macrophages: Histiocytes Old and New Macrophages are classically tissue-resident cells, or “histiocytes.” Many different specialized populations

of macrophages exist in different sites. Observations from human HSC transplantation and monocytopenic states suggest that as in mice, these are likely to be developmentally old cells that can persist for long periods without replenishment from the bone marrow (7, 42– 44). The label “histiocyte” is perhaps still useful to denote these developmentally older populations from more recently recruited monocyte-derived cells. Location matters, and the tissue environment induces specific gene regulation in resident histiocytes, regardless of their origin (27). Recent advances have led to much clearer definitions between long-term resident histiocytes, DCs, and more transient populations of monocyte-derived cells (Fig. 3). Macrophages are operationally distinguished from DCs by their much lower ability to simulate naive T cells (4). There are also many differences in gene expression (9, 25, 26). Useful markers in human skin such as CD163, factor XIIIA, and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1) identify macrophage populations that are completely nonoverlapping with DCs, identified by CD11c or CD1c expression (8, 45). In addition to histiocytes and DCs, populations of monocytederived cells such as CD14+ or “interstitial-type” DCs are also found in human skin. These were originally classified as DCs owing to their ability to migrate from explanted skin (46), but transcriptomic profiling shows close relationships with the human dermal macrophage and mouse monocyte-derived macrophages (9, 10). As predicted, these monocyte-derived macrophages are indeed poor allostimulators, produce IL-10, and can provide regulatory function (47). Confusingly, CD141 may also be induced on monocyte-derived cells, but they remain distinct from CD141+ DCs particularly through the expression of CD14, SIRPα, and other markers (11, 47). In vivo human data show that monocyte-derived macrophages are rapidly repopulated after HSC transplantation, in contrast to resident histiocytes (7, 9). Monocyte-derived cells are also rapidly turned over in murine tissues and may be found in afferent lymph (10, 19, 20). Together, these observations support a model that recently derived monocyte-macrophages are mobile, in contrast to the long-term histiocyte population. Immobility and the progressive accumulation of lipofuscin and pigment that gives macrophages autofluorescence in the flow cytometer are physical signs of longevity that are experimentally tractable to identify histiocytes and to separate them from monocyte-derived cells (7, 9). One difference between mouse and human is that it has been difficult to demonstrate homeostatic proliferation in human macrophage populations. Observations



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FIGURE 3 Summary of human DC, monocyte, and macrophage origins in relation to steady-state surface markers. The most distinctive DC phenotypes are displayed by pDCs and CD141+ cDC1 cells. There is more overlap between cDC2 and monocyte-derived cells that may coexist in tissues. Monocyte-derived macrophages also share many markers with resident histiocytes. The relative size of arrows from the precursor populations indicates the estimated steady-state flux. CMoP, common monocyte progenitor; mac, macrophage; mo, monocyte.

in quiescent skin suggest that if proliferation does occur, it is less than one-tenth of the level observed in LCs (∼2 to 3%) (7, 43). These observations need to be extended to inflammatory situations such as Th2 environments that have been observed to stimulate murine macrophage proliferation (48). The relatedness of monocytes, monocyte-derived macrophages, and tissue histiocytes by unsupervised clustering of their gene expression signatures is reminiscent of the classic picture of the MPS as a monocytebased system but also strong evidence of the separateness of the DC lineage, members of which cluster very distinctly (9, 31). However, care must be made in extrapolating these static similarities to population dynamics. The plasticity of monocytes allows them take

on genuine DC properties during inflammation (49–51). We concur with others that in experimental work the term “monocyte-derived DC” should be used to describe such populations (12, 28, 52).

Dendritic Cells DCs were first isolated from the mouse spleen as nonadherent branching cells that were 100-fold more potent at activating naive T cells compared with adherent macrophages (4). “Resident” populations of DCs that are derived from blood-borne precursors are found in lymphoid tissues, while “migratory” DCs found in peripheral tissues take up exogenous antigens and migrate to draining lymph nodes, performing a surveillance role (6).

