Changing Views of the Role of Langerhans Cells

REVIEW

Changing Views of the Role of Langerhans Cells Nikolaus Romani1, Patrick M. Brunner2 and Georg Stingl2 Langerhans cells (LCs) have long been considered to be the major sensitizing cells in the skin by initiating productive immunity in naive resting T cells. This picture has changed over the past decade. We now know (i) that the skin also harbors other types of dendritic antigen-presenting cells and (ii) that the genetically driven removal of the LC population results in increased T-cell immunity against haptens and infectious agents. It is not clear at present whether the situation in genetically modified mice is in any way indicative of the actual in vivo function of LCs. Exciting and challenging years lie ahead of the LC research community. Journal of Investigative Dermatology (2012) 132, 872–881; doi:10.1038/ jid.2011.437; published online 5 January 2012

INTRODUCTION In 1868, a medical student Paul Langerhans (Figure 1) reported on the occurrence of a dendritically shaped cell population within the human epidermis, which now bears his name (Langerhans, 1868). He had detected them by impregnating tissue sections with gold chloride, which, at that time, was a recognized marker for nerves and nervous tissue. Langerhans was therefore convinced that he had discovered cutaneous nerves/nerve endings, a view that was held by several other investigators in the century to come (Ferreira-Marques, 1951; Niebauer and Sekido, 1965). Although the advent of electron microscopy in skin research ultimately disproved the ‘‘nerve’’ theory of Langerhans cells (LCs), it is noteworthy that now, in 2011, LCs are known to be surrounded by sensory nerve endings, which, by the secretion of neuropeptides, can modulate LC immune function (Hosoi et al., 1993).

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Department of Dermatology and Venereology, Innsbruck Medical University, Innsbruck, Austria and 2Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria

This article is dedicated to the memory of Ralph M. Steinman, who received the 2011 Nobel Prize in Medicine. Correspondence: Georg Stingl, Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, Vienna 1090, Austria. E-mail: [email protected] Abbreviations: DTR, diphtheria-toxin receptor; LC, Langerhans cell; TLR, Tolllike receptor Received 4 October 2011; revised 28 October 2011; accepted 29 October 2011; published online 5 January 2012

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A second theory that prevailed for quite some time claimed that LCs are ontogenetically related to melanocytes. The reason for this assumption was not only the common dendritic configuration of LCs and melanocytes, but also the observation that, in vitiligo, LCs often occupy the space of destroyed melanocytes (Birbeck et al., 1961). Later, by the demonstration of LCs in neural crest–deprived, i.e., melanocyte-free, mice (Breathnach et al., 1968), it became clear that the ‘‘melanocyte theory’’ of LC origin was not tenable. The same was true for the ‘‘keratinocyte’’ theory, which suggested a common ontogeny between LCs and keratinocytes (Reams and Tompkins, 1973). At present, however, we fully appreciate that there is a vivid and, apparently, bidirectional communication between these two cell systems. Keratinocytes elaborate cytokines, growth factors, and hormones that influence LC phenotype and function (Romani et al., 2010a). Notably, there also exists a relationship between LC numbers and the extent of keratinocyte proliferation and differentiation (Potten and Allen, 1976; Schweizer and Marks, 1977). The observation of reduced LC numbers in parakeratotic psoriatic plaques (Ashworth and Mackie, 1986; Placek et al., 1988) is in keeping with this concept. It will certainly be interesting and important to determine whether keratinocytes, when they undergo transformation, lose chemoattracting properties for LC precursors and, if so, the underlying molecular mechanisms. Conversely, it remains to be determined why the induction of squamous metaplasia in the tracheal epithelium and the urothel, e.g., by a vitamin A–free diet, coincides with the migration of LCs into the epidermis (Wong and Buck, 1971). An entirely new line of thinking about the derivation of LCs emerged when the unique cytoplasmic organelles of these cells, i.e., the pentalaminar Birbeck granule, were detected within histiocytes of lung and bone lesions of patients suffering from what was called at that time ‘‘histiocytosis X’’ (Basset et al., 1966). Subsequently, it became clear that these neoplastic cells exhibit essentially all of the phenotypic features of LCs (Nezelof et al., 1973), and it was only logical to rename the disease ‘‘LC histiocytosis’’. One hundred years after Langerhans seminal discovery, the concept of a histogenetically heterogeneous epidermis prevailed, consisting of epithelial keratinocytes, neuroectodermal melanocytes, and mesenchymal LCs (Wolff, 1972). Why ‘‘mesenchymal’’? This is not entirely clear, but, in the late 1960s, histiocytes were probably considered to be of mesenchymal origin. LANGERHANS CELLS AS IMMUNOCYTES—THE EARLY YEARS After the mid-1970s, a series of observations led to a radical change in our understanding of LC biology. These cells were & 2012 The Society for Investigative Dermatology

