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Developmental origin of pre-DC2
Developmental Origin of Pre-DC2 Bianca Blom, Suzanne J. W. C. Ligthart, Remko Schotte, and Hergen Spits ABSTRACT: It is generally accepted that dendritic cells can be generated from either myeloid or lymphoid derived progenitors. Ample information has been collected on the development and nature of myeloid DC type 1 (DC1). In contrast, our current understanding on the origin and function of the lymphoid derived DC type 2 (DC2) is still limited but is increasing rapidly. Here we will summarize recent findings on the developmental origin of the precursor of DC2 (pre-DC2). The presence of pre-DC2 has been revealed in bone marrow, fetal liver, and cord blood, where they develop from hematopoietic stem cells (HSC) most likely via an intermediate pro-DC2 stage. Both in human and mouse, development of pre-DC2 depends on the cytokine FLT3-ligand (FLT3-L). In addition, transcription factors such as Spi-B and members of the basic helix-loop helix (bHLH) family have been shown to be involved in the proper differentiation of HSC into preDC2. The human thymus contains a population of cells that closely resembles the peripheral pre-DC2, including ABBREVIATIONS DC1 dendritic cell type 1 DC2 dendritic cell type 2 pre-DC2 precursor of type 2 dendritic cell/type I interferon producing cell Th1 T helper 1 cell BDCA-2 blood dendritic cell antigen 2 GM-CSF granulocyte/macrophage-colony stimulating factor IL-3 Interleukin-3 IFN interferon
INTRODUCTION Dendritic cells (DCs) are highly efficient antigen presenting cells, which can potently induce cell mediated immune responses, as well as delete or anergise autoreactive T cells [1, 2]. Although several distinct types of DCs, DC type 1 (DC1) and DC type 2 (DC2) have been
Division of Immunology, Plesmanlaan 121, Amsterdam, The Netherlands. Address reprint requests to: Dr. Bianca Blom, Division of Immunology, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands; E-mail: [email protected]. Received July 22, 2002; accepted September 27, 2002. Human Immunology 63, 1072–1080 (2002) © American Society for Histocompatibility and Immunogenetics, 2002 Published by Elsevier Science Inc.
interferon (INF)-a production after viral stimulation. Some phenotypic differences have been observed however. Furthermore, we have shown that the thymic microenvironment is able to support development of pre-DC2 from HSC in vivo. A thymus independent pathway of pre-DC2 development exists as well, although at present it is not clear where these extrathymic pre-DC2 are generated. In regard of the absence of a phenotypic defined pro-DC2 population in the thymus, we speculate that development of thymic pre-DC2 may differ from peripheral pre-DC2. The challenge of the near future will be to determine the role of pre-DC2 during thymic T cell development. Human Immunology 63, 1072–1080 (2002). © American Society for Histocompatibility and Immunogenetics, 2002. Published by Elsevier Science Inc. KEYWORDS: (pre-)DC2; FLT3-L; thymus;
TNF-␣ FLT3-L NK cell HSC bHLH Id TCR pT␣ TF
differentiation;
CD34;
tumor necrosis factor-alpha FLT3-ligand natural killer cell hematopoietic stem cells basic helix loop helix inhibitor of DNA binding T cell receptor pre-TCR-alpha transcription factor
identified, it remains elusive whether these contribute differentially to those events. A longstanding debate on the nomenclature of the precursors of DC2 is ongoing, and these cells have been termed plasmacytoid T cells, plasmacytoid monocytes, pre-DC2, type 1 interferon producing cells (IPC), plasmacytoid DC (PDC). Previously, the precursors of DC2 were denominated plasmacytoid T cells, since pathologists had observed the presence of plasma cells that express the T cell marker CD4, but lack intracellular immunoglobulins within the T cell zones of human lymph nodes [3]. Several years ago, Yong-Jun Liu iden0198-8859/02/$–see front matter PII S0198-8859(02)00745-0
Developmental Origin of Pre-DC2
tified the enigmatic plasmacytoid T cells as precursors for dendritic cells. Culture with IL-3 and CD40L induces the maturation of these precursors into DC that prime naı¨ve CD4⫹ T cells for Th2 differentiation, hence the name DC2. Recently it has become clear that DC possess certain flexibility to respond towards different environmental stimuli. Indeed, precursors of DC2 (pre-DC2) that are stimulated with virus become DC that induce naı¨ve T cells to differentiate into a regulatory type of IL-10 producing T cells. Thus, the strict correlation of DC2 inducing Th2 polarization and DC1 inducing Th1 polarization has lost its credibility. The term plasmacytoid DC to denote the pre-DC2 is incorrect in our opinion, because they are not DC. In addition, the mature progeny of the pre-DC2 is not plasmacytoid. It is evident that DC2 are different from monocyte-derived DC (DC1). Importantly, the mechanisms of generation of these DC are clearly different. In analogy to the classification of B cells (B1 and B2 cells) and T cells (Th1 and Th2), we like to propose the name DC2 to denote DC type 2, in parallel to DC type 1 (DC1, monocyte derived DC). Thus, the DC1/DC2 nomenclature reflects different cell types, but not the function of these cells. The immediate precursor of DC2 would then be designated pre-DC2. Classification of the different DCs is based on their developmental origin, phenotypical characteristics, and anatomical localisation [4]. In human, DC1 can develop from CD34⫹ stem cells [5, 6], or peripheral blood CD14⫹ monocytes [7–9] or CD11c⫹ blood precursors [10] with granulocyte/macrophage– colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) or tumor necrosis factor ␣ (TNF-␣). At their immature stage, CD11c⫹ precursors are efficient to uptake and process antigen (reviewed in [11]). Furthermore, depending on the maturation stimulus (reviewed in [11]), DC1 are able to produce IL-12, which then polarise naı¨ve CD4⫹ T cells into interferon-␥ (IFN-␥) producing T helper (Th)1 cells [12]. In human, DC2 derive from FLT3-ligand (FLT3-L) cultured CD34⫹ stem cells [13] or from CD11c⫺ blood precursors with IL-3 and CD40-L [14] or virus [15]. The CD11c⫺ precursors, also referred to as pre-DC2, are characterized by high level expression of the IL-3R␣ chain [16], and the recently identified blood dendritic cell antigen-2 (BDCA-2) and BDCA-4 [17]. The capability of antigen uptake by pre-DC2 is poor [14]. Upon maturation the DC2 efficiently stimulate naı¨ve T cells to proliferate [14]. Dependent on the manner in which the pre-DC2 are stimulated, the mature DC2 stimulate either a Th1-like or Th2 differentiation [12, 18, 19]. Viral stimulation of pre-DC2, which coincides with the production of high amounts of IFN-␣ [15, 20] and TNF-␣ [18], induces the maturation into DC2
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that promote differentiation of Th cells with regulatory characteristics (IFN-␥ and IL-10 producing cells) [18]. IL-3 and CD40-L stimulation of pre-DC2, which does not induce significant amounts of IL-12, leads to the differentiation of mature DC2 that polarize naı¨ve T cells into Th2 cells (IL-4, IL-5 producing cells) [12]. Since their discovery in the 1980s, many groups have done extensive analysis on the origin, developmental requirements, and function of DC1. Therapeutical applications using DC1 in vaccination strategies and antitumor therapy have been extensively investigated [21, 22]. The discovery of pre-DC2 and studies on their origin and developmental requirements has only been initiated over the past 5 years. Therefore, considering clinical applications using DC2 will require more interventions [23]. The recent discovery of pre-DC2 in mice will help to shed more light on origin, requirements, and function of these cells. This article will summarize recent findings addressing these issues. Lymphoid Versus Myeloid Origin of DC Originally it was postulated in the mouse that DCs are either of the myeloid or the lymphoid lineage, based on the reciprocal presence of the myeloid CD11b (Mac-1) or the lymphoid CD8␣ markers, respectively [24]. Support for the lymphoid origin of CD8␣⫹CD11b⫺ DC came from findings that in the thymus these type of DC are most prominent [24]. Moreover, CD8␣⫹CD11b⫺ DC derive from an intrathymic lymphoid restricted progenitor cell, that upon adoptive transfer exclusively gives rise to the lymphoid T, B, and natural killer (NK) cells, but not to myeloid cells [25–27]. The concept that CD8␣ defines lymphoid DC has been challenged by several groups, who conclude that in fact both CD8␣⫹ and CD8␣⫺ DC can be derived from both lymphoid and myeloid committed precursors [28, 29]. Furthermore, CD8␣⫺ DC can become CD8␣⫹ DC involving a differentiation step [30]. Together, these findings strongly support the notion that CD8␣⫹ and CD8␣⫺ represent separate differentiation or maturation stages of the same DC population. Nonetheless the studies of Martin et al. [28] and Traver et al. [29] indicate that DC can be either of lymphoid or myeloid origin. A common precursor for all DC types was identified recently, which generates all DC subpopulations, but is devoid of myeloid and lymphoid developmental potential [31]. These cells are therefore committed to the DC lineage, but whether the committed DC precursors identified by del Hoyo et al. [31] are of lymphoid or myeloid origin or derive directly from a common lymphoid/myeloid precursor is unknown. In humans, DC1 and DC2 subsets are discriminated by the expression of the myeloid marker CD11c. Myeloid DC1 are CD11c⫹, whereas lymphoid DC2 are CD11c⫺.
