Dendritic Cell Development in Long-Term Spleen Stromal Cultures
http://www.100md.com
《干细胞学杂志》
School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, Australia
Key Words. Dendritic cells ? Stromal cells ? Hematopoiesis ? Differentiation
Correspondence: H.C. O’Neill, Ph.D., School of Biochemistry and Molecular Biology, Building #41, Linnaeus Way, Australian National University, Canberra, ACT 0200, Australia. Telephone: 61-2-6125-4720; Fax: 61-2-6125-0313; e-mail: Helen.ONeill@anu.edu.au
ABSTRACT
The study of the dendritic cell (DC) lineage and of the signals and factors regulating cell development and function is an area of intense interest to immunologists. DCs are highly endocytotic and the most efficient antigen-presenting cells for the immune system. They are therefore the ultimate controllers of the immune response and an extremely important target in the development of strategies for immunotherapy. Multiple DC subsets have now been defined in many organs on the basis of cell surface marker expression and function . DC subsets can also interact with a range of immune cells, presenting antigen to na?ve or memory T cells and inducing activation of natural killer cells . They also present unprocessed antigen to B cells and modulate B-cell responses . Immature DCs in peripheral tissues are thought to be the predominant DC population in the immune steady-state. They are highly endocytotic but not activated in terms of immunostimulatory capacity. Full maturation or activation depends on exposure to pathogens, inflammatory cytokines, or necrotic cells . Only activated DCs can induce a T-cell response, and different subsets of DCs appear to be responsible for induction of either tolerance or immunity . At this stage, it is unclear whether functional division among DC subsets reflects cell specialization during development from progenitors or functional plasticity of fully differentiated DCs. The answer to this question depends on direct analysis of the development of DCs from progenitors. While there are several ways to study DC development, the procedures involved are only possible in animal models. This review considers current approaches to the study of lineage commitment in DCs. Reference is made primarily to the murine DC lineage(s), although many similarities are known to exist between human and murine DCs .
Difficulties Associated with the Study of Dendritic Cell Development
DCs are scattered in most organs of the body, including lung, gut, skin, and lymphoid tissues. Unlike other leucocytes, these cells are not present in high numbers in blood. They are widely distributed in most peripheral tissues, but their numbers are so small as to make isolation an extremely difficult task. There are very few markers specific for the DC lineage, although the CD11c marker in mouse has been particularly useful . Cells are identified on the basis of combined expression of a number of markers common to other leucocytes. Langerhans cells in skin represent a distinct lineage of DCs and are identifiable by expression of Langerin . It is well accepted that DCs in peripheral tissues, including Langerhans cells in skin, are immature DCs that take up antigen and mature as they traffic to lymph nodes where they encounter T cells. Lymphoid tissues also contain endogenous populations of immature DCs that are distinguishable from antigen-carrying DCs entering from peripheral tissues . It is not yet known whether endogenous immature DCs in lymphoid tissues differ from immature DCs in peripheral tissue sites. Cells in both sites could have an important role in the maintenance of peripheral tolerance, but it is not known exactly how cells differentiate to acquire those distinctly different functional properties. While endogenous spleen DCs are thought to be blood-derived, at this stage it is unclear whether they undergo differentiation within the spleen from endogenous progenitors or arrive in a more mature state, perhaps as blood-derived DC precursors .
Many labs have developed procedures for fractionation of DC subsets of increasingly higher purity. Methods for cell isolation depend on known cell properties or known cell surface marker expression and are fraught with problems of cell contamination . Another problem is that once cells are isolated from tissue and exposed to in vitro culture, they immediately become activated, which can lead to alterations in functional capacity and marker expression . For experimentation, isolated cell fractions enriched for DC precursors are commonly expanded in vitro by culture in medium containing a cocktail of growth factors, usually containing granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), and tumor necrosis factor alpha (TNF- ). However, cytokine-supported cultures yield a heterogeneous mixture of cells, including DCs and in some circumstances monocytes and macrophages. Most cytokine-supplemented cultures are not suitable for studying the intermediate stages of DC development, since progenitors are quickly driven to become mature DCs, which undergo limited proliferation and survive for only a short period of time. These in vitro culture conditions do not maintain progenitor cell populations and provide little opportunity to study intermediate stages in development. The pathways that connect intermediate stages in DC development with progenitors and mature cells remain difficult to analyze by studying the population of cells resulting from tissue fractionation or in vitro culture.
Ways to Study Dendritic Cell Hematopoiesis
The study of DC development from progenitors is inherently difficult because progenitors are present in tissues in very low numbers, and specific markers for progenitors have not yet been defined. Characterization of DC progeny is all the more difficult because it is dependent on the production of identifiable progeny DCs. Progenitor or stem cells are also intimately tied to a niche or microenvironment that provides a complex arrangement of soluble factors, as well as cell–cell and cell–matrix interactions that are essential for maintenance of stem cells and their eventual differentiation . The microenvironments in which DCs develop are, so far, undefined. It is likely that a number of signaling events apart from cytokines combine to drive progenitor cells to differentiate into DCs. While growth factors like Flt3Land GM-CSF can support DC development both in vivo and in vitro, these cytokines do not have an essential or even a specific role in lineage commitment . The study of progenitors requires a suitable model system in which to study their differentiation into functional DCs. There are three main approaches to the study of hematopoiesis: subset analysis, lineage analysis, and production of cells in long-term cultures. A more thorough understanding of developmental pathways could stem from the application of all three approaches.
Subset analysis involves identification of cells on the basis of differential expression of multiple cell surface markers and predictions about their lineage relationship. This is usually the first approach, and it uses isolated cells, flow cytometry, and any available antibodies. Multiple DC subsets have been identified in murine lymphoid tissues on the basis of expression of markers including CD11c, CD11b, CD205, CD80, CD86, B220, MHC-II (major histocompatibility complex-2), CD40, CD8 , and CD4. Currently there are three known subsets in spleen , two in thymus , and five in lymph node . More recently, the distinct lineage of murine plasmacytoid DCs was identified in multiple organs on the basis of B220 expression . DC subsets in murine spleen are located in two distinct locations: some are located in the marginal zone, and others, notable by expression of CD205 and CD8, are located in T-cell areas . Marker combinations have been identified which can distinguish Langerhans cells, thymic lymphoid DCs, plasmacytoid DCs, and the distinct populations of myeloid CD8a+ and CD8a–DCs. Definition of the functional capacity of the many known DC subsets lags behind phenotypic definition. Furthermore, the developmental origin of each of the different subsets is one of the most controversial areas of DC biology. To date, very little is known about the lineage relationship among these DC subsets.
