Le cancer du col utérin est précédé par des lésions prénéoplasiques. Celles-ci sont associées dans plus de 95% des cas à une infection par un papillomavirus (HPV). Un phénomène fréquent durant la cancérogenèse cervicale est l'intégration du génome dun HPV oncogène dans lADN cellulaire. Celle-ci entraîne une expression sélective de gènes codant pour des oncoprotéines virales (appelées E6 et E7) capables d'inactiver les produits de certains gènes suppresseurs de tumeurs (p53, p21, pRb) ou dinteragir avec dautres protéines cellulaires impliquées dans le contrôle du cycle cellulaire. Des travaux antérieurs du laboratoire daccueil suggèrent que le développement du cancer du col utérin est associé à une faible capacité de présentation dantigènes au système immunitaire, comme le démontre la rareté et le déficit fonctionnel des cellules de Langerhans (LC, cellules dendritiques ayant une fonction professionnelle de présentation antigénique au niveau de la peau et des muqueuses) dans les lésions (pré)cancéreuses cervicales. Ces altérations pourraient empêcher une réponse immunitaire efficace et faciliter la persistance du virus ainsi que la progression tumorale. Il est actuellement bien admis que les kératinocytes (cellules cibles de linfection par HPV) sont susceptibles dinfluencer les réactions immunitaires au niveau de la peau et des muqueuses épidermoïdes par lintermédiaire de facteurs solubles, les chémokines (CCL20, contrôlant linfiltration des LC immatures au sein de lépithélium) ou de contacts membranaires (E-cadhérine). Les kératinocytes infectés par HPV pourraient se différencier des cellules normales pour la production de ces facteurs, ce qui pourrait contribuer aux altérations des cellules de Langerhans/cellules dendritiques (LC/DC) observées dans les lésions (pré)cancéreuses cervicales. Le fait que la molécule dadhésion E-cadhérine intervienne dans lattachement des LC aux kératinocytes suggère limportance de cette molécule dadhésion dans la rétention des CL au sein de lépithélium cervical. Les objectifs de ce travail ont été détudier linfluence des oncogènes viraux sur lexpression de facteurs immunitaires et dexaminer les conséquences de linhibition de E6 et de E7 sur lexpression de la E-cadhérine et de CCL20, qui jouent un rôle important dans limmunosurveillance au niveau des épithélia via leur action sur les cellules de Langerhans. En accord avec notre hypothèse, nous avons montré une diminution de lexpression de la E-cadhérine dans les lésions (pré)néoplasiques du col par rapport à lépithélium exocervical normal (Hubert et coll. 2005). Par des expériences dARN interférence (siRNA), nous avons également démontré limplication de loncoprotéine virale E7 dans linhibition de lexpression de la E-cadhérine membranaire (Caberg et coll. 2008) et limplication des oncoprotéines virales E6 et E7 dans la diminution de la sécrétion de la chémokine CCL20 dans des kératinocytes transformés par HPV16 (Caberg et coll. 2008).

