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Three subsets of lymphocytes exist in the human immune system: B cells, T cells, and NK cells (see Section “Cells of the Innate Immune System”). The adaptive immune response is mediated by T and B lymphocytes. The unique role of these cells is the ability to recognize antigenic specificities in all their diversity. All lymphocytes derive from a common bone marrow stem cell. This finding has been exploited in various clinical settings, with attempts to restore the entire lymphocyte pool by bone marrow or stem cell transplantation.
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B cells mature in the fetal liver and adult bone marrow. They produce antibody-protein complexes that bind specifically to particular molecules defined as antigens. As a consequence of recombinatorial events in different Ig gene segments (V or variable; D or diversity; J or joining), each B cell produces a different antibody molecule (eFig. 10-3.1). Some of this antibody is present on the surface of the B cell, conferring the unique ability of that B cell to recognize a specific antigen. B cells then differentiate into plasma cells, the actual antibody-producing and -secreting cells. Plasma-cell secreted Ig comprise the dimer IgA, the monomers IgD, IgE, and IgG as well as the pentamer IgM that mediate humoral immune responses. In general, antibodies bind to microbial agents and neutralize them or facilitate uptake of the pathogen by phagocytes that destroy them. Briefly, IgA can be found in mucosal tissues, saliva, tears, or breast milk and prevents colonization by various pathogens. IgD functions mainly as an antigen receptor on B cells and, as recently discovered, activates mast cells and basophils to produce antimicrobial factors.116 IgE binds to allergens on mast cells and basophils and can thereby trigger histamine release and allergic reactions including anaphylaxis and urticaria. In addition, some evidence exists that it can protect against parasitic and helminthic infections. IgG provides the majority of antibody responses that contribute to the immune defense against extracellular pathogens. It is the only antibody that is capable of crossing the placenta in order to protect the fetus. Finally, IgM is available either surface-bound on B cells or as secreted form and eliminates microbes in the early stages of humoral immunity before there is sufficient IgG production. Antibodies are also responsible for mediating certain pathologic conditions in skin. In particular, antibodies against self-antigens (mostly IgG, but also IgA) lead to autoimmune disease, typified in the pathogenesis of pemphigus and bullous pemphigoid (see Chapter 37 for more details about B cells and antibody production).
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T cells mature in the thymus, where they are selected to live or to die. Those T cells that will have the capacity to recognize foreign antigens are positively selected and can enter the circulation. Those T cells that react to self are negatively selected and destroyed. T cells have the unique ability to direct other cells of the immune system. They do this, in part, by releasing cytokines. For example, T cells contribute to cell-mediated immunity (CMI), required to eliminate intracellular pathogens, by releasing cytokines that activate macrophages and other T cells. T cells release cytokines that activate NK cells and permit the growth, differentiation, and activation of B cells.
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T cells can be classified and subdivided in different ways: (1) on the basis of the T cell receptor; (2) on the basis of the accessory molecules CD4 and CD8; (3) on the basis of their virginity, i.e., their activation status (naive, memory, effector T cells); and (4) on the basis of their functional role in the immune response, which is often linked to the cytokine secretion property of the respective cell population. We have used the abbreviations Th1 and Th2 to distinguish CD4+ helper T cell subtypes but, as discussed below, many of the functional attributes, including cytokine production, of Th cells are not as clearly defined as previously thought and some cytokine profiles are also attributable to CD8+ cytotoxic T cells (Tc) (see Section “Functionality”).
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T-Cell Antigen Receptor (TCR)
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The T-cell antigen receptor (TCR) is a complex of molecules consisting of an antigen-binding heterodimer (α/β or γ/δ chains) that is noncovalently linked with five CD3 subunits [(1) γ, (2) δ, (3) ε, (4) ζ, or (5) η). The TCR chains have amino acid sequence homology with structural similarities to Ig heavy and light chains. The genes encoding TCR molecules are encoded as clusters of gene segments (V, J, D, C, or constant) that rearrange during T-cell maturation (eFig. 10-3.1). Together with the addition of nucleotides at the junction of rearranged gene segments, this recombinatorial process, which involves the enzymes recombinase activating gene 1 and 2, results in a heterogeneity and diversity of the antigen recognition unit that is broad enough to allow for a successful host defense. TCR α/β or TCR γ/δ molecules must be paired with CD3 molecules to be inserted into the T-cell surface membrane117 (see Fig. 10-4). The TCR chains form the actual antigen-binding unit, whereas the CD3 complex mediates signal transduction, which results in either productive activation or nonproductive silencing of the T lymphocyte. Most T cells express α/β TCRs, which typically bind antigenic peptides presented by MHC molecules. Immunity provided by
/β T cells includes Th1, Th2, Th17 and T reg responses (see Section “
Functionality”). In contrast, only a small subset of T cells express γ/δ TCRs. These T cells have the capacity to directly bind pathogen-derived glycoproteins or nonclassical MHC molecules. It has been shown that γ/δ T cells in men and mice predominantly display a tissue-associated TCR repertoire as well as a memory phenotype, both probably due to chronical stimulation by nonpeptide antigens within the tissue. Importantly, they act early during immune response and are therefore termed “innate-like effectors.” Previous studies conducted in mice infected with
Listeria monocytogenes or
Nippostrongylus brasiliensis revealed that
/δ T cells discriminate early between these pathogens and react by IFN-γ versus IL-4 production, skewing
/β T-cell responses in a Th1 or Th2 direction, respectively.
118 Meanwhile, growing evidence exists that human and murine
/δ T cells also have the capacity to produce IL-17 during bacterial or viral infections and thereby significantly contribute to the early innate immune defense.
119–121
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Accessory Molecules122,123
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During their maturation in the thymus thymocytes start to express the molecules that allow T cells to display their unique functional capacity, which is to specifically recognize antigen in an MHC-restricted fashion (see Section “
General Principles of Antigen Presentation”). These are the TCR and the accessory molecules CD4 and CD8. The latter stabilize the interaction of the TCR with the MHC-linked peptide antigen. Whereas CD4 binds to MHC class II molecules, CD8 acts as an adhesive by binding to MHC class I molecules. Thymocyte development follows a strict selection process. First, lymphoid progenitor cells enter the thymus and develop into CD25
+CD4
−CD8
− (double-negative, DN) thymocytes. Upon successful generation of functional TCR-b and pre-TCR-α receptors, further development to CD4
+CD8
+ (double-positive, DP) thymocytes with fully functional TCR-αβ chains is initiated. Following low-avidity TCR recognition of self-peptide/MHC molecules, DP thymocytes receive signals for survival and further differentiate into single positive (SP) thymocytes. These positively selected mature thymocytes constitute only 3%–5% of all thymocytes and are either CD4
+CD8
− MHC class II–restricted cells or CD8
+CD4
− MHC class I-restricted cells. Subsequently, they leave the thymus and migrate to the peripheral lymphoid tissues (lymph nodes, spleen, Peyer's patches, etc.). On the contrary, thymocytes that show self-reactivity undergo apoptosis in order to avoid autoimmunity (negative selection), and DP thymocytes that do not receive TCR signals die due to neglect. This process is most active in early infancy and childhood but continues with decreasing output well into adult life.
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The original observation that CD4+ T cells are critical for helping B cells to produce antibodies by triggering their differentiation into plasma cells in the humoral response coined the term “T helper cells” (Th cells). During the past years these lymphocytes have been characterized extensively. To our current knowledge, CD4+ T cells represent a heterogeneous cell population with diverse function depending on environmental requirements that play a central role in humoral and cell-mediated immunity. Effector CD4+ T cells protect against pathogens mainly by their production of Th1, Th2, or Th17 cytokines (i.e., IFN-γ, IL-4, IL-17) and influence immune responses through both “helper” and “effector” functions. In contrast, regulatory CD4+ T cells have the capacity to downregulate disproportionate effector responses to (self-) antigen (see Section “Functionality”).
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CD8+ Cytotoxic T Cells
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In responding to an intracellular pathogen (e.g., a virus) the T cell must lyse the infected cell. To do so, it must be able to recognize and respond to antigenic peptides encoded by this pathogen and displayed on the cell surface. For this to occur, antigens arising in the cytosol are cleaved into small peptides by a complex of proteases, called the proteasome. The peptide fragments are then transported from the cytosol into the lumen of the endoplasmic reticulum, where they associate with MHC class I molecules. These peptide–class I complexes are exported to the Golgi apparatus and then to the cell surface (see Section “General Principles of Antigen Presentation”). The maturation of a CD8+ T cell to a killer T cell requires not only the display of the antigenic signal but also the delivery of helper signals from CD4+ T cells, for which the functional interaction between CD40 on the APC and CD40L on the CD8+ T cell can substitute.
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Two distinct subsets of cytotoxic T cells have been identified and can be differentiated by the mechanism by which they kill targets
124; the end result being the induction of a programed cell death known as
apoptosis.
125,126 The first mechanism of cytotoxicity involves the interaction of two cell surface proteins, FasL (CD95L) on the T cells and Fas (CD95) on the target. Ligation of these molecules delivers a signal through Fas that induces the apoptosis cascade in the target. The second mechanism involves the release of cytoplasmic granules present in such T cells. These granules contain perforin, which induces a pore in the target, and granzymes, serine esterases that, when injected into cells, trigger the apoptotic pathway. Such granules also contain granulysin, a protein with a broad spectrum of antimicrobial activity against bacteria, fungi, and parasites.
