CD2 is a transmembrane molecule and a “non-canonical” member of the signaling lymphocyte activation molecule (SLAM) family of receptors that is expressed on T cells and NK cells. Its ligands, mouse CD48 and human CD58, are widely expressed on hematopoietic cells including antigen-presenting cells (APCs) and T cells. Previous studies indicated that CD2 promotes T-cell receptor (TCR) signaling when it is engaged by its ligands displayed on APCs. However, the supporting experimental data were rather controversial, and there is no general agreement about the role of CD2 in T cell activation. To study the function of CD2 and its ligands in T cells, we examined T cell functions in newly generated mouse strains lacking CD2 or CD48 in the C57BL/6 background. Compared to wild-type (WT) mice, T cells from CD2-deficient (“knock-out”; KO) mice had severe activation defects. Surprisingly, expression of CD48 on T cells, not on APCs, was also necessary for optimal T cell responses. We found evidence of CD2 interacted with CD48 in cis on T cells and observed their co-localization by confocal microscopy and fluorescence resonance energy transfer (FRET). The only exception was CD2-dependent cytotoxicity, which required CD48 both on T cells and on APCs. Mechanistic studies using mass spectrometry and structure-function analyses revealed that the cis interactions between CD2 and CD48 on T cells boosted TCR signaling, an effect that correlated with the capacity of CD2 to recruit the kinase Lck. Similarly, our further study revealed that the cis interactions between CD2 and CD58 on human T cells were also necessary for maximal TCR signaling and T cell activation. Taken together, our studies provide clear evidence that cis interactions between CD2 and its ligands on T cells are important in TCR signaling and T cell activation. Modulation of these cis interactions can be a promising approach to suppress or enhance T cell activation in a therapeutic setting. / CD2 est une molécule transmembranaire et un membre “ non-canonique ” de la famille de la famille SLAM (« signaling lymphocyte activation molecule ») exprimée à la surface des lymphocytes T et des cellules NK (« natural killer »). Les ligands de CD2, CD48 chez la souris et CD58 chez l’humain, sont exprimés de manière ubiquitaire sur les cellules hématopoïétiques, y compris sur les cellules présentatrices d’antigène (CPA) et lymphocytes T. Des études antérieures ont indiqué que CD2 est impliqué dans la signalisation des récepteurs TCR (« T-cell receptor ») en réponse à son engagement par CD48 sur le CPA; cependant, les données expérimentales qui supportent ce modèle sont plutôt contradictoires et aucun accord n’a été trouvé sur les rôle de CD2 dans l’activation de lymphocytes T. Pour étudier la fonction de CD2 et ses ligands, nous avons examiné les fonctions des lymphocytes T chez des souches de souris dépourvues de CD2 ou CD48 nouvellement générées à partir du “fond génétique” C57BL/6. Par rapport aux souris de type sauvage (WT; « wild-type »), les lymphocytes T de souris CD2-déficientes (« knock-out »; KO) présentent des sévères défauts d’activation. Il est intéressant de noter que l’expression de CD48 sur les lymphocytes T, mais non sur les CPA, était aussi nécessaire pour les réponses des lymphocytes T. Nous avons également démontré que CD2 interagit en cis avec CD48 sur les cellules T et avons observé leur co-localisation par microscopie confocale et FRET (« fluorescence resonance energy transfer) ». La seule exception était la cytotoxicité CD2- dépendante, qui nécessitait l’expression de CD48 à la fois sur les lymphocytes T et sur les CPA. L’étude des mécanismes par la spectrométrie de masse et les analyses structurefonction ont démontré que les interactions en cis entre CD2 et CD48 permettent de stimuler la signalisation du TCR, ce qui corrèle avec la capacité de CD2 à recruter la kinase Lck. De manière similaire, notre étude plus approfondie a démontré que les interactions en cis entre CD2 et CD58 sur les lymphocytes T humains sont nécessaires pour la signalisation maximale du TCR et l’activation cellulaire T. L’ensemble de nos études ont mis en évidence que les interactions en cis entre CD2 et ses ligands sur les lymphocytes T jouent un rôle important dans la signalisation du TCR et l’activation de ces cellules. La modulation de ces interaction en cis pourrait être une approche potentielle pour augmenter ou interférer avec l’activation des lymphocytes T dans un contexte thérapeutique.