Nous avons découvert que les DC pathologiques (LCH DC) qui s’accumulent dans les granulomes de patients atteints d’histiocytose langerhansienne (LCH) produisent l’IL-17A (Coury et al, Nat Med 2008). In vitro, les LCH DC fusionnent en cellules géantes (MGC) sous l’influence de leur production autocrine d’IL-17A. In vivo, les granulomes de LCH DC et MGC détruisent les tissus. Pendant la thèse, nous avons étudié les transcriptomes des monocytes, DC, traitées ou non par l’IL-17A et des LCH DC. L’IL-17A induit BCL2A1, un membre de la famille Bcl-2 qui prolonge la survie des DC. Elle induit aussi les chémokines CCL20 et CCL2, qui regroupent les DC avant leur fusion. L’induction du récepteur nucléaire LXR-α, de protéines impliquées dans le métabolisme, le transport et le stockage des lipides témoignent d’un profond remodelage lipidique. Après la confirmation de ces régulations par PCR et western, les goutelettes lipidiques sont quantifiées à l’huile rouge, puis l’analyse lipidomique révèle l’augmentation de phospholipides, triglycérides, esters de cholestérol et cholestérol par l’IL-17A dans les DC qui sont aussi capables de capturer du palmitate extracellulaire fluorescent. Les transcriptomes des DC traitées à l’IL-17A et des LCH DC sont similaires. La simvastatine qui bloque la synthèse de cholestérol tue les LCH DC. Ce travail établit pour la première fois que l’IL-17A affecte profondément le métabolisme lipidique des DC, une activité qui pourrait avoir d’importantes applications dans les maladies chroniques inflammations associées à une dérégulation lipidique comme l’athérosclérose, l’obésité, la tuberculose et la LCH / We found that the pathological DCs (DC LCH), accumulating in the granulomas of patients affected with Langerhans cell histiocytosis (LCH), produce IL-17A (Coury et al, Nat Med 2008). In vitro, LCH DCs form multinucleated giant cells (MGC) by a fusion process, under the influence of their IL-17A autocrine production. In vivo, granulomas, which are mainly composed of LCH DCs and MGCs, destroy tissues of the patients. During the PhD, we studied the transcriptomes of monocytes, DCs, treated or not with IL-17A, and LCH DCs. IL-17A induceed BCL2A1, a member of the Bcl-2 family that prolonged DC survival. It also induced the CCL2 and CCL20 chemokines that clustered DC, a process required to license DC fusion. The inductions of LXR-α nuclear receptors, proteins involved in metabolism, transport and storage of lipids signed a deep lipid remodeling induced by IL-17A in DCs. We confirmed these regulations by PCR and western studies, then the lipid droplets were quantified after Oil Red-O staining. Further lipidomic analysis revealed an increase of phospholipids, triglycerides, cholesteryl esters and cholesterol by IL-17A in DCs, which are also able to capture extracellular fluorescent palmitate. Transcriptomes of DCs treated with IL-17A and LCH DCs were similar. Simvastatin, which inhibits the synthesis of cholesterol, killed LCH DC. For the first time, this work establishes that IL-17A profoundly affects the lipid metabolism of DCs, an activity that may have important applications in chronic inflammations associated with lipid deregulations such as atherosclerosis, obesity, tuberculosis and LCH

La vaccination du tissu cutané présente un fort potentiel, car elle permet le ciblage de l’antigène aux populations de cellules dendritiques uniques et spécialisées de la peau, et le recrutement de cellules inflammatoires du sang.Les cellules T helper folliculaires (TFH) jouent un rôle crucial dans l’établissement de la réponse humorale. Cependant, les interactions cellulaires et moléculaires qui gouvernent leur induction dans un contexte de vaccination restent à élucider.Mon projet de thèse a eu pour but de mieux comprendre les mécanismes d’induction des réponses TFH et humorales, par l’étude des événements précoces ayant lieu aux sites d’immunisation et d’induction de l’immunité adaptative après vaccination cutanée. L’utilisation de modèles murins nous a permis d’évaluer la contribution de différentes populations de la peau, du ganglion, et du sang dans la mise en place de ces réponses après immunisation intradermique avec un antigène particulaire présentant l’antigène modèle p24 du VIH. Cette étude a révélé un rôle crucial des cellules de Langerhans et des cellules dendritiques migratoires de la peau dans l’induction de réponses TFH et humorales.Nous avons ensuite évalué la capacité de différentes formulations d’adjuvants à polariser la réponse TFH et humorale contre un antigène de l’enveloppe du VIH à fort potentiel vaccinal. L’utilisation de l’émulsion IFA favorise l’induction des cellules TFH et induit la production d’anticorps neutralisants des souches du VIH.Ces résultats soulignent l’importance de cibler les DCs de la peau par l’utilisation de voies de vaccination pertinentes et l’utilisation d’adjuvants capables de favoriser la réponse cellulaire TFH. / Skin vaccination is of great interest, as it enables targeting of the antigen to unique and specialized dendritic cell populations of the skin, as well as recruitment of inflammatory blood cells.T follicular helper (TFH) cells play a critical role in the setting of the humoral response. However, the cellular and molecular interactions that underlie their induction after vaccination remain unknown.My thesis project aimed at understanding the immune mechanisms by which skin vaccination could favor the induction of TFH and humoral immune responses by studying the early events that take place in tissue and lymph node.Using mice models, we evaluated the relative contributions of various populations from the skin, lymph node and blood in the setting of TFH cell responses after intradermal immunization with nanoparticles coated with p24 antigen from HIV. This revealed a crucial role of Langerhans cells and skin migratory dendritic cells in the induction of TFH and germinal center responses.We then evaluated the ability of different adjuvant formulations to polarize the TFH and humoral response against a promising vaccine antigen from HIV envelope protein. Emulsifying the antigen in IFA favors the induction of TFH cells and induces the production of neutralizing antibodies able to block viral infection.This work highlights the relevance of targeting skin dendritic cells by using relevant vaccination routes and adjuvant formulation able to induce TFH cell responses.