124,127 In this manner, cytotoxic T cells can directly kill microbial invaders. Besides contributing to host defense against infection and tumors, cytotoxic T cells can also contribute to tissue injury. For example, cytotoxic T cells exist which recognize self-antigens of melanocytes and thus may contribute to the pathogenesis of vitiligo.
128
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Double-negative (DN) T cells comprise only 1%–5% of the peripheral T-cell population of mice and men. DN T cells can be detected in lymphoid and nonlymphoid tissues. Their developmental origin is still under investigation, but several results suggest that both intra- and extrathymical maturation pathways may exist.129,130 Early findings already described a non-MHC restricted-natural suppressor activity of murine DN T-cell lines,131 although cytokine analysis revealed a marked IFN-γ and TNF-α., but no IL-2, IL-4, IL-10, or IL-13 production.132,133 Meanwhile there is ample evidence of the regulatory function of DN T cells in vitro and in vivo.132–134 In contrast to naturally occurring CD4+CD25+ regulatory T cells (see Section “Functionality”), human DN T reg cells seem to exert their suppressive function in an antigen-specific fashion.133 Interestingly, the capacity of DN T reg cells to suppress syngeneic CD8+ and CD4+ effector cells arises from their Fas/Fas L-mediated cytotoxicity.134 DN T cells use their TCR complex to acquire allo-MHC peptides from APC via trogocytosis (acquisition of membrane-bound proteins) and then kill CD8+ T cells that recognize the same allo-MHC peptides.135 In vivo experiments in murine transplantation models confirmed a cell-to-cell contact-dependent, antigen-specific killing of CD8+ effectors by DN T cells that effectively prolonged skin allograft survival132 and, in addition, plays a role in preventing graft-versus-host disease.136 Similarly, a protective role of DN T cells has been proposed for autoimmune diseases137 and cancer development.136
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After positive selection in the thymus, mature T cells with low affinity for self-peptide/MHC molecules are released into the blood stream and form the long-lived pool of naive T cells. In order to survive, naive T cells require IL-7 signaling and a low level of self-reactivity entertained by constant TCR engagement with self-p/MHC molecules.145
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Recent studies identified fibroblastic reticular cells in secondary lymphoid organs as essential source of IL-7 and the CCR7 ligand CCL19.
146 High expression of CCR7 and CD62L on naive T cells ensures their homing to LN and, at the same time, enables their IL-7-mediated survival. Under homeostatic conditions, a stable population size of naive T cells can thereby be maintained. The transcription factor FoxO1 has been identified as important regulator for the expression of CCR7, CD62L, and the a chain of the IL-7 receptor (CD127) on naive T cells. FoxO1-deficient mice fail to home to secondary lymphoid organs and show only very low levels of CD127, which, in turn, leads to a decrease of naive T cells in these mice.
147 On robust activation, naive T cells undergo a process of expansion and differentiate into effector cells with potent pathogen-eliminating functions.
148 A great proportion of effector cells dies off within a few weeks, but few cells are selected to enter the memory pool according to their capacity to access and use of survival signals.
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Two types of CD45RO+ memory T cells can be generated: central memory and effector memory T cells.
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Central Memory T Cells
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Similar to naive T cells, long-lived central memory T cells express the lymph node homing receptors CD62L and CCR7, which allow their circulation through peripheral blood and secondary lymphoid organs. They are responsible for secondary or long-term responses to antigen and might be involved in long-term maintenance of effector memory cells.149 The pool of memory T cells increases gradually with age at the expense of their naive counterparts. In contrast to naive T cells, memory T cells undergo cell division within an interval of 2–3 weeks, which is balanced by an almost equivalent number of cell death.150 The homeostatic expansion and survival of central memory T cells crucially depend on the responsiveness to IL-7 and IL-15, mediated via surface expression of CD127 (IL7Rα) and CD122 (IL-15R), respectively.151 Central memory T cells exhibit only modest effector functions, but, upon rechallenge with a given antigen, they can develop into effector T cells.152
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It appears that the strength of the antigenic signal determines the ultimate fate of a naive T cell, as robust TCR signaling may result in the generation of effector memory T cells.
149 Contrary to central memory T cells, effector memory cells are excluded from secondary lymphoid organs, but home to peripheral tissues and are responsible for immediate protection against challenge. Following the peak of the immune response, most of these cells disappear from the blood and central memory T cells appear instead. It seems that effector memory cells represent a transitory population rather than a distinct cell type, ending with the development of central memory T cells.
153 Conversely, central memory T cells convert into effector cells and subsequently into effector memory T cells in the presence of antigen.
153 Recent studies in mice demonstrated the importance of IL-2 signaling for the survival and differentiation of long-term effector memory cells, as IL-2Rα-deficient T cells maintain the phenotype of central memory T cells and do not differentiate into effector memory T cells upon secondary antigen challenge.
154 In contrast, IL-15 seems to play a negligible role in promoting effector memory differentiation during primary immune responses, but it is apparently essential for the survival of effector memory T cells after pathogen clearance.
154
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Very recently, two new T-cell subsets have been described: Th22 (T22) cells and Th9 (T9) cells.
156,157 Th22 cells were identified in human peripheral blood as skin-homing CCR6+ CCR4+ CCR10+ CLA+ memory T cells that produce IL-22, but no IL-17 or IFN-γ.
156 That somehow came as a surprise, as IL-22 has so far been associated with Th17 cells only. In vivo, Th22 cells could be isolated from the epidermis of inflammatory skin diseases such as psoriasis, atopic eczema, and allergic contact dermatitis.
158 In vitro, their generation from naive T cells was reported to be dependent on IL-6 and TNF-α.., but independent from the Th17-specific transcription factor RORγt.
156 Th9 cells produce IL-9 upon stimulation with TGF-β and IL-4 and do not express any of the established transcription factors for T-cell differentiation, suggesting a new lineage of T cells. IL-9, originally associated with Th2-dominated responses, is known to be involved in immune responses to helminths and to contribute to the pathogenesis of asthma. Lately, is has also been described that T-cell derived IL-9 may mediate immune suppression, as it is functionally important for allograft survival.
159 Future experiments will tell whether Th22 and Th9 T cells truly represent distinct T-cell subsets with lineage-specific transcription factors.
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With regard to the functional capacities of various T-cell subsets, it was originally assumed that CD4+ cells predominantly subserve helper functions and that CD8+ cells act as killer cells. Many exceptions to this rule are now known to exist; for example, both CD4+ and CD8+ regulatory cells are found, but CD4+ cells are still commonly referred to as helper T cells (Th cells) and CD8+ cells as cytotoxic T cells (Tc cells).
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During an immune response, naive Th/Tc cells can differentiate into several functional classes of cells: (1) Th1 cells (type 1 T cells); (2) Th2 cells (type 2 T cells); (3) Th17 cells; (4) natural killer T cells (NKT); (5) regulatory T cells (T reg); and (6) T follicular helper (Tfh) cells (Fig. 10-4). Originally, all these T-cell subsets have mainly been defined as CD4+ Th cells. In the meantime we have learned that both CD4+ Th and CD8+ Tc cells can produce cytokines allowing their classification into these distinct T-cell subsets. The functional commitment of effector T-cell populations is controlled by the expression of lineage-specific transcription factors, but individual T cells can also express cytokines that are not lineage-specific. It therefore remains to be determined whether T cells display heterogeneity within a lineage or whether each distinct cytokine-expression pattern already reflects a separate lineage. It seems that T cells, although already polarized, still possess a high degree of functional plasticity that allows further differentiation depending on various factors such as the strength of antigenic signaling, cytokines, or interactions with other cells encountered in their microenvironment.155
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T Helper 1/T Helper 2 Paradigm
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T cells that produce IL-2, IFN-γ, and TNF are termed Th1 cells. They are the main carriers of cell-mediated immunity (CMI). Other T cells produce IL-4, IL-5, IL-6, IL-13, and IL-15. These are termed Th2 cells and are primarily responsible for extracellular immunity (see below).160,161 Many factors influence whether an uncommitted T cell develops into a mature Th1 or Th2 cell. The cytokines IL-12 and IL-4, acting through signal transducer and activator of transcription (STAT) 4 and 6, respectively, are key determinants of the outcome, as are antigen dose, level of costimulation, and genetic modifiers. Certain transcription factors have causal roles in the gene-expression programs of Th1 and Th2 cells. For example, the T-box transcription factor T-bet is centrally involved in Th1 development, inducing both transcriptional competence of the IFN-γ locus and selective responsiveness to the growth factor IL-12.162 By contrast, the zinc-finger transcription factor GATA-3 seems to be crucial for inducing certain key attributes of Th2 cells, such as the transcriptional competence of the Th2 cytokine cluster, which includes the genes encoding IL-4, IL-5, and IL-13.163,164
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In murine models of intracellular infection, resistant versus susceptible immune responses appear to be regulated by these two T-cell subpopulations.165–167 Th1 cells, primarily by the release of IFN-γ, activate macrophages to kill or inhibit the growth of the pathogen and trigger cytotoxic T-cell responses, which results in mild or self-curing disease. In contrast, Th2 cells facilitate humoral responses and inhibit some cell-mediated immune responses, which results in progressive infection. These cytokine patterns are cross-regulatory. The Th1 cytokine IFN-γ downregulates Th2 responses. The Th2 cytokines IL-4 and IL-10 downregulate both Th1 responses and macrophage function. The result is that the host responds in an efficient manner to a given pathogen by making either a Th1 or Th2 response. Sometimes, the host chooses an inappropriate cytokine pattern, which results in clinical disease.
