Abstract
Dendritic cells (DC) are critical to the induction and regulation of the innate and adaptive immune responses. They have been implicated in the pathogenesis of many autoimmune and chronic inflammatory diseases as well as contributing to the development of tumours by their lack of appropriate function. As such, understanding human DC biology provides the insight needed to develop applications for their use in the treatment of diseases. Currently, studies on mouse DC outnumber those on human cells; however, the comparison between mouse and human models has been somewhat misleading due to the basic biological and practical differences between the two models. In this review, we summarise the current understanding of human DC subtypes by describing the phenotype of the populations and how this relates to function. We also hope to clarify the differences in nomenclature between the human and mouse models that have arisen by way of the different experimental models.
Key words
1 Introduction
Dendritic cells (DC) were first identified functionally as a haematopoietic-derived cell population able to initiate a primary T-lymphocyte response. They were identified initially in mouse lymphoid tissues (1) and then soon after as interdigitating cells in rat and human tissues (2–4). Studies in mice and humans have diverged dramatically over the years. This may reflect fundamental differences but in part relate to the difficulties involved in working on humans compared to inbred mouse strains. The tissue sources used are different due to ethical constraints, comparable reagents are unavailable and the motivations differ. Generally, the primary reason for human studies is to translate the research into clinical outcomes for patients, whereas mouse studies focus on proving or disproving a scientific question – a subtle but important difference. Research in the human system can be focused on diagnostically relevant or therapeutically feasible protocols. Many experiments performed in the mouse models are unfortunately very difficult or impossible to corroborate in humans. Studies in an inbred mouse strain may, for example, be very different in the outbred human. Consequently, some of the assumptions made about human DC are transposed from small animal experiments that have not been confirmed in man and may not necessarily apply.
Human DC subtypes have been primarily defined using peripheral blood, although a large body of data is available describing the immunohistological analysis of DC populations in human tissues. Unlike in the mouse, few studies have isolated DC from the tissues to analyse the functional capacity of these cells. This chapter will discuss the common subpopulations of human DC, whilst an accompanying chapter (Chapter 3) concentrates on the purification of the major human blood DC (BDC) populations. Broadly speaking human DC have been segregated on the basis of (1) location, e.g. peripheral tissues, lymphoid tissues, blood, (2) source, e.g. in vitro monocyte-derived, CD34-derived, or (3) phenotype, e.g. Lin−HLA-DR+CD11c+/−. A functional classification (e.g. inflammatory, tolerogenic, migratory, tissue resident) is more popular in the mouse. In humans, DC were first identified as HLA-DR+ interstitial cells in the kidney (2, 3). Following the identification of the tissue-resident HLA-DR+ cells in the kidney, different populations of DC were described in the tonsil (5–7), thymus (8), liver (9), interstitium of non-lymphoid organs (interstitial DC) (10, 11), and bone marrow (12–14). However, it was recognised early that studies of human DC populations would require firstly lineage-specific surface markers and secondly, a convenient source and means of purifying this rare population. The ability to generate DC in vitro, with many of the properties of the DC manufactured in vivo, initiated an explosion of DC research as these cells were relatively easy to come by. However, again comparisons with the natural DC populations highlighted some obvious differences between ex vivo- and in vitro-obtained cells (15, 16). Recently, microarray analyses enabled an extensive comparative mRNA analysis of mouse and human DC populations and went some way towards reconciling the differences (16, 17).
Many groups, including this laboratory, have attempted to develop surface markers to expedite the study and isolation of human DC populations (Table 1.1). However, the generation of new antibodies has been slow and even today, there are very few antibodies that make a singular contribution to the identification of human DC populations (18). The use of informative panels particularly lineage-negative (Lin−; CD3, CD14, CD19, CD20, CD56) panels remains the standard. Despite these difficulties, five populations of allostimulatory leucocytes have been distinguished in the peripheral blood (19), tonsil (7), and spleen (20).
