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Dendritic cell development: A personal historical perspective
Molecular Immunology 119 (2020) 64–68
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Revised manuscript
Dendritic cell development: A personal historical perspective
T
Ken Shortman* The Walter and Eliza Hall Institute, Melbourne, Australia
A B S T R A C T
Dendritic cells(DCs) were once considered as a single cell type closely related developmentally to macrophages. Now we recognise several subtypes of DCs and have outlined several different pathways that potentially lead to their development. This article outlines some of the research findings that led to these changes in perspective, from the point of view of one of the participants.
1. The DC picture around 1990 A clear model of dendritic cell (DC) biology and function had emerged by the 1990s, primarily from the pioneering work of Ralph Steinman and colleagues (as reviewed in Steinman, 1991). DCs were considered as a single group of antigen-presenting cells, able to activate naïve T cells and so crucial for initiating immune responses. They were considered as part of the mononuclear phagocyte system, but differed from related macrophages in their bias towards processing antigens for MHC presentation, as opposed to a macrophage bias to antigen degradation. Langerhans cells had been established as the model of migratory DCs, migrating from the skin to lymph nodes on pathogen contact and developing into mature DCs able to activate T cells (Schuler and Steinman, 1985). DCs had been found to be widely distributed in tissues (Hart and Fabre, 1981), but their low incidence made them difficult to isolate. A culture system for generating DC-like antigen presenting cells from blood monocytes under the influence of granulocyte/macrophage colony stimulating factor (GM-CSF) (Sallusto and Lanzavecchia, 1994; Romani et al., 1994) provided a convenient model for study of DC biology, and further emphasised a monocyte/macrophage/DC relationship. A simple developmental relationship between DCs and macrophages seemed likely, and this was later endorsed by the evidence for a common macrophage/DC committed progenitor termed MDP (Fogg et al., 2006). This was the straightforward model that my laboratory and other groups then complicated by the discovery of functionally and developmentally distinct subsets of DCs. 2. The switch from T cell to DC development I was initially interested in lymphocyte rather than DC development. In collaboration with Roland Scollay, I was studying T cell development in the thymus (Shortman et al., 1990). By seeking
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progressively earlier CD4−CD8− thymocytes we were hoping to identify the earliest T precursors (now termed ETPs) that had arrived in the thymus from bone marrow (BM), so making a connection with other aspects of haematopoiesis. This proved difficult, but eventually with Li Wu an early precursor was isolated (Wu et al., 1991). Despite this T cell focus, thymic DCs kept emerging from our studies and shifted our attention. A compelling instance came during our attempt to develop successive stages of T cell development in culture from our isolated ETPs. This was doomed to fail, since we were unaware at that time of the Notch signalling requirement for T cell development. Instead of T lineage cells, we produced with high efficiency clones of DCs from these apparently lymphoid committed precursors (Saunders et al., 1996). I then adopted a new philosophy: “If all you can grow in your garden are weeds, then study the weeds!”. DCs were my weeds, so my laboratory proceeded to study thymic DCs in some detail. 3. DC subsets Approaching the DC field from a thymus perspective immediately revealed some features of the DC surface that differed from the conventional picture, namely the expression of many typical lymphoid markers. In particular, a high proportion but not all of mouse thymic DCs were found to express CD8, and this was found to extend to a smaller proportion of DCs in spleen and lymph nodes (Vremec et al., 1992, 2000). This CD8 on mouse DCs was produced by the DCs themselves, rather than being picked up from associated T cells, and was an αα homodimer rather than the αβ heterodimer of typical of T cells. Furthermore, a different subset of mouse DCs was found to express CD4 (Vremec et al., 2000). Confusingly, this pattern of lymphoid marker expression differed from the pattern on human DCs, which typically expressed CD4 but not CD8. The clear phenotypic segregation of mouse CD8+ from CD8− DCs
Corresponding author at: The Walter and Eliza Hall Institute, 1G Royal Parade Parkville, Victoria, 3050, Australia. E-mail address: [email protected].
https://doi.org/10.1016/j.molimm.2019.12.016 Received 18 September 2019; Received in revised form 2 December 2019; Accepted 20 December 2019 0161-5890/ © 2019 Elsevier Ltd. All rights reserved.