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Figure 1. A group of Langerhans cell researchers visiting the grave of Paul Langerhans in Funchal, Madeira, Portugal in 2005. From left to right: Georg Stingl, Ralph Steinman, Rick Granstein, Marcel Teunissen (kneeling), Mark Udey, Lasse Braathen, Mieke Mommaas, Geoffrey Rowden, Kunihiko Tamaki, and Paul Bergstresser. Someone (Niki Romani) had to take the photograph. Both Kunihiko Tamaki and Ralph Steinman have died since this photograph was taken.

then recognized as bone marrow–derived leukocytes (Katz et al., 1979; Volc-Platzer et al., 1984) with an immunophenotype similar to antigen-presenting monocyte–macrophages and dendritic cells, respectively (Stingl et al., 1980). At the functional level, it was demonstrated convincingly that epidermal cells enriched for LCs, but not those depleted of LCs, were potent stimulators of antigen (haptens, soluble proteins, alloantigens)-specific T-cell proliferation in an major histocompatibility complex II-dependent manner (Stingl et al., 1978; Braathen and Thorsby, 1980). These in vitro findings were complemented by in vivo data that also implied an immunological role for these cells. In contact eczema, Silberberg et al. (1976) found a close apposition of LCs and lymphocytes and, subsequently, reduction and damage of LCs. The Dallas group, on the other hand, provided evidence for a direct relationship between epidermal LC density and the successful induction of contact hypersensitivity (Toews et al., 1980) and allograft immunity (Streilein et al., 1982). Particularly important was the observation that the attempted hapten sensitization via LC-depleted skin was not an immunological null event, but rather resulted in antigen-specific unresponsiveness. The sensitizing power of LCs was also underscored by the stunning observation that LCs induced contact hypersensitivity even under experimental conditions that normally would result in tolerization (Ptak et al., 1980). Altogether, these data implied that antigenic contact of LCs would invariably result in a productive immune response. This idea was maintained for quite some time, although its consequences, i.e., constant tissue inflammation as a result of immunity to self and non-self, would not be compatible with skin homeostasis and integrity. In the mid-1980s, evidence was presented indicating that the

immunostimulatory capacity of LCs depends on the quality and quantity of external signals and stimuli. Schuler and Steinman (1985) reported that freshly isolated LCs appear as round cells, express only modest amounts of major histocompatibility complex antigens and costimulatory molecules on their surfaces, and are only poor stimulators of primary immune responses. After several days of coculture with their epidermal symbionts, however, they had assumed a highly dendritic configuration, expressed high amounts of major histocompatibility complex and costimulatory molecules, and evoked vigorous responses in naive resting T cells. It soon became clear that the delivery of ‘‘danger’’ signals (Matzinger, 1994) such as highly reactive haptens leads to a marked increase in the production and release of proinflammatory and immunostimulatory cytokines by keratinocytes (Enk and Katz, 1992) and that some of these cytokines, e.g., GM-CSF and IL-1, are essential for the viability and immunostimulatory function of cultured murine epidermal LCs (Witmer-Pack et al., 1987; Heufler et al., 1988). What did this finding tell us about in vivo circumstances? A large number, because under stress conditions (e.g., skin explants), of LCs undergo the same phenotypic changes as do single epidermal cell cultures in vitro (Larsen et al., 1990). Together with the finding by Romani et al. (1989) that freshly isolated LCs present large protein antigens more efficiently to T-cell clones than do cultured LCs, it was believed that LCs pick up and process antigenic moieties in situ and, upon receipt of danger signals only, start to mature phenotypically and functionally and migrate to the T-cell zones of regional lymph nodes. Once arrived, they would ultimately come into prolonged contact with antigen-specific, naive resting T cells, leading to their activation. This concept also implied that, under homeostatic, non-dangerous conditions, LCs would be, at best, exposed to innocuous substances (e.g., self-proteins) and this encounter would neither entice them to migrate nor induce immunological maturation. LANGERHANS CELLS AS IMMUNOCYTES—NEW DEVELOPMENTS As described above, by about the end of the last century a large body of evidence for an immunogenic role of LCs had accumulated. LCs were considered as being responsible for inducing essentially all immune responses that originate in the skin, especially in the epidermis. New developments in LC research, however, have markedly changed the way we see LCs. Two concepts have now been challenged: (1) that LCs are the major antigen-presenting cells in skin and (2) the fact that LCs are invariably immunogenic needs reconsideration. Important accelerators for LC research