Rituximab (R) is increasingly incorporated in reduced intensity conditioning (RIC) regimens for allogeneic hematopoietic cell transplantation (alloHCT) in patients with B-cell malignancies, not only to improve disease control, but also to prevent graft-versus-host disease (GVHD). There are no randomized prospective data to validate this practice, although single center data and the CIBMTR analysis have shown promising results. We aimed at validation of these findings in a large registry study. We conducted a retrospective analysis using the EBMT registry of 3,803 adult patients with B-cell malignancies undergoing alloHCT (2001–2013) with either rituximab (R-RIC-9%) or nonrituximab (RIC-91%) reduced intensity regimens respectively. Median age and median follow up were 55 years (range 19.1–77.3) and 43.2 months (range 0.3–179.8), respectively. There was no difference in transplant outcomes (R-RIC vs RIC), including 1-year overall survival (69.9% vs 70.7%), 1-year disease-free survival (64.4% vs 62.2%), 1-year non-relapse mortality (21% vs 22%), and day-100 incidence of acute GVHD 2-4° (12% vs 12%). In summary, we found that addition of rituximab in RIC regimens for B-cell malignancies had no significant impact on major transplant outcome variables. Of note, data on chronic GVHD was not available, limiting the conclusions that can be drawn from the present study.

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

Graft-versus-host disease (GvHD) of the skin is a severe allo-immune reaction and complication following allogeneic stem cell transplantation. Over the past years, intensive pre-clinical research has led to an improved understanding of the pathophysiology of acute and to a lesser extend chronic GvHD. This has translated into the approval of several new agents for the treatment of both forms of GvHD. This review summarizes the most recent advances in underlying pathomechanisms, clinical trials and newly approved agents for GvHD, with a special focus on skin involvement.

A boca é local de frequentes complicações relacionadas ao transplante de células tronco hematopoiéticas (TCTH) tais como xerostomia, disgeusia, disfagia, mucosite, infecções oportunistas e doença do enxerto contra hospedeiro (DECH). Sabe-se que estas complicações podem comprometer a qualidade de vida do paciente e interferir na morbidade pós-TCTH. O dentista é o profissional da saúde que deverá intervir no momento correto para tratar e minimizar esses efeitos secundários do TCTH. Para tanto é importante conhecermos o momento em que cada complicação ocorre para que a intervenção seja pronta e eficiente. O objetivo principal deste estudo foi identificar e quantificar as alterações bucais em indivíduos submetidos ao TCTH em cinco momentos consecutivos desde antes do início do condicionamento pré-TCTH até o dia 100 pós-TCTH. Como objetivos secundários buscamos investigar possíveis relações entre a severidade da mucosite oral e a manifestação da DECH com dados demograficos (sexo, idade), com o status de saude bucal (por meio dos índices IHO-S, CPOD, número de dentes cariados) e com a realização de adequação bucal pré-TCTH, e ainda, somente para a DECH, também foi investigada a possível relação entre esta doença com infecção sistêmica por citomegalovírus e com a manifestação de mucosite oral severa. Foram incluídos no estudo 27 indivíduos com doenças hematológicas do Serviço de Transplante de Medula Óssea do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), com idade 12 anos que receberam TCTH alogênico. Os indivíduos foram examinados em cinco momentos consecutivos. No primeiro momento, pré-TCTH, foi realizada a coleta de índices de saúde bucal e aplicação de questionário sobre o histórico de tratamentos odontológico prévios. Nos momentos de 10, 20, 60 e 100 dias pós-TCTH foram avaliadas as manifestações bucais presentes. A incidência de mucosite oral foi de 82,6% considerando todos os momentos avaliados. Mucosite oral severa, ou seja, graus 3 e 4 (OMS) foi observada em 57,9% dos pacientes avaliados nos momentos 2 e 3. Dez (37%) pacientes apresentaram GVHD em algum órgão, e destes, 8 (80%) apresentaram GVHD de boca. Infecção sistêmica por CMV foi diagnosticada em 6 (22,2%) pacientes. Concluímos que entre as queixas levantadas, dor bucal e disfagia foram as mais referidas. O período de maior incidência das complicações bucais foi nos segundo e terceiro momentos, ou seja, D+10 e D+20, representando deste forma, o período de maior morbidade