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intrathymic origin differing from the BM myeloid progenitor origin of their splenic counterparts. When Carlos Ardavin and Li Wu transferred our ETP into an irradiated mouse thymus, T lineage thymocytes and thymic DCs were generated in a similar ratio to that found in the intact thymus (Ardavin et al., 1993), suggesting a common linked origin for both. Later elegant studies from Goldschneider’s laboratory (Donsky and Goldschneider, 2003) pointed to two developmentally separate thymic DC populations, one representing 50 % of thymic DCs resulting from ongoing seeding of DC from the bloodstream and including the CD8− subset, and the other with early thymus seeding characteristics similar to prothymocytes and representing mainly CD8+ DCs. However, despite the demonstrated potential of our ETPs to produce DCs under irradiated recipient conditions, most subsequent work on the mouse adult steady state thymus, including crucial lineage marker studies, has pointed to a conventional myeloid origin unlinked to T cell development, even for the thymic cDC1 subset (Rodenwald et al., 1999; Radtke et al., 2000; Luche et al., 2011; Lyszkiewicz et al., 2015). The only study pointing to a distinct lymphoid origin for much of the steady state thymic cDC1 population is our own work using immunoglobulin heavy chain (IgH) DJ gene rearrangements as an internal marker of lymphoid origin, and where thymic but not splenic cDC1 showed such gene rearrangements (Corcoran et al., 2003). However, the PCR assays for such gene rearrangements were not quantitative and despite our controls signals from contaminant lymphoid DNA sources cannot be excluded. Overall, the development of thymic DCs needs further investigation, not only because of the lymphoid versus myeloid origin issue, but also to determine if there is an intrathymic early precursor origin for many thymic cDC1, as opposed to an ongoing bloodstream origin, regardless of the nature of this precursor. Both the postnatal developing thymus and the steady state adult thymus need to be analysed.
pointed to the existence of discrete DC subtypes, an unwanted complication to the previous straightforward model of DC biology. This concept of separate DC subsets gained more credence when the CD8+ and CD8− subsets were shown, by our laboratory and other groups, to have differing immunological functions. We first noted the production of different cytokines in response to different stimuli (Hochrein et al., 2001). Other functional differences were found (reviewed in Shortman and Heath, 2010), including the special capacity of the CD8+ subset to cross-present exogenous antigens on MHC class I and so selectively activate CD8+ T cells (den Haan et al., 2000; Pooley et al., 2001; Schnorrer et al., 2006). However, the significance of such DC subsets for the human immune system was frequently questioned, since human DCs lacked a CD8+ population. In addition, to this day there is no demonstrated function for CD8αα on mouse DCs. Mireille Lahoud, Irene Caminschi and I began to search for surface receptors that might explain functional differences between DC subsets, which led us to the dead cell receptor Clec9A, largely specific for CD8+ DCs (Caminschi et al., 2008). This receptor was also found by the Reis e Sousa laboratory, who termed it DNGR1 (Sancho et al., 2008). The new chemokine receptor XCR1 was then found to have an even clearer specificity for CD8+ DCs (Crozat et al., 2010). Importantly, these new surface receptors served to mark similar populations in the human as well as the mouse DC systems. This, followed by detailed gene expression analyses, led to identification of common human and mouse DC subsets, now designated as cDC1 and cDC2 (Guilliams et al., 2014). The final extension of the DC family came from studies on human rather than mouse immune responses. A new class of cells producing large quantities of type 1 interferon in response to viral infection was identified, shown to have a plasma cell like form but then to adopt a dendritic morphology on further activation; they were finally termed plasmacytoid DCs (pDCs) (reviewed in Liu, 2001). An equivalent mouse pDC population was then sought and found by many laboratories, including by Meredith O’Keeffe in my laboratory (Asselin-Paturel et al., 2001; O’Keeffe et al., 2002).
6. Pre-DCs Tracking the myeloid pathway of DC development from early BM precursors proved difficult, although Cattral and colleagues were making progress (Diao et al., 2004). Shalin Naik and I adopted a reverse strategy, to hunt downstream for the immediate precursors of DC in mouse spleen. Since we aimed to understand steady state DC development, we used transfer of putative precursors into non-irradiated recipients, and assayed soon after transfer to identify these late progenitors. This led to the identification of what we termed “pre-cDCs”, since they formed cDC1 and cDC2 but not pDC (Naik et al., 2006). They are now simply termed pre-DCs. Importantly, these immediate DC progenitors were clearly not monocytes, so this separated the pathway leading to steady state spleen DCs from the established monocyte to DC pathway. This work was extended by the Nussenzweig laboratory (Liu et al., 2009), who made the connection to BM by showing that these pre-DCs were the precursors that migrated from BM through the bloodstream to produce the steady state DC population in peripheral lymphoid organs. Our earlier work had hinted that the pre-DC population included precursors already committed to the cDC1 or cDC2 subtype. Subsequent work from the Ginhoux laboratory (Schlitzer et al., 2015) and the Murphy laboratory (Grajales-Reyes et al., 2015) demonstrated that such commitment had already occurred in BM. Although the role of bloodstream-born pre-DCs in establishing the DC populations in the periphery is well accepted, the contribution of other possible DC precursors needs to be accessed. A BM population resembling pDCs by some surface markers but distinct from true pDCs has been described (Schlitzer et al., 2009; O’Keeffe et al., 2012), and found to have a capacity produce pDCs and cDCs, depending on environmental conditions. The extent to which this potential precursor contributes to the peripheral DC population is still not clear.
