Die akute Graft-versus-Host Erkrankung (GvHD), insbesondere die Darm GvHD, stellt weiterhin eine der Hauptursachen für Mortalität und Morbidität nach allogener SZT dar. Aktivierte, alloreaktive Spender T-Zellen infiltrieren dabei über die Blutbahn die intestinale Lamina Propria. Erst kürzlich konnten wir zeigen, dass neben der vaskulären Migration ein Teil der Spender T-Zellen auch direkt aus den PP in die angrenzende Lamina Propria migrieren. Um Faktoren, die diese direkte Migration fördern, zu untersuchen und die direkt migrierenden T-Zellen genauer zu charakterisieren, verwendeten wir ein MHC-inkompatibles Mausmodell zur Induktion einer akuten GvHD. Durch RNA Sequenzierung und Massenspektrometrie lasermikrodissezierter Darmschleimhautproben konnte eine starke Expression der Chemokine CXCL9, CXCL10, CXCL11, CCL3, CCL4 und CCL5 während der akuten intestinalen GvHD aufgezeigt werden. Neben CCL4 und XCL1 wiesen verschiedene Faktoren der T-Zellaktivierung, wie CD3ζ, LAT, Lck und ZAP70, sowie Faktoren der zytoskelettalen Reorganisation, wie Dock2, Coro1α und Parvin-γ, eine vermehrte Expression insbesondere nahe der PP auf. Die Expression der migrationsfördernden Faktoren Coro1α und Parvin-γ in Spender T-Zellen nahe der PP konnte anschließend mittels histologischen Immunfluoreszenzfärbungen bestätigt werden. Durchflusszytometrische Analysen konnten weiterhin eine vermehrte Expression von CCR5, CCR9 und Intgerin α4β7 auf den vornehmlich Tbet+ Spender T-Zellen nahe der PP nachweisen. Funktionelle in vitro Migrationsversuche zeigten abschließend, dass in vivo aktivierte Spender T-Zellen eine gerichtete Migration in Richtung auf CXCL11 und zu späterem Zeitpunkt auch auf CCL4 vollziehen können. Zusammenfassend zeigt diese Arbeit die Bedeutung zahlreicher Chemokine für das sequenzielle T-Zell-Homing während der akuten intestinalen GvHD. Neben der insbesondere durch Faktoren der zytosekeletalen Reorganisation vermittelten amoeboiden Migration kann auch eine mesenchymale Fortbewegung über Faktoren wie CCR5, CCR9 und Integrin α4β7 die direkte Migration der T-Zellen fördern. Den direkt migrierenden vornehmlich TH1 polarisierten Zellen folgen weitere, CD27 und Integrin αLβ2 exprimierende, zytotoxische T-Zellen aus der Blutbahn. Die direkt migrierenden Zellen könnten als Initiator und Potentiator der intestinalen T-Zell Infiltration wirken und müssen für zukünftige therapeutische Strategien nicht nur der Darm GvHD, sondern der intestinalen Inflammation im Allgemeinen mitberücksichtigt werden. / Acute graft-verus-host disease (GvHD), especially intestinal GvHD, remains one of the main causes of mortality and morbidity after allogeneic hematopoietic stem cell transplantation. In this process activated alloreactive donor T cells infiltrate the intestinal lamina propria via the bloodstream. Our group could recently show that besides the vascular migration route some donor T cells migrate directly from the Peyer’s patches into the adjacent lamina propria. To investigate factors that could promote such a direct migration, and to characterize these direct migrating T cells we applied a major mismatch mouse model to induce acute GvHD. Using RNA sequencing and mass spectrometry of lasermicrodissected lamina propria samples, we detected a strong upregulation of the chemokines CXCL9, CXCL10, CXCL11, CCL3, CCL4 and CCL5 during acute intestinal GvHD. Alongside CCL4 and XCL1, several factors of T cell activation, such as CD3ζ, LAT, Lck und ZAP70, as well as factors of cytoskeletal reorganization, such as Dock2, Coro1α und Parvin-γ, showed higher expression near the Peyer’s patches. Subsequently, we validated the expression of Coro1α and Parvin-γ on donor T cells near the Peyer’s patches with histological immunofluorescence stainings. Flow cytometry analysis further revealed high expression of CCR5, CCR9 and Intgerin α4β7 on the predominantly Tbet+ donor T cells near the Peyer’s patches. Conclusively, in vitro migration assays showed that in vivo activated donor T cells can directly migrate towards CXCL11 and subsequently also towards CCL4. The present study shows the relevance of several chemokines for the sequential T-cell homing during acute intestinal GvHD. Besides the amoeboid migration mode, which is particularly driven by cytoskeletal reorganization, a mesenchymal movement using factors, such as CCR5, CCR9 and Integrin α4β7, can promote the direct migration of donor T cells. The directly migrating cells, which are predominantly of a TH1 phenotype, are followed by cytotoxic T cells, expressing CD27 and Integrin αLβ2 (LFA-1), from the systemic circulation. Thus, these directly migrating cells may act like an initiator and potentiator for the intestinal T cell infiltration and must be considered for new therapeutic strategies not only of GvHD but of intestinal inflammation in general