Lineage analysis involves ex vivo isolation of a cell subset containing progenitor cells and transfer of these into a syngeneic host that may have been sublethally irradiated to destroy host hematopoietic cells. This can be done effectively if donor and host express different allelic markers delineating the different cells. Progeny cells are identified by antibody staining to detect cell surface markers that indicate the lineage of donor-derived cells. The drawback with these studies is that information obtained is limited by knowledge of the starting cell population. It is difficult to clearly delineate lineage when the progenitor population contains more than one progenitor type. The most definitive experiments involve isolation of a pure starting population of otherwise rare cells. Another requirement is that a sufficient number of progeny cells is produced so that it is possible to detect them with certainty among the cells of the host. Using the lineage approach, both DCs and T cells were shown to develop in thymus from thymic CD4lo lymphoid precursors following intrathymic injection of cells into syngeneic host mice . This finding led to the hypothesis for a separate lymphoid-like lineage of DCs in thymus. Recently this approach was used to demonstrate plasticity in DC development. DCs of different phenotype, previously thought to be representative of distinct myeloid and lymphoid lineage subtypes, could be derived from both the common myeloid and the common lymphoid progenitor subsets in bone marrow following transplantation into host mice . This study predicted the existence of a common DC precursor derived from both myeloid and lymphoid progenitors and questioned hypotheses about the existence of separate myeloid and lymphoid lineages of DCs.
The third approach to cell development is the study of hematopoiesis in long-term stroma-dependent cultures. In these cultures, hematopoiesis is dependent on the establishment of a layer of stromal cells that support stem cell survival, self-renewal, and differentiation . Hematopoiesis was first achieved in vitro in long-term bone marrow cultures in which granulopoiesis predominated . Whitlock et al. also developed a long-term culture (LTC) system that supported production of cells representing early stages in B-cell development. Similarly, thymic organ cultures have been used to study the development of T cells in contact with the thymic stromal cell environment . In each of these culture systems, early progenitors are maintained in a complex cellular environment conducive to lineage commitment. The main advantage of these cultures is that the microenvironment supports self-renewal and maintenance of a population of progenitor cells which would otherwise be impossible to isolate. Long-term cultures mimic the normal developmental process within tissue niches. The niche established in a long-term culture supports a variety of interactions between stem cells, stromal cells, extracellular matrix, and growth and differentiation factors . Cells develop under more normal physiological conditions than in cultures supplemented with cytokines alone. This approach has limitations in that it represents in vitro development and any findings need to subsequently be tested in vivo. However, cell development and function can be observed in vitro, and cells can be isolated for study at various time points.
This lab has developed a long-term culture system that produces DCs. While some success has been obtained in establishing cultures from cells derived from a number of lymphoid sites, by far the most productive long-term cultures have been those derived from spleen . Areadily identifiable common class of immature DCs has been found to develop in spleen long-term cultures . The ease with which these cultures are established and their consistent production of DCs of a common phenotype suggest that an equivalent normal process of DC hematopoiesis may occur in spleen. Our prediction is that spleen contains DC progenitors that develop into DCs in situ and are supported by stromal cells and other factors. An endogenous population of spleen immature DCs could develop from progenitor cells resident in spleen. This hypothesis is under further test.
LONG-TERM SPLEEN CULTURES PRODUCING DENDRITIC CELLS
Nonadherent cells produced in long-term cultures have been characterized over many years as DCs on the basis of multiple parameters, including morphology, cell surface phenotype, and antigen-presenting capacity . The size distribution of nonadherent cells collected from long-term cultures has also been measured using flow cytometry to record light scatter properties of cells. LTC produces two clear subpopulations of "small" and "large" cells, as defined by Forward and Side scatter profiles . These have been referred to as small LTC-DC and large LTC-DC (Fig. 3). While the division into subsets on the basis of size appears arbitrary, it represents the best division to delineate a phenotypically homogeneous population of fully differentiated large-sized DCs. The small-cell subset is not so homogeneous and reflects less-differentiated cells with only some cells expressing the markers of DCs .
Figure 3. The major cell subsets produced in long-term cultures (LTCs). Abbreviations: FSC, forward scatter; SSC, side scatter.
The cell surface phenotype of small and large cells produced in long-term cultures has been analyzed using flow cytometry and antibodies specific for a range of surface markers. The collective data from many different long-term lines maintained over a 4-year period are shown in Table 1. These data clearly delineate the phenotype of the small and large cells produced in long-term culture and also demonstrate the consistency in the percentage of positive cells and mean fluorescence achieved over many repeat experiments. The cell population produced in long-term cultures has remained remarkably constant in terms of cell size and surface marker expression over many years and over many lines established from different strains of mice .
Table 1. Marker expression on small and large subsets of cells produced in long-term cultures
The majority of cells produced in long-term cultures are large-sized cells with a clear DC phenotype of CD11c+CD11b+CD80+CD86+MHC-II–/lo (Table 1; Fig. 3). Absence of CD40, B200, and CD8–reflects precursor or immature myeloid-like DCs (Table 1) . Numerous tests have shown these cells to be highly endocytotic and immunostimulatory for T cells . Small DCs represent a minor subset of cells expressing variable levels of CD117 (c-kit), CD80, CD11c, and CD11b. They are only weakly endocytotic and do not stimulate T cells . A minor (1%–3%) subset of large cells produced in LTC is MHC-II+ and is thought to represent DCs activated in culture . Large LTC-DCs collected from stromal cultures can be weakly activated if they are cultured directly on plastic surfaces. This leads to upregulation of CD86 and MHC-I, although not of MHC-II . These cells also respond weakly to activation with known DC-activating agents like lipopolysaccharide (LPS), TNF- , and CD40 ligand. While these factors typically induce upregulation of CD86, MHC-I, CD44, and MHC-II in bone marrow–derived or monocyte-derived DCs, LTC-DCs respond to these particular stimuli by upregulation of MHC-I and CD86 but not MHC-II or CD80 . The significance of this response is under further investigation. One hypothesis is that DCs in different sites may have different functional capacity with respect to activation consistent with exposure to different types of pathogens or activators. This would be consistent with site-specific immune responses and compartmentalization of the immune system to best meet the invasion of a range of different pathogens.
In relation to known DC subsets, large LTC-DCs represent fully developed myeloid DCs that are unusual in that they do not express MHC-II and CD40. An in vivo equivalent cell type can be characterized in mouse spleen (unpublished data). In terms of cell surface phenotype, these cells resemble recently described murine CD11c+CD11b+MHC-II–CD40–blood DC precursors (Fig. 3) . A similar subset of DCs in spleen has also been described, which can activate marginal zone B cells to respond to bloodborne bacterial antigen . DCs produced in long-term cultures are phenotypically and functionally distinct from the "gold standard" CD11c+CD11b+MHC-II+ DCs generated in cytokine-supported cultures. The commonly described DC subsets isolated by cell enrichment from the spleen and from lymph nodes are also different in that they have high MHC-II expression. Indeed, many of the procedures used to isolate DCs would eliminate subsets of cells resembling LTC-DCs on the basis of no MHC-II expression. However, it is difficult to determine from combined studies whether MHC-II levels are low to medium or high on spleen DC subsets, and, in fact, contradictory reports exist . DCs produced in the LTC system described here also differ from other cytokine-dependent DC-producing lines, like the D1 line described by Rescigno et al. . D1 cells are myeloid DCs that express high levels of immunostimulatory markers, including MHC-II. These levels also increase after activation of cells with LPS and other bacterial activators . Low expression of MHC-II in LTC-DC could be a result of in vitro culture conditions, however. LTC-DCs have been shown to synthesize the MHC-II invariant chain by reverse transcription polymerase chain reaction (RT-PCR) (unpublished data). It is possible that with their extremely high endocytotic capacity, cells lose surface MHC-II due to rapid turnover of the plasma membrane. DCs with a high rate of endocytosis have been shown to reabsorb and degrade MHC-II/peptide complexes faster than weakly endocytotic DCs .