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Of particular interest to immunologists is the delineation of factors that influence the T-cell cytokine pattern. The innate immune response is one important factor involved in determining the type of T-cell cytokine response.
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The ability of the innate immune response to induce the development of a Th1 response is mediated by release of IL-12, a 70-kDa heterodimeric protein.168 For example, in response to various pathogens, APCs including DCs and macrophages release IL-12, which acts on NK cells to release IFN-γ. The presence of IL-12, IL-2, and IFN-γ, with the relative lack of IL-4, facilitates Th1 responses. In contrast, in response to allergens or extracellular pathogens, mast cells or basophils release IL-4, which in the absence of IFN-γ leads to differentiation of T cells along the Th2 pathway. It is intriguing to speculate that keratinocytes may also influence the nature of the T-cell cytokine response. Keratinocytes can produce IL-10, particularly after exposure to UVB radiation.96 The released IL-10 can specifically downregulate T1 responses, thus facilitating the development of Th2 responses.
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Not every T-cell-mediated immune response and/or disease can be easily explained by the T1/T2 paradigm. Certain T-cell subpopulations are characterized by the secretion of IL-17. These cells are therefore termed Th17 cells. It was originally assumed that Th1 and Th17 cells arise from a common T1 precursor, but it now appears that Th17 cells are a completely separate and early lineage of effector CD4+ T cells produced directly from naive CD4+ T cells. This was proven by the identification of the Th17-specific transcription factor ROR (RAR-related orphan nuclear receptor) that regulates the expression of IL-17, IL-23R, and CCR6 in Th17 cells.170 The expression of CCR6 is unique for Th17 cells amongst T cells and regulates their migration into epithelial sites depending on its ligand CCL20.171 Recently, it has been demonstrated that Th17 cells may originate from a small subset of CD4+ T cells bearing the NK-cell-associated C-type lectin NKP-1A (CD161), which are present in cord blood and newborn thymus.172 Differentiation of human Th17 cells strongly depends on IL-23, a member of the IL-12 family, as well as on IL-1β, IL-6, and low doses of TGF-β173,174; murine Th17-lineage commitment is mainly induced by IL-6 and TGF-β. Importantly, the induction of Th17 cells from naive precursors may be inhibited by IFN-γ and IL-4, using a cross-regulatory mechanism between Th1, Th2, and Th17 cells. One of the main physiological roles of Th17 cells is to promote protection against fungi, protozoa, viruses, and various extracellular bacteria, but Th17 cells have also been linked to a growing list of autoimmune and inflammatory diseases such as neuroinflammatory disorders, asthma, lupus erythematosus, rheumatoid arthritis, Crohn's disease and, most notably, psoriasis.99,175 Very recent evidence exists that Th17 cells might also play a role in antitumor immunity.176 Importantly, IL-17 expression is not restricted to CD4+ cells only, but has also been detected in CD8+ T cells.177 Th17 cells exert their function by producing effector cytokines including IL-17A, IL-17F, IL-22, and IL-26. Whereas IL-17 is believed to contribute to the pathogenesis of these diseases by acting as potent proinflammatory mediator, IL-22 has been described as a multifunctional cytokine with inflammatory as well as protective properties. In vitro stimulation of normal keratinocytes with IL-22, for example, results in inhibition of keratinocyte differentiation followed by epidermal hyperplasia and upregulated expression of proinflammatory genes in these cells.178
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Natural Killer T (NKT) Cells179
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NKT cells are a distinctive T-cell population with low frequency ranging from 0.01% to 1% of T cells in peripheral blood. They have properties of NK cells but, at the same time, express TCR α/β that, in human beings, consists of an invariant a chain (Vα24-Jα18) preferentially paired with a Vb11 chain. Phenotypically, NKT cells are also defined by the expression of CD45RO and CD161, indicating their effector/memory function. These cells specifically recognize certain tumor-cell-associated or bacterial glycolipids in the context of CD1 molecules and are therefore implicated in tumoricidal and bactericidal host responses (see Section “
CD1-Dependent Antigen Presentation”). On antigenic stimulation, NKT cells produce large quantities of cytokines, particularly IL-4 and IL-10, and can use them to suppress Th1 responses. The biologic relevance of these in vitro data can be deduced from the observation that depletion of NKT cells can aggravate and accelerate Th1-mediated autoimmune diseases in mice, such as insulin-dependent diabetes, multiple sclerosis, and inflammatory bowel disease.
180
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An important type of immunomodulatory T cells that controls immune responses are the so-called regulatory T cells (T reg cells), formerly known as T suppressor cells.181 T reg cells are induced by immature APCs/DCs and play key roles in maintaining tolerance to self-antigens in the periphery. Loss of T reg cells is the cause of organ-specific autoimmunity in mice that results in thyroiditis, adrenalitis, oophoritis/orchitis, etc. T reg cells are also critical for controlling the magnitude and duration of immune responses to microbes. Under normal circumstances, the initial antimicrobial immune response results in the elimination of the pathogenic microorganism and is then followed by an activation of T reg cells to suppress the antimicrobial response and prevent host injury. Some microorganisms (e.g., Leishmania parasites, mycobacteria) have developed the capacity to induce an immune reaction in which the T reg component dominates the effector response. This situation prevents elimination of the microbe and results in chronic infection.
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The best-characterized T reg subset is the CD4+/CD25+/CTLA-4+/GITR+ (glucocorticoid-induced TNF receptor family-related gene)/FoxP3+ lymphocytes.182 The transcription factor FoxP3 is specifically linked to the suppressor function, as evidenced by the findings that mutations in the FoxP3 gene cause the fatal autoimmune and inflammatory disorder of scurfy in mice and IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) in humans. The cytokines TGF-β and IL-10 are thought to be the main mediators of suppression.
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During the past years the situation has become even more complicated, because, at least under certain conditions, subsets with different phenotypes have been associated with regulatory functions such as CD4+, CD8+, and NKT cells. Accordingly, the existence of T reg cells coexpressing IL-17 and FoxP3 has been described.183 CD8+ cells can also be activated to become suppressor cells by antigenic peptides that are presented in the context of an MHC class Ib molecule [Qa1 in mice; human leukocyte antigen E (HLA-E) in humans]. CD8+ T reg cells suppress T cells that have intermediate affinity for self or foreign antigens and are primarily involved in self–nonself discrimination. In addition, recent data provides evidence for a suppressive function of human FoxP3-, TGf-β-producing γ/δ T cells.184
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T Follicular Helper (Tfh) Cells
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Tfh cells represent a distinct subset of CD4+ T cells found in limited numbers, especially in B-cell areas of lymph nodes and spleen.
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Homing and long-term residency in B-cell follicles of these newly described T cells is secured by their surface expression of CXCR5. They have a crucial role in orchestrating T-cell-dependent effector and memory B-cell responses, produce IL-21 and express inducible T-cell costimulator (ICOS) and programed cell death 1 (PD-1) as costimulatory and coinhibitory molecules, respectively. Specific differentiation of Tfh cells was associated to the transcription factor Bcl6 as well as to the cytokines IL-6 and IL-21.185–187
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Lymphocytes in Normal and Diseased Skin
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As opposed to normal mouse skin, in which a resident population of dendritic epidermal T cells uniformly equipped with a nonpolymorphic, canonical
δ TCR exists, the lymphocytes of normal human skin are mainly located in the dermis and predominantly express the
β TCR rather than the γ/δ TCR. While the majority of epidermal T cells exhibit the CD8
+/CD4
− phenotype, dermal T cells are mainly CD4
+/CD8
−, belong to the CD45RO memory population, express the addressins CLA (cutaneous lymphocyte antigen) and CCR4 which they use for skin-homing purposes,
188 and are largely devoid of CCR7 and L-selectin, i.e., addressins promoting the homing of lymphocytes to the lymphoid organs.
152,189 This situation is true also for homeostatic conditions which means that a cutaneous pool of effector memory cells is already in place when danger is imminent. Some of these effector memory T cells have a rather long life span and have been found in different skin conditions, for example, at sites of HSV infection of mice
190,191 and men
192 as well as in clinically resolved, hyperpigmented fixed drug eruptions.
193
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Normal human skin contains approximately 1 million T cells per cm2, 2%–3% of which reside within the epidermis,194 primarily in the basal and suprabasal layers. The T cells of the dermis are preferentially clustered around postcapillary venules of the superficial plexus high in the papillary dermis and are often situated just beneath the dermal–epidermal junction and within, or in close proximity to, adnexal appendages such as hair follicles and eccrine sweat ducts.
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The process of T-cell trafficking to the skin is guided by a series of receptor–ligand interactions between cells. It is of note that DCs are capable of imprinting homing receptor expression on T cells,195 which means that T cells programed by skin and/or skin-derived DCs will preferentially return to the skin. One such moiety is the glycoprotein cutaneous lymphocyte antigen (CLA) that defines a subset of memory T cells that home to skin. It is a glycosylated form of P-selectin–glycoprotein ligand 1 that is expressed constitutively on all human peripheral blood T cells. The level of CLA on cells is regulated by an enzyme, α (1,3)-fucosyltransferase VII, which modifies P-selectin glycoprotein ligand 1. In this manner, CLA+ cells bind to both E-selectin and P-selectin, whereas CLA− cells bind P-selectin, but not E-selectin.196,197 The chemokine–chemokine receptor system is the other major regulator and coordinator of leukocyte migration to the skin (see Chapter 12).