No description available.

L’allogreffe de cellules hématopoïétiques est une stratégie thérapeutique importante dans les hémopathies malignes. La maladie du greffon contre l’hôte (GvH) en est une complication majeure menaçant le pronostic vital. Elle est due à la reconnaissance des antigènes du receveur par les lymphocytes T du donneur et à l’activation de ceux-ci, à l’origine de dommages tissulaires. L’altération de la barrière intestinale joue un rôle critique dans la GvH. La famille des protéines de choc thermique (HSP)90 comporte cinq membres dont trois cytosoliques dénommés HSP90, et un localisé dans le réticulum endoplasmique (RE), Gp96, qui peut être sécrété en cas de stress. Nous montrons dans nos travaux de thèse que la 17AAG, un inhibiteur des HSP90, réduit la mortalité liée à la GvH dans un modèle murin. Cet effet est associé à une augmentation de la réponse au stress du RE dans les cellules épithéliales intestinales comme en atteste l’augmentation de l’épissage du facteur de transcription XBP-1, corrélée à une diminution du dommage tissulaire intestinal. Ces résultats permettent d’envisager une place pour la 17AAG ou d’autres inhibiteurs de HSP90 dans la prévention de la GvH chez l’homme. D’autre part, nous montrons que Gp96 est sécrétée dans le sérum de patients développant une GvH aiguë sévère avec atteinte intestinale. Nous suggérons de valider la pertinence de Gp96 comme biomarqueur de GvH intestinale dans une étude de plus grande ampleur. Enfin, nous trouvons que Gp96 s’associe avec le composant 3 du complément, une protéine impliquée dans l’immunité innée et adaptative, et inhibe certaines de ses fonctions. Les conséquences fonctionnelles de cette association sont discutées. / Allogeneic hematopoietic cell transplantation is a treatment for certain disorders including hematologic malignancies. Graft-versus-host-disease (GvHD) is a major, life-threatening complication. It is due to the recognition of recipient antigens by donor T cells, which activate and damage tissues. Intestinal barrier alteration plays a critical role in GvHD. Heat shock proteins (HSP)90 include five members, three cytosolic members named HSP90, and one member localized in endoplasmic reticulum (ER) called Gp96 and able to gain extracellular level in case of stress. We show in our thesis that 17AAG, a HSP90 inhibitor, reduces GvHD mortality in a mouse model. This effect is associated with an increase in ER stress pathway in intestinal epithelial cells as figured by transcription factor XBP-1 splicing, correlated to a decrease in intestinal tissue damage. These results suggest that 17AAG could be considered in GvHD prevention in human. Moreover, we show that Gp96 is secreted in serum of patients developing an acute GvHD with intestinal involvement. We propose to validate the relevance of Gp96 as an intestinal GvHD biomarker is a larger study. Finally, we find that Gp96 associate with complement component 3, a protein involved in innate and adaptive immunity, and inhibit some of its functions. Functional consequences of this association are discussed.