Spleen also contains subsets of DCs that resemble LTC-DCs in terms of their absence of the CD8 and CD205 markers . Low to nondetectable expression of CD205 in LTC-DCs is indicative of DCs residing outside the T-cell area of spleen , suggesting that LTC-DCs may represent a marginal zone population of DCs. CD8 was originally thought to be a stable marker of spleen DC subsets, but it is now thought to fluctuate with activation and differentiation of cells. CD8–DCs can develop into CD8 + DCs in a process that also involves upregulation of CD205 and is thought to be associated with maturation and movement of DCs into the T-cell areas. Alternatively, there is also evidence for a CD8 + blood precursor of spleen CD8 +DCs, which suggests that separate lineages of CD8 +cells may exist .
Large DCs produced in long-term cultures also express high levels of the FcII/III receptor and are highly endocytotic . This is consistent with immature DCs that have a high capacity to endocytose antigen in preparation for processing and presentation to Tcells . The capacity of cells to present antigen leading to T-cell activation has been demonstrated in vitro using both the conalbumin-specific D10.G4.1 Th cell clone and hen egg lysosome (HEL)–specific T cells isolated from T-cell receptor 3A9 (TCR)-transgenic (Tg) mice . Since CD4+T cells derived from 3A9 TCR-Tg mice are na?ve, LTC-DCs have a distinct antigen-presenting capacity for unprimed T cells, which is a defining property of DCs. LTC-DCs pulsed with tumor cell membranes can induce a specific antitumor CD8+ cytotoxic T-cell response following adoptive transfer into mice . This response has also been shown to be protective and to reduce mortality among tumor-bearing mice.
DCs derived from most long-term cultures have no or weak capacity to stimulate allogeneic T cells in a mixed lymphocyte reaction . It is possible that incapacity to stimulate T cells in mixed lymphocyte reaction (MLR) is related to low expression of immunostimulatory molecules on cells. While LTC-DCs have high expression of CD80, CD86, and MHC-I, they have low or no expression of other immunostimulatory markers such as MHC-II, CD40, and CD40L. Some DC subsets have been shown to induce tolerance rather than immunity . It is not yet known whether the limited CD4+ T-cell stimulation by LTC-DCs is immunogenic or whether it leads to abortive T-cell proliferation and apoptosis of cells, but this is under further investigation. However, this inability to stimulate an MLR could be due to the immature nature of LTC-DCs and a lack of MHC-II expression on most cells. There could also be differences in the activation threshold of T-cell clones or TCR-Tg T cells, compared with the heterogeneously responding T-cell population used in MLR. Recent studies have also demonstrated that the response in T cells generated by DCs in an allogeneic MLR may depend on the presence of syngeneic DCs that present allogeneic antigens to the responding leucocyte population . Another consideration is that LTC-DCs have a distinct functional capacity for stimulation of B cells or natural killer cells, in line with their location in spleen and exposure to bloodborne pathogens .
In summary, DCs produced in long-term spleen cultures represent immature or precursor CD8–CD205–/lo DCs. These could be representative of marginal zone type DCs that show some weak expression of CD205. Long-term cultures could provide an environment that promotes DC differentiation to the immature DC state but limits development of cells whose end result might well have been to reside in T-cell areas. Alternatively, low expression of CD205 could place these cells on the periphery of T-cell areas. As early immature or precursor DCs lacking expression of MHC-II and CD40, they could also represent a DC subclass with distinct functional capacity for mediating early natural killer or B-cell immune responses. These hypotheses are under further test.
CULTURED STROMAL CELLS SUPPORT DENDRITIC CELL DEVELOPMENT FROM PROGENITORS
The spleen long-term cultures described here are ideal for the study of differential gene expression, since progenitors and their progeny are maintained within the same culture environment. Subtracted cDNA libraries have been generated; these contain sequences that are differentially expressed in either "small minus large" or "large minus small" subsets of LTC-DCs . Differential screening was used to select clones expressed in either the small or the large LTC-DC populations for sequencing and identification. Known genes isolated from subtracted libraries relate to different stages in DC development and support previous findings regarding the function of the small and large cell subsets (Fig. 3) . This lab has had very good success with this procedure, with no overlapping clones detected in the two cell subsets. Large LTC-DCs express a number of immunologically important genes, including CD86, CCR1, osteopontin, and lysozyme. Small LTC-DCs resemble progenitors expressing genes relating to organization of the cytoskeleton and regulation of antigen processing. Novel transcripts have been isolated from both small and large LTC-DC–subtracted libraries that could encode novel proteins important in DC development.
The individual small- and large-cell subsets are also highly suitable for gene profiling using Affymetrix microarrays. Multiple cell sorts have been performed to isolate subsets of small and large cells from long-term cultures and RNA isolated for probe preparation. Procedures have been developed to prepare labels from small quantities of RNA isolated from the rare small-cell subset. This type of analysis should identify genes that are differentially expressed between progenitors and DCs generated in spleen long-term cultures and should help to identify factors and genes important in early DC development.
CONCLUSION: A MODEL FOR DENDRITIC CELL DEVELOPMENT IN SPLEEN
This work was supported by grants to H.O. from the National Health and Medical Research Council of Australia, the Australian Research Council, the Clive & Vera Ramaciotti Foundation, and the Australian National University Faculties Research Grant Scheme. H.W.and B.Q.were supported by Australian Postgraduate Awards. J.A.was supported by an ANU Graduate Scholarship. G.D. was supported by a scholarship from the Fonds Nature et Technologies-Fonds de laRecherche en Santé du Québec.
REFERENCES
Shortman K, Liu Y-J. Mouse and human dendritic cell subtypes. Nat Rev Immunol 2002;2:151–161.
Ardavin C, Martinez del Hoyo G, Martin P et al. Origin and differentiation of dendritic cells. Trends Immunol 2001;21:691–700.
Ferlazzo G, Tsang M, Moretta L et al. Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells. J Exp Med 2002;195:343–351.
Wykes M, Pombo A, Jenkins C et al. Dendritic cells interact directly with na?ve B lymphocytes to transfer antigen and initiate class switching in a primary T-dependent response. J Immunol 1998;161:1313–1319.
Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med 1999;5:1249–1255.
Steinman RM. The control of immunity and tolerance by dendritic cell. Pathol Biol (Paris) 2003;51:59–60.
Metlay JP, Witmer-Pack MD, Agger R et al. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J Exp Med 1990;171:1753–1771.
Valladeau J, Ravel O, Dezutter-Dambuyant C et al. Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytotic receptor that induces the formation of Birbeck granules. Immunity 2000;12:71–81.
Wilson N, El-Sukkari D, Belz G et al. Most lymphoid organ dendritic cell types are phenotypically and functionally immature. Blood 2003;102:2187–2194.