DC subtypes in the different human tissues are inevitably defined first according to their phenotype, indicated by specific markers (Table 1.2). Using the markers for nomenclature is a simple and, provided commonly available markers are employed, consistent manner to describe a population compared to alternative descriptive or functional names which may hide the specific function of such cell type. Phenotypes should not change but our current knowledge as to DC biology may change dramatically. Human DC subtypes fit broadly into the subset divisions outlined in (Table 1.3). The common experimental practice that uses monocytes as DC precursors has continued the debate as to the relationship between monocytes, DC, and the myeloid lineage. Within each of these subpopulations are further segregation of DC populations that have particular phenotypic or functional markers and these are described below in more detail.
2 Subdivision by Cell Surface Phenotype of DC from Different Tissue Sources
2.1 Bone Marrow
CD34 + precursors: CD34 is one of the markers for haematopoietic stem cells in blood, bone marrow (BM), and cord blood. CD34+ cells are capable of differentiating into the full range of haematopoietic cell lineages. Transplantation of ex vivo expanded human umbilical cord blood CD34+ cells into Rag2− / −γ− / − mice reconstitutes lymphocyte and dendritic cells (21). Previously, we demonstrated DC in human bone marrow that had specific antigen-presenting capacity and T-cell stimulatory function (22). Within blood, there are CD34+HLA-DR+Lin−CD11c− with low, but measurable allostimulatory capacity (21). These cells do not express CD80/CD86, but are CD40+ (21). Whilst they may contribute to antigen-presenting capacity (particularly given the in vitro studies using cytokine-derived CD34+-DC), these are not generally included in DC populations. Nonetheless, it has emphasised the point that CD34 mAb should be used experimentally to exclude these cells from Lin−HLA-DR+ populations.
2.2 Blood DC
Blood provides a delivery system for distributing BM-derived DC to lymphoid and other tissues. It may also contain at least in some circumstances, DC derived from other tissues, e.g. vascularised organs or grafts (22).
Freshly isolated human peripheral blood HLA-DR+Lin-CD11c+ DC are able to stimulate allo- and antigen-specific T-cell responses. With careful isolation, these cells are CD80− but do express CD86. Minimal culture (37°C, human AB serum) of DC results in increased expression of HLA-DR and upregulation of activation markers such as CMRF-44 and CMRF-56. Human pDC from the peripheral blood are in an immature state and require activation with TLR ligands to induce maturation associated with the ability to present antigen. However, these BDC do not divide in order to become functionally mature. On the other hand, mouse peripheral blood DC are unable to stimulate antigen-specific responses without prior activation (23). This apparent difference has led to the general assumption that peripheral blood DC are precursor cells (24). Whilst this may be the case for mouse peripheral blood DC, this is probably not the case in humans. Whether this process is differentiation of a precursor population or an appropriate response to stimuli depends on one’s definition of “precursor”. The same thought process must also be applied to other monocyte populations circulating in blood. DC can be derived from common myeloid or lymphoid progenitors. In vivo evidence for a human DC lineage differentiation pathway from haematopoietic stem cells has followed clinical transplant studies in xenogenic models (25).
A classification of human blood DC includes the cells outlined in Table 1.3 and separates into five subpopulations (Fig. 1.1 ). The HLA-DR+Lin−CD11c+ BDC populations divide into three allostimulatory cell populations: CD1c+, CD141+, and CD16+ cells. Blood CD1c+ myeloid DC originate from bone marrow, they circulate in the blood and migrate constantly to the second lymphoid organs and peripheral tissues as resting interstitial DC. These cells express myeloid markers such as CD13, CD33, CD11c, CD1c, but are Lin−. The majority of myeloid DC are CD14− but a minor population expresses low levels of CD14 and are considered to be a myeloid DC precursor (12). It is conceivable that CD1c+ myeloid DC may develop from CD14+ monocytes in vivo. CD1c+ DC comprise about 0.6% of peripheral blood mononuclear cells or approximately 19% of the HLA-DR+Lin− population.