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For obvious logistical reasons the analysis of human lymphoid and tissue DCs has proceeded more slowly than the study of human blood. In human blood, three populations of DCs are readily described: plasmacytoid DCs (pDCs) and two populations of “myeloid” DCs (Fig. 3). The term “myeloid” is equivalent to “classical” or “conventional” in mice. Its usage in humans is quite specific and based on the expression of antigens typically seen on granulocytes or monocytes, including CD13, CD33, CD11b, and CD11c. These markers are lower or absent on pDCs, which are also morphologically and functionally distinct, providing a major source of type I interferon in response to viral infection (reviewed in reference 53). All DCs carry antigens typically associated with lymphoid differentiation, such as CD4 (54). MHC-II and CD11c are not useful for separating DCs from monocytes in humans, but the series of BDCA antigens is extremely powerful (23, 24). Myeloid DCs separate into a major CD1c+ (BDCA-1) population and a minor CD141+ (BDCA-3) population. The minor population is homologous with the mouse CD8+/CD103+ DC lineage, has high expression of XCR1 and CLEC9A, and has natural cross-presenting capacity (55, 56). In a recent nomenclature system this “conventional” DC lineage has been called cDC1, while human CD1c+ DCs together with mouse conventional CD4+ or CD11b+ DCs are cDC2 (28). An alternative system is simply to name these cells CD141+ myeloid DCs and CD1c+ myeloid DCs, although, as previously noted, CD141+ is quite a promiscuous antigen (30). It has been suggested that more-universal markers are XCR1 and CLEC9A for cDC1 and SIRPα for cDC2 (57). The conservation of pDCs is apparent in many different mammalian species, and in humans they express CD123, CD303 (BDCA-2; CLEC4C), and CD304 (BDCA-4; neuropilin). In tissues it is possible to detect equivalent populations of migratory myeloid DCs. Lymphoid organs also contain CD1c+ and CD141+ DCs in addition to pDCs and LCs in skin-draining locations (58–61).

Langerin-Positive DCs The expression of Langerin on non-LCs was first highlighted in the mouse by several groups working with models of inducible LC depletion, in which Langerin + CD103+EpCAM– DCs were observed in the complete absence of LCs (62). Langerin expression had already been observed in CD8+ lymph node-resident DCs (63), and it became clear that Langerin is expressed by murine CD8+/CD103+ cDC1s. In contrast, a small component of CD1c+ cDC2s express Langerin in human tissues in vivo (64). This population is distinct from LCs, and

the human CD141+ cDC1 population in the steady state, but may be a precursor of LCs after inflammation (65).

Langerhans Cells LCs are an unusual population of migratory myeloid DCs situated in the epidermis and mucosae (66). They express Langerin and CD1a at high levels and contain Birbeck granules by electron microscopy (63). LCs can be stimulated to leave the epidermis, forming migratory DCs detectable in skin-draining lymph nodes. LCs appear to play multiple roles in immunity, including the presentation of lipid antigens (67), inactivation of pathogens (68), and induction of Th17 responses (69) and graft-versus-host disease (70), in addition to the maintenance of tolerance in the steady state (14). LCs are derived from myeloid precursors during fetal life (71, 72) that differentiate under the influence of the colony-stimulating factor-1 receptor (CSF-1R; CD115), cytokine IL-34 (73, 74), and autocrine production of transforming growth factor β (75). Proliferation of epidermal LCs was first reported in humans (76), but elegant experiments in mice subsequently proved that selfrenewal was sufficient to provide local homeostasis (77). LCs were the first example of an MMC that was not continuously replenished from the bone marrow. Limb transplantation and bone marrow failure syndromes confirm their independence of bone marrow-derived precursors in humans (43, 44, 78). Interestingly, it has been difficult to reproduce mouse data showing that LCs survive HSC transplantation, and most are replaced by donor cells within a few months, even with nonmyeloablative conditioning and in the absence of graft-versushost disease (79, 80).