Martinez del Hoyo G, Martin P, Hernandez Vargas H et al. Characterization of a common precursor population for dendritic cells. Nature 2002;415:1043–1047.
O’Keeffe M, Hochrein H, Vremec D et al. Dendritic cell precursor populations of mouse blood: identification of the murine homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors. Blood 2003;101:1453–1459.
Martin P, Del Hoyo GM, Anjuere F et al. Characterization of a new subpopulation of mouse CD8alpha+ B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential. Blood 2002;100:383–390.
Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem cell biology. Cell 1997;88:287–298.
Orkin SH, Zon LI. Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nat Immunol 2002;3:323–328.
Sitnicka E, Bryder D, Theilgaard-Monch K et al. Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool. Immunity 2002;17:463–472.
O’Neill HC, Wilson HL. Limitations with in vitro production of dendritic cells using cytokines. J Leukoc Biol 2004;75:600–603.
Vremec D, Pooley J, Hochrein H et al. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol 2000;164:2978–2986.
Henri S, Vremec D, Kamath A et al. The dendritic cell populations of mouse lymph nodes. J Immunol 2001;167:741–748.
Nakano H, Yanagita M, Gunn M. CD11c+B220+Gr-1+ cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J Exp Med 2001;194:1171–1178.
Leenen P, Radosevic K, Voerman J et al. Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and sub-population turnover. J Immunol 1998;160:2166–2173.
Ardavin C, Wu L, Li C-L et al. Thymic dendritic cells and Tcells develop simultaneously in the thymus from a common precursor population. Nature 1993;362:761–763.
Manz MG, Traver D, Miyamoto T et al. Dendritic cell potentials of early lymphoid and myeloid progenitors. Blood 2001;97:3333–3341.
Dexter T, Testa N. In vitro methods in haemopoiesis and lymphopoiesis. J Immunol Methods 1980;38:177–190.
Dexter T, Lajtha L. Proliferation of haemopoietic stem cells in vitro. Br J Haematol 1974;28:525–530.
Whitlock C, Robertson D, Witte O. Murine B cell lymphopoiesis in long term culture. J Immunol Methods 1984;67:353–369.
Suniara RK, Jenkinson EJ, Owen JJ. An essential role for thymic mesenchyme in early T cell development. J Exp Med 2000;191:1051–1066.
Tsai R, Kittappa R, McKay R. Plasticity, niches, and the use of stem cells. Dev Cell 2002;2:707–712.
Ni K, O’Neill HC. Long-term stromal cultures produce dendritic-like cells. Br J Haematol 1997;97:710–725.
Ni K, O’Neill HC. Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: production of cells with myeloid/dendritic characteristics. In Vitro Cell Dev Mol Biol 1998;34:298–307.
Ni K, O’Neill HC. Spleen stromal cells support haemopoiesis and in vitro growth of dendritic cells from bone marrow. Br J Haematol 1999;105:58–67.
O’Neill HC. Ni, K, Wilson H. Long-term stroma-dependent cultures are a consistent source of immunostimulatory dendritic cells. Immunol Cell Biol 1999;77:434–441.
Wilson H, Ni K, O’Neill HC. Proliferation of dendritic cells in long term culture is not dependent on granulocyte macrophage-colony stimulating factor. Exp Hematol 2000;28:193–202.
Wilson HL, Ni K, O’Neill HC. Identification of progenitor cells in long-term spleen stromal cultures that produce immature dendritic cells. Proc Natl Acad Sci U S A 2000;97:4784–4789.
Wilson H, O’Neill HC. Dynamics of dendritic cell development from precursors maintained in stroma-dependent long-term cultures. Immunol Cell Biol 2003;81:144–151.
Ni K, O’Neill HC. Development of dendritic cells from GM-CSF–/–mice in vitro: GM-CSF enhances production and survival of cells. Dev Immunol 2001;8:133–146.
Ni K, O’Neill HC. Improved FACS analysis confirms generation of immature dendritic cells in long-term stromal-dependent spleen cultures. Immunol Cell Biol 2000;78:196–204.
Hsieh SM, Pan SC, Hung CC et al. Kinetics of antigen-induced phenotypic and functional maturation of human monocyte-derived dendritic cells. J Immunol 2001;167:6286–6291.
Quah B, Nik, O’Neill HC. In vitro hematopoiesis produces a distinct class of immature dendritic cells from spleen progenitors with limited T cell stimulation capacity. Int Immunol 2004;16:567–577.
Szabolcs P, Moore MA, Young JW. Expansion of immunostimulatory dendritic cells among the myeloid progeny of human CD34+ bone marrow precursors cultured with c-kit ligand, granulocyte-macrophage colony-stimulating factor, and TNF-alpha. J Immunol 1995;154:5851–5861.
Adachi Y, Toki J, Ikebukuro K et al. Immature dendritic cells (CD11c+ CD3–B220–cells) present in mouse peripheral blood. Immunobiology 2002;206:354–367.
Balazs M, Martin F, Zhou T et al. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 2002;17:341–352.
Kamath AT, Pooley J, O’Keeffe MA. The development, maturation, and turnover rate of mouse spleen dendritic cell populations. J Immunol 2000;165:6762–6770.
Rescigno M, Sutherland M, Gold M et al. Dendritic cell survival and maturation are regulated by different signalling pathways. J Exp Med 1998;188:2175–2180.
Hofer S, Rescigno M, Granucci F et al. Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide. Microbes Infect 2001;3:259–265.
Kampgen E, Koch N, Koch F et al. Class II major histocompatibility complex molecules of murine dendritic cells: synthesis, sialylation of invariant chain, and antigen processing capacity are down-regulated upon culture. Proc Natl Acad Sci U S A 1991;88:3014–3018.
Vremec D, Shortman K. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation and differences among thymus, spleen, and lymph nodes. J Immunol 1997;159:565–573.
Pulendran B, Lingappa J, Kennedy MK et al. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J Immunol 1997;159:2222–2231.
Martinez del Hoyo G, Martin P, Arias C et al. CD8alpha+ dendritic cells originate from the CD8alpha–dendritic cell subset by a maturation process involving CD8alpha, DEC-205, and CD24 upregulation. Blood 2002;99:999–1004.
Wang Y, Zhang Y, Yoneyama H et al. Identification of CD8alpha+CD11c–lineage phenotype-negative cells in the spleen as committed precursor of CD8alpha dendritic cells. Blood 2002;100:569–577.
O’Neill HC, Jonas N, Wilson H et al. Immunotherapeutic potential of dendritic cells generated in long-term stroma-dependent cultures. Cancer Biother Radiopharm 1999;14:263–276.
Mosier M. Dendritic cells in immunity and tolerance: Do they display opposite functions? Immunity 2003;19:5–8.
Bedford P, Garner K, Knight SC. MHC class II molecules transferred between allogeneic dendritic cells stimulate primary mixed leukocyte reactions. Int Immunol 1999;11:1739–1744.