4. Early speculation on DC subset development The existence of discrete DC subtypes immediately posed questions about their origin. Did they all derive from the same progenitor in BM, or were different developmental pathways involved? A major advance in haematology at that time was the isolation by the Weissman laboratory of common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) (Kondo et al., 1997; Akashi et al., 2000). I proposed, without direct evidence, that the CD8+ DCs bearing lymphoid markers were of lymphoid (CLP) origin whereas the more conventional CD8− DCs were of myeloid (CMP) origin. This stands as the first suggestion that some members of the DC family could be of lymphoid origin. However, when directly tested by the Weissman laboratory (Traver et al., 2000: Manz et al., 2001) and by Li Wu in my laboratory (Wu et al., 2001; D’Amico and Wu, 2003) this theory proved to be wrong. On transfer into irradiated recipient mice both the CMP and the CLP progenitor populations had the capacity to produce CD8+ and CD8− DCs, as well as pDCs. Clearly the nature of the precursor population did not determine the surface phenotype of the DC produced! Results from this extreme emergency haematopoiesis model left open the question of the origin of these DC subtypes in steady state. Subsequent work has made it clear that in steady state the majority of DCs in spleen and lymph nodes, both the CD8+ DCs (cDC1) and CD8− DCs (cDC2), derive from myeloid precursors. 5. Thymic DC development
7. Cytokines driving DC production
The thymus differs from peripheral lymphoid organs in having a much higher proportion of the CD8+ (cDC1) DC subset. A key question was whether thymic DCs, in particular the thymic cDC1 subtype, had an
The finding that the immediate precursors of steady state lymphoid 65
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organ DCs were not monocytes fitted with the emerging picture of two distinct DC developmental systems, one generating steady state DCs, the other monocyte-derived stream functioning mainly in response to inflammation (Reviewed in Shortman and Naik, 2007; Merad et al., 2013). The generation in culture of DC-like antigen presenting cells from monocytes in response to GM-CSF was seen as a model of inflammatory DC development. The finding by the Immunex laboratories Maraskovcsky et al. (1996) that Flt3-ligand (FL) promoted the expansion of the DCs normally found in steady state lymphoid tissues added to this picture of separate DC developmental pathways, and FL was established as a major regulator of DC homeostasis (Reviewed in Merad et al., 2013). Since DCs had been considered as non-dividing end cells, this effect of FL was initially assumed to be at the level of DC development in BM. In support of this, an effective culture system for generating cDC1, cDC2 and pDC from mouse BM was developed, with FL the only exogenous cytokine required (Naik et al., 2005). However, the Nussenzweig laboratory found that peripheral DCs were capable of proliferative expansion and showed that much of the effect of FL on DC numbers was at this level (Waskow et al., 2008). They claimed FL did not promote DC development at the BM level, despite the expression of Flt3 by many BM precursors. In contrast, recent studies by Lin and Naik (In preparation) have shown that FL does promote DC development at the BM progenitor level if the appropriate progenitors are studied. Accordingly, it now seems FL promotes DC development at both the early BM and later peripheral DC levels.
development of DCs from pre-DCs and the proliferative expansion of early DCs driven by FL all indicated that important developmental events occurred in the periphery. Lew and colleagues (Lin et al., 2008) showed that only about half the CD8+DC (cDC1) in mouse spleen had developed the capacity to cross-present exogenous antigens on MHC I, pointing either to separate sub-lineages or to an ongoing developmental process. Sathe and I, together with two other laboratories, then showed that cDC1 are not born with cross-presentation capacity; rather it is induced during peripheral development, by factors including GM-CSF and TLR ligands (Reviewed in Dresch et al., 2012). Doubtless other crucial DC functions develop in the periphery in steady state within cells we already class as fully formed DCs, even before any overt DC activation initiates further changes. 10. pDC development pDCs were always uncomfortable members of the DC family, since they resemble B cells as much as DCs in their physiology and antigen presentation functions. However, their developmental history seemed to unite them with DCs, particularly their common origin from the myeloid pathway via the CDP (Naik et al., 2007; Onai et al., 2007). Further details of pDC specification down this pathway were later described (Onai et al., 2013). In view of the subsequent finding of a type 1 interferon-producing non-pDC population, similar to pDC by some surface markers but lacking CCR9 expression (Schlitzer et al., 2009; O’Keeffe et al., 2012), we must question how many of the previously described myeloid derived pDCs were actually this new pDC-like cell. Many of the reports that pDC on activation can develop into cDC, including our own (O’Keeffe et al., 2002), might be due to this pDC-like cell. As discussed in 4, CLP have a demonstrated capacity to produce both pDC and cDC on transfer into irradiated recipients, and later work demonstrated how this potential might be biased to pDC production (Chen et al., 2013). There were several hints that this capacity to produce pDC via a lymphoid route might be expressed in steady state mice (Vogt et al., 2009). Lineage marker studies then strongly pointed to a lymphoid origin for many pDCs. Kincade’s laboratory (Pelayo et al., 2005) used the expression of green fluorescent protein under the RAG-1 promoter as a lymphoid origin marker and so demonstrated that many pDCs displayed this indicator of past lymphoid orientation; this marker had the advantage of direct single cell readout, although it was potentially lost by dilution with extensive proliferation. In collaboration with Lynn Corcoran (Corcoran et al., 2003) we used IgH gene DJ rearrangements to delineate a proportion of pDCs that appeared to be of lymphoid precursor origin; this provided an indelible origin marker, but involved non-quantitative assays on bulk pDC preparations, so was susceptible to contamination by other cells of lymphoid origin. Using our FL stimulated BM culture system and employing both markers of a lymphoid past, Sathe and I then found two routes to pDC development, one apparently lymphoid, one apparently myeloid (Sathe et al., 2013; Sathe et al., 2014). Our general conclusion was that pDCs were derived from both lymphoid and myeloid pathways (reviewed in Shortman et al., 2013). Since then more technologically advanced studies have concluded that the majority of “true” mouse pDCs (type 1 interferonproducing cells with a CD11c+CD45RA+CD11b−CCR9+ surface phenotype) are likely of lymphoid origin (Rodrigues et al., 2018; Dress et al., 2019). The developmental origin of human pDCs, whose immunological properties have often been poorly aligned with mouse pDCs, remains to be determined.