Indiana University-Purdue University Indianapolis (IUPUI) / Graft-versus-host disease (GVHD) remains the major complication after allogeneic hematopoietic stem cell transplantation (HSCT), resulting from immunological attack on target organs such as gastrointestinal (GI) tract, liver and skin from donor allogeneic T cells. The most common treatment for GVHD is immunosuppressive drugs such as corticosteroids, which may result in many side effects including the loss of the beneficial graft-versus-leukemia (GVL) effect and increased infection rates. However, GVHD-specific drugs have yet to be implemented. Here we show that by targeting on a novel pathogenic CD4+ T cell subpopulation that our lab previously found in patients with GI GVHD, we can develop new avenues to treat GVHD. This novel population is characterized as CD146+CCR5+ T cells, co-expressing IL-17A and IFN-γ. We found that the inducible T-cell costimulator (ICOS), which has been reported to be important for human Th17 differentiation in vitro, is critical for the development of this nonconventional T Helper 1 (Th1*)-polarized CD146+CCR5+ conventional T cells (Tconvs) population. Furthermore, we found that ICOS can induce the generation of Th1*-polarized CD146+CCR5+ regulatory T cells (Tregs) population, lowering the frequencies of phenotypic markers of functional Tregs. Our data also showed that inhibiting the major transcriptional factor of Th17, RAR-related orphan receptor gamma t (RORγt), could prevent the development of CD146+CCR5+ Tconvs in vitro. Our results demonstrate how pathogenic CD146+CCR5+ T cells are induced through ICOS or RORγt, suggesting new targets for GVHD treatment. We anticipate our assay to be a starting point for the development of novel GVHD-specific drugs. For example, the treatments that focus on inhibiting RORγ would have fewer side effects than general immunosuppressive drugs that GVHD patients use today and inhibit GVHD while sparing the GVL effect. Furthermore, we expect the CD146+CCR5+ Tconvs and/or Tregs can be used as GVHD biomarkers. These biomarkers may guide preemptive treatments such as RORγt inhibitor.

Allogeneic haematopoietic stem cell transplantation (alloHSCT) is an established therapy for many haematological disorders. Unfortunately, the new donor-derived immune system may damage host cells (graft-versus-host disease (GvHD)), causing significant morbidity and mortality. Since regulatory T cells (Tregs) can modulate immune responses, it was hypothesised that Treg numbers in the haematopoietic stem cell grafts and/or peripheral blood may influence the development of GvHD and other transplant-related complications. In this project, a prospective observational clinical study of putative Tregs in human alloHSCT was performed in Oxford. Flow cytometry and methylation-specific qPCR assays were developed to quantify putative Tregs and lymphocyte populations within the grafts and post-transplant blood samples. Although low CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell numbers were not associated with increased incidence of GvHD, low proportions of CD25(+)FOXP3(+)CD127(-/dim) cells in the graft (as a percentage of total CD4(+) T cells) were independently associated with poor engraftment, increased non-relapse mortality and inferior overall survival. Similarly, falling CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell counts over the first three months post-transplant were associated with higher non-relapse mortality and inferior overall survival. In view of these novel findings, strategies that increase CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T cells in alloHSCT may improve clinical outcomes. One possible route for increasing Tregs is through cellular therapy. This project therefore tested the hypothesis that CD4(+)CD25(+)FOXP3(+) Tregs can be produced in vitro from conventional CD4(+) T cells. In the presence of TGFβ and Azacitidine, FOXP3 was expressed in the majority of activated CD4(+) T cells. These cells also had a demethylated FOXP3 TSDR enhancer which is specific to natural Tregs. However, most of these cells produced pro-inflammatory cytokines, for example, TNFα. Therefore, under these conditions, FOXP3 expression was not sufficient to produce a Treg phenotype. It is proposed that current focus for generating Tregs for human clinical trials should be directed towards improving isolation and expansion of ex vivo isolated Tregs.