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FIGURE 1 Intrathymic and extrathymic development of the dendritic cell type 2 (DC2) lineage from hematopoietic stem cells (HSC). Bone marrow derived HSC are able to give rise to cells of all hematopoietic lineages. In the periphery, the DC2 lineage possibly develops from CD34⫹CD45RA⫺ early progenitor cells, to CD34⫹CD45RA⫹ late progenitor cells, to CD34dimIL-3R␣high pro-DC2, to CD34⫺IL3R␣high pre-DC2, and finally to DC2. The thymus is seeded by CD34⫹CD45RA⫹ late progenitor cells, which give rise predominantly to T cells. In addition, DC and natural killer cells can be found in the thymus. The intrathymic DC2 lineage presumably develops independent of an intermediate CD34dimIL-3R␣high pro-DC2 stage (see also Figure 2).
The notion that pre-DC2 are of lymphoid origin was supported by findings that genes originally found to be expressed only in developing T and B cells, such as pre-T-cell receptor ␣ (pT␣), 5, and Spi-B, are expressed in pre-DC2 as well, but not in myeloid cells [32, 33]. Notably, overexpression of the naturally occurring, dominant negative factors Id2 or Id3 (which inhibit the transcriptional activity of basic-helix-loop-helix transcription factors) block pre-DC2 development, like that of T cells [34, 35] and B cells [36], but not myeloid DC development [37]. This indicates that pre-DC2 are more closely related to T and B cells, than to myeloid cells. Development of Human Pre-DC2 From Hematopoietic Stem Cells It is generally accepted that all hematopoietic cells are ultimately derived from CD34-expressing hematopoietic stem cells (HSC). Early during human fetal life HSC reside in the fetal liver, but around week 20 of gestation the bone marrow becomes the major resident site. After birth CD34⫹ HSC can be found in the bone marrow and in the circulation. Significant numbers of HSC are
present in the umbilical cord blood, where on average 1%–3% of the mononuclear cells express the CD34 antigen. Recirculating CD34⫹ progenitor cells can be found in the peripheral blood of adults, although their numbers are extremely low (⬍0.5%). Multipotent progenitor cells are enriched within the CD34high CD45RA⫺ early progenitor subset and have the ability to differentiate into erythrocytes, granulocytes, monocytes, T cells, B cells, NK cells, and DC (Figure 1). Upon differentiation of HSC expression of CD34 is gradually lost. Cells that express low levels of CD34, and in addition phenotypically resemble pre-DC2 (CD4⫹CD45RA⫹IL-3R␣highHLA-DRint) can be detected in cord blood, fetal liver, and fetal bone marrow [13]. It is likely that these cells are the direct precursors for pre-DC2 and, therefore, are designated progenitors of pre-DC2 (pro-DC2, Figure 1). Not only phenotypically, but also functionally these cells appear similar to preDC2, because they are able to respond to viral stimulation by producing high amounts of IFN-␣. Moreover, CD34dimIL-3R␣high pro-DC2 differentiate into mature DC, when stimulated with IL-3 and CD40-L expressing
Developmental Origin of Pre-DC2
fibroblast L cells [13]. The resulting mature DC2 are able to induce the proliferation of allogeneic naı¨ve CD4⫹ T cells. These findings together indicate that CD34dimIL-3R␣high pro-DC2 are cells that have most likely already committed to the pre-DC2 lineage. Formal proof however that these cells have lost the ability to differentiate into cells of other lineages has not been provided. CD34highCD45RA⫺ cells, enriched for multipotent stem cells, do not express any marker characteristic of pre-DC2. Notably, the CD34highCD45RA⫺ early progenitor cells do not produce IFN-␣ in response to viral stimulation [13]. To address whether pre-DC2 can be generated in vitro from CD34highCD45RA⫺ early progenitor cells, we explored the effect of several different factors in culturing of these cells. These studies revealed that FLT3-L is the only cytokine that supported differentiation into pre-DC2 [13]. Part of the cultured cells expresses high levels of IL-3R␣, which was maximal after 3 weeks of culture reaching 10% of the total cells. Interestingly, the capacity to produce high amounts of IFN-␣ upon viral stimulation was contained exclusively within this IL-3R␣high population. The IL-3R␣neg/dim cells did not produce significant levels of IFN-␣ in response to virus [13]. Support for the importance of FLT3-L in the generation of pre-DC2 in vivo came from two studies where volunteers were injected with FLT3-L, and an increase in the numbers of pre-DC2 in the peripheral blood was observed [38] [39]. A plausible explanation for these findings is that FLT3-L induces differentiation of HSC into pre-DC2. In addition, FLT3-L may induce survival and expansion of the pro-DC2. In one of these studies [38], volunteers were injected with granulocyte– colony-stimulating factor (G-CSF) in parallel. In clinical settings of autologous stem cell transplantation, G-CSF is administered to mobilize HSC residing in the bone marrow into the peripheral blood prior to transplantation. The increased levels of HSC in the peripheral blood facilitates hematopoietic recovery following re-infusion. Administration of G-CSF resulted in a similar increase in numbers of peripheral pre-DC2 as observed with FLT3-L, suggesting that G-CSF may also increase differentiation of pre-DC2 [38]. However, in vitro culture of CD34⫹CD45RA⫺ early progenitor cells with G-CSF did not result in the differentiation of preDC2, nor did it enhance the FLT3-L induced pre-DC2 development [13]. An explanation for the observed GCSF effect in vivo on pre-DC2 in the study of Pulendran et al. [38] might be the mobilization of the pro-DC2 from the bone marrow into the periphery. Taken together, both FLT3-L and G-CSF are promising cytokines for therapy using pre-DC2, since large numbers of preDC2 can be obtained. Importantly, these cytokines do not display toxic side effects for patients.