8. DC progenitors in bone marrow With Shalin Naik our laboratory began a search for the DC precursors in the BM myeloid pathway upstream of the pre-cDC. This search was simplified by using the FL-driven culture system that modelled closely DC development in BM (Naik et al., 2005). We identified a precursor clonally restricted to producing cDCs and pDCs that we termed pro-DC (Naik et al., 2007). At the same time the Manz laboratory isolated directly from BM a precursor clonally restricted to cDC and pDC production that they termed a common DC progenitor or CDP (Onai et al., 2007). These proved to be equivalent precursors and the term CDP rather than pro-DC became generally accepted. A central finding in these studies was that CDP showed very little capacity to generate monocytes or macrophages, indicating that the pathway to macrophage production branched off further upstream. Priyanka Sathe and I then looked in mouse BM for DC precursors upstream of the CDP, expecting to find the previously described MDP (Fogg et al., 2006), restricted to DC and macrophage production. Our FL dependent clonal assays failed to reveal a significant frequency of precursors with this restricted potential (Sathe et al., 2014), although with a cloning efficiency of no more than 30% we could have missed precursors with special culture requirements. However recent lineage tracking studies in mice have not revealed evidence for a common restricted macrophage/DC precursor (Naik et al., 2013). In contrast, the Nussenzweig laboratory has produced clonal evidence for an MDP population in humans (Lee et al., 2015). The clear clonal evidence for MDP in the original publication (Fogg et al., 2006) was obtained from cultures driven with relatively high levels of GM-CSF. Even in the presence of FL, this produces a strong bias to production of the monocyte derived DC type, rather than cDC1 or cDC2 (Sathe et al., 2014). The DC potential of mouse MDP might therefore be restricted to monocyte derived DC, the lineages leading to cDC1 and cDC2 having a different origin. The significance of the MDP population in mice and in humans should be re-examined using newer single precursor cell technologies.
11. Final comments
9. DC development in the periphery
The studies surveyed here served to establish some basic features of the complex cellular developmental pathways that lead to the group of cells we call DCs. I have omitted molecular studies of the crucial transcription factors that determine the development of distinct DC
It was initially assumed that all DC development would occur in BM, the DC exiting as short-lived end cells. As described in 6 and 7, the final 66
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subtypes, since these have been largely been carried out by other groups and have been reviewed elsewhere (Merad et al., 2013; Midler and Jung, 2014; Satpathy et al., 2012). The early cellular studies have shown that there are different functional subtypes of DCs and potentially several different developmental routes to each one. I have used the term “convergent differentiation” to indicate that different developmental routes may lead to a given functional state. The product cells sharing this immunological function may appear to be similar but are often distinct. For example, efficient antigen processing and presentation, and even cross-presentation, can be achieved by cells derived from CDPs or derived from monocytes. Whether they both should be called DCs is disputed (Guilliams et al., 2014; Lutz et al., 2017). Similarly, cells with the striking type 1 interferon production capability and much of the surface phenotype of pDCs can be formed via a lymphoid or a myeloid route. Whether the two routes are able to produce identical cells is unclear. Much of the previous work, including my own, has focussed on the pathways leading to adult “steady state” DC populations. While important, this is essentially a state where the immune system is not engaged in some of its basic defence functions. DC developmental pathways are likely to change from enfant to adult, after infection or inflammation, and during emergency haematopoiesis following BM transplantation. It will be important to understand DC developmental pathways under these conditions, as well as in steady state A limitation of past research has been the need to work with precursor populations such as CDP, CMP or MDP, isolated as bulk populations despite evidence of heterogeneity within each. A much more precise and detailed mapping of DC developmental pathways is now possible by single precursor cell surface phenotype and RNA expression analysis, by employing new endogenous lineage markers and introduced bar-code markers, as detailed by later contributions to this volume. References Akashi, K., Traver, D., Miyamoto, T., Weissman, I.L., 2000. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404, 193–197. Ardavin, C., Wu, L., Li, C.L., Shortman, K., 1993. Thymic dendritic cells and T cells develop simultaneously within the thymus from a common precursor population. Nature 362, 761–763. Asselin-Paturel, C., Boonstra, A., Dalod, M., Durand, I., Yessaad, N., Dezutter-Dambuyant, C., Vicari, A., O’Garra, A., Biron, C., Briere, F., Trinchieri, G., 2001. Mouse type 1 IFNproducing cells are immature APCs with plasmacytoid morphology. Nat. Immunol. 2, 1144–1150. 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Crozat, K., Guiton, R., Contreras, V., Feuillet, V., Dutertre, C.A., Ventre, E., Thein-Phong, V.M., Baranek, T., Storset, A.K., Marvel, J., Boudinot, P., Hosmalin, A., SchwartzCornil, I., Dalod, M., 2010. The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells. J. Exp. Med. 207, 1283–1292. D’Amico, A., Wu, L., 2003. The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3. J. Exp. Med. 198, 293–303. Diao, J., Winter, E., Chen, W., Cantin, C., Cattral, M.S., 2004. Characterization of distinct conventional and plasmacytoid dendritic cell-committed precursors in murine bone marrow. J. Immunol. 173, 1826–1833. Donsky, E., Goldschneider, I., 2003. Two developmentally distinct populations of dendritic cells inhabit the adult mouse thymus: demonstration by differential importation of hematogenous precursors under steady state conditions. 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Revised manuscript
Dendritic cell development: A personal historical perspective
T
Ken Shortman* The Walter and Eliza Hall Institute, Melbourne, Australia
A B S T R A C T
Dendritic cells(DCs) were once considered as a single cell type closely related developmentally to macrophages. Now we recognise several subtypes of DCs and have outlined several different pathways that potentially lead to their development. This article outlines some of the research findings that led to these changes in perspective, from the point of view of one of the participants.