Wilson H, O’Neill HC. Identification of differentially expressed genes representing dendritic cell precursors and their progeny. Blood 2003;102:1661–1669.(Helen C. O’Neill, Heather)
Key Words. Dendritic cells ? Stromal cells ? Hematopoiesis ? Differentiation
Correspondence: H.C. O’Neill, Ph.D., School of Biochemistry and Molecular Biology, Building #41, Linnaeus Way, Australian National University, Canberra, ACT 0200, Australia. Telephone: 61-2-6125-4720; Fax: 61-2-6125-0313; e-mail: Helen.ONeill@anu.edu.au
ABSTRACT
The study of the dendritic cell (DC) lineage and of the signals and factors regulating cell development and function is an area of intense interest to immunologists. DCs are highly endocytotic and the most efficient antigen-presenting cells for the immune system. They are therefore the ultimate controllers of the immune response and an extremely important target in the development of strategies for immunotherapy. Multiple DC subsets have now been defined in many organs on the basis of cell surface marker expression and function . DC subsets can also interact with a range of immune cells, presenting antigen to na?ve or memory T cells and inducing activation of natural killer cells . They also present unprocessed antigen to B cells and modulate B-cell responses . Immature DCs in peripheral tissues are thought to be the predominant DC population in the immune steady-state. They are highly endocytotic but not activated in terms of immunostimulatory capacity. Full maturation or activation depends on exposure to pathogens, inflammatory cytokines, or necrotic cells . Only activated DCs can induce a T-cell response, and different subsets of DCs appear to be responsible for induction of either tolerance or immunity . At this stage, it is unclear whether functional division among DC subsets reflects cell specialization during development from progenitors or functional plasticity of fully differentiated DCs. The answer to this question depends on direct analysis of the development of DCs from progenitors. While there are several ways to study DC development, the procedures involved are only possible in animal models. This review considers current approaches to the study of lineage commitment in DCs. Reference is made primarily to the murine DC lineage(s), although many similarities are known to exist between human and murine DCs .
Difficulties Associated with the Study of Dendritic Cell Development
DCs are scattered in most organs of the body, including lung, gut, skin, and lymphoid tissues. Unlike other leucocytes, these cells are not present in high numbers in blood. They are widely distributed in most peripheral tissues, but their numbers are so small as to make isolation an extremely difficult task. There are very few markers specific for the DC lineage, although the CD11c marker in mouse has been particularly useful . Cells are identified on the basis of combined expression of a number of markers common to other leucocytes. Langerhans cells in skin represent a distinct lineage of DCs and are identifiable by expression of Langerin . It is well accepted that DCs in peripheral tissues, including Langerhans cells in skin, are immature DCs that take up antigen and mature as they traffic to lymph nodes where they encounter T cells. Lymphoid tissues also contain endogenous populations of immature DCs that are distinguishable from antigen-carrying DCs entering from peripheral tissues . It is not yet known whether endogenous immature DCs in lymphoid tissues differ from immature DCs in peripheral tissue sites. Cells in both sites could have an important role in the maintenance of peripheral tolerance, but it is not known exactly how cells differentiate to acquire those distinctly different functional properties. While endogenous spleen DCs are thought to be blood-derived, at this stage it is unclear whether they undergo differentiation within the spleen from endogenous progenitors or arrive in a more mature state, perhaps as blood-derived DC precursors .
Many labs have developed procedures for fractionation of DC subsets of increasingly higher purity. Methods for cell isolation depend on known cell properties or known cell surface marker expression and are fraught with problems of cell contamination . Another problem is that once cells are isolated from tissue and exposed to in vitro culture, they immediately become activated, which can lead to alterations in functional capacity and marker expression . For experimentation, isolated cell fractions enriched for DC precursors are commonly expanded in vitro by culture in medium containing a cocktail of growth factors, usually containing granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), and tumor necrosis factor alpha (TNF- ). However, cytokine-supported cultures yield a heterogeneous mixture of cells, including DCs and in some circumstances monocytes and macrophages. Most cytokine-supplemented cultures are not suitable for studying the intermediate stages of DC development, since progenitors are quickly driven to become mature DCs, which undergo limited proliferation and survive for only a short period of time. These in vitro culture conditions do not maintain progenitor cell populations and provide little opportunity to study intermediate stages in development. The pathways that connect intermediate stages in DC development with progenitors and mature cells remain difficult to analyze by studying the population of cells resulting from tissue fractionation or in vitro culture.
Ways to Study Dendritic Cell Hematopoiesis
The study of DC development from progenitors is inherently difficult because progenitors are present in tissues in very low numbers, and specific markers for progenitors have not yet been defined. Characterization of DC progeny is all the more difficult because it is dependent on the production of identifiable progeny DCs. Progenitor or stem cells are also intimately tied to a niche or microenvironment that provides a complex arrangement of soluble factors, as well as cell–cell and cell–matrix interactions that are essential for maintenance of stem cells and their eventual differentiation . The microenvironments in which DCs develop are, so far, undefined. It is likely that a number of signaling events apart from cytokines combine to drive progenitor cells to differentiate into DCs. While growth factors like Flt3Land GM-CSF can support DC development both in vivo and in vitro, these cytokines do not have an essential or even a specific role in lineage commitment . The study of progenitors requires a suitable model system in which to study their differentiation into functional DCs. There are three main approaches to the study of hematopoiesis: subset analysis, lineage analysis, and production of cells in long-term cultures. A more thorough understanding of developmental pathways could stem from the application of all three approaches.
Subset analysis involves identification of cells on the basis of differential expression of multiple cell surface markers and predictions about their lineage relationship. This is usually the first approach, and it uses isolated cells, flow cytometry, and any available antibodies. Multiple DC subsets have been identified in murine lymphoid tissues on the basis of expression of markers including CD11c, CD11b, CD205, CD80, CD86, B220, MHC-II (major histocompatibility complex-2), CD40, CD8 , and CD4. Currently there are three known subsets in spleen , two in thymus , and five in lymph node . More recently, the distinct lineage of murine plasmacytoid DCs was identified in multiple organs on the basis of B220 expression . DC subsets in murine spleen are located in two distinct locations: some are located in the marginal zone, and others, notable by expression of CD205 and CD8, are located in T-cell areas . Marker combinations have been identified which can distinguish Langerhans cells, thymic lymphoid DCs, plasmacytoid DCs, and the distinct populations of myeloid CD8a+ and CD8a–DCs. Definition of the functional capacity of the many known DC subsets lags behind phenotypic definition. Furthermore, the developmental origin of each of the different subsets is one of the most controversial areas of DC biology. To date, very little is known about the lineage relationship among these DC subsets.