do tratamento. Não houve associação entre a severidade de mucosite oral e idade, sexo, fonte de células, regime de condicionamento, número de dentes cariados, IHO-S, CPOD e preparo bucal pré-TCTH. Para a DECH a única relação encontrada foi para fonte de células, tendo sido observada menor chance de ocorrer DECH quando a fonte de células foi o sangue periférico. / The mouth is a well-known site of complications of the hematopoietic stem cell transplantation (HSCT) such as dry mouth, dysgeusia, dysphagia, mucositis, opportunistic infections and graft versus host disease (GVDH). It is known that these complications can compromise the patients quality of life and morbidity post-HSCT. The dentist is the health professional who should interfere at the right time to treat and minimize these side effects of HSCT. Thus, it is important to know the time at which each complication occurs to be dynamic and efficient. The main objective of this study was to identify and quantify the oral complications in patients treated with HSCT in five consecutive moments starting before conditioning chemotherapy until day 100 post-HSTC. As secondary objectives we seek to investigate possible relationships between the severity of oral mucositis and the manifestation of GVHD with demographic data (gender, age), with the oral health status (IHO-S, CPOD, number of decayed teeth) and dental treatment previously HSCT, and, only for GVHD, was also investigated the possible relationship between this disease with systemic cytomegalovirus infection and the manifestation of severe oral mucositis. It was included in the study 27 patients with hematologic diseases who were admitted in the Unit of Bone Marrow Transplantation, Hospital of Clinics, Faculty of Medicine, University of Sao Paulo (HC-FMUSP), 12 years old whom received allogeneic HSCT. The subjects were examined in five consecutive moments. At the first moment, before HSCT, the oral health índex evaluation and a questionnaire about history of previous dental treatments were performed. Besides that, 10, 20, 60 and 100 days after HSCT they were evaluated for oral manifestations. Oral mucositis incidence was 82,6% and 57,9% of these patients presented severe mucositis. Ten (37%) patients had GVHD in any organ, and of these, 8 (80%) had oral GVHD. Infection by CMV was diagnosed in 6 (22.2%) patients. We conclude that among the complaints raised, mouth pain and dysphagia were the most mentioned. The period of increased incidence of oral complications was the second and third times (D +10 and +20), representing the increased morbidity period. There was no association between the severity of oral mucositis and age, sex, cell source, conditioning regimen, number of decayed teeth, IHO-S, CPOD and dental treatment pre-HSCT. For GVHD the only relation found was with source of cells, in which, GVHD was less likely to occur when the source of cells was peripheral blood.

A Doença Enxerto Contra Hospedeiro Associada à Transfusão (DECH-AT) é uma reação transfusional rara, porém fatal, que ocorre devido à presença de células T no sangue doado e que pode ser prevenida por meio da irradiação do sangue do doador e de seus componentes antes da transfusão. Assim, o controle de qualidade associado à irradiação do sangue é necessário para se garantir a qualidade do produto transfundido. Neste trabalho é proposta a caracterização e a adaptação da resposta do dosímetro Fricke para uso na dosimetria da irradiação de sangue, mais especificamente o Fricke Xilenol Gel (FXG). Este é um dosímetro químico radiocrômico, que apresenta as vantagens de ser tecido equivalente e de permitir a inferência espacial da dose absorvida dentro da faixa de doses usados na prevenção DECH-AT. Dessa maneira, de modo a possibilitar a inferência de dose absorvida em todo o intervalo utilizado na prevenção da DECH-AT (25 a 50 Gy), o FXG foi caracterizado e adaptado para aplicações dosimétricas envolvendo a irradiação de sangue e derivados. Os resultados com o novo dosímetro apontaram adequabilidade para toda a faixa necessária de doses absorvidas, com sensibilidade e desvanecimento temporal satisfatórios para aplicações rotineiras. Além disso, pela metodologia proposta neste trabalho, foi possível determinar as distribuições espaciais das doses absorvidas com o dosímetro proposto de uma maneira rápida e simples, mostrando assim que este dosímetro apresenta características convenientes para o controle de qualidade para a dosimetria da irradiação de