CD141+-DC are a very rare type of DC and only in 0.04% of PBMC (or 3% of the DC population), they are CD11cdimCD1c−CD4+CD123– and Lin− (27). Clec9 is a new marker strongly expressed on both human and mouse CD141+ DC (28, 29). Isolating these cells from human peripheral blood is discussed in an accompanying paper (Chapter 3). Human CD141+ DC expressed high levels of toll-like receptor (TLR) 3 but lacked TLRs 4, 5, and 7 whilst CD1c+ DC expressed the TLRs1–8 and TLR10.
Plasmacytoid DC (pDC) are a specific type of DC that are found in an immature form in the peripheral blood but in higher numbers in inflamed tissue and tonsil (30). They are the major IFN-α/β producers upon stimulation. pDC show plasmacytoid morphology, express pre-T-cell receptor α-chain, and are CD123++CD4+CD11c–CD303+CD304+. CD303, whilst a useful marker on fresh blood pDC, is downregulated upon culture in vitro and the antibody blocks secretion of IFN-α (27). ILT-7 was recently identified as a human pDC-specific marker, which is negative in other DC subtypes (31–33). pDC express TLR7 and TLR9 and differentiate into mature antigen-presenting cells in response to TLR7 and TLR9 ligands. Blood pDC also express L-selectin and migrate to second lymphoid organs through the high endothelial venules around which they congregate (34). pDC and myeloid DC exhibit some important differences in terms of antigen uptake, presentation, and T-cell stimulatory activity. In human pDC activated with IL-3 are unable to uptake FITC-dextran, in contrast to monocyte-derived DC (35). pDC have limited ability to prime naïve CD4+ T cells but are able to induce IL-10-producing regulatory T cells upon activation (36, 37). pDC are also found to be capable of cross-presentation antigens to CD8+ T cells (38, 39).
The CD16+ BDC population represents the largest HLA-DR+Lin− population in the blood comprising up to 50% of the CD11c+ DC. The literature sometimes confuses these cells as a monocyte subpopulation or DC precursor (28). Care must be taken to ensure that the CD14+ monocytes are removed from the gating strategies during analysis or from the Lin− preps during preparation. The CD16+CD14− cell population expresses 6-Sulpho LacNAc, an O-linked carbohydrate modification of PSGL-1 recognised by binding to the MDC-8 mAb (16). These cells are CD1c–CD11c+C5aR+CD45RA+HLA-DR+. They are the principal source of TNF-α and IL-12p70 when blood leucocytes are stimulated with TLR4 ligand LPS or CD40 ligands (43, 44). Most importantly, these cells express receptors for the inflammatory mediators C3a and C5a but not the skin homing molecule, CLA. In xenogenic models, these cells specifically migrate to C5a (42). They are able to produce allogeneic proliferative responses of naïve CD4+ T lymphocytes but produce poor autologous T-cell responses compared to MDC-8− cells. The CD16+ DC are a proinflammatory cell type possibly indicating similarities with monocyte-derived DC (MoDC). From the gene microarray data, CD16+-DC cluster with neutrophils and monocytes but not with CD1c+-DC and pDC.
2.3 Monocytes
The lineage marker most commonly used for monocytes is CD14. Peripheral blood monocytes express high levels of CD14 and these CD14+ cells can be subdivided by their expression of CD16. The CD14++CD16− population of monocytes is the predominant population of monocytes in human peripheral blood. These cells are thought to correspond to the tissue-resident cells that are the precursors to tissue macrophages, Kupffer cells, osteoclasts, microglia, etc. (43). The CD14+CD16+ population forms less than 10% of the normal CD14+ monocyte population in healthy individuals. However, these cells have the properties of proinflammatory cells that, on activation, may correspond to a similar cell type as the monocyte-derived DC (44). Careful attention to detail is required when analysing human peripheral blood monocyte subpopulations (Table 1.3). CD14++ monocytes have been routinely used as a DC precursor following the demonstration that they can differentiate into DC-like cells in vitro (45, 46). Consequently CD14++ cells have sometimes been referred to in the literature as DC1 precursors (47). However, the evidence indicates that monocytes are most likely precursors of a DC that differentiates in inflammatory situation and that they are not the major precursors of steady-state DC.