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Development of Mouse Pre-DC2 From HSC Recently, the potential mouse equivalent of human preDC2 was identified. While functionally these cells represent the major IFN-␣ producing cells after viral stimulation in the mouse, phenotypically differences were observed compared to human pre-DC2. The mouse preDC2 are characterized by expression of B220, Gr-1, and CD11c [40 – 42]. These antigens have been commonly used to define B cells, granulocytes, or myeloid dendritic cells, respectively. This may explain why cells with this phenotype (B220⫹CD19⫺Gr-1⫹CD11c⫹) were not identified previously. Furthermore, mouse pre-DC2 express CD4 and Ly6C, but do not express CD11b or CD8␣ [42]. At the functional level some differences between human and mouse pre-DC2 are observed. Unlike human pre-DC2, the equivalent cells in mouse hardly respond to IL-3, which correlates with the lack of high level IL-3R␣ expression [41]. Their survival does increase in response to viral stimulation or CpG oligonucleotides [41, 42]. Furthermore, in contrast to human pre-DC2 which are not able to produce significant amounts of IL-12, mouse pre-DC2 secrete IL-12 after virus or CpG oligonucleotide stimulation [42]. Whether mouse pre-DC2 derive exclusively from lymphoid or myeloid committed progenitor cells has not been determined. Mouse pre-DC2 are generated from the common DC precursor identified recently by del Hoyo et al. [31]. Similar to the generation of human pre-DC2 [13], development of mouse pre-DC2 from bone marrow derived HSC depends on FLT3-L [43]. Treatment of mice with FLT3-L results in a striking increase in the number of pre-DC2 in the bone marrow and spleen [44]. Notably, similar to the development of human pre-DC2, the myeloid growth factor GM-CSF completely blocked their development in vitro [13, 43]. Thus, although some phenotypical and functional differences are observed between human and mouse pre-DC2, their developmental requirements appear to be conserved during evolution. Intrathymic and Extrathymic Development of Pre-DC2 The thymus is the main site where T cells develop from CD34⫹ progenitor cells via sequential stages involving rearrangement of T-cell receptor (TCR) genes and selection of correctly processed TCR proteins [27, 45– 47]. It is well established that thymic DCs mediate the selection process termed negative selection [48, 49]. This involves interaction of immature T cells and DC, which results in removal of T cells expressing TCRs recognizing selfantigens. The thymus contains different types of DC. In humans, at least two types of myeloid related dendritic cells have been identified, one of which reveals an immature DC phenotype (HLA-DRintCD11c⫹CD13⫹CD33⫹),
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while the other displays a more mature phenotype (HLADRhighCD11c⫹) [33, 50]. Furthermore, in the thymus cells that closely resemble peripheral pre-DC2 are observed, including IFN-␣ production after viral stimulation [32, 33, 50]. Some differences between thymic and peripheral pre-DC2 are observed, however, with respect to expression of the lymphoid markers CD2, CD5, and CD7. Whereas all thymic pre-DC2 express CD2, CD5, and CD7, their peripheral counterparts do not or only partially express these markers [32]. All pre-DC and mature DC subsets are located predominantly in the medulla, although some pre-DC2 were detectable in the corticomedullary border and the cortex [32, 33, 50]. Freshly isolated thymic pre-DC2 cultured in the presence of IL-3 and CD40-L upregulate CD83 [32], which marker is expressed on all mature DC. However, whether pre-DC2 differentiate into mature DC under the influence of the thymic environment in vivo has not been resolved. Coexpression of CD83 on the IL-3R␣high thymic cells has not been observed [32]. The thymus is seeded by multipotent progenitor cells that are able to give rise to T cells, B cells, NK cells, and DCs. It has been observed that both myeloid and lymphoid DC can be generated from intrathymic progenitor cells. Spits et al. [37] has reported that part of the thymic CD34⫹CD1a⫺ subset, which is enriched for multipotent progenitor cells, can develop into pre-DC2 on a stromal cell layer [37]. Previously it was demonstrated that culture of thymic precursor cells in GM-CSF and TNF-␣ directs myeloid DC1 development [51, 52]. More recently a CD34dim thymic progenitor subset has been identified, which expresses the GM-CSF receptor, but lacks expression of the IL-7R␣ chain [53]. These CD34dimGM-CSFR⫹ progenitor cells generate myeloid DC1, but have lost the potential to develop into T cells, and have reduced potential to develop into NK cells, when compared with CD34dimGM-CSFR⫺ progenitors (Figure 1). The capacity of the CD34dimGM-CSFR⫹ progenitor cells to develop into pre-DC2 has not been determined. However, considering the fact these cells express almost undetectable levels of pT␣, compared with the CD34dimGM-CSFR⫺ subset and pre-DC2 [53], it is likely to assume that CD34dimGM-CSFR⫹ progenitor cells are myeloid DC1 committed progenitors. The question whether pre-DC2 are generated in the thymus in vivo or develop elsewhere and then migrate into the thymus was addressed recently. Weijer et al. [54] revealed that upon injection of human CD34⫹ cells directly into a human thymic transplant, grafted subcutaneously into recombination activating gene (RAG)-2/␥common double deficient mice, a proportion of injected cells differentiate into pre-DC2. These findings suggest that the thymic microenvironment is able to support the development of pre-DC2. Whether these cells
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FIGURE 2 CD34dimIL-3R␣high dendritic cells (pro-DC2) are found in fetal liver, but not in fetal thymus. Fetal liver and thymus were depleted of all lineage positive cells (CD3⫹, CD14⫹, CD19⫹, CD56⫹, glycophorin⫹) by magnetic bead depletion. Then cells were stained with a FITC-conjugated cocktail of antibodies against lineage markers (CD3, CD14, CD19, CD56), PE-conjugated anti-IL-3R␣, and antigen presenting cell conjugated anti-CD34. Illustrated are CD34/IL3R␣ flowcytometric dotplots of (A) fetal liver and (B) fetal thymus electronically gated on lineage negative cells.