L’histiocytose langerhansienne (HL) est la plus fréquente des histiocytoses, liée à l’accumulation de cellules pathologiques de phénotype langerhansien. La découverte de la mutation somatique BRAFV600E dans environ 50% des cas à ouvert un nouveau champ d’investigation pour tirer bénéfice de ce statut moléculaire pour la prise en charge des patients.Tout d’abord, nous avons montré l’efficacité des inhibiteurs de BRAF sans rapporter de résistance dans les formes actives d’HL, en particulier dans les formes multisystémiques avec atteinte des organes à risque (MS OR+) du nourrissons, confirmant le rôle driver de cette mutation dans l’HL. Toutefois, après l’arrêt du traitement administré durant 2 à 6 mois, de nombreuses récidives ont été constatées.Ensuite, nous avons montré que pour les enfants atteints d’HL, la mutation BRAFV600E était significativement associée aux formes MS OR+, retrouvée dans 87,8% de ces cas. Comparés aux patients non mutés BRAF, les patients avec la mutation BRAFV600E présentaient un taux de résistance plus élevé à la chimiothérapie de première ligne velbé - corticoïde (21,9% contre 3,3%), un taux plus élevé de réactivation à 5 ans (42,8% contre 28,1%) et un taux de séquelles supérieur (27,9% contre 12,6%).Par ailleurs, nous avons montré que, pour les HL BRAFV600E mutées, la quantification de BRAFV600E dans l’ADN libre circulant par PCR digitale en gouttelette était un biomarqueur pertinent pour les cas d’HL MS OR+ et les présentations résistantes au traitement de première ligne.Enfin, après un criblage de points chauds mutationnels d’une série d’échantillons tissulaires d’HL ayant permis de mettre en évidence un cas avec la mutation somatique PIK3CAE542K, 9 couples d’échantillons tumeur/constitutionnel ont été étudiés par séquençage d’exome. Cela nous a permis de mettre en évidence une mutation récurrente (n=2) de BRAF au niveau du site d’épissage 5’ (donneur) de l’intron 12. Selon l’analyse de l’ARN, cette mutation conduirait à l’insertion de 3 acides aminés (LLR) dans le domaine kinase de la protéine mutée, dont l’analyse fonctionnelle est en cours. / Langerhans cell histiocytosis (LCH) is the most common type of histiocytosis owing to accumulation of pathologic CD1a+ CD207+ histiocytes. The identification of BRAFV600E in more than half of patients with LCH has launched a new field of investigation to study potential patient’s management benefits and implications from this molecular status.First, in BRAFV600E mutated LCH, we reported the effectiveness of BRAF inhibitors. Efficacy with no resistance to vemurafenib was reported in all cases with active LCH disease, especially for multi-system LCH with risk organ (MS RO+) involvement, confirming the driver status of this mutation in LCH. However, after discontinuation of this treatment administered during 2-6 months, many recurrences were observed.Then, we showed that children with BRAFV600E mutated LCH manifested more severe disease, comprised 87.8% of patients with MS RO+ involvement. Compared to patients with wild-type BRAF, patients with BRAFV600E more commonly displayed resistance to combined vinblastine and corticosteroid therapy (21.9% vs. 3.3%), showed a higher 5-year reactivation rate (42.8% vs. 28.1%) and had more long-term permanent consequences (27.9% vs. 12.6%).Moreover, we showed that BRAFV600E quantification in circulating cell-free DNA by droplet digital PCR is a relevant biomarker to monitor response to therapy for MS RO+ LCH and RO- LCH children who failed to respond to first line chemotherapy.Finally, after the screening of LCH biopsy (n=86) for the BRAF, KRAS, NRAS and PI3KCA most common mutations, leading to highlight one case with the PIK3CAE542K somatic mutation, 9 paired tumor-normal samples from children with LCH were analyzed by whole exome sequencing. Data showed a new BRAF recurrent mutation (n=2) in the 5′ splice sites of the intron 12. According to RNA analysis, this mutation would lead to the insertion of 3 amino acids (LLR) in the smaller N-terminal lobe of the BRAF kinase domain. Functional analysis is ongoing.