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Skin Homing of Memory T Cells
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Of particular importance for skin homing of memory T cells, independent of their polarization, is the interaction of CCL17 and CCL22 with CCR4 and of CCL27 with its counterreceptor CCR10 on CLA+ T cells. CCL17 is synthesized by activated keratinocytes, DCs and endothelial cells of the skin, while CCL22 is mainly of macrophage and DC origin. The CCR10 ligand, CCR27, appears to be exclusively produced by epidermal keratinocytes.198 Although it was originally assumed that functionally different T-cell subsets can be distinguished from each other by their chemokine receptor expression pattern and their responsiveness to the respective chemokines, the situation is less clear now. Reportedly, T1 cells selectively bear CXCR3 and CCR5, T2 cells preferentially exhibit CCR8 and CCR3, and T17 as well as T reg express CCR6, allowing them to respond to the keratinocyte- and endothelial cell-derived chemokine CCL20.199,200
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From all that has been said so far, one can surmise that the accumulation of T cells in skin is not stochastic. This is indeed the case as exemplified by the dominance of CD8+ T cells in skin lesions, but not in the peripheral blood of patients with lepromatous leprosy201 as well as by the clonality of the T-cell population in cutaneous T-cell lymphoma, in which a single V gene usage is found to predominate in different skin lesions from the same individual.202,203 A limited TCR V gene usage has also been reported to be present in skin lesions of leprosy,204 psoriasis,205 basal cell carcinoma, and countless other reactions in which T cells are present.
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The most direct indication of relevant T-cell populations in skin is determination of the number of antigen-specific T cells. It has been documented that 1 in 1,000 to 1 in 10,000 T cells in the peripheral blood, but only 1 in 50 to 1 in 100 T cells recognize the antigen causing the disease at sites of inflammation.206,207 Thus, there is as much as a 100-fold enrichment of antigen-reactive T cells at the site of cutaneous inflammation.
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With regard to survival and/or expansion of T cells of human skin/epidermis, it appears that IL-2, IL-7, and IL-15 play important roles.208 Notably, the latter two T-cell growth factors can be produced by human epidermal cells, and all of them are frequently overexpressed in T cell-rich skin lesions, for example, in patients with tuberculoid leprosy. For a long period of time, the Th1/Th2 paradigm was used to explain the pathogenesis and, more often, the course of infectious, inflammatory and, even, neoplastic skin diseases. Leprosy and leishmaniasis are outstanding examples of diseases in which the clinical manifestations are decisively determined by the dominance of either Th1 or Th2 cells. With the identification of new function-based T-cell subpopulations (e.g., T0 cells, Th17 cells, Th22 cells), this classification is too rigid and no longer tenable. In fact, we come to realize that the T-cell pathogenesis of certain diseases that we had originally considered to belong into either the Th1 (e.g., psoriasis, allergic contact dermatitis) or the Th2 world (atopic dermatitis) is very complex and sometimes even stage-specific. Th17 and/or Th22 cells are apparently major players in psoriasis158 and allergic contact dermatitis.177 In atopic dermatitis, the acute lesions harbor not only Th2, but also Th17 and Th22 cells; in the chronic stage, however, Th1 cells seem to predominate. In syphilis, perhaps not only Th1 cells, but also CD8+IFN-γ-producing Th17 cells do confer immunologic resistance to T. pallidum.209,210 Th17 cells may also be important in the pathogenesis of Borrelia burgdorferi-induced Lyme arthritis, which was long attributed to be a solely Th1 cell-mediated response.211,212 In patients with cutaneous T cell lymphoma (CTCL), Th2 responses dominate the inflammatory infiltrate of the skin, especially at late stages.213 In early lesions, however, infiltrating CD3+CD45RO+CLA+CCR4+ T cells also express IFN-γ and IL-17 (see Chapter 145). In basal cell carcinomas the presence of a Th2-dominated environment with an increased expression of IL-4 and IL-10 as well as tumor-surrounding T reg cells may be responsible for tumor growth214 (see Chapter 115). In alopecia areata, recent data suggest a role for Th1 cells.215
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Antigen Presenting Cells
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While lymphocytes are the only cells capable of recognizing antigenic moieties, the recognition process per se, at least as far as T cells are concerned, is dependent on the presence of antigen-presenting cells (APC). Unlike B cells, T cells cannot recognize soluble protein antigen per se; their antigen receptor (TCR) is designed to recognize antigen-derived peptides bound to MHC locus-encoded molecules expressed by APCs. Most CD8+ T cells, destined to become cytotoxic T cells, recognize the endogenous antigen in association with MHC class I molecules.216 Because most nucleated cells transcribe and express MHC class I genes and gene products, it is evident that many cell types can serve as APCs for MHC class I-restricted antigen presentation and/or as targets for MHC class I-dependent attack by T cells. For the antigen-specific activation of CD4+ T cells, exogenous antigen-derived peptides are usually presented in the context of MHC class II molecules.216 In this situation, peptides are generated in the endocytic, endosomal/lysosomal pathway and are bound to MHC class II molecules. The resulting MHC-peptide complex is expressed at the APC surface for encounter by the TCR of CD4+ T cells. In the MHC class II-dependent antigen presentation pathway, dendritic cells (DCs), including Langerhans cells (LCs) and dermal dendritic cells (DDCs), B cells, and activated monocytes/macrophages are the major APC populations. Among these, DCs act as professional APC, i.e., are capable of migration and stimulating antigen-specific responses in naive, resting T cells.
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General Principles of Antigen Presentation
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Major Histocompatibility Complex Class I-Restricted Antigen Presentation: Classic Pathway217,218
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Immediately after their biosynthesis, MHC class I heavy and light (β2-microglobulin) chains are inserted into the membranes of the endoplasmic reticulum. The third subunit of the functional MHC class I complex is the peptide itself. The major sources of peptides for MHC class I loading are cytosolic proteins, which can be targeted for their rapid destruction through the catalytic attachment of ubiquitin. These cytosolic proteins can be self-proteins, viral particles, or neoantigens (altered self-proteins). Cytosolic proteinaceous material undergoes enzymatic digestion by the proteasome to yield short peptide chains of 8–12 amino acids, an appropriate length for MHC class I binding. In its basic conformation, the proteasome is a constitutively active “factory” for self-peptides. IFN-γ, by replacing or adding certain proteasomal subunits, induces “immunoproteasomes,” presumably to fine-tune the degradation activity and specificity to the demands of the immune response. The processed peptides are translocated to the endoplasmic reticulum by the transporter associated with antigen processing (TAP), an MHC-encoded dimeric peptide transporter. With the aid of chaperons (calnexin, calreticulin, tapasin), MHC class I molecules are loaded with peptides, released from the endoplasmic reticulum, and transported to the cell surface. Several infectious agents with relevance to skin biology have adopted strategies to subvert MHC class I presentation, and thus the surveillance of cell integrity, by interfering with defined molecular targets. Important examples of such interference are the inhibition of proteasomal function by the Epstein–Barr virus-encoded EBNA-1 protein, the competition for peptide–TAP interactions by a herpes simplex virus protein, and the retention or destruction of MHC class I molecules by adenovirus- and human cytomegalovirus-encoded products.
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Alternative Pathway (Cross-Presentation)
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Under certain conditions, exogenous antigen can reach the MHC class I presentation pathway. Significant evidence for this cross-presentation first came from in vivo experiments in mice demonstrating that viral, tumor, and MHC antigens can be transferred from MHC-mismatched donor cells to host bone marrow-derived APCs to elicit antigen-specific cytotoxic T-cell responses that are restricted to self-MHC molecules.219 In vitro studies have defined that exosomes (i.e., small secretory vesicles of approximately 100 nm in diameter secreted by various cell types, including tumor cells), heat shock proteins, immune complexes, and apoptotic cells (taken up via CD36 and αvβ3 or αvβ5 integrins) can all serve as vehicles for the delivery of antigen to DCs in a manner that permits the cross-presentation of antigen. In all in vitro systems in which a direct comparison has been made, DCs, including LCs, but not monocytes/macrophages, were capable of cross-presentation.220,221 Three distinct pathways are currently exploited by which antigen can access MHC class I molecules of DCs: (1) a recycling pathway for MHC class I in which antigen is loaded in the endosome; (2) a pathway by which retrograde transport of the antigen from the endosome to the endoplasmic reticulum facilitates entry into the classic MHC class I antigen presentation pathway; and (3) an endosome to the cytosol transport pathway, which again allows antigen processing via the classic MHC class I antigen presentation pathway.