sangue e de hemocomponentes. / The Transfusion Associated Graft Versus Host Disease (TA-GVHD) is a rare transfusion reaction, however fatal, which develops due to the presence of donor T lymphocytes in the donated blood and that can be avoided by the irradiation of the donated blood blood and blood components prior to transfusion. Thus, the associated quality control of blood irradiation is necessary to guarantee the quality of the transfused product. In this work it is proposed the characterization and adaptation of the response of a Fricke dosimeter to be used for dosimetry of blood irradiation, more especiafically the Fricke Xylenol Gel (FXG). This is a radiochromic chemical dosimeter, which presents advantages to be tissue equivalent and allows the spatial absorbed dose inference. In this manner, in a way to possibilitate the absorbed dose inference in the full interval used for the prevention of the TA-GVHD (25 to 50 Gy) the FXG was characterized and adapted for dosimetry applications involving blood and blood components irradiation. The results with the new dosimeter showed adequability for the necessary absorbed doses, with satisfactory sensibility and time fading for routine applications. Furthermore, according to the methodology proposed in this work, it was possible to determine the spatial absorbed dose distributions with the new dosimeter in an fast and simple way, showing that this dosimeter presents convenient characteristics for dosimetry quality control of irradiated the blood and blood components.

A boca é local de frequentes complicações relacionadas ao transplante de células tronco hematopoiéticas (TCTH) tais como xerostomia, disgeusia, disfagia, mucosite, infecções oportunistas e doença do enxerto contra hospedeiro (DECH). Sabe-se que estas complicações podem comprometer a qualidade de vida do paciente e interferir na morbidade pós-TCTH. O dentista é o profissional da saúde que deverá intervir no momento correto para tratar e minimizar esses efeitos secundários do TCTH. Para tanto é importante conhecermos o momento em que cada complicação ocorre para que a intervenção seja pronta e eficiente. O objetivo principal deste estudo foi identificar e quantificar as alterações bucais em indivíduos submetidos ao TCTH em cinco momentos consecutivos desde antes do início do condicionamento pré-TCTH até o dia 100 pós-TCTH. Como objetivos secundários buscamos investigar possíveis relações entre a severidade da mucosite oral e a manifestação da DECH com dados demograficos (sexo, idade), com o status de saude bucal (por meio dos índices IHO-S, CPOD, número de dentes cariados) e com a realização de adequação bucal pré-TCTH, e ainda, somente para a DECH, também foi investigada a possível relação entre esta doença com infecção sistêmica por citomegalovírus e com a manifestação de mucosite oral severa. Foram incluídos no estudo 27 indivíduos com doenças hematológicas do Serviço de Transplante de Medula Óssea do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), com idade 12 anos que receberam TCTH alogênico. Os indivíduos foram examinados em cinco momentos consecutivos. No primeiro momento, pré-TCTH, foi realizada a coleta de índices de saúde bucal e aplicação de questionário sobre o histórico de tratamentos odontológico prévios. Nos momentos de 10, 20, 60 e 100 dias pós-TCTH foram avaliadas as manifestações bucais presentes. A incidência de mucosite oral foi de 82,6% considerando todos os momentos avaliados. Mucosite oral severa, ou seja, graus 3 e 4 (OMS) foi observada em 57,9% dos pacientes avaliados nos momentos 2 e 3. Dez (37%) pacientes apresentaram GVHD em algum órgão, e destes, 8 (80%) apresentaram GVHD de boca. Infecção sistêmica por CMV foi diagnosticada em 6 (22,2%) pacientes. Concluímos que entre as queixas levantadas, dor bucal e disfagia foram as mais referidas. O período de maior incidência das complicações bucais foi nos segundo e terceiro momentos, ou seja, D+10 e D+20, representando deste forma, o período de maior morbidade do tratamento. Não houve associação entre a severidade de mucosite oral e idade, sexo, fonte de células, regime de condicionamento, número de dentes cariados, IHO-S, CPOD e preparo bucal pré-TCTH. Para a DECH a única relação encontrada foi para fonte de células, tendo sido observada menor chance de ocorrer DECH quando a fonte de células foi o sangue periférico. / The mouth is a well-known site of complications of the hematopoietic stem cell transplantation (HSCT) such as