develop via a CD34dim pro-DC2 (ILhigh ⫹ ⫹ 3R␣ CD4 CD45RA HLA-DRint) intermediate stage, like was observed in cord blood, fetal liver, and fetal bone marrow, remains elusive. Fact is that CD34dimIL-3R␣high pro-DC2 are not detected in fresh fetal or postnatal thymus (Figure 2) [13]. This suggests that thymic pre-DC2 may develop via a pathway that is different from pre-DC2 generated in fetal liver, cord blood, or bone marrow. No-
Developmental Origin of Pre-DC2
tably, pre-DC2 can develop in the absence of the thymic environment, since they are also observed after intravenous injection of human CD34⫹ cells in RAG-2/␥common double deficient mice lacking a human thymic transplant. In those mice, human pre-DC2 are detected in the liver, peripheral blood, spleen, and bone marrow [54]. Isolation of these pre-DC2 and viral stimulation in vitro induced the production of IFN-␣, strongly suggesting that the mouse environment sustains development of functionally competent pre-DC2. Although it remains elusive where and how these pre-DC2 develop, it is evident from these findings that different developmental pathways, i.e., intrathymic and extrathymic, exist. Transcriptional Control of Pre-DC2 Development Differentiation of HSC towards a particular cell lineage requires an orchestrated program of developmental steps driven by the expression of lineage specific and nonspecific genes activated by transcription factors (TFs). One way to address the importance of critical genes is to manipulate the level of expression of their TFs and to analyze the developmental outcome. An interesting family of TFs that has been revealed to be essential in the development of lymphoid cells is the basic-helix-loopedhelix (bHLH) family of TFs [55]. The lack of the E2A bHLH proteins (encoded by the genes E12 and E47) leads to a severe block in both T-cell [56] and B-cell development [57–59]. Consistent with these findings, overexpression of inhibitor of DNA binding (Id) proteins [60], which are related to the bHLH proteins but lack the basic DNA binding domain and therefore act as dominant negative HLH factors, block T- and B-cell development [61, 62]. To address the importance of bHLH factors in the development of pre-DC2, we have reported that Id2 or Id3, when overexpressed in CD34⫹ progenitor cells by retroviral gene transfer, can inhibit pre-DC2 development [37], like that of T and B cells [34 –36]. In this context it will be interesting to investigate which bHLH factors are induced upon FLT3-L induced pre-DC2 development [13, 43], driving preDC2 differentiation. The Ets family of DNA-binding proteins is a highly conserved group of TFs, which have regulatory functions during hematopoietic development [63, 64]. A subfamily of the Ets-domain containing family is formed by the Spi factors, which include among others PU.1 (Spi-1) [65] and Spi-B [66]. PU.1 and Spi-B are coexpressed in B cells, with low level expression in early thymocytes [65– 67]. In addition, PU.1 is expressed in granulocytes, macrophages and erythrocytes [68 –70]. Whereas Spi-B deficient mice are viable, and exhibit impaired B cell functions [71], PU.1 deficient mice have severe hematopoietic defects leading to premature death between day 18.5 [72] of gestation and 3 weeks after birth, if main-
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tained in antibiotics [73]. The PU.1 function regulates early B cell development by directly controlling expression of the IL-7R␣ gene [74]. In vitro, high level Spi-B expression can substitute for PU.1 in early B cell development [74]. In vivo, however, Spi-B cannot functionally replace PU.1 in lymphoid development, although it does rescue myeloid development [75]. Interestingly, Spi-B is also expressed in human pre-DC2, as was observed after a subtractive library screen with monocyte-derived DC [33]. Expression is contained in IL-3 and CD40-L matured DC2 inting at a possible contribution of Spi-B during development of pre-DC2. Overexpression by retroviral mediated gene transfer of Spi-B in human CD34⫹ progenitor cells, however, does not affect the development of FLT3-L induced differentiation of pre-DC2 [76]. Notably, constitutive expression of Spi-B severely impairs T cell and NK cell development, suggesting that Spi-B expression is required to be downregulated for proper T cell development to occur. This notion is supported by the fact that Spi-B transcripts are downregulated during transition of uncommitted CD34⫹CD1a⫺ into T cell committed CD34⫹CD1a⫹ precursor cells [76]. CONCLUSION It has been convincingly demonstrated that both human and mouse pre-DC2 can be generated from HSC upon culture with FLT3-L [13, 43]. Whether this cytokine is responsible for the induction of differentiation or also the maintenance or survival of pro-DC2 in culture has not been resolved. Furthermore, it remains elusive what the underlying molecular mechanism is that drives this differentiation or survival. Using the current knowledge on the phenotype of the mouse pre-DC2 makes analysis of genetically modified animals submissive for detailed studies on the generation, homing and function of these cells. We [32] and others [33, 40 – 42, 50] have reported that the thymus contains pre-DC2. However, it is not clear what the functional contribution is of these cells in the thymus. We have shown that the thymic microenvironment is able to support the development of CD34⫹ precursor cells into pre-DC2 [54]. In addition, intrathymic progenitor cells are able to differentiate into preDC2 [37]. It remains elusive whether these cells develop in the thymus via a CD34dimIL-3R␣high pro-DC2 intermediate stage, since pro-DC2 are not observed in the fetal or postnatal thymus (Figure 2). Taken together, these findings support the notion that pre-DC2 play a role during T cell development. Culture of pre-DC2 in vitro with IL-3 and CD40-L, or stimulation with virus results in the maturation of these cells into DC2. These in vitro generated mature DC2 express CD83, which marker is expressed on all mature
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DC. However, in vivo coexpression of CD83 on IL3R␣high cells has not been observed. This may suggest that either mature DC2 in vivo loose IL-3R␣ expression, or do not upregulate CD83 expression. It will be a challenge to identify mature DC2 in vivo and to explore the function of these cells during innate and adaptive immune responses.
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27. Shortman K, Vremec D, Corcoran LM, Georgopoulos K, Lucas K, Wu L: The linkage between T-cell and dendritic cell development in the mouse thymus. Immunol Rev 165:39, 1998. 28. Martin P, del Hoyo GM, Anjuere F, Ruiz SR, Arias CF, Marin AR, Ardavin C: Concept of lymphoid versus myeloid dendritic cell lineages revisited: both CD8alpha(⫺) and CD8alpha(⫹) dendritic cells are generated from CD4(low) lymphoid-committed precursors. Blood 96:2511, 2000. 29. Traver D, Akashi K, Manz M, Merad M, Miyamoto T, Engleman EG, Weissman IL: Development of CD8alphapositive dendritic cells from a common myeloid progenitor. Science 290:2152, 2000. 30. Martinez del Hoyo G, Martin P, Arias CF, Marin AR, Ardavin C: CD8alpha⫹ dendritic cells originate from the CD8alpha⫺ dendritic cell subset by a maturation process involving CD8alpha, DEC-205, and CD24 up-regulation. Blood 99:999, 2002. 31. del Hoyo GM, Martin P, Vargas HH, Ruiz S, Arias CF, Ardavin C: Characterization of a common precursor population for dendritic cells. Nature 415:1043, 2002. 32. Res PC, Couwenberg F, Vyth-Dreese FA, Spits H: Expression of pTalpha mRNA in a committed dendritic cell precursor in the human thymus. Blood 94:2647, 1999. 33. Bendriss-Vermare N, Barthelemy C, Durand I, Bruand C, Dezutter-Dambuyant C, Moulian N, Berrih-Aknin S, Caux C, Trinchieri G, Briere F: Human thymus contains IFN-alpha-producing CD11c(⫺), myeloid CD11c(⫹), and mature interdigitating dendritic cells. J Clin Invest 107:835, 2001. 34. Heemskerk MH, Blom B, Nolan G, Stegmann AP, Bakker AQ, Weijer K, Res PC, Spits H: Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3. J Exp Med 186:1597, 1997. 35. Blom B, Heemskerk MH, Verschuren MC, van Dongen JJ, Stegmann AP, Bakker AQ, Couwenberg F, Res PC, Spits H: Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loophelix protein Id3 in committed T cell progenitors. Embo J 18:2793, 1999. 36. Jaleco AC, Stegmann AP, Heemskerk MH, Couwenberg F, Bakker AQ, Weijer K, Spits H: Genetic modification of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3. Blood 94:2637, 1999. 37. Spits H, Couwenberg F, Bakker AQ, Weijer K, Uittenbogaart CH: Id2 and Id3 inhibit development of CD34(⫹) stem cells into predendritic cell (pre-DC)2 but not into pre-DC1. Evidence for a lymphoid origin of pre-DC2. J Exp Med 192:1775, 2000. 38. Pulendran B, Banchereau J, Burkeholder S, Kraus E, Guinet E, Chalouni C, Caron D, Maliszewski C, Davoust J, Fay J, Palucka K: Flt3-ligand and granulocyte colony-
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53. de Yebenes VG, Carrasco YR, Ramiro AR, Toribio ML: Identification of a myeloid intrathymic pathway of dendritic cell development marked by expression of the granulocyte macrophage-colony- stimulating factor receptor. Blood 99:2948, 2002. 54. Weijer K, Uittenbogaart CH, Voordouw A, Couwenberg F, Seppen J, Blom B, Vyth-Dreese FA, Spits H: Intrathymic and extrathymic development of human plasmacytoid dendritic cell precursors in vivo. Blood 99:2752, 2002. 55. Massari ME, Murre C: Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol Cell Biol 20:429, 2000. 56. Bain G, Engel I, Robanus Maandag EC, te Riele HP, Voland JR, Sharp LL, Chun J, Huey B, Pinkel D, Murre C: E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas. Mol Cell Biol 17:4782, 1997. 57. Bain G, Maandag EC, Izon DJ, Amsen D, Kruisbeek AM, Weintraub BC, Krop I, Schlissel MS, Feeney AJ, van Roon M, et al: E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 79:885, 1994. 58. Zhuang Y, Soriano P, Weintraub H: The helix-loop-helix gene E2A is required for B cell formation. Cell 79:875, 1994. 59. Bain G, Robanus Maandag EC, te Riele HP, Feeney AJ, Sheehy A, Schlissel M, Shinton SA, Hardy RR, Murre C: Both E12 and E47 allow commitment to the B cell lineage. Immunity 6:145, 1997. 60. Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H: The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49, 1990. 61. Kim D, Peng XC, Sun XH: Massive apoptosis of thymocytes in T-cell-deficient Id1 transgenic mice. Mol Cell Biol 19:8240, 1999. 62. Sun XH: Constitutive expression of the Id1 gene impairs mouse B cell development. Cell 79:893, 1994. 63. Graves BJ, Petersen JM: Specificity within the ets family of transcription factors. Adv Cancer Res 75:1, 1998. 64. Wasylyk B, Hahn SL, Giovane A: The Ets family of transcription factors. Eur J Biochem 211:7, 1993. 65. Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA: The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell 61:113, 1990.