A histiocitose de células de Langerhans (HCL) é uma neoplasia mieloide inflamatória comumente afetando pacientes pediátricos e apresenta frequentemente mutações ativadoras somáticas em genes da via MAPK, incluindo BRAF e MAP2K1. Vários estudos sugerem que as células lesionais da HCL podem recrutar e modular células inflamatórias e cujas citocinas parecem fornecer sinais recíprocos de sobrevivência celular. Para o presente estudo foram selecionados 15 casos de HCL (10 crianças, 5 adultos), sendo as amostras de tecido avaliadas através de imunoistoquímica utilizando marcadores para macrófagos (CD68 e CD163), células dendríticas maduras (CDm) (CD83 e CD208), linfócitos T regulatórios (LTregs) (CD4, CD25 e FOXP3) e linfócitos citotóxicos (LCs) (CD56, CD57, perforina e granzima B). Além disso, marcadores de células B (CD20), células T (CD3, CD8) e confirmatórios de HCL foram analisados. Todos os casos de HCL foram positivos para S100, CD1a, CD207 e CD4; enquanto que Bcl-2 e Ciclina D1 foram positivos em 13/15 (86,7%) casos. No microambiente imune intralesional, macrófagos M2 (CD68+/CD163+), seguidos por LTregs, foram as populações celulares mais predominantes. Em quantidade significativamente menor, foram observadas CDm, seguidas por escassos LCs. Considerando a população linfoide, linfócitos T CD3+ foram mais numerosos do que linfócitos B CD20+. Dentro dos linfócitos T, linfócitos T CD4+ foram mais numerosos do que linfócitos T CD8+ (p<0,05). Nossos resultados sugerem que a infiltração de células imunes na HCL, provavelmente através de mecanismos pró-tumorais, inflamatórios e/ou imunossupressores mediados por citocinas, pode promover o desenvolvimento e sobrevivência das células lesionais da HCL, fornecendo uma justificativa para a combinação de imunoterapia e terapia gênica (BRAF) na HCL / Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia often affecting children with constitutively somatic activating mutations in MAPK pathway genes including BRAF and MAP2K1. Several studies suggest that LCH cells can recruit and modulate inflammatory cells and whose cytokines appear provide reciprocal survival signals. For the present study, 15 cases of LCH (10 children, 5 adults) were selected, and the tissue samples were evaluated through immunohistochemistry using markers for macrophages (CD68 and CD163), mature dendritic cells (mDC) (CD83 and CD208), regulatory T-cells (Tregs) (CD4, CD25 and FOXP3) and cytotoxic lymphocytes (CLs) (CD56, CD57, perforin and granzime B). Moreover, B-cell (CD20), T-cell (CD3, CD8) and LCH markers were analyzed. All LCH cases were positive for S100, CD1a, CD207 and CD4, while Bcl-2 and Cyclin D1 were positive in 13/15 cases (86.7%). In the immune microenvironment, M2-polarized macrophages (CD68+/CD163+), followed by LTregs, were the predominant cell populations. In a significantly lower amount, mDC were observed, followed by scarce CLs. Moreover, CD3+ Tcells than CD20+ B-cells were more numerous (p>0.05), the former presenting a higher number of CD4+ than CD8+ T-cells (p<0.05). Our results suggest that immune cell infiltration in LCH, probably through cytokine-mediated pro-tumoral, inflammatory and/or immunosupressive mechanisms, can promote LCH cell development and survival, providing a rationale for combining immunotherapy and BRAF-targeted therapy in LCH

Dendritic cells (DCs), a hematopoietic cell type belonging to the sub-group of cells called antigen presenting cells (APCs), inhabit a central role in innate and adaptive immunity. Although the DC family is very heterogeneous, all members share unique features. Most importantly, DCs can stimulate an immune response. This is due to the cells’ ability to capture and process antigens and to maturate in the presence of danger signals presented by pathogens. Maturation in turn results in the migration of DCs from the tissue they reside in to the draining lymph nodes, as well as in the subsequent presentation of the acquired antigens to T cells. In the skin, which is one of the most immunogeneic organs, DCs are present in sizable numbers in both the epidermis and the dermis. This study focused on two types of DCs: epidermal Langerhans cells (LCs) and dermal DCs (DDCs). While much is understood about LCs, far less is known about the role that DDCs play in skin immunity. Therefore one purpose of this study was to characterize DDCs and to compare their phenotype and functions to that of LCs. This study used two different methods to characterize human skin resident immune cells with