EXPERIMENTAL APPROACHES TO ANALYZING MYELOID DIFFERENTIATION Hematopoiesis has provided one of the seminal model systems of developmental biology. The key concepts are that stem cells are self-renewing and pluripotent and that differentiation proceeds by a progressive restriction of cell potential, through multiple rounds of proliferation. The focus of almost all research has been to define the nodes in this process, the points at which cell potential bifurcates, through the isolation of clonogenic progenitors that give rise to daughter cells each with nonoverlapping developmental potential. Combined with flow cytometry, the major fruit of this labor is the purification of cells with progressively restricted potential, manifest by a number of in vitro or in vivo assays. There are a number of limitations of this approach that are pertinent



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to unraveling the development and differentiation of MMCs.

Not All Swans Are White Experiments subjecting purified progenitors to any interrogation of their potential can only rule in a particular cell fate potential; they cannot exclude it. Even when a comparator population is used to verify the performance of a readout, a cell that fails in one assay may have the same potential under more sophisticated conditions, for example, the use of stromal cells or new growth factors. This problem has led to the appearance of a number of black swans over the years as new potentials are revealed through improvements in the sensitivity of readouts. Xenografting in immunodeficient mice is no exception, as the expression of cell potential is still limited by an arbitrary range of cross-reactive hematopoietins (81).

Uncertainty Principles In Vitro A further problem in analyzing single cells with dual potential is that it may be difficult or impossible to find a set of conditions that allow simultaneous differentiation along more than one lineage. A progenitor may exist in one of several states of potential, but often this can only be interrogated by collapsing the potential to a singular outcome through defined culture conditions. Critically, attempts to define a “universal” readout are likely to increase the chance of missing a potential outcome and creating an unseen black swan. An interesting metaproblem of the same nature is that antibodies to growth factor receptors are frequently used to sort populations and can potentially interfere with growth factor signaling during the analysis of progeny. Thus, it has been suggested that high expression of CD115 used to isolate restricted DC progenitors may blunt the ability of these cells to develop into monocytes (5). Recent efforts to define DC progenitors in humans have all capitalized on growth factor expression to isolate prospective cell populations (82, 83).

Monocyte and DC Potential The black swan and simultaneous differentiation problems both impede the study of monocyte and DC development in humans. First, conditions permissive for the development of human DCs in vitro and in animal models have only recently been defined (56, 82, 84); and second, the phenotypic readouts are subtle, and robust definitions of what constitutes a DC or monocytederived cell have been hard to formulate, particularly due to the extreme plasticity of monocytes in adopting DC-like phenotypes (51).

In terms of defining the “true” DC potential of an assay, the appearance of pDCs and XCR1+CLEC9A+ myeloid DCs (cDC1) is instrumental. This is because CD1c+ myeloid DCs (cDC2) are close in phenotype to monocyte-derived DCs and it may be difficult to discern if cDC2 phenotype output is simply a reflection of monocyte potential; this is especially true when granulocyte-macrophage CSF (GM-CSF) and IL-4 are present, conditions that promote the formation of monocytederived DCs (85). It is necessary to perform quite detailed transcriptional profiling in order to ascertain the true lineage of culture outputs (82). The ability of CD1c+ DCs to form LC-like cells in response to transforming growth factor β may be useful in distinguishing them from monocyte-derived DCs (65, 86).