Discovery of langerin. The key discovery that set off a veritable boom in LC research was made by Jenny Valladeau and Sem Saeland about 12 years ago. They followed up on early descriptions of an LC-specific molecule, the ‘‘Lag’’ antigen (Kashihara et al., 1986), and found and characterized a C-type lectin receptor that was specifically expressed in and on LCs; they therefore named it ‘‘langerin’’ (CD207; www.jidonline.org

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Valladeau et al., 1999, 2000). Langerin is also a major molecular constituent of Birbeck granules. Excellent antibodies against langerin became available, allowing a direct and simple identification of LCs outside the familiar environment of the epidermis. Genetically modified mice. Progress in genetic engineering led to the generation of mice that had GFP (green fluorescent protein) coupled to langerin as a reporter dye for easy and straightforward identification and sorting of langerin-expressing cells (Figure 2; Kissenpfennig et al., 2005). Equally important, mice were devised in which langerin-expressing cells could be removed selectively by means of diphtheria toxin (Bennett et al., 2005; Kissenpfennig et al., 2005) or where LC never even appeared—also based on diphtheria toxin technology (Kaplan et al., 2005). Non-LC dendritic cell in skin. Undoubtedly, LCs were the first skin dendritic cells to be discovered and characterized. For a long time, they were therefore considered to be the main, if not the only, dendritic cell of the skin. However, this turned out to be incorrect. In the beginning of the 90s, dermal dendritic cells were discovered (Lenz et al., 1993; Nestle et al., 1993): an important additional cutaneous dendritic cell had entered the stage. Since then, much knowledge has been gathered about these cells, but their true identity is not as crystal clear as that of classical LCs. In normal healthy epidermis, LCs are the only type of antigen-presenting cells. This makes definitions easy. The dermis, in contrast, contains different hematopoietic cells, including closely related ones such as dendritic cells and macrophages. Their exact ontogenetic relationship has not been completely clarified, but we now have some good markers at hand, such as CD163, which allow separation of dermal dendritic cells from macrophages (Zaba et al., 2007; Teunissen et al., 2011). Another degree of complexity was added in 2007, when a novel type of dendritic cell was detected in the murine dermis (Bursch et al., 2007; Ginhoux et al., 2007; Poulin et al., 2007). Until then, the rare langerin þ cells in the steady-state dermis had been considered LCs that just happened to be in transit from the epidermis through the dermis toward lymph

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Figure 2. Epidermal sheets from mouse ear skin. Thirty years ago, Langerhans cells were visualized by the classical ATPase/ADPase enzyme reaction (left). At present, Langerhans cells can be observed directly, without any antibodies, by means of green fluorescent protein (GFP) expressed along with langerin (right; black/white mode). Although the cells still look alike, our views on their biology have changed considerably (GFP photo by Florian Sparber).