1. The DC picture around 1990 A clear model of dendritic cell (DC) biology and function had emerged by the 1990s, primarily from the pioneering work of Ralph Steinman and colleagues (as reviewed in Steinman, 1991). DCs were considered as a single group of antigen-presenting cells, able to activate naïve T cells and so crucial for initiating immune responses. They were considered as part of the mononuclear phagocyte system, but differed from related macrophages in their bias towards processing antigens for MHC presentation, as opposed to a macrophage bias to antigen degradation. Langerhans cells had been established as the model of migratory DCs, migrating from the skin to lymph nodes on pathogen contact and developing into mature DCs able to activate T cells (Schuler and Steinman, 1985). DCs had been found to be widely distributed in tissues (Hart and Fabre, 1981), but their low incidence made them difficult to isolate. A culture system for generating DC-like antigen presenting cells from blood monocytes under the influence of granulocyte/macrophage colony stimulating factor (GM-CSF) (Sallusto and Lanzavecchia, 1994; Romani et al., 1994) provided a convenient model for study of DC biology, and further emphasised a monocyte/macrophage/DC relationship. A simple developmental relationship between DCs and macrophages seemed likely, and this was later endorsed by the evidence for a common macrophage/DC committed progenitor termed MDP (Fogg et al., 2006). This was the straightforward model that my laboratory and other groups then complicated by the discovery of functionally and developmentally distinct subsets of DCs. 2. The switch from T cell to DC development I was initially interested in lymphocyte rather than DC development. In collaboration with Roland Scollay, I was studying T cell development in the thymus (Shortman et al., 1990). By seeking
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progressively earlier CD4−CD8− thymocytes we were hoping to identify the earliest T precursors (now termed ETPs) that had arrived in the thymus from bone marrow (BM), so making a connection with other aspects of haematopoiesis. This proved difficult, but eventually with Li Wu an early precursor was isolated (Wu et al., 1991). Despite this T cell focus, thymic DCs kept emerging from our studies and shifted our attention. A compelling instance came during our attempt to develop successive stages of T cell development in culture from our isolated ETPs. This was doomed to fail, since we were unaware at that time of the Notch signalling requirement for T cell development. Instead of T lineage cells, we produced with high efficiency clones of DCs from these apparently lymphoid committed precursors (Saunders et al., 1996). I then adopted a new philosophy: “If all you can grow in your garden are weeds, then study the weeds!”. DCs were my weeds, so my laboratory proceeded to study thymic DCs in some detail. 3. DC subsets Approaching the DC field from a thymus perspective immediately revealed some features of the DC surface that differed from the conventional picture, namely the expression of many typical lymphoid markers. In particular, a high proportion but not all of mouse thymic DCs were found to express CD8, and this was found to extend to a smaller proportion of DCs in spleen and lymph nodes (Vremec et al., 1992, 2000). This CD8 on mouse DCs was produced by the DCs themselves, rather than being picked up from associated T cells, and was an αα homodimer rather than the αβ heterodimer of typical of T cells. Furthermore, a different subset of mouse DCs was found to express CD4 (Vremec et al., 2000). Confusingly, this pattern of lymphoid marker expression differed from the pattern on human DCs, which typically expressed CD4 but not CD8. The clear phenotypic segregation of mouse CD8+ from CD8− DCs
Corresponding author at: The Walter and Eliza Hall Institute, 1G Royal Parade Parkville, Victoria, 3050, Australia. E-mail address: [email protected].
https://doi.org/10.1016/j.molimm.2019.12.016 Received 18 September 2019; Received in revised form 2 December 2019; Accepted 20 December 2019 0161-5890/ © 2019 Elsevier Ltd. All rights reserved.