THE MYELOID-BASED MODEL OF MOUSE MMC DIFFERENTIATION Many critical details of murine MMC development have recently been elaborated. As alluded to previously, it is now appreciated that primitive myelopoiesis contributes almost exclusively to microglia, which arise directly from yolk sac macrophages, and that LCs and most tissue macrophages also have a primitive origin via fetal liver monocytes (12, 17, 18). Definitive hematopoiesis continues to supply monocytes and DCs throughout the life of an animal, and unraveling a discrete pathway of DC development in the mouse has been a significant advance in the field of hematopoiesis (6). The model that has emerged is firmly myeloid based, in which granulocyte-macrophage progenitors (GMPs) give rise to macrophage and DC progenitors (MDPs), which have lost granulocyte potential (87, 88). MDPs in turn give rise to a monocyte precursor (35) and common DC precursor (CDP) (89, 90). The latter has restricted DC potential and is the key staging post of the DC lineage where monocyte potential should disappear. pDCs are said to arise directly from CDPs, but classical or conventional DCs descend from an intermediate pre-cDC population that can be found in the blood (91, 92) (Fig. 1B). This model appeals to the reductionist by proposing sequential bifurcations of cell potential consistent with textbook models of hematopoiesis. However, a number of inconsistencies are emerging. The MDP has recently come under fresh scrutiny with evidence that its ability to generate pDCs and XCR1+ cDC1s (markers of “true” DC potential) is much less than its monocyte potential and that it may still retain some ability to form granulocytes (93). The identity of the CDP as a clonogenic

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progenitor has also been questioned, with reports that populations originally classified as CDPs are primed to produce either classical DCs (cDC1 and cDC2) or pDCs according to their level of CLEC9A and CD115 expression (94, 95). Pre-DC function has also been observed in a pDC-like cell that is distinct from the pre-DC (96). Furthermore, the pre-DC itself shows evidence of precommitment to either cDC1 or cDC2 progeny (97). The concept of precommitment or lineage priming is not new to stem cell biology but is at variance with models based on discrete cell fate “decisions” at the nodes of clonogenic progenitors (98). Lentiviral barcoding reveals that lineage priming may bias the entire progeny of early progenitors, with a significant tranche apparently devoted to the generation of DCs (99). A final aspect of the myeloid-based model that has been difficult to assimilate with other mouse and human data is the lack of consideration for lymphoid pathways of DC development (100, 101). The original concept that pDCs might have a lymphoid heritage stems not only from their morphology and surface markers but also from the fact that rearranged IgH and T-cell receptor loci are found at high frequency (102, 103). Rather than “convergent development” from two different branches of hematopoiesis, it appears more likely that lymphoid and myeloid potential are conjoined until late stages of differentiation.

MYELOID AND LYMPHOID OR LYMPHOMYELOID? The terms “myeloid” and “lymphoid” dominate hematopoiesis. “Myeloid” is used here in the broad sense of bone marrow derived, encompassing erythropoiesis, megakaryopoiesis, granulopoiesis, and the production of monocytes and macrophages; indeed anything that does not include small lymphoid cells. In the classical model of hematopoiesis, the earliest dichotomy occurs between myeloid and lymphoid, based on the description of common myeloid (erythro-mega-granulocytemacrophage) and common lymphoid (B and T) progenitors (104, 105). However, in assays that supported both myeloid and lymphoid differentiation, lymphomyeloid progenitors lacking erythro-megakaryocyte potential were observed, suggesting that the specification of lymphoid potential is a more continuous process (81, 106). Many groups have since described “lymphoid-primed multipotent progenitors” (LMPPs) in mice and humans, in support of this concept (85, 107) (Fig. 4). From an epigenetic standpoint, the gradual, rather than abrupt, loss of lymphoid

potential concords with progressive methylation of myeloid regulatory loci (108). From the point of trying to decipher monocyte and DC development, these findings are absolutely key as it is no longer necessary to invoke an unorthodox blending of myeloid and lymphoid potential if instead they coexist. The DC embodies innate myeloid phenotype with an ability to interact with adaptive immune system lymphocytes, and difficulties in mapping its precise origin may be intimately linked to problems with the myeloid-lymphoid dichotomy of classical hematopoiesis. A graphical illustration of this is the structure of hematopoiesis derived from an unbiased analysis of transcription factor expression by the “known” hematopoietic lineages and their intermediates: this places the DC lineage as neither myeloid nor lymphoid but arising directly from HSCs (109).