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66. Su GH, Ip HS, Cobb BS, Lu MM, Chen HM, Simon MC: The Ets protein Spi-B is expressed exclusively in B cells and T cells during development. J Exp Med 184:203, 1996. 67. Anderson MK, Hernandez-Hoyos G, Diamond RA, Rothenberg EV: Precise developmental regulation of Ets family transcription factors during specification and commitment to the T cell lineage. Development 126:3131, 1999. 68. Galson DL, Hensold JO, Bishop TR, Schalling M, D’Andrea AD, Jones C, Auron PE, Housman DE: Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis. Mol Cell Biol 13:2929, 1993. 69. Chen HM, Zhang P, Voso MT, Hohaus S, Gonzalez DA, Glass CK, Zhang DE, Tenen DG: Neutrophils and monocytes express high levels of PU: 1 (Spi-1) but not Spi-B. Blood 85:2918, 1995. 70. Hromas R, Orazi A, Neiman RS, Maki R, Van Beveran C, Moore J, Klemsz M: Hematopoietic lineage- and stagerestricted expression of the ETS oncogene family member PU.1. Blood 82:2998, 1993. 71. Su GH, Chen HM, Muthusamy N, Garrett-Sinha LA, Baunoch D, Tenen DG, Simon MC: Defective B cell receptor-mediated responses in mice lacking the Ets protein, Spi-B. Embo J 16:7118, 1997. 72. Scott EW, Simon MC, Anastasi J, Singh H: Requirement of transcription factor PU: 1 in the development of multiple hematopoietic lineages. Science 265:1573, 1994. 73. McKercher SR, Torbett BE, Anderson KL, Henkel GW, Vestal DJ, Baribault H, Klemsz M, Feeney AJ, Wu GE, Paige CJ, Maki RA: Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. Embo J 15:5647, 1996. 74. DeKoter RP, Lee HJ, Singh H: PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity 16:297, 2002. 75. Dahl R, Ramirez-Bergeron DL, Rao S, Simon MC: Spi-B can functionally replace PU.1 in myeloid but not lymphoid development. Embo J 21:2220, 2002. 76. Schotte R, Rissoan MC, Bendriss-Vermare N, Bridon JM, Duhen T, Weijer K, Brie`re F, Spits H: The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T, B, and NK cell development. Blood 2002 Sep 19; epub ahead of print.
INTRODUCTION Dendritic cells (DCs) are highly efficient antigen presenting cells, which can potently induce cell mediated immune responses, as well as delete or anergise autoreactive T cells [1, 2]. Although several distinct types of DCs, DC type 1 (DC1) and DC type 2 (DC2) have been
Division of Immunology, Plesmanlaan 121, Amsterdam, The Netherlands. Address reprint requests to: Dr. Bianca Blom, Division of Immunology, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands; E-mail: [email protected]. Received July 22, 2002; accepted September 27, 2002. Human Immunology 63, 1072–1080 (2002) © American Society for Histocompatibility and Immunogenetics, 2002 Published by Elsevier Science Inc.
interferon (INF)-a production after viral stimulation. Some phenotypic differences have been observed however. Furthermore, we have shown that the thymic microenvironment is able to support development of pre-DC2 from HSC in vivo. A thymus independent pathway of pre-DC2 development exists as well, although at present it is not clear where these extrathymic pre-DC2 are generated. In regard of the absence of a phenotypic defined pro-DC2 population in the thymus, we speculate that development of thymic pre-DC2 may differ from peripheral pre-DC2. The challenge of the near future will be to determine the role of pre-DC2 during thymic T cell development. Human Immunology 63, 1072–1080 (2002). © American Society for Histocompatibility and Immunogenetics, 2002. Published by Elsevier Science Inc. KEYWORDS: (pre-)DC2; FLT3-L; thymus;
TNF-␣ FLT3-L NK cell HSC bHLH Id TCR pT␣ TF
differentiation;
CD34;
tumor necrosis factor-alpha FLT3-ligand natural killer cell hematopoietic stem cells basic helix loop helix inhibitor of DNA binding T cell receptor pre-TCR-alpha transcription factor
identified, it remains elusive whether these contribute differentially to those events. A longstanding debate on the nomenclature of the precursors of DC2 is ongoing, and these cells have been termed plasmacytoid T cells, plasmacytoid monocytes, pre-DC2, type 1 interferon producing cells (IPC), plasmacytoid DC (PDC). Previously, the precursors of DC2 were denominated plasmacytoid T cells, since pathologists had observed the presence of plasma cells that express the T cell marker CD4, but lack intracellular immunoglobulins within the T cell zones of human lymph nodes [3]. Several years ago, Yong-Jun Liu iden0198-8859/02/$–see front matter PII S0198-8859(02)00745-0
Developmental Origin of Pre-DC2
tified the enigmatic plasmacytoid T cells as precursors for dendritic cells. Culture with IL-3 and CD40L induces the maturation of these precursors into DC that prime naı¨ve CD4⫹ T cells for Th2 differentiation, hence the name DC2. Recently it has become clear that DC possess certain flexibility to respond towards different environmental stimuli. Indeed, precursors of DC2 (pre-DC2) that are stimulated with virus become DC that induce naı¨ve T cells to differentiate into a regulatory type of IL-10 producing T cells. Thus, the strict correlation of DC2 inducing Th2 polarization and DC1 inducing Th1 polarization has lost its credibility. The term plasmacytoid DC to denote the pre-DC2 is incorrect in our opinion, because they are not DC. In addition, the mature progeny of the pre-DC2 is not plasmacytoid. It is evident that DC2 are different from monocyte-derived DC (DC1). Importantly, the mechanisms of generation of these DC