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intrathymic origin differing from the BM myeloid progenitor origin of their splenic counterparts. When Carlos Ardavin and Li Wu transferred our ETP into an irradiated mouse thymus, T lineage thymocytes and thymic DCs were generated in a similar ratio to that found in the intact thymus (Ardavin et al., 1993), suggesting a common linked origin for both. Later elegant studies from Goldschneider’s laboratory (Donsky and Goldschneider, 2003) pointed to two developmentally separate thymic DC populations, one representing 50 % of thymic DCs resulting from ongoing seeding of DC from the bloodstream and including the CD8− subset, and the other with early thymus seeding characteristics similar to prothymocytes and representing mainly CD8+ DCs. However, despite the demonstrated potential of our ETPs to produce DCs under irradiated recipient conditions, most subsequent work on the mouse adult steady state thymus, including crucial lineage marker studies, has pointed to a conventional myeloid origin unlinked to T cell development, even for the thymic cDC1 subset (Rodenwald et al., 1999; Radtke et al., 2000; Luche et al., 2011; Lyszkiewicz et al., 2015). The only study pointing to a distinct lymphoid origin for much of the steady state thymic cDC1 population is our own work using immunoglobulin heavy chain (IgH) DJ gene rearrangements as an internal marker of lymphoid origin, and where thymic but not splenic cDC1 showed such gene rearrangements (Corcoran et al., 2003). However, the PCR assays for such gene rearrangements were not quantitative and despite our controls signals from contaminant lymphoid DNA sources cannot be excluded. Overall, the development of thymic DCs needs further investigation, not only because of the lymphoid versus myeloid origin issue, but also to determine if there is an intrathymic early precursor origin for many thymic cDC1, as opposed to an ongoing bloodstream origin, regardless of the nature of this precursor. Both the postnatal developing thymus and the steady state adult thymus need to be analysed.
pointed to the existence of discrete DC subtypes, an unwanted complication to the previous straightforward model of DC biology. This concept of separate DC subsets gained more credence when the CD8+ and CD8− subsets were shown, by our laboratory and other groups, to have differing immunological functions. We first noted the production of different cytokines in response to different stimuli (Hochrein et al., 2001). Other functional differences were found (reviewed in Shortman and Heath, 2010), including the special capacity of the CD8+ subset to cross-present exogenous antigens on MHC class I and so selectively activate CD8+ T cells (den Haan et al., 2000; Pooley et al., 2001; Schnorrer et al., 2006). However, the significance of such DC subsets for the human immune system was frequently questioned, since human DCs lacked a CD8+ population. In addition, to this day there is no demonstrated function for CD8αα on mouse DCs. Mireille Lahoud, Irene Caminschi and I began to search for surface receptors that might explain functional differences between DC subsets, which led us to the dead cell receptor Clec9A, largely specific for CD8+ DCs (Caminschi et al., 2008). This receptor was also found by the Reis e Sousa laboratory, who termed it DNGR1 (Sancho et al., 2008). The new chemokine receptor XCR1 was then found to have an even clearer specificity for CD8+ DCs (Crozat et al., 2010). Importantly, these new surface receptors served to mark similar populations in the human as well as the mouse DC systems. This, followed by detailed gene expression analyses, led to identification of common human and mouse DC subsets, now designated as cDC1 and cDC2 (Guilliams et al., 2014). The final extension of the DC family came from studies on human rather than mouse immune responses. A new class of cells producing large quantities of type 1 interferon in response to viral infection was identified, shown to have a plasma cell like form but then to adopt a dendritic morphology on further activation; they were finally termed plasmacytoid DCs (pDCs) (reviewed in Liu, 2001). An equivalent mouse pDC population was then sought and found by many laboratories, including by Meredith O’Keeffe in my laboratory (Asselin-Paturel et al., 2001; O’Keeffe et al., 2002).
6. Pre-DCs Tracking the myeloid pathway of DC development from early BM precursors proved difficult, although Cattral and colleagues were making progress (Diao et al., 2004). Shalin Naik and I adopted a reverse strategy, to hunt downstream for the immediate precursors of DC in mouse spleen. Since we aimed to understand steady state DC development, we used transfer of putative precursors into non-irradiated recipients, and assayed soon after transfer to identify these late progenitors. This led to the identification of what we termed “pre-cDCs”, since they formed cDC1 and cDC2 but not pDC (Naik et al., 2006). They are now simply termed pre-DCs. Importantly, these immediate DC progenitors were clearly not monocytes, so this separated the pathway leading to steady state spleen DCs from the established monocyte to DC pathway. This work was extended by the Nussenzweig laboratory (Liu et al., 2009), who made the connection to BM by showing that these pre-DCs were the precursors that migrated from BM through the bloodstream to produce the steady state DC population in peripheral lymphoid organs. Our earlier work had hinted that the pre-DC population included precursors already committed to the cDC1 or cDC2 subtype. Subsequent work from the Ginhoux laboratory (Schlitzer et al., 2015) and the Murphy laboratory (Grajales-Reyes et al., 2015) demonstrated that such commitment had already occurred in BM. Although the role of bloodstream-born pre-DCs in establishing the DC populations in the periphery is well accepted, the contribution of other possible DC precursors needs to be accessed. A BM population resembling pDCs by some surface markers but distinct from true pDCs has been described (Schlitzer et al., 2009; O’Keeffe et al., 2012), and found to have a capacity produce pDCs and cDCs, depending on environmental conditions. The extent to which this potential precursor contributes to the peripheral DC population is still not clear.