HUMAN MONOCYTE AND DC DIFFERENTIATION Serious analysis of human hematopoiesis began with in vitro colony-forming assays defining erythroid-, megakaryocytic-, granulocyte-, and “macrophage”forming units, thus providing the first example of readouts that could interrogate either granulocyte or monocyte potential (110). CD34 is a key marker of the human progenitor/stem cell compartment. Most multilineage potential is contained within the CD38lo fraction of CD34+ cells, while CD38+ cells contain committed cells. The CD38+CD10+ fraction contains lymphoid potential, while GMPs fall in the CD38+CD45RA+ fraction. The importance of CSF-1R in identifying progenitors with monocyte but not granulocyte potential was recognized early (111). This population potentially represents MDP-like cells within the GMP, but to our knowledge a human equivalent of the common monocyte progenitor has not been identified. In addition, within the GMP, a discrete population of CD123hi (IL-3 receptor) cells was noted to give rise to pDCs (112). CD123hi cells form a distinct peninsula of the GMP cloud that has been observed in a number of studies (113). Upon in vitro exposure to Flt3 ligand, stem cell factor, GM-CSF, and stromal cells, the CD123hi population of GMPs contains pDC and myeloid cDC1 and cDC2 potential but lacks monocyte potential (82). In this study, molecular profiling was required to differentiate between monocytes and cDC2 because CD1c+ cDC2s can acquire CD14 expression in vitro and CD14 monocytes can differentiate into CD1c+ cDC2-like cells. These results show that good DC potential resides in the



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FIGURE 4 Revised structure of human hematopoiesis showing common lymphomyeloid origin of MMCs including DCs and monocytes. Color coding of progenitor populations is according to their expression of CD38 and CD45RA (inset). B/NK, B- and NK-cell progenitor; EMP, erythromyeloid progenitor; EoBa, eosinophil-basophil progenitor; ETP, early thymic progenitor; GM(D)P, granulocyte-monocyte (DC) progenitor; MEP, megaerythroid progenitor; MLP, multi-lymphoid progenitor; MDP, monocyte-DC progenitor.

CD123hi population, but the statement that these cells are “DC restricted” should be qualified to the specified conditions. Under these conditions, GMPs generated both monocytes and DCs. However, strong monocytic differentiation with M-CSF was not tested, and it might therefore be argued that monocyte potential was not completely excluded from the CD123hi fraction. It was also previously shown that CD38+CD10+ lymphoid progenitors have the ability to develop into HLA-DR+CD1a+ DC-like cells and pDCs (100–102). More-recent studies suggest that the CD38–CD45RA+ LMPPs (or multi-lymphoid progenitors), which can give rise to monocytes, will also contain genuine DC potential (85). Whether these lymphoid pathways funnel into the CD123hi population is unknown. Also, it is possible that accessory pathways of DC differentiation exist, as the authors who first described pDC potential in the CD123hi population also noted LC-like potential in the

CD123lo GMP fraction (112); this may indicate the presence of CD1c+ cDC2s (65, 86). The same authors who defined a CDP also reported pre-cDCs in human blood by scrutinizing small populations of CD34–, lineage-negative cells expressing Flt3, GM-CSF, and M-CSF receptors (83). The pre-cDCs that were defined did not express high CD123 and had no pDC potential. The transition between CD123hi CDP and CD123lo pre-cDC is not immediately obvious, and other transitional CD34–, lineage-negative intermediates may exist. Bone marrow transplantation and human DC deficiency states indicate that continual replenishment from blood-borne precursors is required for tissue populations of CD141+ and CD1c+ DCs (7, 43, 44). Comparison with the mouse suggests that these cells are derived from rare pre-DC populations although the potential precursor role of blood DCs has not been excluded (114).