are clearly different. In analogy to the classification of B cells (B1 and B2 cells) and T cells (Th1 and Th2), we like to propose the name DC2 to denote DC type 2, in parallel to DC type 1 (DC1, monocyte derived DC). Thus, the DC1/DC2 nomenclature reflects different cell types, but not the function of these cells. The immediate precursor of DC2 would then be designated pre-DC2. Classification of the different DCs is based on their developmental origin, phenotypical characteristics, and anatomical localisation [4]. In human, DC1 can develop from CD34⫹ stem cells [5, 6], or peripheral blood CD14⫹ monocytes [7–9] or CD11c⫹ blood precursors [10] with granulocyte/macrophage– colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) or tumor necrosis factor ␣ (TNF-␣). At their immature stage, CD11c⫹ precursors are efficient to uptake and process antigen (reviewed in [11]). Furthermore, depending on the maturation stimulus (reviewed in [11]), DC1 are able to produce IL-12, which then polarise naı¨ve CD4⫹ T cells into interferon-␥ (IFN-␥) producing T helper (Th)1 cells [12]. In human, DC2 derive from FLT3-ligand (FLT3-L) cultured CD34⫹ stem cells [13] or from CD11c⫺ blood precursors with IL-3 and CD40-L [14] or virus [15]. The CD11c⫺ precursors, also referred to as pre-DC2, are characterized by high level expression of the IL-3R␣ chain [16], and the recently identified blood dendritic cell antigen-2 (BDCA-2) and BDCA-4 [17]. The capability of antigen uptake by pre-DC2 is poor [14]. Upon maturation the DC2 efficiently stimulate naı¨ve T cells to proliferate [14]. Dependent on the manner in which the pre-DC2 are stimulated, the mature DC2 stimulate either a Th1-like or Th2 differentiation [12, 18, 19]. Viral stimulation of pre-DC2, which coincides with the production of high amounts of IFN-␣ [15, 20] and TNF-␣ [18], induces the maturation into DC2
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that promote differentiation of Th cells with regulatory characteristics (IFN-␥ and IL-10 producing cells) [18]. IL-3 and CD40-L stimulation of pre-DC2, which does not induce significant amounts of IL-12, leads to the differentiation of mature DC2 that polarize naı¨ve T cells into Th2 cells (IL-4, IL-5 producing cells) [12]. Since their discovery in the 1980s, many groups have done extensive analysis on the origin, developmental requirements, and function of DC1. Therapeutical applications using DC1 in vaccination strategies and antitumor therapy have been extensively investigated [21, 22]. The discovery of pre-DC2 and studies on their origin and developmental requirements has only been initiated over the past 5 years. Therefore, considering clinical applications using DC2 will require more interventions [23]. The recent discovery of pre-DC2 in mice will help to shed more light on origin, requirements, and function of these cells. This article will summarize recent findings addressing these issues. Lymphoid Versus Myeloid Origin of DC Originally it was postulated in the mouse that DCs are either of the myeloid or the lymphoid lineage, based on the reciprocal presence of the myeloid CD11b (Mac-1) or the lymphoid CD8␣ markers, respectively [24]. Support for the lymphoid origin of CD8␣⫹CD11b⫺ DC came from findings that in the thymus these type of DC are most prominent [24]. Moreover, CD8␣⫹CD11b⫺ DC derive from an intrathymic lymphoid restricted progenitor cell, that upon adoptive transfer exclusively gives rise to the lymphoid T, B, and natural killer (NK) cells, but not to myeloid cells [25–27]. The concept that CD8␣ defines lymphoid DC has been challenged by several groups, who conclude that in fact both CD8␣⫹ and CD8␣⫺ DC can be derived from both lymphoid and myeloid committed precursors [28, 29]. Furthermore, CD8␣⫺ DC can become CD8␣⫹ DC involving a differentiation step [30]. Together, these findings strongly support the notion that CD8␣⫹ and CD8␣⫺ represent separate differentiation or maturation stages of the same DC population. Nonetheless the studies of Martin et al. [28] and Traver et al. [29] indicate that DC can be either of lymphoid or myeloid origin. A common precursor for all DC types was identified recently, which generates all DC subpopulations, but is devoid of myeloid and lymphoid developmental potential [31]. These cells are therefore committed to the DC lineage, but whether the committed DC precursors identified by del Hoyo et al. [31] are of lymphoid or myeloid origin or derive directly from a common lymphoid/myeloid precursor is unknown. In humans, DC1 and DC2 subsets are discriminated by the expression of the myeloid marker CD11c. Myeloid DC1 are CD11c⫹, whereas lymphoid DC2 are CD11c⫺.
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FIGURE 1 Intrathymic and extrathymic development of the dendritic cell type 2 (DC2) lineage from hematopoietic stem cells (HSC). Bone marrow derived HSC are able to give rise to cells of all hematopoietic lineages. In the periphery, the DC2 lineage possibly develops from CD34⫹CD45RA⫺ early progenitor cells, to CD34⫹CD45RA⫹ late progenitor cells, to CD34dimIL-3R␣high pro-DC2, to CD34⫺IL3R␣high pre-DC2, and finally to DC2. The thymus is seeded by CD34⫹CD45RA⫹ late progenitor cells, which give rise predominantly to T cells. In addition, DC and natural killer cells can be found in the thymus. The intrathymic DC2 lineage presumably develops independent of an intermediate CD34dimIL-3R␣high pro-DC2 stage (see also Figure 2).