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Major Histocompatibility Complex Class II-Restricted Antigen Presentation216
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MHC class II molecules predominantly bind peptides within endosomal/lysosomal compartments. Sampling peptides in these subcellular organelles allow class II molecules to associate with a broad array of peptides derived from proteins targeted for degradation after internalization by fluid phase or receptor-mediated endocytosis, macropinocytosis, or phagocytosis. One of the striking structural differences between MHC class I and class II molecules is the conformation of their peptide-binding grooves. Whereas MHC class I molecules have binding pockets to accommodate the charged termini of peptides and thus selectively associate with short peptides, the binding sites of MHC class II molecules are open at both ends. Thus, MHC class II molecules bind peptides with preferred lengths of 15–22 amino acids but can also associate with longer moieties. An important chaperone for MHC II molecules and responsible for the correct folding and the functional stability of MHC II molecules is the type II transmembrane glycoprotein invariant chain (Ii; CD74). Ii also prevents class II molecules from premature loading by peptides intended for binding to MHC class I molecules in the endoplasmic reticulum and participates in the sorting of MHC II toward the endocytic pathway.222 Depending on the cell type and the activation status of a cell, the half-life of class II–peptide complexes varies from a few hours to days. It is particularly long (more than 100 hours) on DCs that have matured into potent immunostimulatory cells of lymphoid organs on encounter with an inflammatory stimulus in nonlymphoid tissues. The very long retention of class II–peptide complexes on mature DCs ensures that only the peptides generated at sites of inflammation will be displayed in lymphoid organs for T cell priming. Cytokines have long been known to regulate antigen presentation by DCs. In fact, proinflammatory (TNF-α, IL-1, IFN-γ) and anti-inflammatory (IL-10, TGF-β1) cytokines regulate presentation in MHC class II molecules in an antagonistic fashion. Mechanistically, regulatory effects include the synthesis of MHC components and proteases, and the regulation of endolysosomal acidification.223,224
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CD1-Dependent Antigen Presentation225,226
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Besides peptides, self-glycosphingolipids and bacterial lipoglycans may also act as T-cell-stimulatory ligands. Molecules that bind and present these moieties belong to the family of nonpolymorphic, MHC class I- and II-related CD1 proteins. CD1 molecules are structurally close to MHC class I molecules, but functionally related to MHC class II molecules. In the skin, members of the CD1 family are expressed mainly by LCs and DDCs. The CD1 isoforms CD1a, CD1b, CD1c, and CD1d sample both recycling endosomes of the early endocytic system and late endosomes and lysosomes to which lipid antigens are delivered. Unlike in the MHC class II pathway, antigen loading in the CD1 pathway occurs in a vacuolar acidification-independent fashion. T cells expressing a Vα24-containing canonic TCR, NKT cells, and CD4−/CD8− T cells include the most prominent subsets of CD1-restricted T cells. CD1-restricted T cells play important roles in host defense against microbial infections. Accordingly, human subjects infected with M. tuberculosis showed stronger responses to CD1c-mediated presentation of a microbial lipid antigen than control subjects, and activation of CD1d-restricted NKT cells with a synthetic glycolipid antigen resulted in improved immune responses to several infectious pathogens. Thus, the CD1 pathway of antigen presentation and glycolipid-specific T cells may provide protection during bacterial and parasite infection, probably by the secretion of proinflammatory cytokines, the direct killing of infected target cells, and B cell help for Ig production.
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DCs are the only APC capable of interacting with naive T cells. Depending on the DC activation status (i.e., mature versus immature), this cellular contact will result in either productive or nonproductive T-cell responses. Originally, DCs were identified in peripheral lymphoid organs in mice (lymphoid DC).227 A few years later the presence of DC in nonlymphoid tissue (nlDC) was first demonstrated as evidenced by the expression of Fc and C3 receptors as well as MHCII antigens on epidermal LC.228–230 This finding anchored LC as cells of the immune system.
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DCs populate nearly every mammalian tissue under homeostatic (indigenous DC) and inflammatory (inflammatory DC) conditions (Fig. 10-6). Both indigenous and inflammatory DCs ultimately derive from hematopoietic stem and progenitor cells (HSPC) in the bone marrow. HSPCs give rise to progenitor cells that can further differentiate into one or more DC subsets.231,232 DC precursors can be found in multiple locations throughout the body such as the bone marrow, the thymus as well as the peripheral lymphoid organs including the blood.233–235 These blood-derived DC precursors populate nonlymphoid tissues and organs using specific chemokine receptor–ligand pathways (e.g., CCR2-CCL2, CCR5-CCL5, CCR6-CCL20).236–239 Upon arrival in the periphery, they either undergo a process of differentiation or maintain their density by self-renewal.234 Inflammatory DCs are mainly mobilized into the tissues from peripheral blood precursors upon receipt of danger signals. They probably do not constitute a DC subpopulation per se, but rather represent an activated state of a given DC.
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Within the periphery, differentiated DCs accumulate in extravascular areas and survey their surroundings for microbial invasion, always prepared for antigen capture. Under homeostatic conditions, the overwhelming majority of DCs are in an immature state that allows them to efficiently take up antigen (e.g., serum proteins, extracellular matrix components, dead cells) with the help of specific receptor sites (e.g., Langerin, macrophage mannose receptor, C-type lectin receptor DEC-205, low-affinity IgG receptor CD32/FcγRII, high-affinity IgE receptor FceRI, the thrombospondin receptor CD36, DC-SIGN), but does not endow them with immunostimulatory properties for naive resting T cells. DCs apparently increase their efficacy in antigen-uptake by repetitively extending and retracting their dendrites through intercellular spaces (dSEARCH: dendrite surveillance extension and retracting cycling habitude).240 Antigen-engulfment triggers DC maturation, which is followed by DC detachment from neighboring cells and trafficking to draining lymph nodes dependent on CCR7 signaling.241–243
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DC trafficking from nonlymphoid to lymphoid tissues occurs, in a limited fashion, also under homeostatic conditions,244,245 but is much more enhanced upon the delivery of danger signals. During this journey, DCs have to overcome several obstacles such as vessel walls, connective tissue, basement membranes, or other anatomical barriers. To be capable of traveling, DCs are equipped with distinct proteolytic enzymes such as matrix metalloproteinase 2 (MMP-2) and MMP-9 that lead to the degradation of extracellular matrix proteins.246–248 Interstitial DC migration is partly controlled by tissue inhibitors of metalloproteinases (TIMPs), which inhibit MMP activity under nondanger conditions. However, upon maturation of DCs, TIMP expression is downregulated and MMPs exert their function.249 In the LN, DCs rapidly extend their dendrites in a “probing” way thereby establishing physical contacts with adjacent T cells, as in vivo two-photon intravital microscopy of inguinal lymph nodes of mice has revealed.250,251 The display of MHC-peptide complexes on the DC surface delivers the “first signal” to T cells thereby starting communication, i.e., the triggering of the TCR by the APC-bound peptide-MHC complex. Upon activation, DCs display an upregulated and prolonged surface expression of MHCII as compared with nonactivated APC. Although this event may be sufficient to induce the proliferation of primed T cells, it is insufficient for the productive activation of naive T cells. The occurrence of the latter requires the receipt of “second signals,” which are also delivered by professional APCs. In fact, antigen-specific T cells that encounter MHC-expressing cells that cannot deliver second signals (e.g., MHC class II-induced keratinocytes, endothelial cells, fibroblasts) enter a state of anergy.252 Second signals, which include secreted cytokines and membrane-bound costimulatory molecules, determine the magnitude and quality of primary and secondary T-cell responses. Upon contact with the DC-derived cytokine IL-12, for example, T cells turn into type 1 IFN-γ-producing cells, whereas DC-derived IL-23 may skew T-cell responses in the type 17 direction (see Section “Functionality”). Upon danger stimuli, DCs produce a variety of additional cytokines such as IL-1β, TNF-α, TGF-β, or IL-6 that all have the potential to polarize distinct T-cell responses. Costimulatory molecules on DCs are upregulated during the process of maturation induced by surface receptors triggered by ligands secreted or presented by other somatic cells or, alternatively, by microbial products (danger signals).253 The best-defined costimulatory molecules are the two members of the B7 family, B7.1/CD80 and B7.2/CD86. LCs/DCs in situ do not express or express only minute amounts of these costimulatory molecules, but greatly upregulate these moieties during maturation. Other costimulatory molecules include the ICAM-1 that binds to LFA-1 and LFA-3, the ligand of T cell-expressed CD2. Other important ligand–receptor pairs that positively affect T-cell activation by DCs include heat-stable antigen CD24/CD24L, CD40/CD40L, CD70/CD27L, OX40 (CD134)/OX40L, and receptor activator of nuclear factor κB (RANK)/RANKL. Another costimulatory molecule of great importance is the membrane-bound glycoprotein CD83. It is significantly upregulated during DC maturation and enhances CD8+ T cell proliferation upon binding to an as yet unknown CD83 ligand on T cells whose expression is strictly dependent on CD28-mediated costimulation.254,255
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Recent evidence suggests that DCs/LCs themselves can actively induce immune tolerance. The main mechanism to maintain immune tolerance is deletion of T cells with high affinity to self-peptide/MHC complexes in the thymus by inducing apoptosis (negative selection). Another variation of tolerance is T cell-anergy induced by contact with APC that do not provide second signals. Finally, DCs, at least in their immature state, preferentially activate T reg cells.256 When antigen is targeted to these nonactivated DCs in vivo, antigen-specific hyporesponsiveness occurs.257–261 This finding has therapeutic implications for the treatment of autoimmune diseases.262
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Mechanisms responsible for the tolerance-inducing property of nonactivated DCs, although not entirely understood, include (1) a reduced expression of MHC-antigen complexes263 and costimulatory molecules264 on the cell surface; (2) expression of the coinhibitory receptor ligands programed cell death-ligand 1 (PD-L1/B7-H1) and, to a lesser extent, PD-L2 (B7-DC)265–267; (3) the secretion of immunosuppressive cytokines such as IL-10,268 which fits well to the finding of T reg induction by UV-irradiated, IL-10-producing T reg cells269; (4) the expression of immunoinhibitory enzymes such as indoleamine 2,3-dioxygenase270; and (5) the receipt of signals interfering with the maturation and migration of DCs, for example, neuropeptides such as CGRP271 and vasoactive intestinal peptide,272 or the engagement of the CD47/SHPS-1 signal transduction cascade.273,274 It appears that these different factors are not equally operative in all situations. LCs, for example, can activate self-antigen-specific CD8 T cells in the steady state, which leads to chronic skin disease,275 and, at the same time, LCs are dispensable for276 or can even downregulate277 the induction of CHS.