regard to their number and distribution. First, a stable analytical immunohistochemistry-based method was developed and applied to a substantial number of healthy skin donors. This enabled a quantitative analysis of skin DC types and skin resident T cells at different anatomical locations in situ. A novel method to count dermal cell populations in situ was developed that resulted in the first published quantification of APCs, DDCs, as well as T cells in human dermis. Second, the traditional form of the emigration assay, which selectively enriches vital cells capable of ex vivo emigration from the skin, was upgraded toward a stable analytical method to separate epidermal LCs from DDCs. In this way, both skin DC types became accessible in sufficient numbers to allow for a comparison of phenotypes and functions in vitro. The resulting phenotypic observations clearly showed that both, LCs and DDCs are not fully mature after their emigration ex vivo and that both can be transformed into a phenotypically more mature state by treating them with inflammatory cytokines. What’s more, LCs are also functionally in an immature state after their emigration. They efficiently took up antigen, showed a low capacity to trans-migrate in response to chemokines, and demonstrated a low capacity to stimulate allogeneic T cells in a mixed leukocyte reaction (MLR). For the first time this study observed all these main APC functions not only for LCs but additionally for DDCs. As these observations were made in relation to LCs of the same donor, it could be concluded that DDCs are functionally more mature than LCs after emigration. DDCs showed a lower antigen uptake capacity than LCs but were superior in terms of their migratory and stimulatory capacity. However, treatment with cytokines could skew LC functions toward functional capacities observed for DDCs, i.e., it decreased LCs’ Ag uptake and increased their migratory and stimulatory capacity, whereas the cytokine treatment did not alter DDCs’ functional capacities. After improving immuno-histochemistry and the emigration assay using healthy skin samples, these newly developed techniques were implemented in clinical trials to observe the number, distribution and migratory capacity of skin DCs and T cells in patients undergoing allogeneic hematopoietic cell transplantation (aHSCT). Such a study is of importance because the turnover of DCs and T cells is closely associated with the occurrence of acute graft-versus-host disease (aGvHD), the major cause of morbidity and mortality after aHSCT. Due to the study design used, this study concisely demonstrate that at the onset of aGvHD, different DC types accumulate along with effector T cells in skin lesions of aGvHD but not in uninvolved skin of the same patient. These results suggest that in addition to donor T cells LCs and DDCs play a role during the early phase of cutaneous aGvHD directly within the site of inflammation. The view of many authors that DC depletion in the transplant recipient, especially in target organs, is a promising approach for aGvHD prophylaxis and therapy is further underscored by these results. One targeting strategy to inhibit GvHD by eliminating recipient DCs may be the use of DC specific monoclonal antibodies. Alemtuzumab (anti-CD52) is a monoclonal antibody and has proven effective in preventing aGvHD after aHSCT. It may, despite depleting donor T cells, also work by targeting recipient DCs. To determine whether the last mechanism of action is significant, a second clinical study investigated the effects of intravenous alemtuzumab on DCs by comparing the number of these cells in skin and blood of patients before and after a 4-week course of alemtuzumab treatment. The result was that although skin DCs weakly express the target antigen CD52 the number of these cells was not consistently reduced by alemtuzumab. In contrast, circulating blood DCs have a stronger CD52 expression and were significantly reduced by the treatment. In conclusion, this work provides new insights into the phenotypical and functional characteristics of human skin DCs, as well as into the fate of these cell types during aHSCT. The investigation of the APC system during aGvHD as carried out here will help to understand the process of aGvHD in more detail. All these efforts may hopefully support the development of new approaches for therapy and prevention of this major limitation of aHSCT and may help to improve this only curative therapy for several life-threatening diseases.