The notion that pre-DC2 are of lymphoid origin was supported by findings that genes originally found to be expressed only in developing T and B cells, such as pre-T-cell receptor ␣ (pT␣), 5, and Spi-B, are expressed in pre-DC2 as well, but not in myeloid cells [32, 33]. Notably, overexpression of the naturally occurring, dominant negative factors Id2 or Id3 (which inhibit the transcriptional activity of basic-helix-loop-helix transcription factors) block pre-DC2 development, like that of T cells [34, 35] and B cells [36], but not myeloid DC development [37]. This indicates that pre-DC2 are more closely related to T and B cells, than to myeloid cells. Development of Human Pre-DC2 From Hematopoietic Stem Cells It is generally accepted that all hematopoietic cells are ultimately derived from CD34-expressing hematopoietic stem cells (HSC). Early during human fetal life HSC reside in the fetal liver, but around week 20 of gestation the bone marrow becomes the major resident site. After birth CD34⫹ HSC can be found in the bone marrow and in the circulation. Significant numbers of HSC are
present in the umbilical cord blood, where on average 1%–3% of the mononuclear cells express the CD34 antigen. Recirculating CD34⫹ progenitor cells can be found in the peripheral blood of adults, although their numbers are extremely low (⬍0.5%). Multipotent progenitor cells are enriched within the CD34high CD45RA⫺ early progenitor subset and have the ability to differentiate into erythrocytes, granulocytes, monocytes, T cells, B cells, NK cells, and DC (Figure 1). Upon differentiation of HSC expression of CD34 is gradually lost. Cells that express low levels of CD34, and in addition phenotypically resemble pre-DC2 (CD4⫹CD45RA⫹IL-3R␣highHLA-DRint) can be detected in cord blood, fetal liver, and fetal bone marrow [13]. It is likely that these cells are the direct precursors for pre-DC2 and, therefore, are designated progenitors of pre-DC2 (pro-DC2, Figure 1). Not only phenotypically, but also functionally these cells appear similar to preDC2, because they are able to respond to viral stimulation by producing high amounts of IFN-␣. Moreover, CD34dimIL-3R␣high pro-DC2 differentiate into mature DC, when stimulated with IL-3 and CD40-L expressing
Developmental Origin of Pre-DC2
fibroblast L cells [13]. The resulting mature DC2 are able to induce the proliferation of allogeneic naı¨ve CD4⫹ T cells. These findings together indicate that CD34dimIL-3R␣high pro-DC2 are cells that have most likely already committed to the pre-DC2 lineage. Formal proof however that these cells have lost the ability to differentiate into cells of other lineages has not been provided. CD34highCD45RA⫺ cells, enriched for multipotent stem cells, do not express any marker characteristic of pre-DC2. Notably, the CD34highCD45RA⫺ early progenitor cells do not produce IFN-␣ in response to viral stimulation [13]. To address whether pre-DC2 can be generated in vitro from CD34highCD45RA⫺ early progenitor cells, we explored the effect of several different factors in culturing of these cells. These studies revealed that FLT3-L is the only cytokine that supported differentiation into pre-DC2 [13]. Part of the cultured cells expresses high levels of IL-3R␣, which was maximal after 3 weeks of culture reaching 10% of the total cells. Interestingly, the capacity to produce high amounts of IFN-␣ upon viral stimulation was contained exclusively within this IL-3R␣high population. The IL-3R␣neg/dim cells did not produce significant levels of IFN-␣ in response to virus [13]. Support for the importance of FLT3-L in the generation of pre-DC2 in vivo came from two studies where volunteers were injected with FLT3-L, and an increase in the numbers of pre-DC2 in the peripheral blood was observed [38] [39]. A plausible explanation for these findings is that FLT3-L induces differentiation of HSC into pre-DC2. In addition, FLT3-L may induce survival and expansion of the pro-DC2. In one of these studies [38], volunteers were injected with granulocyte– colony-stimulating factor (G-CSF) in parallel. In clinical settings of autologous stem cell transplantation, G-CSF is administered to mobilize HSC residing in the bone marrow into the peripheral blood prior to transplantation. The increased levels of HSC in the peripheral blood facilitates hematopoietic recovery following re-infusion. Administration of G-CSF resulted in a similar increase in numbers of peripheral pre-DC2 as observed with FLT3-L, suggesting that G-CSF may also increase differentiation of pre-DC2 [38]. However, in vitro culture of CD34⫹CD45RA⫺ early progenitor cells with G-CSF did not result in the differentiation of preDC2, nor did it enhance the FLT3-L induced pre-DC2 development [13]. An explanation for the observed GCSF effect in vivo on pre-DC2 in the study of Pulendran et al. [38] might be the mobilization of the pro-DC2 from the bone marrow into the periphery. Taken together, both FLT3-L and G-CSF are promising cytokines for therapy using pre-DC2, since large numbers of preDC2 can be obtained. Importantly, these cytokines do not display toxic side effects for patients.
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Development of Mouse Pre-DC2 From HSC Recently, the potential mouse equivalent of human preDC2 was identified. While functionally these cells represent the major IFN-␣ producing cells after viral stimulation in the mouse, phenotypically differences were observed compared to human pre-DC2. The mouse preDC2 are characterized by expression of B220, Gr-1, and CD11c [40 – 42]. These antigens have been commonly used to define B cells, granulocytes, or myeloid dendritic cells, respectively. This may explain why cells with this phenotype (B220⫹CD19⫺Gr-1⫹CD11c⫹) were not identified previously. Furthermore, mouse pre-DC2 express CD4 and Ly6C, but do not express CD11b or CD8␣ [42]. At the functional level some differences between human and mouse pre-DC2 are observed. Unlike human pre-DC2, the equivalent cells in mouse hardly respond to IL-3, which correlates with the lack of high level IL-3R␣ expression [41]. Their survival does increase in response to viral stimulation or CpG oligonucleotides [41, 42]. Furthermore, in contrast to human pre-DC2 which are not able to produce significant amounts of IL-12, mouse pre-DC2 secrete IL-12 after virus or CpG oligonucleotide stimulation [42]. Whether mouse pre-DC2 derive exclusively from lymphoid or myeloid committed progenitor cells has not been determined. Mouse pre-DC2 are generated from the common DC precursor identified recently by del Hoyo et al. [31]. Similar to the generation of human pre-DC2 [13], development of mouse pre-DC2 from bone marrow derived HSC depends on FLT3-L [43]. Treatment of mice with FLT3-L results in a striking increase in the number of pre-DC2 in the bone marrow and spleen [44]. Notably, similar to the development of human pre-DC2, the myeloid growth factor GM-CSF completely blocked their development in vitro [13, 43]. Thus, although some phenotypical and functional differences are observed between human and mouse pre-DC2, their developmental requirements appear to be conserved during evolution. Intrathymic and Extrathymic Development of Pre-DC2 The thymus is the main site where T cells develop from CD34⫹ progenitor cells via sequential stages involving rearrangement of T-cell receptor (TCR) genes and selection of correctly processed TCR proteins [27, 45– 47]. It is well established that thymic DCs mediate the selection process termed negative selection [48, 49]. This involves interaction of immature T cells and DC, which results in removal of T cells expressing TCRs recognizing selfantigens. The thymus contains different types of DC. In humans, at least two types of myeloid related dendritic cells have been identified, one of which reveals an immature DC phenotype (HLA-DRintCD11c⫹CD13⫹CD33⫹),
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while the other displays a more mature phenotype (HLADRhighCD11c⫹) [33, 50]. Furthermore, in the thymus cells that closely resemble peripheral pre-DC2 are observed, including IFN-␣ production after viral stimulation [32, 33, 50]. Some differences between thymic and peripheral pre-DC2 are observed, however, with respect to expression of the lymphoid markers CD2, CD5, and CD7. Whereas all thymic pre-DC2 express CD2, CD5, and CD7, their peripheral counterparts do not or only partially express these markers [32]. All pre-DC and mature DC subsets are located predominantly in the medulla, although some pre-DC2 were detectable in the corticomedullary border and the cortex [32, 33, 50]. Freshly isolated thymic pre-DC2 cultured in the presence of IL-3 and CD40-L upregulate CD83 [32], which marker is expressed on all mature DC. However, whether pre-DC2 differentiate into mature DC under the influence of the thymic environment in vivo has not been resolved. Coexpression of CD83 on the IL-3R␣high thymic cells has not been observed [32]. The thymus is seeded by multipotent progenitor cells that are able to give rise to T cells, B cells, NK cells, and DCs. It has been observed that both myeloid and lymphoid DC can be generated from intrathymic progenitor cells. Spits et al. [37] has reported that part of the thymic CD34⫹CD1a⫺ subset, which is enriched for multipotent progenitor cells, can develop into pre-DC2 on a stromal cell layer [37]. Previously it was demonstrated that culture of thymic precursor cells in GM-CSF and TNF-␣ directs myeloid DC1 development [51, 52]. More recently a CD34dim thymic progenitor subset has been identified, which expresses the GM-CSF receptor, but lacks expression of the IL-7R␣ chain [53]. These CD34dimGM-CSFR⫹ progenitor cells generate myeloid DC1, but have lost the potential to develop into T cells, and have reduced potential to develop into NK cells, when compared with CD34dimGM-CSFR⫺ progenitors (Figure 1). The capacity of the CD34dimGM-CSFR⫹ progenitor cells to develop into pre-DC2 has not been determined. However, considering the fact these cells express almost undetectable levels of pT␣, compared with the CD34dimGM-CSFR⫺ subset and pre-DC2 [53], it is likely to assume that CD34dimGM-CSFR⫹ progenitor cells are myeloid DC1 committed progenitors. The question whether pre-DC2 are generated in the thymus in vivo or develop elsewhere and then migrate into the thymus was addressed recently. Weijer et al. [54] revealed that upon injection of human CD34⫹ cells directly into a human thymic transplant, grafted subcutaneously into recombination activating gene (RAG)-2/␥common double deficient mice, a proportion of injected cells differentiate into pre-DC2. These findings suggest that the thymic microenvironment is able to support the development of pre-DC2. Whether these cells
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FIGURE 2 CD34dimIL-3R␣high dendritic cells (pro-DC2) are found in fetal liver, but not in fetal thymus. Fetal liver and thymus were depleted of all lineage positive cells (CD3⫹, CD14⫹, CD19⫹, CD56⫹, glycophorin⫹) by magnetic bead depletion. Then cells were stained with a FITC-conjugated cocktail of antibodies against lineage markers (CD3, CD14, CD19, CD56), PE-conjugated anti-IL-3R␣, and antigen presenting cell conjugated anti-CD34. Illustrated are CD34/IL3R␣ flowcytometric dotplots of (A) fetal liver and (B) fetal thymus electronically gated on lineage negative cells.