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MONOCYTE-DERIVED CELLS IN INFLAMMATION The derivation of macrophages from monocytes in vitro has been studied for many years and was one of the founding observations leading to the MPS model. A “classical” highly immune-activated macrophage phenotype is seen after exposure to gamma interferon, while “alternative” activation with IL-4 was developed as a model of resident macrophage differentiation (3). These two phenotypes, known as M1 and M2, respectively, have been regarded as two poles of macrophage differentiation with contrasting functions. More-recent studies have highlighted the complexity of monocyte activation using a greater range of stimuli and employing transcriptomic analysis to derive modules of coregulated genes and their regulators (29). The isolation of primary human macrophages from different sites will now facilitate a more systematic comparison of ex vivo populations with in vitro-derived cells (7, 29, 115, 116). The observation that monocytes could differentiate into myeloid DCs with the ability to stimulate naive T cells was a significant experimental breakthrough facilitating a great number of studies in humans (2). However, recognition of naturally occurring DCs in vivo has led to a reappraisal of the monocyte-derived DC as probably more closely linked with inflammatory monocyte function than to steady-state DC biology. Latterly, the importance of monocyte-derived DCs in resistance to infection has been fully recognized in the mouse, thus validating the human in vitro data (49, 50), and inflammatory DCs have been described in humans in several pathological settings (115–118). In addition to monocyte-derived DCs, the direct recruitment of DC-lineage cells to inflammatory infiltrates remains to be investigated. The problem of separating monocyte-origin and DC-derived inflammatory cells is highlighted by in vitro studies showing that inflammation drives convergent transcriptional programs probably leading to very similar phenotypes (51). Indicators of recent monocyte origin include S100A8/9, recognized by antibody MAC387, a marker long used to describe “monocyte-macrophages” by pathologists (119). Further information is also required to understand the repopulation of LCs following an inflammatory insult. In humans, LCs are replaced by bone marrow-derived cells after HSC transplantation and graft-versus-host disease (79, 80). A two-phase kinetic of LC recovery was observed many years ago in humans in serial skin biopsies of delayed-type hypersensitivity reactions (120). In mice, more detailed analysis shows an initial infiltra-

tion by classical monocytes that express low levels of Langerin (121) followed by a second wave of more longlived LC precursors (122, 123). Langerin+ cells may be derived from monocytes and CD34+ progenitors in vitro (124–126). Recent experiments indicate that monocytes express only a low level of Langerin in comparison with CD1c+ DCs, which rapidly form LC-like cells with Birbeck granules (65, 86). Together, these results suggest that the DC differentiation pathway may contribute to long-term LC repopulation following inflammation.

GENETIC CONTROL OF THE HUMAN MONOCYTE AND DC LINEAGES The genetic control of human monocyte and DC development has been illuminated in a number of ways, using defined growth factors, knockdown of specific genes, and xenograft models (56, 102). Humans with genetic defects of monocyte and DC development have also been described (reviewed in reference 127). DC production from human progenitors is promoted by Flt3 ligand, GM-CSF, IL-4, and the presence of stromal cells (82, 84). Administration of Flt3 ligand to humans expands DCs in vivo (83). GM-CSF is implicated in DC development in mice. This function has not been thoroughly explored in humans, but blockade of GM-CSF signaling by spontaneously occurring autoantibodies (or rare receptor mutations) dramatically inhibits alveolar macrophage development, causing pulmonary alveolar proteinosis (128). Human monocyte and DC development becomes severely impaired in humans with heterozygous GATA2 mutation (129). GATA2 is required for stem cell selfrenewal, and the hemizygous state preferentially affects mononuclear cell development, resulting in a combined DC, monocyte, B, and NK lymphoid (DCML) deficiency (130). The clinical picture of immunodeficiency is variable, but susceptibility to viral infections and mycobacteria is common (131). Flt3 ligand is hugely elevated, probably in response to a fundamental disturbance of stem cell equilibrium rather than as a result of DC deficiency. Ikaros mutation also causes a severe pancytopenia that includes monocytes and probably DCs (132). Neutropenia may involve genes implicated in MMC development in mice, such as GFI1, but monocytes and DCs have not been examined systematically in these disorders (133). Finally, WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome, due to gain-of-function mutation of CXCR4, causes neutropenia and global mononuclear deficiency by preventing leukocytes from leaving the marrow. Clinical