Lineage analysis involves ex vivo isolation of a cell subset containing progenitor cells and transfer of these into a syngeneic host that may have been sublethally irradiated to destroy host hematopoietic cells. This can be done effectively if donor and host express different allelic markers delineating the different cells. Progeny cells are identified by antibody staining to detect cell surface markers that indicate the lineage of donor-derived cells. The drawback with these studies is that information obtained is limited by knowledge of the starting cell population. It is difficult to clearly delineate lineage when the progenitor population contains more than one progenitor type. The most definitive experiments involve isolation of a pure starting population of otherwise rare cells. Another requirement is that a sufficient number of progeny cells is produced so that it is possible to detect them with certainty among the cells of the host. Using the lineage approach, both DCs and T cells were shown to develop in thymus from thymic CD4lo lymphoid precursors following intrathymic injection of cells into syngeneic host mice . This finding led to the hypothesis for a separate lymphoid-like lineage of DCs in thymus. Recently this approach was used to demonstrate plasticity in DC development. DCs of different phenotype, previously thought to be representative of distinct myeloid and lymphoid lineage subtypes, could be derived from both the common myeloid and the common lymphoid progenitor subsets in bone marrow following transplantation into host mice . This study predicted the existence of a common DC precursor derived from both myeloid and lymphoid progenitors and questioned hypotheses about the existence of separate myeloid and lymphoid lineages of DCs.
The third approach to cell development is the study of hematopoiesis in long-term stroma-dependent cultures. In these cultures, hematopoiesis is dependent on the establishment of a layer of stromal cells that support stem cell survival, self-renewal, and differentiation . Hematopoiesis was first achieved in vitro in long-term bone marrow cultures in which granulopoiesis predominated . Whitlock et al. also developed a long-term culture (LTC) system that supported production of cells representing early stages in B-cell development. Similarly, thymic organ cultures have been used to study the development of T cells in contact with the thymic stromal cell environment . In each of these culture systems, early progenitors are maintained in a complex cellular environment conducive to lineage commitment. The main advantage of these cultures is that the microenvironment supports self-renewal and maintenance of a population of progenitor cells which would otherwise be impossible to isolate. Long-term cultures mimic the normal developmental process within tissue niches. The niche established in a long-term culture supports a variety of interactions between stem cells, stromal cells, extracellular matrix, and growth and differentiation factors . Cells develop under more normal physiological conditions than in cultures supplemented with cytokines alone. This approach has limitations in that it represents in vitro development and any findings need to subsequently be tested in vivo. However, cell development and function can be observed in vitro, and cells can be isolated for study at various time points.
This lab has developed a long-term culture system that produces DCs. While some success has been obtained in establishing cultures from cells derived from a number of lymphoid sites, by far the most productive long-term cultures have been those derived from spleen . Areadily identifiable common class of immature DCs has been found to develop in spleen long-term cultures . The ease with which these cultures are established and their consistent production of DCs of a common phenotype suggest that an equivalent normal process of DC hematopoiesis may occur in spleen. Our prediction is that spleen contains DC progenitors that develop into DCs in situ and are supported by stromal cells and other factors. An endogenous population of spleen immature DCs could develop from progenitor cells resident in spleen. This hypothesis is under further test.
LONG-TERM SPLEEN CULTURES PRODUCING DENDRITIC CELLS
Nonadherent cells produced in long-term cultures have been characterized over many years as DCs on the basis of multiple parameters, including morphology, cell surface phenotype, and antigen-presenting capacity . The size distribution of nonadherent cells collected from long-term cultures has also been measured using flow cytometry to record light scatter properties of cells. LTC produces two clear subpopulations of "small" and "large" cells, as defined by Forward and Side scatter profiles . These have been referred to as small LTC-DC and large LTC-DC (Fig. 3). While the division into subsets on the basis of size appears arbitrary, it represents the best division to delineate a phenotypically homogeneous population of fully differentiated large-sized DCs. The small-cell subset is not so homogeneous and reflects less-differentiated cells with only some cells expressing the markers of DCs .
Figure 3. The major cell subsets produced in long-term cultures (LTCs). Abbreviations: FSC, forward scatter; SSC, side scatter.
The cell surface phenotype of small and large cells produced in long-term cultures has been analyzed using flow cytometry and antibodies specific for a range of surface markers. The collective data from many different long-term lines maintained over a 4-year period are shown in Table 1. These data clearly delineate the phenotype of the small and large cells produced in long-term culture and also demonstrate the consistency in the percentage of positive cells and mean fluorescence achieved over many repeat experiments. The cell population produced in long-term cultures has remained remarkably constant in terms of cell size and surface marker expression over many years and over many lines established from different strains of mice .
Table 1. Marker expression on small and large subsets of cells produced in long-term cultures
The majority of cells produced in long-term cultures are large-sized cells with a clear DC phenotype of CD11c+CD11b+CD80+CD86+MHC-II–/lo (Table 1; Fig. 3). Absence of CD40, B200, and CD8–reflects precursor or immature myeloid-like DCs (Table 1) . Numerous tests have shown these cells to be highly endocytotic and immunostimulatory for T cells . Small DCs represent a minor subset of cells expressing variable levels of CD117 (c-kit), CD80, CD11c, and CD11b. They are only weakly endocytotic and do not stimulate T cells . A minor (1%–3%) subset of large cells produced in LTC is MHC-II+ and is thought to represent DCs activated in culture . Large LTC-DCs collected from stromal cultures can be weakly activated if they are cultured directly on plastic surfaces. This leads to upregulation of CD86 and MHC-I, although not of MHC-II . These cells also respond weakly to activation with known DC-activating agents like lipopolysaccharide (LPS), TNF- , and CD40 ligand. While these factors typically induce upregulation of CD86, MHC-I, CD44, and MHC-II in bone marrow–derived or monocyte-derived DCs, LTC-DCs respond to these particular stimuli by upregulation of MHC-I and CD86 but not MHC-II or CD80 . The significance of this response is under further investigation. One hypothesis is that DCs in different sites may have different functional capacity with respect to activation consistent with exposure to different types of pathogens or activators. This would be consistent with site-specific immune responses and compartmentalization of the immune system to best meet the invasion of a range of different pathogens.
In relation to known DC subsets, large LTC-DCs represent fully developed myeloid DCs that are unusual in that they do not express MHC-II and CD40. An in vivo equivalent cell type can be characterized in mouse spleen (unpublished data). In terms of cell surface phenotype, these cells resemble recently described murine CD11c+CD11b+MHC-II–CD40–blood DC precursors (Fig. 3) . A similar subset of DCs in spleen has also been described, which can activate marginal zone B cells to respond to bloodborne bacterial antigen . DCs produced in long-term cultures are phenotypically and functionally distinct from the "gold standard" CD11c+CD11b+MHC-II+ DCs generated in cytokine-supported cultures. The commonly described DC subsets isolated by cell enrichment from the spleen and from lymph nodes are also different in that they have high MHC-II expression. Indeed, many of the procedures used to isolate DCs would eliminate subsets of cells resembling LTC-DCs on the basis of no MHC-II expression. However, it is difficult to determine from combined studies whether MHC-II levels are low to medium or high on spleen DC subsets, and, in fact, contradictory reports exist . DCs produced in the LTC system described here also differ from other cytokine-dependent DC-producing lines, like the D1 line described by Rescigno et al. . D1 cells are myeloid DCs that express high levels of immunostimulatory markers, including MHC-II. These levels also increase after activation of cells with LPS and other bacterial activators . Low expression of MHC-II in LTC-DC could be a result of in vitro culture conditions, however. LTC-DCs have been shown to synthesize the MHC-II invariant chain by reverse transcription polymerase chain reaction (RT-PCR) (unpublished data). It is possible that with their extremely high endocytotic capacity, cells lose surface MHC-II due to rapid turnover of the plasma membrane. DCs with a high rate of endocytosis have been shown to reabsorb and degrade MHC-II/peptide complexes faster than weakly endocytotic DCs .