dry mouth, dysgeusia, dysphagia, mucositis, opportunistic infections and graft versus host disease (GVDH). It is known that these complications can compromise the patients quality of life and morbidity post-HSCT. The dentist is the health professional who should interfere at the right time to treat and minimize these side effects of HSCT. Thus, it is important to know the time at which each complication occurs to be dynamic and efficient. The main objective of this study was to identify and quantify the oral complications in patients treated with HSCT in five consecutive moments starting before conditioning chemotherapy until day 100 post-HSTC. As secondary objectives we seek to investigate possible relationships between the severity of oral mucositis and the manifestation of GVHD with demographic data (gender, age), with the oral health status (IHO-S, CPOD, number of decayed teeth) and dental treatment previously HSCT, and, only for GVHD, was also investigated the possible relationship between this disease with systemic cytomegalovirus infection and the manifestation of severe oral mucositis. It was included in the study 27 patients with hematologic diseases who were admitted in the Unit of Bone Marrow Transplantation, Hospital of Clinics, Faculty of Medicine, University of Sao Paulo (HC-FMUSP), 12 years old whom received allogeneic HSCT. The subjects were examined in five consecutive moments. At the first moment, before HSCT, the oral health índex evaluation and a questionnaire about history of previous dental treatments were performed. Besides that, 10, 20, 60 and 100 days after HSCT they were evaluated for oral manifestations. Oral mucositis incidence was 82,6% and 57,9% of these patients presented severe mucositis. Ten (37%) patients had GVHD in any organ, and of these, 8 (80%) had oral GVHD. Infection by CMV was diagnosed in 6 (22.2%) patients. We conclude that among the complaints raised, mouth pain and dysphagia were the most mentioned. The period of increased incidence of oral complications was the second and third times (D +10 and +20), representing the increased morbidity period. There was no association between the severity of oral mucositis and age, sex, cell source, conditioning regimen, number of decayed teeth, IHO-S, CPOD and dental treatment pre-HSCT. For GVHD the only relation found was with source of cells, in which, GVHD was less likely to occur when the source of cells was peripheral blood.

A doença do enxerto contra o hospedeiro (DECH) é uma das maiores causas de mortalidade e morbidade pós-transplante de células-tronco hematopoiéticas. A DECH, em sua manifestação crônica (DECHc), ainda não tem sua fisiopatologia totalmente esclarecida; entretanto, o envolvimento do sistema imunológico, por meio de respostas imunes inatas e adaptativas é bem estabelecido na literatura. A DECHc afeta múltiplos órgãos, incluindo as glândulas salivares, o que tem, como causa imediata, a xerostomia. Essas alterações são largamente desconhecidas e sub-relatadas. Assim, esse estudo indagou se o sintoma de xerostomia na DECHc é decorrente de alterações morfológicas e funcionais das glândulas salivares. Para responder essa pergunta analisamos de forma qualitativa e por meio da morfometria, espécimes de biopsias de glândulas salivares labiais de pacientes com DECHc e com sintoma de xerostomia. Foram utilizados como controles, espécimes de biopsias de glândulas salivares de pacientes diagnosticados com Síndrome de Sjögren (SSp) (que é um modelo clássico de xerostomia) e de pacientes com líquen plano oral (LPO) (cujas manifestações clínicas orais podem se assemelhar às da DECHc) e queixa de xerostomia. Também foram analisadas, por meio de ensaio multiplex, as citocinas relacionadas à resposta imune Th17 (importante via imunológica na patogenia da DECHc) na saliva de 21 pacientes de DECHc, 27 de SSp, 10 de LPO e 15 voluntários saudáveis. Os principais achados morfológicos nas glândulas salivares dos pacientes de DECHc foram a extensa fibrose, fibroplasia periductal, atrofia ductal e acinar; alterações vasculares representadas pela congestão e formação de trombos hialinos; e infiltrado inflamatório intersticial difuso de aspecto leve a moderado. As glândulas salivares de SSp, entretanto, apresentaram um infiltrado inflamatório na forma de focos de linfócitos ao redor dos ductos excretores de intensidade moderada a severa; os ductos excretores apresentaram-se atróficos, ectásicos, exibindo metaplasia oncocítica e fibroplasia