2.4 Non-lymphoid Tissue DC
DC have been identified in most human non-lymphoid tissue (9, 48–51). Their presence in the skin has major historical importance as the original description of the Langerhans cell (LC). There are two major DC subpopulations in the skin: LC and Dermal DC. LC reside mainly in the epidermis where they constantly monitor for the presence of foreign antigens. LC express Langerin (CD207), CD1a, and E-Cadherin and contain specific intracellular or membrane-bound structures, referred to as Birbeck granules, that are not found in other DC subsets (49). Murine LC are capable of cross-presentation of antigen to CD8+ T cells but is yet to be described for human cells (50). Under steady-state conditions, immature LC migrate continuously into lymph nodes without further maturation and induce T-cell tolerance (51). They are not continuously replenished by migratory blood precursors, but rather renew through local proliferation under steady condition (52). LC might not be essential for priming T cells to foreign antigens that enter the skin, but instead, may induce immune tolerance. Dermal DC are distinguished from LC by their complement of C-type lectins (CD209+CD206+CD207−) and a more activated phenotype (53). The difference in the receptor expression indicates that the two skin DC populations may recognise and present different microbial antigens (54, 55).
2.5 Lymphoid Tissue DC
Tonsil: Tonsil is the most available lymphoid tissue although it must be remembered firstly that they are gut-associated lymphoid tissue and not lymph nodes and secondly that most tonsils available ethically, are those removed due to prolonged inflammation. The Lin−HLA-DR+ DC population of tonsil can be subdivided into five populations of which four are interdigitating DC. Isolation of the tonsil DC populations needs to minimise further cellular activation during the procedure from this generally already inflamed tissue. The phenotypes of the populations were HLA-DR++CD11c+CD83+ CMRF-56+, HLA-DR+ CD11c+ CD13+, HLA-DR+ CD11c+ CD13−, HLA-DR+ CD11c− CD123+, and HLA-DR+ CD11c− CD123− (7). The relationship to each of these populations requires functional studies. Activation markers such as NKp46 further divide the Lin−HLA-DR+CD123+ population of tonsil pDC (5, 7, 56).
Lymph node: DC expressing CD103, the integrin α chain, reside in the human intestinal lymph node (LN). CD103+-DC efficiently induce gut homing receptor CCR9 on responding T cells with regulatory function (iTreg) via a retinoic acid receptor-dependent mechanism (57). The ability of CD103+-DC to induce CCR9 expression is maintained in patients with Crohn’s disease indicating a role for CD103+-DC in the intestinal homeostasis and inflammation. It might represent a potential targeting of human intestinal inflammatory disease.
Thymus: Human thymus DC are CD1a–CD3–CD4+CD8– cells and express high levels of CD123 on the membrane and are able to develop into mature DC upon culture with IL-3 and CD40 ligation (58, 59).
Spleen: In human spleen, four DC subsets have been described. Most DC are CD11c+HLA-DR+Lin− cells that express CD54 and low levels of CD40 and CD86 but not CD80 or CD83. There is a smaller population of CD11c− DC. This suggests that the majority of human splenic DC have an immature phenotype similar to CD11c+ BDC (20). There did not appear to be CD16+ DC and CD141 was not tested. The CD11c+ DC were subdivided based on their distribution: marginal zone DC, B-cell zone DC, and T-cell zone DC. Splenic DC expressing high levels of co-stimulatory molecules could be found in some donors but the authors argued that these donors may have had bacterial infection.
2.6 In Vitro Derived
The most common source of DC for human studies has been the cells derived in vitro from monocytes or CD34+ haematopoietic stem cells. These derived cells are not identical to cells in vivo or purified ex vivo (15, 16, 60), nor are the products of cultures derived from cells purified by different technologies or in the presence of different media supplements. Claims attributing specific functions to specific subsets should be treated with suitable reservation as to direct correlation with in vivo functions.