4. Early speculation on DC subset development The existence of discrete DC subtypes immediately posed questions about their origin. Did they all derive from the same progenitor in BM, or were different developmental pathways involved? A major advance in haematology at that time was the isolation by the Weissman laboratory of common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) (Kondo et al., 1997; Akashi et al., 2000). I proposed, without direct evidence, that the CD8+ DCs bearing lymphoid markers were of lymphoid (CLP) origin whereas the more conventional CD8− DCs were of myeloid (CMP) origin. This stands as the first suggestion that some members of the DC family could be of lymphoid origin. However, when directly tested by the Weissman laboratory (Traver et al., 2000: Manz et al., 2001) and by Li Wu in my laboratory (Wu et al., 2001; D’Amico and Wu, 2003) this theory proved to be wrong. On transfer into irradiated recipient mice both the CMP and the CLP progenitor populations had the capacity to produce CD8+ and CD8− DCs, as well as pDCs. Clearly the nature of the precursor population did not determine the surface phenotype of the DC produced! Results from this extreme emergency haematopoiesis model left open the question of the origin of these DC subtypes in steady state. Subsequent work has made it clear that in steady state the majority of DCs in spleen and lymph nodes, both the CD8+ DCs (cDC1) and CD8− DCs (cDC2), derive from myeloid precursors. 5. Thymic DC development
7. Cytokines driving DC production
The thymus differs from peripheral lymphoid organs in having a much higher proportion of the CD8+ (cDC1) DC subset. A key question was whether thymic DCs, in particular the thymic cDC1 subtype, had an
The finding that the immediate precursors of steady state lymphoid 65
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organ DCs were not monocytes fitted with the emerging picture of two distinct DC developmental systems, one generating steady state DCs, the other monocyte-derived stream functioning mainly in response to inflammation (Reviewed in Shortman and Naik, 2007; Merad et al., 2013). The generation in culture of DC-like antigen presenting cells from monocytes in response to GM-CSF was seen as a model of inflammatory DC development. The finding by the Immunex laboratories Maraskovcsky et al. (1996) that Flt3-ligand (FL) promoted the expansion of the DCs normally found in steady state lymphoid tissues added to this picture of separate DC developmental pathways, and FL was established as a major regulator of DC homeostasis (Reviewed in Merad et al., 2013). Since DCs had been considered as non-dividing end cells, this effect of FL was initially assumed to be at the level of DC development in BM. In support of this, an effective culture system for generating cDC1, cDC2 and pDC from mouse BM was developed, with FL the only exogenous cytokine required (Naik et al., 2005). However, the Nussenzweig laboratory found that peripheral DCs were capable of proliferative expansion and showed that much of the effect of FL on DC numbers was at this level (Waskow et al., 2008). They claimed FL did not promote DC development at the BM level, despite the expression of Flt3 by many BM precursors. In contrast, recent studies by Lin and Naik (In preparation) have shown that FL does promote DC development at the BM progenitor level if the appropriate progenitors are studied. Accordingly, it now seems FL promotes DC development at both the early BM and later peripheral DC levels.
development of DCs from pre-DCs and the proliferative expansion of early DCs driven by FL all indicated that important developmental events occurred in the periphery. Lew and colleagues (Lin et al., 2008) showed that only about half the CD8+DC (cDC1) in mouse spleen had developed the capacity to cross-present exogenous antigens on MHC I, pointing either to separate sub-lineages or to an ongoing developmental process. Sathe and I, together with two other laboratories, then showed that cDC1 are not born with cross-presentation capacity; rather it is induced during peripheral development, by factors including GM-CSF and TLR ligands (Reviewed in Dresch et al., 2012). Doubtless other crucial DC functions develop in the periphery in steady state within cells we already class as fully formed DCs, even before any overt DC activation initiates further changes. 10. pDC development pDCs were always uncomfortable members of the DC family, since they resemble B cells as much as DCs in their physiology and antigen presentation functions. However, their developmental history seemed to unite them with DCs, particularly their common origin from the myeloid pathway via the CDP (Naik et al., 2007; Onai et al., 2007). Further details of pDC specification down this pathway were later described (Onai et al., 2013). In view of the subsequent finding of a type 1 interferon-producing non-pDC population, similar to pDC by some surface markers but lacking CCR9 expression (Schlitzer et al., 2009; O’Keeffe et al., 2012), we must question how many of the previously described myeloid derived pDCs were actually this new pDC-like cell. Many of the reports that pDC on activation can develop into cDC, including our own (O’Keeffe et al., 2002), might be due to this pDC-like cell. As discussed in 4, CLP have a demonstrated capacity to produce both pDC and cDC on transfer into irradiated recipients, and later work demonstrated how this potential might be biased to pDC production (Chen et al., 2013). There were several hints that this capacity to produce pDC via a lymphoid route might be expressed in steady state mice (Vogt et al., 2009). Lineage marker studies then strongly pointed to a lymphoid origin for many pDCs. Kincade’s laboratory (Pelayo et al., 2005) used the expression of green fluorescent protein under the RAG-1 promoter as a lymphoid origin