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Dendritic Cells of Normal and Diseased Skin
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In essentially unperturbed normal human skin we find several APC including epidermal Langerhans cells (LC) and dermal dendritic cells278 (DDC). LCs and DDCs are lineage-negative (Lin−), bone marrow-derived leukocytes, which phenotypically and functionally resemble other DCs present in most, if not all, lymphoid and nonlymphoid tissues.279 As gatekeepers of the immune system, they control the response to events perturbing tissue/skin homeostasis. In other species such as mice an additional DC subset has been described recently, namely CD103+CD207+ cells, which in humans have yet to be identified.280–282 Healthy skin also harbors other cells which at least theoretically could subserve APC function, such as basophils and mast cells. While these cells have been shown to play a role in the modulation of cutaneous immune responses, their functions as APC remain to be defined.
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Under inflammatory conditions, DC types that are not residents of the normal cutaneous environment appear in the skin. These include DCs from the plasmacytoid lineage, so-called plasmacytoid DC (pDCs) and inflammatory dendritic skin cells (IDSC), which originate from myeloid precursors and phenotypically resemble myeloid DCs (mDC) of the peripheral blood.
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In 1868, the medical student Paul Langerhans, driven by his interest in the anatomy of skin nerves, identified a population of dendritically shaped cells in the suprabasal regions of the epidermis after impregnating human skin with gold salts.284 These cells, which later were found in virtually all stratified squamous epithelia of mammals, are now eponymously referred to as Langerhans cells (Fig. 10-7).
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The expression of the Ca2+-dependent lectin Langerin (CD207) is currently the single best feature discriminating LCs from other cells. Langerin is a transmembrane molecule associated with and sufficient to form Birbeck granules, the prototypic, and cell type-defining organelles of LCs (see Fig. 10-7). Birbeck granules are pentilaminar cytoplasmic structures frequently displaying a tennis racket shape at the ultrastructural level. The additional presence of Langerin on the cell surface coupled with its binding specificity for mannose suggests that Langerin is involved in the uptake of mannose-containing pathogens by LCs. However, the disruption of the Langerin gene in experimental animals does not result in a marked loss in LC functionality.285 Additional molecules besides Langerin allow the identification of LCs within normal unperturbed epidermis. These include CD1a; the MHC class II antigens HLA-DR, HLA-DQ, and HLA-DP; and CD39, a membrane-bound, formalin-resistant, sulfhydryl-dependent adenosine triphosphatase (ATPase).
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The tissue distribution of LC varies regionally in human skin. On head, face, neck, trunk, and limb skin, the LC density ranges between 600 and 1,000/mm2. Comparatively low densities (approximately 200/mm2) are encountered in palms, soles, anogenital and sacrococcygeal skin, and the buccal mucosa. The density of human LCs decreases with age, and LC counts in skin with chronic actinic damage are significantly lower than those in skin not exposed to UV light. HLADR+/ATPase+ DCs can be identified in the human epidermis by 6–7 weeks of estimated gestational age. These cells must originate from hemopoietic progenitor cells in the yolk sac or fetal liver, the primary sites of hemopoiesis during the embryonic period. Until week 14 of estimated gestational age (EGA), these cells acquire the full phenotypic profile of LC in a stepwise fashion.286 The relative numeric stability of LC counts during later life must be achieved by a delicate balance of LC generation and immigration into the epidermis and LC death and emigration from the epidermis. Within the epidermis, LCs are anchored to surrounding keratinocytes by E-cadherin-mediated homotypic adhesion.287 This anchoring and the display of TGF-β1 also prevent terminal differentiation and migration, thus securing intraepidermal residence for the cells under homeostatic conditions. Two nonmutually exclusive pathways of LC repopulation of the epidermis may exist: (1) LC division within the epidermis, and (2) the differentiation of LCs from skin-resident or blood-borne precursors. Evidence for the first possibility is the demonstration of cycling/mitotic LCs in the epidermis,288 although it remains to be established whether this cell division alone suffices for maintaining the epidermal LC population.
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The observation that the half-life of LCs within unperturbed murine epidermis is around 2–3 months289 suggests a significant turnover of the epidermal LC population even under noninflammatory conditions. In seeming contradiction stands the observation that the LC population of human skin grafted onto a nude mouse remains rather constant for the life of the graft, despite epidermal proliferation and the absence of circulating precursors for human LCs. Moreover, epidermal LCs in mice whose bone marrow was lethally irradiated and subsequently transplanted are only partially replaced by LCs of donor origin,290 whereas DCs in other organs are efficiently exchanged for donor DCs.238 Together, these observations suggest that a precursor cell population resides in the dermis that is engaged constantly in the self-renewal of the epidermal LC population under noninflammatory conditions. The prime candidate LC precursors are dermal CD14+/CD11c+ cells that have the potential to differentiate in vitro into LCs in a TGF-β1-dependent fashion.291
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Under inflammatory conditions (e.g., UV radiation exposure, graft-versus-host disease), an additional pathway of epidermal LC recruitment becomes operative. In this situation, LC precursors enter the tissue, and their progeny populate the epidermis in a fashion dependent on chemoattraction mediated by LC-expressed chemokine receptors CCR2 and CCR6,239 the ligands of which are secreted by endothelial cells and keratinocytes. Thus, CCR6 and its ligand MIP-3α/CCL20 may be essential for epidermal LC localization in vivo, as postulated previously in studies of LCs differentiated from human progenitor cells in vitro.108 The action of MIP-3α/CCL20 may be assisted or replaced under noninflammatory situations by the chemokine BRAK/CXCL14, which is constitutively produced by keratinocytes.292 The differentiation stage of the biologically relevant circulating LC precursors entering inflamed skin in vivo remains to be resolved. However, evidence exists that common myeloid progenitors, granulocyte–macrophage progenitors, monocytes, and even common lymphoid progenitors can give rise to the emergence of an epidermal LC population in experimental animals.293,294
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Compelling evidence exists from in vitro and in vivo studies that LCs play a pivotal role in the induction of adaptive immune responses against antigens introduced into and/or generated in the skin (immunosurveillance). This is best illustrated by the early observation that LC-containing, but not LC-depleted, epidermal cell suspensions pulse-exposed to either soluble protein antigens or haptens elicit a genetically restricted, antigen-specific, proliferative T cell response.295 Inaba et al296 found that freshly isolated LCs (“immature” LCs) can present soluble antigen to primed MHC class II-restricted T cells but are only weak stimulators of naive, allogeneic T cells. In contrast, LCs purified from epidermal cell suspensions after a culture period of 72 hours or LCs purified from freshly isolated murine epidermal cells and cultured for 72 hours in the presence of GM-CSF and IL-1 (“mature” LC) are extremely potent stimulators of primary T cell-proliferative responses to alloantigens,296 soluble protein antigens,297 and haptens.297 Immature LCs, however, far excel cytokine-activated LCs in their capacity to take up and process native protein antigens.298 Accordingly, immature rather than mature LCs express antigen uptake receptors. Mature LCs, although fully capable of presenting preprocessed peptides, have lost their capacity to process and present native protein antigens.298
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Upon perturbance of skin homeostasis (e.g., TLR ligation, contact with chemical haptens, hypoxia), LCs gain access to antigen/allergen encountering the epidermis by distending their dendrites through epidermal tight junctions, thereby demonstrating strikingly remarkable cooperation between keratinocytes and LC.299 After a few hours, LCs begin to enlarge, to display increased amounts of surface-bound MHC class II molecules, and to migrate downward in the dermis, where they enter afferent lymphatics and, finally, reach the T-cell zones of draining lymph nodes.300 During this process, LCs undergo phenotypic changes similar to those that occur in single epidermal cell cultures,301 i.e., downregulation of molecules or structures responsible for antigen uptake and processing as well as for LC attachment to keratinocytes (e.g., Fc receptors, E-cadherin) and upregulation of moieties required for active migration and stimulation of robust responses of naive T cells (e.g., CD40, CD80, CD83, CD86). The mechanisms governing LC migration are becoming increasingly clear. TNF-α and IL-1β (in a caspase 1-dependent fashion) are critical promoters of this process, whereas IL-10 inhibits its occurrence. Increased cutaneous production and/or release of the proinflammatory cytokines are probably one of the mechanisms by which certain immunostimulatory compounds applied to or injected into the skin [e.g., imiquimod, unmethylated cytosine–phosphate–guanosine (CpG) oligonucleotides] accelerate LC migration. Another example is the topical application of contact sensitizers (e.g., dinitrofluorobenzene), which leads to the activation of certain protein tyrosine kinases, the modification of cellular content and structure of intracytoplasmic organelles (increase in coated pits and vesicles, endosomes and lysosomes, Birbeck granules), and increased in situ motility of