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nodes. Now we know that they represent, at least partly, another population of langerin þ dendritic cells in the dermis. These cells are not radioresistant as their epidermal counterparts, meaning that in bone marrow transplant experiments they are readily replaced by donor cells in contrast to LCs, which remain of recipient origin for virtually the whole life (Merad et al., 2002; Kanitakis et al., 2011). Langerin þ dermal dendritic cells are dependent on Flt-3 ligand in their development, in contrast to LC, which develop independently of Flt-3 ligand but which need CSF-1 (Ginhoux et al., 2012). They also differ phenotypically. The main markers that set langerin þ dermal dendritic cells apart from LCs are CD103, which they express, and EpCam/ CD326, which they do not express. CD103 þ langerin þ dermal dendritic cells have so far been characterized only in the mouse. Although they are very rare in situ and can hardly be spotted by immunofluorescence or immunohistochemistry, they can be readily detected in migrant populations of skin explants (Flacher et al., 2010; Sparber et al., 2010) or, even more so, in analyses of skin-draining lymph nodes, where they make up a substantial proportion (up to 50%) of all langerin þ cells (Tripp et al., 2009). Langerin þ /CD103 þ dendritic cells in the dermis seem to be part of a dendritic cell system that also occurs in other tissues such as lung and gut (Ginhoux et al., 2009; Helft et al., 2010). Langerhans cells are not (always?) immunogenic

Two important insights were gained in the past 10 years with regard to dendritic cell function in general and LC function in particular, leading us no longer to state categorically that ‘‘Langerhans cells are immunogenic.’’ Dendritic cells can function in a tolerogenic way: a new paradigm. Traditionally, dendritic cells were regarded as primarily or solely immunogenic. The late Ralph M. Steinman, who received the 2011 Nobel Prize in Medicine, even named them ‘‘nature’s adjuvant’’ (Steinman et al., 1990). Around 2002/2003, it became feasible to deliver antigens to mice in vivo, in the absence of perturbations that, via the engagement of Toll-like receptor (TLR) or other pattern recognition receptor, would ordinarily signal danger and, thus, cause inflammation. For this purpose, model antigens were coupled to antibodies against C-type lectin receptors expressed by dendritic cells, typically DEC-205/CD205, and mice were immunized with these conjugates. This did not result in a so-called ‘‘null event’’, but rather a state of tolerance developed after an initial wave of T-cell proliferation. Only when inflammatory stimuli such as CD40 ligand or TLR ligands were given along with the antigen did immunity develop (Hawiger et al., 2001; Bonifaz et al., 2002). These seminal experiments, which were conducted largely in Ralph Steinman’s Rockefeller University labs, taught two lessons. First, immune responses can be markedly enhanced when antigens are ‘‘targeted’’ to uptake receptors on dendritic cells and administered together with inflammatory stimuli. This principle can be harnessed for immunotherapy, e.g., against cancer (Bonifaz et al., 2004) and for improving vaccination in general (Romani et al., 2010b; Flynn et al., 2011). Second,

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and more relevant in the context of our discussion, antigens given in the absence of danger signals induce tolerance (Steinman et al., 2003). This is the proposed mechanism for inducing and maintaining peripheral tolerance to selfantigens (Steinman and Nussenzweig, 2002) and to innocuous substances. This concept of tolerance brought about by dendritic cells in the steady state was based on experiments that did not specifically address the role of LCs in this process. Implicitly, of course, LCs and other skin dendritic cells must have been involved because the preferred route of immunization was the skin (mostly mouse footpad).Nevertheless, no special attention was payed to these subsets of dendritic cells. Langerhans cells do not induce productive/protective immunity: a provocative notion. The first ‘‘blow’’ to the belief that LCs are universally immunogenic came when Allan et al. showed in 2003 that in a mouse model of cutaneous herpes virus infection it was not LCs that would present the viral antigen to CD8 þ T cells in the lymph node, but rather lymph node–resident dendritic cells (Allan et al., 2003). The failure of LCs to present in this model could, in part, be explained by their likely destruction in the skin by the cytopathic virus (Bosnjak et al., 2005). As a consequence, lymph node–resident dendritic cells would step in. Indeed, in other mouse models, where cytopathic and non-cytopathic viruses were compared, skin-derived dendritic cells did transport and present the virus to T cells (He et al., 2006). LCs and other dendritic cell subsets of the skin were not distinguished in this study. Similar observations were made in the murine vagina where submucosal dendritic cells, but not LCs, induced protective T helper type 1 responses to herpes simplex virus-2 (Zhao et al., 2003). In addition, in a murine leishmania skin infection model, the parasites were presented in the draining lymph nodes by dendritic cells other than LCs (Ritter et al., 2004). The other initial doubts about a universally immunogenic function of LCs were raised by the first studies using murine cell–depletion models in contact hypersensitivity. Mice that were depleted of all langerin þ cells (diphtheria-toxin receptor model; ‘‘DTR mice’’) showed unimpaired ear swelling responses (Kissenpfennig et al., 2005), and mice that had no LCs from birth (diphtheria toxin expression model; ‘‘DTA mice’’) even developed enhanced ear swelling responses (Kaplan et al., 2005). In this context, it is important to emphasize that in this quickly evolving research field confusion may stem from the rapid sequence of discoveries. Data from 2006 could not yet take into account the existence of langerin þ dermal dendritic cells that were only published in 2007. This potential problem is often overlooked. Using the mentioned models, a substantial number of studies have been published over the past few years. The more recent among them did in fact take into account langerin þ dermal dendritic cells as critical factors in cutaneous immune responses. Evidence accumulates that, at least in mice, LCs appear to fulfill primarily a downmodulatory role, and that it is mainly langerin þ dermal dendritic cells that subserve immunogenic functions. An immune-dampening function of LCs in vivo was recently shown in a mouse