CD14 + derived: We have shown that there are spontaneous generation of DC from monocyte precursors (61). It has also been demonstrated that blood monocytes can give rise to mucosal DC in vivo (62). In vitro monocytes are cultured with IL-4 and granulocyte macrophage colony-stimulating factor (GM-CSF) or monocyte-conditioned medium to induce DC differentiation (45, 46). Immature MoDC can be matured in the presence of LPS or TNF-α or the cocktail containing IL-6, IL-1β, TNF-α, and PGE2.
CD34 + derived: CD34+ haematopoietic stem cells from cord blood, bone marrow, or mobilised peripheral blood are induced to differentiation into DC with GM-CSF, TNF-α (63). CD34+-derived DC can differentiate into a number of phenotypically distinct populations including CD1a– interstitial DC and CD1a+ LC subpopulation (64).
3 Functional Subsets
In the mouse, DC are also commonly classified according to their distinct function, i.e. migratory DC, tissue-resident DC, inflammatory DC. The application of this type of classification to humans will generally be based on assumptions made from murine models. However, within one functional subdivision used in the mouse, human DC will often have differing phenotypes.
3.1 Migratory DC
Migratory DC in the human include tissue-derived DC, BDC, and LC. The most applicable model for studying human DC migration relies on therapeutic haematopoietic stem cell transplantation where donor and host-derived cells are able to be monitored (65).
3.2 Tissue-Resident DC
Human tissue-resident DC have been studied in the transplant arena where HLA-DR+Lin− cells have been identified in the liver (9, 66), heart, cornea (65), and pancreas (67, 68) and this was extended to HLA-DR+BDCA-1+ or HLA-DR+BDCA-2+ cells in the kidney (48).
3.3 Inflammatory DC
When the host is healthy, it is difficult to identify inflammatory DC. Inflammatory DC are a heterogeneous population that includes tumour necrosis factor and inducible nitric oxide synthase (iNOS)-producing DC (Tip-DC), IL-20-producing DC, and IL-23-producing DC, etc. Tip-DC were originally found in the spleen of Listeria monocytogenes-infected mice, but are now found also in human psoriasis plaque with the expression of CD11c+DR+CD40+CD86+CD83+DC-LAMP+ DC-SIGN+ but Langerin− CD14dimCD1c− (69, 70). pDC are also rarely found in healthy skin, but increased in allergic disease of the airway (71) and diseased skin, e.g. atopic excema, psoriasis, and cutaneous lupus erythematodes (72–75).
3.4 Tolerogenic DC
Immature DC and pDC are capable of inducing tolerance through regulatory T-cell differentiation from naïve CD4+ T cells. Tolerogenic properties can be enhanced by a number of mediators and some DC populations (CD1c+) are able to secrete regulatory cytokines such as IL-10 following certain stimuli. In abnormal situations DC with altered phenotypes exist, i.e. in transplant recipients that have been weened from immunosuppressive drugs. These cells can be distinguished phenotypically as expressing ILT-3 and ILT-4 (76) or indoleamine 2,3-dioxygenase (IDO) enzyme. The ability to specifically induce these cells or monitor their generation in transplantation is a clinical relevance to allow clinicians to remove patient’s from long-term immunosuppressive drugs whilst monitoring rejection episodes (77).
4 Clinically Relevant Subsets
4.1 Diagnostic
4.1.1 Bone Marrow
Mobilisation of haematopoietic stem cells (HSC) with G-CSF induces the mobilisation of pDC into the peripheral blood. An increased number of pDC has been associated with not only enhanced engraftment of HSC transplants but also an increase in chronic graft versus host disease (GVHD).