these cells.302 Interestingly, Cumberbatch et al303 reported that, in psoriasis, LCs are impaired in their migratory capacity. This was somewhat unexpected in view of the remarkable overexpression of TNF-α in psoriatic skin. These investigators also found that the failure of TNF-α and/or IL-1β to induce LC migration from uninvolved skin was not attributable to an altered expression of receptors for these cytokines. An important hurdle for emigrating LCs is the basement membrane. During their downward journey, LCs probably attach to it via α6-containing integrin receptors and produce proteolytic enzymes such as type IV collagenase (MMP-9) to penetrate it and to pave their way through the dense dermal network into the lymphatic system. IL-16 also induces LC mobilization. This process could perhaps be operative in atopic dermatitis. In this disease, DCs of lesional skin exhibit surface IgE bound to high-affinity Fc receptors (FceRI), and allergen-mediated receptor cross-linking results in enhanced IL-16 production. Evidence is accumulating that DC migration occurs in an active, directed fashion. Osteopontin is a chemotactic protein that is essential in this regard. It initiates LC emigration from the epidermis and attracts LCs to draining nodes by interacting with an N-terminal epitope of the CD44 molecule.304 The entry into and active transport of cutaneous DCs within lymphatic vessels appears to be mediated by MCPs binding to CCR2 and by secondary lymphoid-organ chemokine/CCL21 produced by lymphatic endothelial cells of the dermis and binding to CCR7 on maturing LCs and DDCs.242,305 Interestingly, CCL21 expression is upregulated in irritant and allergic contact dermatitis, which implicates its regulated impact on DC emigration from the skin.306
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Dermal Dendritic Cells
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Like resident LCs in the epidermis, dermal dendritic cells (DDCs) constitute another resident DC subpopulation in normal and inflamed skin that is capable of activating the immune system upon receipt of danger signals. Located primarily in the vicinity of the superficial vascular plexus, DDCs have been identified by their surface expression of CD1b, CD1c (BDCA-1), CD11c, CD36, CD205, MHCII, as well as the subunit A of the clotting proenzyme factor XIII (FXIIIa).307 They can be distinguished from LCs by the absence of Langerin expression and lack of Birbeck granules. Based on the positive reactivity for FXIIIa, DDCs from dermal single-cell suspensions were originally classified into at least three different subsets: (1) CD1a−/CD14− cells, (2) CD1a−/CD14− cells, and (3) CD1a−/CD14+ cells. Many assays conducted with DDCs during the past years revealed that they possess functional features of both macrophages and DCs, i.e., the capacity of efficient phagocytosis on the one hand as well as antigen-presenting, migratory and T-cell-stimulating capacities on the other hand.308,309
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Further Studies of DDCS
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Investigation of
C-type lectin expression on DDCs demonstrated that CD209 (DC-specific intercellular adhesion molecule 3 grabbing nonintegrin molecule/DC-SIGN) and CD206 (macrophage–mannose receptor/MMR), both previously associated with DDCs, are expressed by macrophages in human leprosy lesions and tonsils as well as in TLR-activated peripheral monocytes in vitro.
36 Supporting these results, recent data obtained from in situ immunofluorescence of DCs in the dermis of normal human skin (NS) imposed the idea that resident DDCs in NS are comprised of two phenotypically distinct subsets: CD209 (DC-SIGN)
+/CD163
+/CD206
+/CD68
+ dermal dendritic-appearing macrophages and CD1c
+/CD11c
+/CD208
+ “true” DCs.
310 Additional evidence exists that FXIIIa is upregulated on DCs in culture and rather reflects a specific marker for macrophages than for DCs.
311,312 On the basis of these findings careful revisions of the current literature will be necessary in order to reevaluate the presence of CD209
+FXIIIa
+ dermal DCs.
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Another DDC subset of normal human skin can be defined by its CD141 (blood dendritic cell antigen 3/BDCA-3) expression. These DCs constitute approximately 10% of all CD11c
+ dermal DCs and do not coexpress CD1c (BDCA-1).
312 They express TLR3, produce IL12p70 and IFN-β, and excel CD1c
+ DCs in their T-cell stimulatory capacity. Recent data obtained from peripheral blood demonstrate their capacity of cross-presenting Ag to cytotoxic T cells after TLR3 ligation and, thus, suggest an important role in activating cytotoxic T-cell responses.
313
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DDCs are derived from a common myeloid precursor cell. Notably, it has been discovered that DDCs proliferate constitutively in situ in murine and human quiescent dermis,
314 which indicates that homeostatic cell division contributes to the maintenance of this skin DC population. Interestingly, UV-induced cutaneous inflammation leads to a circulating dermal DC precursor replacement of locally proliferating DDCs, which in contrast to that of LCs, relies only on CCR2, but not on CCR6-dependent cell migration.
314 Studies on the ontogeny of human APCs in embryonic foreskin revealed that at 9 week EGA MHCII+ DCs can already be distinguished from MHCII+ macrophages by their expression of the DC marker CD1c. These MHCII
high DCs already exhibit the capacity of antigen uptake, upregulate costimulatory molecules, and stimulate T-cell proliferation.
286 Under homeostatic conditions, a predominant proportion of CD1c
+/CD11c
+ DDCs is in an immature state and, accordingly, shows only weak T-cell stimulatory capacity.
312 However, their immunostimulatory potential increases upon maturation/activation. Migration to the draining lymph nodes (LN) is facilitated, as CD1c
+/CD1a
+ dermal DCs from NS express CCR7 and egress the skin in response to CCL19.
315
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LC versus DDC in Skin Immunity
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What is the function of LCs/DDCs in normal skin? Is there a natural flux of LCs/DDCs to the regional lymph nodes? If so, what are the consequences of such an occurrence? Evidence exists that melanin granules captured in the skin accumulate in the regional lymph nodes but not in other tissues. The further observation of only very few melanin granule-containing cells in TGF-β1−/− mice suggests that, under steady-state conditions, epidermal and/or dermal antigens are carried to the regional lymph nodes by TGF-β1-dependent cells (most likely LCs/DDCs) only. It appears that T lymphocytes encountering such APCs in vivo are rendered unresponsive in an antigen-specific manner.259 It is therefore conceivable that immature resident skin DC, i.e., LCs and DDCs, are endowed with tolerogenic skills inhibiting inflammatory T-cell responses in the steady state and, consequently, that absence of pathogenic T-cell autoimmunity and/or lack of reactivity against seemingly innocuous environmental compounds (e.g., aeroallergens) in the periphery is primarily the consequence of an active immune response rather than the result of its nonoccurrence.
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In the past few years, there has been a heavy debate about the relative sensitizing capacity of LCs versus DDCs in skin-derived immune responses. This discussion was initiated by seemingly controversial results obtained with different types of LC-depleted mice undergoing contact sensitization.
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Models for LC-Depleted Mice
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Two main models were constructed: (a) knockin mice linking the diphtheria toxin receptor (DTR) to the Langerin gene locus in order to induce transient LC ablation by administration of diphtheria toxin276,316 and (b) transgenic mice that coordinately express the diphtheria toxin subunit A (DTA) with Langerin resulting in a constitutive and permanent absence of LC.277 Using DTR mice, one group of investigators316 demonstrated a diminished CHS response in LC-free mice, supporting the long-prevailing concept317 that LCs are needed for optimal contact sensitization. These findings are also inline with the demonstration of cross-presentation of keratinocyte antigens (also tumor-associated antigens) by LCs to T cells221 and underscore the role of LCs in cutaneous immunosurveillance. In sharp contrast to these findings, the other researchers found that the lack of LCs affects neither the sensitization nor the elicitation phase of CHS276 or even demonstrated an amplification of the CHS response.277 Together with the observation that DDCs, before LCs, leave the skin following sensitization, migrate to the lymph nodes and populate separate areas than LCs do,276 these results led to the hypothesis that LCs are primarily concerned with downregulatory functions, whereas DDCs are mainly acting as inducers of productive immune responses. The validity of this concept gains support by studies in mice with graft-versus-host disease318 and by the finding that LCs are critical for the induction of regulatory T cells by ultraviolet radiation (UVR).319
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The discrepancies in results obtained with the different LC-depleted mouse models of CHS have not been fully clarified, but may be due to the different timing of LC depletion in relation to hapten treatment. The situation became further complicated by the identification of a Langerin+ cell within the murine dermis. These cells can prime T cells for hapten sensitization280 and display distinct features as compared to LCs concerning anti-CCR2 reactivity, radiosensitivity, and the potential for self-renewal.280–282 But then again: mice are not men and the exact roles of the various DC populations in healthy and diseased human skin have yet to be fully unraveled.
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Inflammatory Dendritic Cells309
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DCs appearing in inflamed skin can be subdivided into two major subpopulations, i.e., (1) inflammatory dendritic epidermal/dermal cells (IDECs/IDDCs) and (2) plasmacytoid dendritic cells (pDCs). The former ones will be referred to as inflammatory dendritic skin cells (IDSCs).