model of cutaneous Leishmania infection (Kautz-Neu et al., 2011), as well as in skin-grafting experiments (Obhrai et al., 2008). A likely underlying mechanism in this setting is the expansion of regulatory T cells induced by LCs (Kautz-Neu et al., 2011). This capacity of LCs was already noted some time ago, and it was shown to depend on receptor activator of NF-kB ligand produced by neighboring keratinocytes (Loser et al., 2006). This may also account for the observed T-regulatory induction by LCs (but not by langerin þ dermal dendritic cells) in the setting of UVB-induced immune suppression (Schwarz et al., 2010). These findings in mouse models were recently corroborated in clinical human samples and with human LCs. Glucocorticoids influenced LCs in such a way that they would induce regulatory T cells in a transforming growth factor-b-dependent manner (Stary et al., 2011). If LCs were to use their T-regulatory-inducing capacity to subserve a tolergenic function in the steady state, i.e., non-inflamed skin, they need to be able to take up and cross-present self-antigens within the intact epidermis as a precondition. Indeed, this was shown unequivocally in a mouse in which the antigen could be inducibly expressed in keratinocytes: LCs isolated from this epidermis readily activated antigen-specific T cells, indicating that they must have captured and processed the antigen in the steady state (Holcmann et al., 2009). Langerin þ dermal dendritic cells were absent in this experimental system (trypsinized epidermis). Conversely, evidence increases for an immunogenic role of langerin þ dermal dendritic cells. The Melbourne group delved into the mouse herpes virus infection model in more detail. In particular, they studied the phase of viral recrudenscence where cytopathic effects of virus are much less pronounced than in primary infection (Bedoui et al., 2009). There, as well as in accompanying experiments with an epidermis-restricted (i.e., expressed under a keratin promoter) antigen, it was clearly the population of CD103 þ /CD8negative/CD205 þ dendritic cells that cross-presented the antigens in the skin-draining lymph nodes. Similar observations were made by the group of Henri et al. (2010). In both studies, LCs contributed only little, if anything, to crosspresentation. This contrasts with LCs freshly isolated from the epidermis, which do cross-present the epidermis-restricted antigen (Stoitzner et al., 2006; Holcmann et al., 2009). The reasons for these differences remain to be determined. Thus, CD103 þ /langerin þ dermal dendritic cells behave functionally similar to their CD103 þ (but only partly langerin þ ) counterparts in other tissues of the mouse. They appear to be the major cross-presenters in the skin (Chang and Kweon, 2010; del Rio et al., 2010; Helft et al., 2010). It will be interesting to see how relevant these data from mouse models will be for the human situation. Human counterparts for the langerin þ dermal dendritic cell have not yet been described, but they may be defined and characterized soon (Teunissen et al., 2011). Thus, LCs may not be operative in cross-presenting viral or tumor antigens in vivo. Nevertheless, they are not inactive! They are able to present antigens to CD4 þ T cells in vivo, as demonstrated in the herpes infection model (Bedoui et al., 2009). This would allow them to induce regulatory T cells as www.jidonline.org