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manifestations of WHIM may overlap with the features of GATA2 mutation, including warts and susceptibility to mycobacteria (134). Biallelic IRF8 mutation in humans was reported to cause a severe defect of monocytes, myeloid DCs, and pDCs, while a heterozygous state induced only changes in CD1c+ DCs (44). A gene dosage effect may be at work because in the mouse there are substantial differences between the hypomorphic BXH2 mouse, which has a specific defect of cDC1, and the IRF8 gene-deleted mouse, which shows multiple lineage deficiencies and expanded granulopoiesis. Monocytopenia appears to be a direct consequence of the IRF8 dependency of Kruppel-like factor 4 (KLF4) (135), while granulopoiesis is promoted by the failure of IRF8 to suppress CCAAT/enhancerbinding protein α (C/EBPα) activity (136). IRF8 also associates with IRF4, a transcription factor implicated in the development of CD1c DCs (cDC2) (137). The variable effect of heterozygous IRF8 mutation on CD1c+ DCs may be mediated through an IRF8/IRF4 interaction, but further clarification of this is required. In cooperation with IRF8, the activator protein 1 (AP1) factor BATF3 plays a key role in DC development in mice and in human in vitro culture (56, 138). Within the DC lineage, the balance of basic helix-loop-helix protein E2-2 and its inhibitor ID2 is critical for the specification of pDCs or classical DCs from DC precursors (53). Mutation of E2-2 is the cause of Pitt-Hopkins syndrome, in which abnormal pDC development has been noted (139). The recently described transcription factor ZBTB46 is specifically expressed in myeloid CD1c+ DCs and their murine counterparts (31). Although it is not required for DC development, ZBTB46 is very useful in lineage marking and lineage-specific DC depletion to demonstrate that cDC2s have specific roles in immunity (97, 140). Monocytes do not express ZBTB46 at rest, but it is induced when they differentiate into DCs (97).

CONCLUSIONS AND FUTURE PERSPECTIVES The MPS provided a conceptual framework for understanding the development of MMCs for nearly half a century. It is becoming clear that monocytes are not the only axis of homeostasis and that long-lived macrophages and a specialized DC lineage contribute to tissue leukocyte populations. The mouse has been an indispensable model for the analysis of hematopoiesis and continues to provide many insights into the pathways of human monocyte and DC development. While the study of monocyte-derived populations in vitro has furnished many exciting experimental results, the true physiolog-

ical context of this work remains incompletely understood but is likely to provide significant insights into inflammatory monocyte differentiation. A distinct DC lineage is well described in humans, but more studies are required to understand the nonredundant role of these cells in immunity and how they can be manipulated for therapeutic purposes. In the near future, single-cell genomics will provide a new level of detail in the analysis of the progenitor cells and of the genomic and transcriptional architecture controlling cell fate (reviewed in reference 141). This is likely to revise classical models of hematopoiesis; rather than successive bifurcations in cell potential, the picture that is emerging is that differentiation is a graded process punctuated by many phenotypic states. Continuous phenotypic variables are likely to reflect gradients in cell potential, which may be in equilibrium and appear stochastic at given points. A further addition to the modern armamentarium is the use of deep phenotyping and unbiased hierarchical clustering to assist the identification of related populations (142). Intermediate populations may indeed harbor cells with intermediate potentials. The diversity of MMCs provides an important regulatory dimension in immunity and the study of their ontogeny endures as a priority area of research. Although many difficulties relating to their plasticity arise, the ability of progenitors to remain flexible in their potential is likely to be critical in allowing hematopoiesis and immunity to adapt to environmental challenges. ACKNOWLEDGMENTS The authors are grateful to M. A. Haniffa for critical review of the manuscript. V.B. is funded by Wellcome Intermediate Clinical Fellowship WT088555MA.

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