marker and so demonstrated that many pDCs displayed this indicator of past lymphoid orientation; this marker had the advantage of direct single cell readout, although it was potentially lost by dilution with extensive proliferation. In collaboration with Lynn Corcoran (Corcoran et al., 2003) we used IgH gene DJ rearrangements to delineate a proportion of pDCs that appeared to be of lymphoid precursor origin; this provided an indelible origin marker, but involved non-quantitative assays on bulk pDC preparations, so was susceptible to contamination by other cells of lymphoid origin. Using our FL stimulated BM culture system and employing both markers of a lymphoid past, Sathe and I then found two routes to pDC development, one apparently lymphoid, one apparently myeloid (Sathe et al., 2013; Sathe et al., 2014). Our general conclusion was that pDCs were derived from both lymphoid and myeloid pathways (reviewed in Shortman et al., 2013). Since then more technologically advanced studies have concluded that the majority of “true” mouse pDCs (type 1 interferonproducing cells with a CD11c+CD45RA+CD11b−CCR9+ surface phenotype) are likely of lymphoid origin (Rodrigues et al., 2018; Dress et al., 2019). The developmental origin of human pDCs, whose immunological properties have often been poorly aligned with mouse pDCs, remains to be determined.
8. DC progenitors in bone marrow With Shalin Naik our laboratory began a search for the DC precursors in the BM myeloid pathway upstream of the pre-cDC. This search was simplified by using the FL-driven culture system that modelled closely DC development in BM (Naik et al., 2005). We identified a precursor clonally restricted to producing cDCs and pDCs that we termed pro-DC (Naik et al., 2007). At the same time the Manz laboratory isolated directly from BM a precursor clonally restricted to cDC and pDC production that they termed a common DC progenitor or CDP (Onai et al., 2007). These proved to be equivalent precursors and the term CDP rather than pro-DC became generally accepted. A central finding in these studies was that CDP showed very little capacity to generate monocytes or macrophages, indicating that the pathway to macrophage production branched off further upstream. Priyanka Sathe and I then looked in mouse BM for DC precursors upstream of the CDP, expecting to find the previously described MDP (Fogg et al., 2006), restricted to DC and macrophage production. Our FL dependent clonal assays failed to reveal a significant frequency of precursors with this restricted potential (Sathe et al., 2014), although with a cloning efficiency of no more than 30% we could have missed precursors with special culture requirements. However recent lineage tracking studies in mice have not revealed evidence for a common restricted macrophage/DC precursor (Naik et al., 2013). In contrast, the Nussenzweig laboratory has produced clonal evidence for an MDP population in humans (Lee et al., 2015). The clear clonal evidence for MDP in the original publication (Fogg et al., 2006) was obtained from cultures driven with relatively high levels of GM-CSF. Even in the presence of FL, this produces a strong bias to production of the monocyte derived DC type, rather than cDC1 or cDC2 (Sathe et al., 2014). The DC potential of mouse MDP might therefore be restricted to monocyte derived DC, the lineages leading to cDC1 and cDC2 having a different origin. The significance of the MDP population in mice and in humans should be re-examined using newer single precursor cell technologies.
11. Final comments
9. DC development in the periphery
The studies surveyed here served to establish some basic features of the complex cellular developmental pathways that lead to the group of cells we call DCs. I have omitted molecular studies of the crucial transcription factors that determine the development of distinct DC
It was initially assumed that all DC development would occur in BM, the DC exiting as short-lived end cells. As described in 6 and 7, the final 66
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subtypes, since these have been largely been carried out by other groups and have been reviewed elsewhere (Merad et al., 2013; Midler and Jung, 2014; Satpathy et al., 2012). The early cellular studies have shown that there are different functional subtypes of DCs and potentially several different developmental routes to each one. I have used the term “convergent differentiation” to indicate that different developmental routes may lead to a given functional state. The product cells sharing this immunological function may appear to be similar but are often distinct. For example, efficient antigen processing and presentation, and even cross-presentation, can be achieved by cells derived from CDPs or derived from monocytes. Whether they both should be called DCs is disputed (Guilliams et al., 2014; Lutz et al., 2017). Similarly, cells with the striking type 1 interferon production capability and much of the surface phenotype of pDCs can be formed via a lymphoid or a myeloid route. Whether the two routes are able to produce identical cells is unclear. Much of the previous work, including my own, has focussed on the pathways leading to adult “steady state” DC populations. While important, this is essentially a state where the immune system is not engaged in some of its basic defence functions. DC developmental pathways are likely to change from enfant to adult, after infection or inflammation, and during emergency haematopoiesis following BM transplantation. It will be important to understand DC developmental pathways under these conditions, as well as in steady state A limitation of past research has been the need to work with precursor populations such as CDP, CMP or MDP, isolated as bulk populations despite evidence of heterogeneity within each. 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