Les Cellules Dendritiques sont un groupe hétérogène de cellules présentatrices d’antigènes, importantes pour le contrôle des réponses innées et adaptatives. Les Cellules Dendritiques Plasmacytoïdes (pCD) en représentent une population unique, aux caractéristiques phénotypiques et fonctionnelles particulières, notamment par leur capacité à produire de grande quantité d’Interféron de type I (IFN). Cette signature IFN marque la physiopathologie du Lupus Erythémateux Systémique (LES), maladie auto-immune systémique. Les mécanismes à l’origine de cette production excessive d’IFN par les pCD restent incomplètement élucidés. Nous montrons, dans notre étude, chez l’homme comme dans un modèle murin que les plaquettes, activées dans le LES, participent à la production d’IFN via le CD40L. Cette production en excès d’IFN, a pour conséquence une maturation et activation d’autres Cellules Dendritiques (CD) entrainant l’activation inappropriée des lymphocytes T. Chez le sujet sain, cette activation inappropriée du système immunitaire adaptatif doit être strictement contrôlée afin d’assurer l’homéostasie du système immunitaire. Il a été montré précédemment que de nombreux lymphocytes aux caractéristiques phénotypiques de type mémoire-effecteur (TEM) peuplent les tissus périphériques, notamment le tissu cutané. Ces TEM sont capables de s’activer et proliférer localement en réponse à un stimulus. Les Cellules de Langerhans (LC) sont des cellules dendritiques résidant au niveau cutané dans l’épiderme. Leur fonction est à ce jour l’objet d’une controverse entre une fonction immuno-stimulante (modèle humain) et une fonction immuno-régulatrice (modèle murin). Nous démontrons dans cette étude que les LC, à l’état basal, chez l’homme, induisent la prolifération de Lymphocytes T régulateurs (Treg) au niveau cutané, capables de bloquer la stimulation inappropriée des TEM cutanés. Cependant en présence d’un stimulus infectieux, les LC induisent préférentiellement la prolifération des TEM en limitant celle des Treg. Les LC semblent être à la fois immuno-régulatrices ou stimulantes en fonction du contexte biologique auquel elles sont confrontées. / Dendritic Cell (DC) are a heterogeneous group of antigen-presenting leukocytes that are important in activation of both the innate and adaptative arms of the immune system. Plasmacytoid Dendritic Cells (pDC) represent a unique population, characterized by their ability to produce large amounts of type I Interferon (IFN). This « IFN signature » is a prominent feature of Systemic Lupus disease (SLE). Mechanisms leading to the excessive production of type I IFN remain largely unknown. Here, in our present study, we demonstrate that platelets are activated in SLE patients by circulating immune complexes and represent a major reservoir of CD40L. Activated platelets potentiate the production of type I IFN by pCD through a CD40L/CD40 interaction. Excessive production of type I IFN by pCD leads to DC activation and maturation and inappropriate activation of auto-reactive T cells.Under steady state condition, inappropriate activation of the immune system must be tightly controlled. It has been previously shown that normal adult human skin contains a large number of resident T cells (TRM) expressing the phenotype of Effector Memory T cells (TEM). These TEMTRM are specific for antigens previously encountered through skin and can be activated and proliferate under specific stimulation. Langerhans Cells (LC) are a group of skin resident DC living in epidermis. There is currently substantial controversy regarding the physiologic role of LC with regard to immunoregulation versus immunostimulation. Here we show that under steady state condition, LC induce the proliferation of a small subset of TRM. These proliferating TRM express the phenotype of TREG and are functional. However this stimulation of TREG could be reversed in the presence of foreign antigen in a dose-dependant fashion, as the addition of a pathogen to LC and TRM led to diminished TREG proliferation and increased TEM proliferation. These findings establish a novel immunological role for LC in human skin, allowing for the constitutive maintenance of tolerance, while also permitting the stimulation of resident immune memory in response to infectious challenge