periductal; as alterações vasculares, por sua vez, se apresentaram em maior proporção na forma de vasculite. No LPO as alterações teciduais foram menos intensas. Quando analisadas as concentrações das citocinas, na DECHc, foram encontradas maiores concentrações de IL-17A, IL-4, IL-17F e IL-10, em relação aos grupos controles, essas citocinas estão envolvidas em mecanismos prófibróticos, o que permitiu a correlação dessa expressão aos eventos escleróticos nas glândulas salivares dos pacientes de DECHc. Entre elas, a IL-17F apresentou uma tendência de aumento em relação a proporção da área de fibrose nas glândulas salivares destes pacientes. A CD40L, que também esteve presente em maior concentração nos pacientes de DECHc, é uma molécula de ligação capaz de amplificar a resposta imunológica no mecanismo de rejeição do enxerto no hospedeiro, além de regular o mecanismo de apoptose e ativar o endotélio para a formação de trombos. As citocinas IL-31, IL-23 e IL-22, também apresentaram relevância na saliva dos pacientes de DECHc, sendo participantes do mecanismo das alterações liquenóides no LPO. Através das análises comparativas foi possível correlacionar a presença de citocinas envolvidas na resposta Th17 com as alterações glandulares e consequente xerostomia nos pacientes de DECHc. / The graft versus host disease (GVHD) is one of the biggest causes of mortality and morbidity after hematopoietic stem cells transplantation. The pathophysiology in the chronical manifestation of the disease (cGVHD), is not entirely elucidated, however the involvement of the immunological system, by means of the innate and adaptive responses are depicted in the literature concerning the disease development. The cGVHD affects multiple organs, including the salivary glands, leading to xerostomia. These alterations are under reported and mostly unknown. Therefore, this study investigated if the symptom of xerostomia in cGVHD is triggered by functional e morphological changes in minor salivary glands. To answer this inquiry specimens of biopsied labial salivary glands from patients of cGVHD and xerostomia were analyzed qualitatively and through morphometry. Specimens of biopsied salivary glands from patients with Sjögren\'s Syndrome (SS) (which is a classic model of xerostomia) and from patients with oral lichen planus (OLP) (whose clinical oral manifestations resemble the cGVHD lesions) were used as controls. Also, the cytokines related to the immunological response Th17 (important immune pathway in cGVHD pathophysiology) in the saliva of 21 cGVHD patients, 27 of SS, 10 patients of OLP and 165 healthy individuals were analyzed using the multiplex assay. The major morphological findings revealed on the salivary glands of cGVHD patients were the extensive fibrosis, periductal fibrosis, ductal and acinar atrophy. Congestion and hyaline thrombi formation were the most important vascular changes shown among these specimens. A diffuse interstitial inflammatory infiltrate was observed, with varied intensity. The SS salivary glands, however, portrayed a focal inflammatory infiltrate, with moderate to severe intensity around the excretory ducts. These ducts exhibited atrophy, ectasia, periductal fibrosis and oncocytic metaplasia. The main vascular change presented in these patients was the manifestation of vasculitis. The salivary glands from the OLP patients showed a lesser amount of alterations. The multiplex assay revealed a higher concentration of the cytokines IL-17A, IL-4, IL-17F and IL-10 in the cGVHD samples, when compared to the other groups. These cytokines are involved within the promotion of fibrosis, which endorsed the association of these secretions with the salivary glands sclerotic mechanisms. The secretion of CD40L was higher in cGVHD samples; this membrane protein is capable of amplifying the immunological response in graft rejection, besides the capacity to regulate apoptosis and activate the endothelium in thrombi formation. The cytokines IL-31, IL-23 and IL-22, also presented a higher concentration in cGHVD patients\' saliva, these secretions are actively involved in the mechanisms of lichenoid lesions in OLP, corroborating the perceived morphological changes. The comparative analysis of the morphological and salivary changes in cGVHD confirmed the correlation of Th17 immunological response within the minor salivary glands injuries and consequent xerostomia in these patients.

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.