Spleen also contains subsets of DCs that resemble LTC-DCs in terms of their absence of the CD8 and CD205 markers . Low to nondetectable expression of CD205 in LTC-DCs is indicative of DCs residing outside the T-cell area of spleen , suggesting that LTC-DCs may represent a marginal zone population of DCs. CD8 was originally thought to be a stable marker of spleen DC subsets, but it is now thought to fluctuate with activation and differentiation of cells. CD8–DCs can develop into CD8 + DCs in a process that also involves upregulation of CD205 and is thought to be associated with maturation and movement of DCs into the T-cell areas. Alternatively, there is also evidence for a CD8 + blood precursor of spleen CD8 +DCs, which suggests that separate lineages of CD8 +cells may exist .
Large DCs produced in long-term cultures also express high levels of the FcII/III receptor and are highly endocytotic . This is consistent with immature DCs that have a high capacity to endocytose antigen in preparation for processing and presentation to Tcells . The capacity of cells to present antigen leading to T-cell activation has been demonstrated in vitro using both the conalbumin-specific D10.G4.1 Th cell clone and hen egg lysosome (HEL)–specific T cells isolated from T-cell receptor 3A9 (TCR)-transgenic (Tg) mice . Since CD4+T cells derived from 3A9 TCR-Tg mice are na?ve, LTC-DCs have a distinct antigen-presenting capacity for unprimed T cells, which is a defining property of DCs. LTC-DCs pulsed with tumor cell membranes can induce a specific antitumor CD8+ cytotoxic T-cell response following adoptive transfer into mice . This response has also been shown to be protective and to reduce mortality among tumor-bearing mice.
DCs derived from most long-term cultures have no or weak capacity to stimulate allogeneic T cells in a mixed lymphocyte reaction . It is possible that incapacity to stimulate T cells in mixed lymphocyte reaction (MLR) is related to low expression of immunostimulatory molecules on cells. While LTC-DCs have high expression of CD80, CD86, and MHC-I, they have low or no expression of other immunostimulatory markers such as MHC-II, CD40, and CD40L. Some DC subsets have been shown to induce tolerance rather than immunity . It is not yet known whether the limited CD4+ T-cell stimulation by LTC-DCs is immunogenic or whether it leads to abortive T-cell proliferation and apoptosis of cells, but this is under further investigation. However, this inability to stimulate an MLR could be due to the immature nature of LTC-DCs and a lack of MHC-II expression on most cells. There could also be differences in the activation threshold of T-cell clones or TCR-Tg T cells, compared with the heterogeneously responding T-cell population used in MLR. Recent studies have also demonstrated that the response in T cells generated by DCs in an allogeneic MLR may depend on the presence of syngeneic DCs that present allogeneic antigens to the responding leucocyte population . Another consideration is that LTC-DCs have a distinct functional capacity for stimulation of B cells or natural killer cells, in line with their location in spleen and exposure to bloodborne pathogens .
In summary, DCs produced in long-term spleen cultures represent immature or precursor CD8–CD205–/lo DCs. These could be representative of marginal zone type DCs that show some weak expression of CD205. Long-term cultures could provide an environment that promotes DC differentiation to the immature DC state but limits development of cells whose end result might well have been to reside in T-cell areas. Alternatively, low expression of CD205 could place these cells on the periphery of T-cell areas. As early immature or precursor DCs lacking expression of MHC-II and CD40, they could also represent a DC subclass with distinct functional capacity for mediating early natural killer or B-cell immune responses. These hypotheses are under further test.
CULTURED STROMAL CELLS SUPPORT DENDRITIC CELL DEVELOPMENT FROM PROGENITORS
The spleen long-term cultures described here are ideal for the study of differential gene expression, since progenitors and their progeny are maintained within the same culture environment. Subtracted cDNA libraries have been generated; these contain sequences that are differentially expressed in either "small minus large" or "large minus small" subsets of LTC-DCs . Differential screening was used to select clones expressed in either the small or the large LTC-DC populations for sequencing and identification. Known genes isolated from subtracted libraries relate to different stages in DC development and support previous findings regarding the function of the small and large cell subsets (Fig. 3) . This lab has had very good success with this procedure, with no overlapping clones detected in the two cell subsets. Large LTC-DCs express a number of immunologically important genes, including CD86, CCR1, osteopontin, and lysozyme. Small LTC-DCs resemble progenitors expressing genes relating to organization of the cytoskeleton and regulation of antigen processing. Novel transcripts have been isolated from both small and large LTC-DC–subtracted libraries that could encode novel proteins important in DC development.
The individual small- and large-cell subsets are also highly suitable for gene profiling using Affymetrix microarrays. Multiple cell sorts have been performed to isolate subsets of small and large cells from long-term cultures and RNA isolated for probe preparation. Procedures have been developed to prepare labels from small quantities of RNA isolated from the rare small-cell subset. This type of analysis should identify genes that are differentially expressed between progenitors and DCs generated in spleen long-term cultures and should help to identify factors and genes important in early DC development.
CONCLUSION: A MODEL FOR DENDRITIC CELL DEVELOPMENT IN SPLEEN
This work was supported by grants to H.O. from the National Health and Medical Research Council of Australia, the Australian Research Council, the Clive & Vera Ramaciotti Foundation, and the Australian National University Faculties Research Grant Scheme. H.W.and B.Q.were supported by Australian Postgraduate Awards. J.A.was supported by an ANU Graduate Scholarship. G.D. was supported by a scholarship from the Fonds Nature et Technologies-Fonds de laRecherche en Santé du Québec.
REFERENCES
Shortman K, Liu Y-J. Mouse and human dendritic cell subtypes. Nat Rev Immunol 2002;2:151–161.
Ardavin C, Martinez del Hoyo G, Martin P et al. Origin and differentiation of dendritic cells. Trends Immunol 2001;21:691–700.
Ferlazzo G, Tsang M, Moretta L et al. Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells. J Exp Med 2002;195:343–351.
Wykes M, Pombo A, Jenkins C et al. Dendritic cells interact directly with na?ve B lymphocytes to transfer antigen and initiate class switching in a primary T-dependent response. J Immunol 1998;161:1313–1319.
Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med 1999;5:1249–1255.
Steinman RM. The control of immunity and tolerance by dendritic cell. Pathol Biol (Paris) 2003;51:59–60.
Metlay JP, Witmer-Pack MD, Agger R et al. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J Exp Med 1990;171:1753–1771.
Valladeau J, Ravel O, Dezutter-Dambuyant C et al. Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytotic receptor that induces the formation of Birbeck granules. Immunity 2000;12:71–81.
Wilson N, El-Sukkari D, Belz G et al. Most lymphoid organ dendritic cell types are phenotypically and functionally immature. Blood 2003;102:2187–2194.