Dendritic cells (DCs), a hematopoietic cell type belonging to the sub-group of cells called antigen presenting cells (APCs), inhabit a central role in innate and adaptive immunity. Although the DC family is very heterogeneous, all members share unique features. Most importantly, DCs can stimulate an immune response. This is due to the cells’ ability to capture and process antigens and to maturate in the presence of danger signals presented by pathogens. Maturation in turn results in the migration of DCs from the tissue they reside in to the draining lymph nodes, as well as in the subsequent presentation of the acquired antigens to T cells. In the skin, which is one of the most immunogeneic organs, DCs are present in sizable numbers in both the epidermis and the dermis. This study focused on two types of DCs: epidermal Langerhans cells (LCs) and dermal DCs (DDCs). While much is understood about LCs, far less is known about the role that DDCs play in skin immunity. Therefore one purpose of this study was to characterize DDCs and to compare their phenotype and functions to that of LCs. This study used two different methods to characterize human skin resident immune cells with regard to their number and distribution. First, a stable analytical immunohistochemistry-based method was developed and applied to a substantial number of healthy skin donors. This enabled a quantitative analysis of skin DC types and skin resident T cells at different anatomical locations in situ. A novel method to count dermal cell populations in situ was developed that resulted in the first published quantification of APCs, DDCs, as well as T cells in human dermis. Second, the traditional form of the emigration assay, which selectively enriches vital cells capable of ex vivo emigration from the skin, was upgraded toward a stable analytical method to separate epidermal LCs from DDCs. In this way, both skin DC types became accessible in sufficient numbers to allow for a comparison of phenotypes and functions in vitro. The resulting phenotypic observations clearly showed that both, LCs and DDCs are not fully mature after their emigration ex vivo and that both can be transformed into a phenotypically more mature state by treating them with inflammatory cytokines. What’s more, LCs are also functionally in an immature state after their emigration. They efficiently took up antigen, showed a low capacity to trans-migrate in response to chemokines, and demonstrated a low capacity to stimulate allogeneic T cells in a mixed leukocyte reaction (MLR). For the first time this study observed all these main APC functions not only for LCs but additionally for DDCs. As these observations were made in relation to LCs of the same donor, it could be concluded that DDCs are functionally more mature than LCs after emigration. DDCs showed a lower antigen uptake capacity than LCs but were superior in terms of their migratory and stimulatory capacity. However, treatment with cytokines could skew LC functions toward functional capacities observed for DDCs, i.e., it decreased LCs’ Ag uptake and increased their migratory and stimulatory capacity, whereas the cytokine treatment did not alter DDCs’ functional capacities. After improving immuno-histochemistry and the emigration assay using healthy skin samples, these newly developed techniques were implemented in clinical trials to observe the number, distribution and migratory capacity of skin DCs and T cells in patients undergoing allogeneic hematopoietic cell transplantation (aHSCT). Such a study is of importance because the turnover of DCs and T cells is closely associated with the occurrence of acute graft-versus-host disease (aGvHD), the major cause of morbidity and mortality after aHSCT. Due to the study design used, this study concisely demonstrate that at the onset of aGvHD, different DC types accumulate along with effector T cells in skin lesions of aGvHD but not in uninvolved skin of the same patient. These results suggest that in addition to donor T cells LCs and DDCs play a role during the early phase of cutaneous aGvHD directly within the site of inflammation. The view of many authors that DC depletion in the transplant recipient, especially in target organs, is a promising approach for aGvHD prophylaxis and therapy is further underscored by these results. One targeting strategy to inhibit GvHD by eliminating recipient DCs may be the use of DC specific monoclonal antibodies. Alemtuzumab (anti-CD52) is a monoclonal antibody and has proven effective in preventing aGvHD after aHSCT. It may, despite depleting donor T cells, also work by targeting recipient DCs. To determine whether the last mechanism of action is significant, a second clinical study investigated the effects of intravenous alemtuzumab on DCs by comparing the number of these cells in skin and blood of patients before and after a 4-week course of alemtuzumab treatment. The result was that although skin DCs weakly express the target antigen CD52 the number of these cells was not consistently reduced by alemtuzumab. In contrast, circulating blood DCs have a stronger CD52 expression and were significantly reduced by the treatment. In conclusion, this work provides new insights into the phenotypical and functional characteristics of human skin DCs, as well as into the fate of these cell types during aHSCT. The investigation of the APC system during aGvHD as carried out here will help to understand the process of aGvHD in more detail. All these efforts may hopefully support the development of new approaches for therapy and prevention of this major limitation of aHSCT and may help to improve this only curative therapy for several life-threatening diseases.