develop via a CD34dim pro-DC2 (ILhigh ⫹ ⫹ 3R␣ CD4 CD45RA HLA-DRint) intermediate stage, like was observed in cord blood, fetal liver, and fetal bone marrow, remains elusive. Fact is that CD34dimIL-3R␣high pro-DC2 are not detected in fresh fetal or postnatal thymus (Figure 2) [13]. This suggests that thymic pre-DC2 may develop via a pathway that is different from pre-DC2 generated in fetal liver, cord blood, or bone marrow. No-
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tably, pre-DC2 can develop in the absence of the thymic environment, since they are also observed after intravenous injection of human CD34⫹ cells in RAG-2/␥common double deficient mice lacking a human thymic transplant. In those mice, human pre-DC2 are detected in the liver, peripheral blood, spleen, and bone marrow [54]. Isolation of these pre-DC2 and viral stimulation in vitro induced the production of IFN-␣, strongly suggesting that the mouse environment sustains development of functionally competent pre-DC2. Although it remains elusive where and how these pre-DC2 develop, it is evident from these findings that different developmental pathways, i.e., intrathymic and extrathymic, exist. Transcriptional Control of Pre-DC2 Development Differentiation of HSC towards a particular cell lineage requires an orchestrated program of developmental steps driven by the expression of lineage specific and nonspecific genes activated by transcription factors (TFs). One way to address the importance of critical genes is to manipulate the level of expression of their TFs and to analyze the developmental outcome. An interesting family of TFs that has been revealed to be essential in the development of lymphoid cells is the basic-helix-loopedhelix (bHLH) family of TFs [55]. The lack of the E2A bHLH proteins (encoded by the genes E12 and E47) leads to a severe block in both T-cell [56] and B-cell development [57–59]. Consistent with these findings, overexpression of inhibitor of DNA binding (Id) proteins [60], which are related to the bHLH proteins but lack the basic DNA binding domain and therefore act as dominant negative HLH factors, block T- and B-cell development [61, 62]. To address the importance of bHLH factors in the development of pre-DC2, we have reported that Id2 or Id3, when overexpressed in CD34⫹ progenitor cells by retroviral gene transfer, can inhibit pre-DC2 development [37], like that of T and B cells [34 –36]. In this context it will be interesting to investigate which bHLH factors are induced upon FLT3-L induced pre-DC2 development [13, 43], driving preDC2 differentiation. The Ets family of DNA-binding proteins is a highly conserved group of TFs, which have regulatory functions during hematopoietic development [63, 64]. A subfamily of the Ets-domain containing family is formed by the Spi factors, which include among others PU.1 (Spi-1) [65] and Spi-B [66]. PU.1 and Spi-B are coexpressed in B cells, with low level expression in early thymocytes [65– 67]. In addition, PU.1 is expressed in granulocytes, macrophages and erythrocytes [68 –70]. Whereas Spi-B deficient mice are viable, and exhibit impaired B cell functions [71], PU.1 deficient mice have severe hematopoietic defects leading to premature death between day 18.5 [72] of gestation and 3 weeks after birth, if main-
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tained in antibiotics [73]. The PU.1 function regulates early B cell development by directly controlling expression of the IL-7R␣ gene [74]. In vitro, high level Spi-B expression can substitute for PU.1 in early B cell development [74]. In vivo, however, Spi-B cannot functionally replace PU.1 in lymphoid development, although it does rescue myeloid development [75]. Interestingly, Spi-B is also expressed in human pre-DC2, as was observed after a subtractive library screen with monocyte-derived DC [33]. Expression is contained in IL-3 and CD40-L matured DC2 inting at a possible contribution of Spi-B during development of pre-DC2. Overexpression by retroviral mediated gene transfer of Spi-B in human CD34⫹ progenitor cells, however, does not affect the development of FLT3-L induced differentiation of pre-DC2 [76]. Notably, constitutive expression of Spi-B severely impairs T cell and NK cell development, suggesting that Spi-B expression is required to be downregulated for proper T cell development to occur. This notion is supported by the fact that Spi-B transcripts are downregulated during transition of uncommitted CD34⫹CD1a⫺ into T cell committed CD34⫹CD1a⫹ precursor cells [76]. CONCLUSION It has been convincingly demonstrated that both human and mouse pre-DC2 can be generated from HSC upon culture with FLT3-L [13, 43]. Whether this cytokine is responsible for the induction of differentiation or also the maintenance or survival of pro-DC2 in culture has not been resolved. Furthermore, it remains elusive what the underlying molecular mechanism is that drives this differentiation or survival. Using the current knowledge on the phenotype of the mouse pre-DC2 makes analysis of genetically modified animals submissive for detailed studies on the generation, homing and function of these cells. We [32] and others [33, 40 – 42, 50] have reported that the thymus contains pre-DC2. However, it is not clear what the functional contribution is of these cells in the thymus. We have shown that the thymic microenvironment is able to support the development of CD34⫹ precursor cells into pre-DC2 [54]. In addition, intrathymic progenitor cells are able to differentiate into preDC2 [37]. It remains elusive whether these cells develop in the thymus via a CD34dimIL-3R␣high pro-DC2 intermediate stage, since pro-DC2 are not observed in the fetal or postnatal thymus (Figure 2). Taken together, these findings support the notion that pre-DC2 play a role during T cell development. Culture of pre-DC2 in vitro with IL-3 and CD40-L, or stimulation with virus results in the maturation of these cells into DC2. These in vitro generated mature DC2 express CD83, which marker is expressed on all mature
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DC. However, in vivo coexpression of CD83 on IL3R␣high cells has not been observed. This may suggest that either mature DC2 in vivo loose IL-3R␣ expression, or do not upregulate CD83 expression. It will be a challenge to identify mature DC2 in vivo and to explore the function of these cells during innate and adaptive immune responses.
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