Martinez del Hoyo G, Martin P, Hernandez Vargas H et al. Characterization of a common precursor population for dendritic cells. Nature 2002;415:1043–1047.
O’Keeffe M, Hochrein H, Vremec D et al. Dendritic cell precursor populations of mouse blood: identification of the murine homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors. Blood 2003;101:1453–1459.
Martin P, Del Hoyo GM, Anjuere F et al. Characterization of a new subpopulation of mouse CD8alpha+ B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential. Blood 2002;100:383–390.
Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem cell biology. Cell 1997;88:287–298.
Orkin SH, Zon LI. Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nat Immunol 2002;3:323–328.
Sitnicka E, Bryder D, Theilgaard-Monch K et al. Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool. Immunity 2002;17:463–472.
O’Neill HC, Wilson HL. Limitations with in vitro production of dendritic cells using cytokines. J Leukoc Biol 2004;75:600–603.
Vremec D, Pooley J, Hochrein H et al. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol 2000;164:2978–2986.
Henri S, Vremec D, Kamath A et al. The dendritic cell populations of mouse lymph nodes. J Immunol 2001;167:741–748.
Nakano H, Yanagita M, Gunn M. CD11c+B220+Gr-1+ cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J Exp Med 2001;194:1171–1178.
Leenen P, Radosevic K, Voerman J et al. Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and sub-population turnover. J Immunol 1998;160:2166–2173.
Ardavin C, Wu L, Li C-L et al. Thymic dendritic cells and Tcells develop simultaneously in the thymus from a common precursor population. Nature 1993;362:761–763.
Manz MG, Traver D, Miyamoto T et al. Dendritic cell potentials of early lymphoid and myeloid progenitors. Blood 2001;97:3333–3341.
Dexter T, Testa N. In vitro methods in haemopoiesis and lymphopoiesis. J Immunol Methods 1980;38:177–190.
Dexter T, Lajtha L. Proliferation of haemopoietic stem cells in vitro. Br J Haematol 1974;28:525–530.
Whitlock C, Robertson D, Witte O. Murine B cell lymphopoiesis in long term culture. J Immunol Methods 1984;67:353–369.
Suniara RK, Jenkinson EJ, Owen JJ. An essential role for thymic mesenchyme in early T cell development. J Exp Med 2000;191:1051–1066.
Tsai R, Kittappa R, McKay R. Plasticity, niches, and the use of stem cells. Dev Cell 2002;2:707–712.
Ni K, O’Neill HC. Long-term stromal cultures produce dendritic-like cells. Br J Haematol 1997;97:710–725.
Ni K, O’Neill HC. Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: production of cells with myeloid/dendritic characteristics. In Vitro Cell Dev Mol Biol 1998;34:298–307.
Ni K, O’Neill HC. Spleen stromal cells support haemopoiesis and in vitro growth of dendritic cells from bone marrow. Br J Haematol 1999;105:58–67.
O’Neill HC. Ni, K, Wilson H. Long-term stroma-dependent cultures are a consistent source of immunostimulatory dendritic cells. Immunol Cell Biol 1999;77:434–441.
Wilson H, Ni K, O’Neill HC. Proliferation of dendritic cells in long term culture is not dependent on granulocyte macrophage-colony stimulating factor. Exp Hematol 2000;28:193–202.
Wilson HL, Ni K, O’Neill HC. Identification of progenitor cells in long-term spleen stromal cultures that produce immature dendritic cells. Proc Natl Acad Sci U S A 2000;97:4784–4789.
Wilson H, O’Neill HC. Dynamics of dendritic cell development from precursors maintained in stroma-dependent long-term cultures. Immunol Cell Biol 2003;81:144–151.
Ni K, O’Neill HC. Development of dendritic cells from GM-CSF–/–mice in vitro: GM-CSF enhances production and survival of cells. Dev Immunol 2001;8:133–146.
Ni K, O’Neill HC. Improved FACS analysis confirms generation of immature dendritic cells in long-term stromal-dependent spleen cultures. Immunol Cell Biol 2000;78:196–204.
Hsieh SM, Pan SC, Hung CC et al. Kinetics of antigen-induced phenotypic and functional maturation of human monocyte-derived dendritic cells. J Immunol 2001;167:6286–6291.
Quah B, Nik, O’Neill HC. In vitro hematopoiesis produces a distinct class of immature dendritic cells from spleen progenitors with limited T cell stimulation capacity. Int Immunol 2004;16:567–577.
Szabolcs P, Moore MA, Young JW. Expansion of immunostimulatory dendritic cells among the myeloid progeny of human CD34+ bone marrow precursors cultured with c-kit ligand, granulocyte-macrophage colony-stimulating factor, and TNF-alpha. J Immunol 1995;154:5851–5861.
Adachi Y, Toki J, Ikebukuro K et al. Immature dendritic cells (CD11c+ CD3–B220–cells) present in mouse peripheral blood. Immunobiology 2002;206:354–367.
Balazs M, Martin F, Zhou T et al. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 2002;17:341–352.
Kamath AT, Pooley J, O’Keeffe MA. The development, maturation, and turnover rate of mouse spleen dendritic cell populations. J Immunol 2000;165:6762–6770.
Rescigno M, Sutherland M, Gold M et al. Dendritic cell survival and maturation are regulated by different signalling pathways. J Exp Med 1998;188:2175–2180.
Hofer S, Rescigno M, Granucci F et al. Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide. Microbes Infect 2001;3:259–265.
Kampgen E, Koch N, Koch F et al. Class II major histocompatibility complex molecules of murine dendritic cells: synthesis, sialylation of invariant chain, and antigen processing capacity are down-regulated upon culture. Proc Natl Acad Sci U S A 1991;88:3014–3018.
Vremec D, Shortman K. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation and differences among thymus, spleen, and lymph nodes. J Immunol 1997;159:565–573.
Pulendran B, Lingappa J, Kennedy MK et al. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J Immunol 1997;159:2222–2231.
Martinez del Hoyo G, Martin P, Arias C et al. CD8alpha+ dendritic cells originate from the CD8alpha–dendritic cell subset by a maturation process involving CD8alpha, DEC-205, and CD24 upregulation. Blood 2002;99:999–1004.
Wang Y, Zhang Y, Yoneyama H et al. Identification of CD8alpha+CD11c–lineage phenotype-negative cells in the spleen as committed precursor of CD8alpha dendritic cells. Blood 2002;100:569–577.
O’Neill HC, Jonas N, Wilson H et al. Immunotherapeutic potential of dendritic cells generated in long-term stroma-dependent cultures. Cancer Biother Radiopharm 1999;14:263–276.
Mosier M. Dendritic cells in immunity and tolerance: Do they display opposite functions? Immunity 2003;19:5–8.
Bedford P, Garner K, Knight SC. MHC class II molecules transferred between allogeneic dendritic cells stimulate primary mixed leukocyte reactions. Int Immunol 1999;11:1739–1744.
Wilson H, O’Neill HC. Identification of differentially expressed genes representing dendritic cell precursors and their progeny. Blood 2003;102:1661–1669.(Helen C. O’Neill, Heather)