Die Arbeit beschreibt das phänotypische Verhalten von kultivierten Langerhanszellen, antigenpräsentierenden Zellen der Epidermis, sowie die Art und Weise ihrer Elimination. Hierfür wurden Zellkulturen von Langerhanszellen durch Migration aus normaler menschlicher Haut gewonnen. Die Langerhanszellen durchlaufen dabei die gleiche funktionelle Entwicklung, wie nach Antigenpräsentation in situ. Ziel der Untersuchung war es, die funktionellen und zellulären Eigenschaften und die Elimination von Langerhanszellen in der Zellkultur zu ermitteln. Die Anzahl viabler Zellen wurde mittels Trypanblauausschluß zu verschiedenen Zeitpunkten der Kultur ermittelt. Außerdem wurden die Zellen mittels Elektronenmikroskopie und Immuncytochemie untersucht. Die Befunde zeigen, daß die Zellen in der Kultur eine Veränderung ihres Phänotyps sowie funktionelle Änderungen im Sinne einer Ausreifung zu antigenpräsentierenden, T-Zell stimulierenden Zellen erfahren. Das in-vitro-Verhalten entspricht dem von Langerhanszellen in vivo nach Kontaktsensibilisierung. Mit Hilfe von eines für Apoptose spezifischen ELISA (= Enzyme-linked immunosorbent assay: eine Nachweisreaktion für Antigene bzw. Antikörper mithilfe von Enzymen) und Elektronenmikroskopie wurde nachgewiesen, daß die Zellen in unseren Kulturen durch Apoptose starben. Es gibt keinen Anhaltspunkt dafür, daß sich die Zellen nicht auch in vivo apoptotisch eliminieren. Die Verlauf der Funktionsmarker weist darauf hin, daß vorwiegend die maturierten Zellen von Apoptose betroffen waren, und die Apoptose über das CD 95/CD 95 L- System gesteuert wurde. Die Versuche zeigten insgesamt, daß Langerhanszellen durch Apoptose aus der Kultur eliminiert werden. Da sich die Zellen nach Migration in vitro wie Langerhanszellen nach Antigenpräsentation in vivo verhalten, scheint die Apoptose ein biologisches Regulativ für die Elimination von funktionell ausgereiften Langerhanszellen darzustellen. / This work describes the phenotypic behavior of cultivated Langerhans-cells, epidermal cells presenting antigenes and how they are eliminated . Therefore cultures of Langerhans-cells won by migration from normal human skin were used. The migrated Langerhans-cells have the same phenotypic features as Langerhans-cells after presentation of antigenes in situ. The aim of this work was to show the functional and cellular features of Langerhans-cells in culture and the way of their elimination. The cells still alive were count at distinct times using the Trypan-blue-exclusion-method. Additionally the cells were examined by electron microscopy and immuncytochemical methods. The findings show, that the cells in culture have the same characteristics of the phenotype and change of their function in the direction of developing to antigen-presenting, T-cell-stimulating cells. The in vitro behaviour is the same as of Langerhans-cells in vivo after contact-sensitization. With the help of an elisa (=Enzyme-linked immunosorbent assay) specific for apoptosis ( Cell Death Detection Elisa = CDDE) and with electron microscopy was shown, that the cultivated cells died by apoptosis. There is no reference point, that the cells do not do the same in vivo. The process of the functional markers shows, that predominantly the matured cells die by apoptosis and that it was controled by the CD 95/CD 95-L -system The investigations showed, that the Langerhans-cells were eliminated by apoptosis of the culture. The cells after migration in vitro behave in the same manner as after presentation of antigen in vivo. This indicates apoptosis to be the biologic regulation for the elimination of functional matured Langerhans-cells.