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discussed above. Underscoring this, LCs can induce contact hypersensitivity all by themselves, i.e., under experimental conditions where the hapten does not reach the dermal dendritic cells (Noordegraaf et al., 2010) or when langerin þ dermal dendritic cells are not present (Honda et al., 2010). The capacity of LCs to process and present to CD4 þ T cells in vivo explains the recent observations that the production of IgG1 antibodies in response to gene gun immunization is dependent on LCs as suggested in one study (Nagao et al., 2009). Another study reminds us that the last word has not been spoken yet over this issue (Stoecklinger et al., 2011). Finally, LCs but not langerin þ dermal dendritic cells turn out to be responsible for the activation of T helper type 17 T cells in response to a fungal infection of the skin (Igyarto et al., 2011). As we will discuss below, there may even be conditions under which LCs can also cross-present and induce major histocompatibility complex I-restricted cytotoxicity to be possibly used for the immunotherapy of cancer. UNRESOLVED QUESTIONS—THE FUTURE OF THE LANGERHANS CELL In spite of the tremendous progress in the field, we are far from completely understanding the biology of LCs. Unresolved questions remain and unexpected new functional features have emerged. Here we present and briefly discuss a number of such issues. LCs in innate immunity

It has been known for a long time that LCs can be infected by HIV-1 (Tschachler et al., 1987). In fact, they have been regarded as being pivotal in disseminating the virus to proliferating CD4 þ memory T cells (Pope et al., 1994; Sugaya et al., 2004). This role of LCs in the promotion of HIV-1 infection was recently challenged by the finding that LCs can bind to the virus via langerin and degrade it in their Birbeck granules, thus preventing viral transmission to T cells (de Witte et al., 2007). This may be the first example of an innate defense mechanism exhibited by LCs. Measles virus may be treated similarly by LCs (van der Vlist et al., 2011). Other effector functions of LCs worth investigating include the synthesis of complement components, the secretion of antimicrobial peptides, and arachidonic acid metabolites. For the latter class of substances, LCs were, indeed, shown to be important producers of prostanoid synthases, and thus responsible for flushing in response to nicotinic acid drugs (Benyo et al., 2006). Imiquimod treatment of basal cell carcinoma of the skin leads to regression and clearance of this tumor. This is mediated by dendritic cells rather than—as would have been expected—by cytotoxic T cells (Stary et al., 2007). These dendritic cells possessed molecules of the lytic machinery, and it would be interesting to explore how LCs behave in this regard. Can they also be induced to make, for instance, perforin and granzyme? Can LCs develop the machinery for intracytoplasmic killing of microorganisms and immune phagocytosis? On the basis of the lack or low expression of TLRs recognizing bacterial molecules (TLRs 2 and 4), Teunissen hypothesizes that LCs may not be the prime defenders against bacteria but, rather, may contribute to 876

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tolerance against commensal bacteria (van der Aar et al., 2007). Clearly, this problem still needs considerable research. Langerhans cells in adaptive immunity

From all that has been said so far, it is clear that LCs are prototypic antigen-presenting cells. Unfortunately, the quality of the immune response induced under different in vivo conditions is far from certain. This is particularly true as far as their role in anti-microbial immunity is concerned. Protection against HSV-2 might be mediated by vaginal submucosal dendritic cells but not LCs (Zhao et al., 2003), and LCs might even be counterproductive in mounting a protective immune response by dampening genital cytotoxic responses following mucosal immunization (Hervouet et al., 2010). Although certain studies with mice, whose langerin-positive cells had been eliminated by genetic manipulation (Kaplan et al., 2005), indicate that LCs, at least under homeostatic conditions, induce antigen-specific unresponsiveness, it is not yet clear whether this is also true when an overwhelming danger signal (e.g., virulent microorganisms) occurs in skin. It is quite likely that such an event would induce maturational steps in LCs and allow these cells to join forces with other dendritic cells in the process of T-cell sensitization. Altogether, it is quite likely that the actual function of an LC in situ is determined by stimuli from the environment and/or their epidermal symbionts. In unperturbed skin or under the influence of hormone drugs (e.g., glucocorticosteroids; Stary et al., 2011), they are most likely concerned with downregulating T-cell responses. In other situations, the immunological outcome may be quite different. LCs can stimulate type 2 T-cell responses (Hauser et al., 1989) and do it in a particularly efficient manner under the influence of thymic stromal lymphopoietic (Ebner et al., 2007). This pathway appears to be operative in atopic dermatitis. The role, if any, of LCs in the activation of Th-17 cells is less clear and, even, controversial (Aliahmadi et al., 2009; Duraisingham et al., 2009; Mathers et al., 2009; Wang et al., 2011). Interestingly, human LCs have been reported to induce distinct CD4 þ T cells that produce IL-22, but not IL-17 (Fujita et al., 2009). The relevance of these findings for our understanding of the immunopathogenesis of diseases such as atopic dermatitis or psoriasis is not yet clear. There are reports that the number of LCs is reduced within lesional psoriatic skin (Ashworth and Mackie, 1986; Bieber et al., 1988; Placek et al., 1988; Jones et al., 1994), although the extent is largely dependent on the mode of quantification. The reduction observed can be reverted by the tumor necrosis factor-a-antagonist adalimumab (Gordon et al., 2005). It was shown that this decrease in LC count distinguishes psoriasis from other chronic inflammatory skin diseases such as lichen planus and inflamed seborrheic keratosis (Gordon et al., 2005), which underlines a possible local anti-inflammatory role of these cells. Consistently, it has been shown previously that tumor necrosis factor-a leads to the exodus of LCs from both murine and human epidermis (Cumberbatch et al., 1994, 1999, 2003; Kimber et al., 2000). In any event, we urgently need molecular biological, as well as functional,