4.1.2 Blood
Despite their rarity, human BDC are the most commonly studied DC populations. Recently developed protocols to enumerate BDC populations enabled the monitoring of human BDC subsets in the healthy and disease populations. This technology relies on flow cytometric-based TruCOUNT assays (78, 79) of whole blood. The four CD14− subsets of human BDC can be assessed from whole blood and the different populations show numerical differences through disease progression (79, 80). The CMRF-44 (IgM) and CMRF-56 (IgG) monoclonal antibodies bind in vitro matured blood DC with 54 and 66% of matured blood DC binding CMRF-44 and CMRF-56 mAb, respectively (81, 82). However, it has been observed that there is an increased number of activated DC in the peripheral blood during GVHD. The ability to monitor the presence of activated DC populations to predict GVHD episodes has proven effective in small studies (83). These studies have the potential to contribute directly to patient management in a variety of clinical scenarios.
4.1.3 Skin
BDCA-1−-DC were found in human psoriatic dermis, which accounts for 90% of CD11c+ dermal-resident DC and belong to inflammatory DC and induce Th1/Th17 polarisation. Etanercept treatment reduces BDCA-1−-DC number but the BDCA-1+-DC remained stable (84). Patients with psoriasis have pDC that are particularly sensitive to the keratinocyte peptide LL37 and responsive with increased IFN-α induction (85).
4.1.4 Cancer Biopsies
Tumours contain an abundance of immature DC and fewer mature DC compared with healthy tissues. A number of cytokines released by tumours recruit these immature DC from peripheral blood and the hypoxia in the tumour microenvironment might support the immature phenotype of DC (86). Tumour DC have been shown to deliver immune suppressive function, meanwhile, they promote tumour angiogenesis by secretion of proangiogenic cytokines.
4.1.5 Transplanted Organs
Human DC are currently exploited for monitoring the outcome of organ and bone marrow transplantation (GVHD and allograft tolerance).
4.2 Therapeutic
Lastly, most, but not all, clinical trials addressing DC tumour vaccination have used in vitro-derived DC preparations generated from either monocytes or CD34+ cord blood cells. (For a detailed summary of DC-based international clinical trials see http://www.mmri.mater.org.au.) However, these trials have not been overly successful and it is commonly agreed that the in vitro-derived cells have failings – the inability to migrate appropriately. The ability to purify BDC from aphaeresis products has provided the potential to initiate clinical trials using these cells (87–92). This description of CMRF-56+ DC populations has enabled a method to be developed suitable for large-scale isolation of DC for clinical application (81, 82).
4.2.1 DC Malignancy
Whilst not a true malignancy, Langerhans Cell Histiocytosis is a rare disease with the abnormal proliferation of Langerhans cells in bone, skin, lung, and stomach. Most cases are found in children (93). Recently, a rare haematopoietic tumour sharing the phenotypic and functional features of pDC was identified and named pDC leukaemia (pDCL), but leukaemic pDC express CD56+ and adaptor protein CD2AP+; it comprises less than 1% of acute leukaemia cases. Some leukaemic pDC produce IFN-α, but lower than their normal counterparts. Leukaemic pDC are able to present viral antigen to CD4+ and CD8+ T cells and prime naïve CD4+ T cells towards Th2 or Th1 pathway. Leukaemic pDC become fully competent antigen-presenting cells after culture with IL-3/CD40L (94, 95). Currently, only allogeneic haematopoietic stem cell transplantation might lead to complete remission.
5 Conclusion
Human DC subpopulations have been described based on phenotypic assessment. Careful flow cytometric analysis ensures that discrete populations are purified or studied. DC populations isolated from peripheral blood or human tissue differ from DC derived in vitro. The DC precursor cells are still not clearly identified in human and appear to differ from mouse cells. Direct comparisons, such as between the DC precursor population in mouse and humans, can be misleading due to differences in the systems. There is now good data indicating the ability to purify five DC populations based on phenotype and function from human peripheral blood. Understanding the functional difference among different DC subsets represents one of the remaining challenges in DC biology.
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Ju, X., Clark, G., Hart, D.N. (2010). Review of Human DC Subtypes. In: Naik, S. (eds) Dendritic Cell Protocols. Methods in Molecular Biology, vol 595. Humana Press. https://doi.org/10.1007/978-1-60761-421-0_1
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