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Inflammatory Dendritic Skin Cells (IDSC)
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It is still unclear whether IDSCs represent a subpopulation of myeloid DCs which, upon danger stimuli, are recruited to the sites of inflammation from the blood, or whether indigenous DDCs are converted into specialized IDSCs that have the capacity to adapt their function according to the kind of danger signal delivered. Supporting the idea of circulating DC precursors infiltrating the skin upon danger signals, potential precursor cells including pre-DCs320,321 or hematopoietic precursor cells234 have been identified.
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Much work on the identification and characterization of epidermal and/or dermal inflammatory DC populations in various skin diseases has lately been provided by different groups.322–325 In the dermis of psoriatic lesions, the number of CD11c+ DCs is 30-fold increased as compared to normal skin.325,326 In contrast to steady-state DDC, these dermal CD11c+ DCs are CD1c−, but produce a number of proinflammatory cytokines (e.g., TNF-α.) and inducible oxide synthetase (iNOS) and were therefore termed TIP-DCs (TNF-α∼ and iNOS-producing DCs). Initially identified in 2003 in a murine model of Listeria monocytogenes infection,327 they have been located in the lamina propria of human gut328 as well as in imiquimod-treated human basal cell carcinoma.324 Imiquimod and the other imidazoquinolines as ligands of TLR7/8 induce strong inflammation and, ultimately, regression of viral acanthomas and other superficial skin neoplasms.329 Upon treatment, TIP-DCs are abundantly present around regressing tumor cell islands330 and, interestingly, can express molecules of the lytic machinery such as perforin, granzyme B, and TRAIL, suggesting their cytotoxic potential. In psoriasis, TIP-DC have the capacity to prime T cells to become Th1, Th17, and a mixture of Th1/Th17 cells, which simultaneously produce IFN-γ and IL-17325 and may contribute to the pathogenesis of the disease. In addition, their pathogenic role is indicated by downregulation of TNF-α, iNOS, and other cytokines they produce, namely, IL-20 and IL-23, upon effective psoriasis treatment.331 Recent work also identified TRAIL on CD11c+ CD1c− TIP-DCs in psoriasis, proposing a proinflammatory, cell-damaging interaction with keratinocytes that express activating TRAIL receptors (death receptor 4 and decoy receptor 2).332
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In the epidermis of atopic dermatitis (AD) skin, the emergence of inflammatory dendritic epidermal cells (IDECs) has been well documented.333 They are characterized by the expression of CD1a, CD1b, CD1c, CD11c, FceRI, CD23, HLA-DR, CD11b, CD206 (MMR/macrophage mannose receptor), and CD36.333,334 In situ staining of costimulatory molecules on epidermal CD1a+ DC in AD skin showed that mainly cells with the phenotype of IDEC display CD80 and CD86, whereas Langerin+ CD1a+ epidermal LC are almost devoid of these molecules.335 CD86 signaling is critical for the stimulatory capacity of IDEC. Evidence exists that, upon engagement of FceRI on IDEC, an immune response triggered by these cells is skewed into the Th1 direction.336 Recent work also located a substantial number of CD1a+ CD11c+ Langerin-DC within the dermis of AD lesions. Interestingly, these cells showed an upregulation of the chemokines CCL17 and CCL18 and can thereby provide a Th2 polarizing environment.323 Importantly, this subset of IDSC does not produce iNOS or TNF-α, thus confirming the presence of different inflammatory DC subsets in different cutaneous pathologies.
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Plasmacytoid Dendritic Cells337
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pDCs are DCs that are characterized by a highly developed endoplasmic reticulum, which results in their plasma cell-like appearance.338 Functionally, pDCs display a unique ability to produce up to 1,000 times more natural IFNs than any other blood mononuclear cell in response to TLR ligands and thus were also named principal type 1 IFN-producing cells.339
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pDCs develop in the bone marrow and are then released into the blood stream. Recent experiments in mice even suggest that pDCs and mDCs might share a common DC precursor cell.
232 Lately, an important regulator of pDC development has been identified, namely the transcription factor E2-2. This E protein is involved in the pDC evolutional pathway not only by controlling the synthesis of other pDC transcription factors like SpiB, but also by regulating genes that induce the production of IFN, for example, IFN-α regulatory factor 7 (IRF7).
340
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Under homeostatic conditions, pDCs are found in peripheral blood (0.2%–0.8% of peripheral blood cells) and T cell-rich areas of secondary lymphatic tissue. Originally characterized as CD4+CD123+ cells, they lack surface expression of lineage markers for B cells, T cells, NK cells and myeloid cells. Over the last decade, pDC-specific markers such as BDCA-2 (CD303), BDCA-4 (Neuropilin-1) and ILT-7 (Ig-like transcript 7) have been identified and are now commonly used for their isolation from peripheral blood or other tissues. While almost absent in healthy tissue, large numbers of pDCs have been identified in certain types of skin inflammation such as viral infections,341 lupus erythematosus,342 psoriasis, allergic contact dermatitis, lichen planus, and atopic dermatitis.343,344 Whereas pDCs enter lymphoid organs from the blood stream through high endothelial venules (HEV) using CD62L and CCR7, their migration into inflamed skin has been linked to different chemokine receptors, such as ChemR23, CXCR3, and CXCR4, and their corresponding chemokines (Chemerin, CXCL9, CXCL10, CXCL11, CXCL12).345–348 Once within the skin, pDCs localize in perivascular clusters with T cells and, depending on their activation/maturation status, they upregulate MHCII, acquire a dendritic cell morphology and prime distinct T-cell responses.337,349
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Immature pDCs lack surface expression of classical costimulatory molecules such as CD80 and CD86 and are therefore incapable of inducing T-cell proliferation. Recently, it has been demonstrated that pDCs constitutively express the inducible costimulator ligand (ICOS-L; B7-H2), which binds to ICOS on T cells and, similar to CD28, generally has costimulatory impact on T-cell effector function. However, ligation of ICOS-L on pDCs leads to the generation of ICOS+FoxP3+ T reg cells.350 Further evidence for a role of nonactivated pDCs in peripheral immune tolerance comes from the observation that pDCs have the capacity to prevent asthmatic reactions to inhaled allergens by induction of T reg cells, which in turn suppress antigen-specific effector T-cell responses.351
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Upon maturation, pDCs upregulate MHCII and costimulatory molecules (CD80, CD86) and acquire dendritic cell morphology. They selectively express TLR7/8 and TLR9 and, upon endosomal ligation of these receptors, acquire the capacity to mount different immune responses through production of a robust amount of type I IFN. Early experiments already demonstrated that pDC-derived IFN-α promotes IFN-γ-dominated Th1 cell responses,349 thereby linking innate and adaptive immunity. This includes activation of mDCs, NK cells, and B cells, which are converted into antibody-secreting plasma cells. In addition, IFN promotes the differentiation of monocytes into mDCs, which again induce a strong CD4+ T-cell-mediated immune response, and increases their ability to cross-present antigen to CD8+ T cells. Recent findings demonstrate that pDCs are prone to detect self-DNA through endosomal TLR9 when complexed with the antimicrobial peptide LL37, which is overexpressed in psoriatic skin lesions. Thus activated, pDCs produce type I interferon and stimulate mDCs which may then trigger a T-cell-mediated autoimmune response and, consequently, may contribute to the development of psoriasis.19 Likewise, pDC-derived IFN-α plays a major role in the maintenance of disease-specific symptoms of systemic lupus erythematosus. Self-nucleic acids form complexes with autoantibodies against nucleic acids and are subsequently transported to endosomal TLR7 and TLR9 in pDCs, which again leads to continuous production of IFN-α.352 As a result, pDC-derived IFN-α. initiates an autoreactive T-cell response primed by activated and matured mDCs. In addition, autoreactive B cells are prompted to differentiate into autoantibody-secreting plasma cells.353 pDCs also have the capacity to acquire effector functions upon TLR7 binding with either synthetic (imiquimod) or natural (HIV, influenza virus) ligands. Again, they upregulate IFN-α and, consequently, surface expression of TNF-related apoptosis-inducing ligand (TRAIL) is induced. TRAIL expression renders them capable of cytotoxic activity toward virus-infected and tumor cells expressing proapoptotic TRAIL receptors.324,354 Accordingly, pDCs were shown to induce TRAIL-dependent apoptosis in HIV-infected TRAIL R1-expressing CD4+ T cells.355 Interestingly, ligation of BDCA-2 inhibits TRAIL production and cytotoxic capacities of pDCs,356 indicating an important functional role of BDCA-2. Meanwhile several studies have also shown the importance of pDC-derived type I IFNs in cancer immunity, autoimmunity and bacterial infections.357 In order to prevent a disproportionate host-harming IFN production, pDCs are equipped with several inhibiting surface receptors including BDCA-2, ILT7, FcεRI358, and NKp44.359
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When cultured with IL-3 and CD40L, pDCs do not produce IFN-α, but upregulate OX40L and prime naive T cells to produce predominantly type II cytokines like IL-4, IL-5, and IL-10.360 Nevertheless, their ability to process/present exogenous antigen appears to be rather limited as compared to their myeloid counterpart, probably due to their limited antigen uptake capacity. Recent evidence, however, suggests that pDCs can phagocytize, process, and present particular forms of exogenous antigen when encapsulated in certain microparticles.361 This implies a new role of pDCs in the induction of adaptive immunity stimulated by phagocytosed exogenous particle-like structures. In fact, clinical trials have been initiated using tumor-associated antigen-pulsed pDCs as immunogens.362