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studies with cells directly isolated from the affected tissues to address these questions appropriately. A most important, yet unresolved, question is whether LCs can be induced to evoke type 1 T-cell responses (Rajkovic et al., 2011) and thus, at least theoretically, be used as vehicles in trans- or epicutaneous approaches for vaccination against microbial and tumor antigens. Such desirable ‘‘needle-free’’ approaches are already being commercialized for vaccination, so far with varying degree of success (Glenn et al., 2007). Antitumor effects in mice could be achieved by epicutaneous immunization with tumor antigen (Stoitzner et al., 2008). Whether they were mediated by LCs alone or together with with langerin þ dermal dendritic cells was not assessed. It is, therefore, not certain how beneficial it would be to address LCs as the primary target in an epicutaneous approach. The open question is still the ability of LCs to crosspresent. The mouse models discussed above suggest strongly that langerin þ dermal dendritic cells, rather than LCs, crosspresent. This notion is supported by recent work from Geijtenbeek’s group (van der Vlist et al., 2011), who infected human LCs, isolated from the epidermis, with measles virus. Although LCs were able to present virus epitopes to CD4 þ T cells, they were unable to cross-present it to CD8 þ T cells. In vitro generated ‘‘LCs’’, in contrast, could readily and efficiently cross-present (Ratzinger et al., 2004; Klechevsky et al., 2008). Only when the antigen was targeted to an antigen uptake receptor on LCs (human DCIR in that case) by means of an antibody conjugated to the antigen could CD8 þ T-cell responses be measured by pentamer staining

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and IFN-g release, thus indicating true cross-presentation (Klechevsky et al., 2010). This validates data by Stoitzner et al. (2006) who have demonstrated unequivocally that murine LCs can cross-present; contamination by langerin þ dermal dendritic cells could be definitively excluded in these experiments. A critical variable that may determine whether LCs can be successfully harnessed to improve vaccination (Ueno et al., 2010) may be the manner of targeting these cells. Several surface receptors are candidates (DEC-205/CD205, langerin, DCIR, Clec9A, and so on), and it is not yet known how targeting one or the other receptor on LCs will shape the ensuing immune responses. The fact that there are important differences has already been shown for dendritic cells other than LCs (Dudziak et al., 2007; Idoyaga et al., 2008). Future research will have to identify methods of targeting and activating LCs that lead to the most efficient (cross-)presentation of vaccine antigens applied to the skin, and thus protect from pathogens and control, or even cure, cancer. Role of LCs in the maintenance of epidermal biology

LCs are prone to primarily populate stratified squamous epithelia. The reason for this affinity is only poorly understood, and the interaction between MIP-3a (CCL20) and CCR6 may be only one factor involved. Having arrived in the epidermis, LCs preferentially occupy a suprabasal position. We know that anchoring of LCs to the surrounding keratinocytes is, at least partly, mediated by E cadherin– dependent homotypic adhesion, but other attachment

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Figure 3. Roles attributed to LCs during the past 143 years. (a) Langerhans cells (LCs) as sensory nerve endings; (b) LCs as ‘‘effete’’ melanocytes; (c) LCs exclusively mediating a productive immune response. (d) Only activated LCs mediate a productive immune response, whereas unperturbed LCs remain inactive; (e) LCs dampen immune responses; (f) LCs as mediators of multiple functions, depending on environmental factors.

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molecules such as claudin probably do also have a role (Zimmerli and Hauser, 2007). These bonds between LCs and keratinocytes must also be dynamic. One has to imagine that LCs localize in the epidermis for years (Merad et al., 2002; Kanitakis et al., 2011), whereas keratinocytes constantly differentiate around them and move up in the epithelial tissue until they ultimately desquamate. The movement of LCs is also a prerequisite for their antigen-capturing capacity. For this purpose, LCs reach out with their dendrites, penetrate through tight junctions, and pick up antigens or pathogens that have successfully passed the stratum corneum barrier but cannot get further because of the tight junctions (Kubo et al., 2009). The intraepidermal movement of LCs has been beautifully visualized (Kissenpfennig et al., 2005; Vishwanath et al., 2006), but the molecular events governing this process are only poorly understood. In any case, we can safely assume that the intraepidermal LC population is a ‘‘stably moving’’ network, always alert and ready to react to external stimuli. The fact that LCs and their epidermal symbionts form a functional unit does not necessarily mean that LCs have a role in epidermal homeostasis. LC-free epidermis in DTR knock-in mice did not show overt alterations (Kissenpfennig et al., 2005), and there were no abnormalities in epidermal structure reported in mice that do not possess epidermal LCs from birth (‘‘DTA mice’’; Kaplan et al., 2005). As epidermal differentiation was not a major issue in these studies, it may well be that more thorough and directed studies of several parameters of keratinocyte differentiation may reveal an influence of LCs. Such an influence was, indeed, suspected many years ago by Potten and Allen (1976), who concluded it from the conspicuous array of keratinocytes and LCs in the epidermis (‘‘epidermal proliferative unit’’). We have already mentioned that LCs can induce T cells to produce IL-22, the main ‘‘fuel’’ for the proliferation of keratinocytes. LCs can therefore influence epidermal homeostasis indirectly via helper T cells. Whether they can do it directly, i.e., without mediation by helper T cells, remains to be determined. Role of Birbeck granules

For many years we liked to call these organelles ‘‘enigmatic’’. They have lost much of their enigmatic character recently. We now know that they are composed of langerin (and certainly other molecules as well). We know that they are part of the endosomal recycling pathway (Mc Dermott et al., 2002; Uzan-Gafsou et al., 2007). Do they fulfill other specialized tasks in addition to serving as ‘‘virus-cracking’’ organelles, as suggested (de Witte et al., 2007)? Interestingly enough, the one and only healthy individual identified to date (by serendipity), who lacks Birbeck granules because of a mutation in the langerin gene, showed no obvious manifestations of this defect (Mommaas et al., 1994; Verdijk et al., 2005). Similarly, pig skin, whose LCs do not contain Birbeck granules (Bautista et al., 2002), has no conspicuous phenotype. There are many other interesting aspects of LC biology (e.g., contribution of LC to the immunogenicity of skin grafts; role of LC in skin aging; LCs as drug targets and so on), but 878

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their detailed description would exceed the scope of this article. Two of the authors of this manuscript have lived through exciting early (NR) and very early (GS) stages of LC research and have contributed seminal evidence for an immunogenic role of LCs. Therefore, it is ‘‘emotionally’’ somewhat difficult for us to accept a thoroughly different, even opposing, view about the function of our ‘‘favorite’’ cells (Figure 3). Scientifically, however, we are thrilled and fascinated by the progress that we observe and that we are privileged to be part of. It is reassuring and ‘‘consoling’’ to learn that LCs remain to be a very important and crucial cell in the immune system of the skin, in spite of changing paradigms. Unless research funding decreases markedly, for which we do not hope, LC research has a bright future. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS This work was supported in part by the Austrian Science Fund (FWF): W1212.

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