The goal of hematopoietic stem cell transplantation (HSCT) is to induce graft-versus-tumor effect (GVT), which is the recognition of and response against tumor- associated antigens (TAAs) by donor immune cells to clear the recipient of residual tumor. A complication of HSCT as a treatment for hematologic malignancies is graft-versus-host disease (GVHD), which is the recognition and reactivity of donor immune cells against healthy tissues. As of now, the differentiation between GVHD and GVT effects has been a hindrance to the development of effective therapies against GVHD. Certain T cell clones may induce both GVHD and GVT effects, making targeted therapy of GVHD difficult. This project was aimed to uncover differences at a molecular level of the T cell recognition site that exist between patients with GVHD and those with GVHD-free survival following allogeneic HSCT. We found that there are inherent differences in the T cell receptor at a molecular level between patients experiencing GVHD and those that are GVHD-free, suggesting the ability of T cells to distinguish tumor cells from self cells. In addition, the intention was to reveal differences in proportions of engrafted donor T cells and stem cells and the effects of these proportions on the severity, outcome, and prognosis of GVHD. We additionally found that a lower proportion of stem cells to T cells was associated with the trend of GVHD, while a higher frequency of T cells engrafted into host may indicate resistance to treatment and a poor prognosis. These data suggest that allogeneic HSCT may be improved by optimizing the proportion of T cells to stem cells in the transplant as well as developing targeted therapy against GVHD-associated T cell clones while rescuing GVT-associated T cell clones.

Introduction: Chronic graft-versus-host disease (cGVHD) is a serious complication following allo-HSCT characterized by immune dysregulation, organ dysfunction, risk for infection, and distressing symptoms. Complications may include scleroderma, hepatic dysfunction and bronchiolitis obliterans. Advances in allo-HSCT for many hematologic dyscrasias (e.g. acute and chronic leukemias, aplastic anemia, and myelodysplastic syndrome) have improved survival which has generated a renewed focus on survivorship issues. Distressing symptoms are noted as negatively impacting quality of life (QoL). The relationship between inflammation and behavioral responses may impact symptoms. Examining patterns and levels of inflammation with symptoms is relevant. Objective: The aims of this study were to examine: 1) levels of symptoms (cGVHD specific, general symptoms, and cluster symptoms [pain, depression and fatigue]), inflammation (cytokines [Interleukin {IL}-1β, IL-6, IL-10, TNF, and INF-γ] and C-reactive protein [CRP]) and QoL in patients diagnosed with cGVHD and 2) relationships between and among symptoms, inflammation and QoL in individuals with cGVHD. Methods: A cross-sectional study design examined 24 individuals (ages 29-79) with cGVHD enrolled from an NCI-designated cancer center after obtaining informed consent. Data were collected using medical record and validated questionnaires. Plasma cytokine levels were measured using BioRad® multiplex assay. C-reactive protein levels were measures using an enzyme-linked immunosorbant assay. Statistical analyses included descriptive statistics and pairwise correlations. Results: A total of 24 participants (58.3% female) with cGVHD enrolled in this study. Multiple, concurrent symptoms were noted. Several pro-inflammatory cytokines were higher in participants with symptoms versus those without symptoms. IL-6 correlated with lack of energy (r= .42; p= .04) and dry mouth (r= .42; p= .04). IL-10 was correlated with difficulty sleeping (r= .43; p= .03). Sexual dysfunction correlated with social well-being (r= -.44; p=.03). Many symptoms negatively correlated with QoL. Conclusion: Findings from this study, one of the first to examine levels of symptoms and inflammatory markers in individuals with cGVHD, demonstrate significant relationships among symptoms, inflammation, and quality of life. The relationship of inflammatory biomarkers with symptoms emphasizes the need for further interdisciplinary research. Better understanding mechanisms associated with symptoms is necessary for the development and testing of targeted interventions to improve QoL for individuals with cGVHD.

Doctor of Philosophy / Department of Anatomy and Physiology / Mark L. Weiss / Graft Versus Host Disease (GVHD) is the major complication following hematopoietic stem cell transplantation. GVHD is activated by immunocompetent T cells presented in the donor grafted tissue. Due to the increased use of bone marrow transplantation to treat diverse malignancies, the incidence of GVHD has shown a notable increase. Depending of the degree of immunological mismatch between donor and host, 50-70% of patients develop GVHD after allogeneic Bone Marrow Transplantation (BMT). Once GVHD develops, mortality reaches up to 50% in humans. Several studies using Mesenchymal Stromal Cells (MSCs) to prevent and treat GVHD have produced controversial results. It is thought that distinct MSCs sources used in those studies might be an important factor that produces different outcomes. For cellular therapy, the most attractive characteristics of MSCs are their reduced immunogenic potential, and their abilities to modulate immune responses. This dissertation addressed the hypothesis that Wharton’s jelly cells (WJCs) would prevent the pathology and death associated with GVHD after BMT. To accomplish this, I created a clinically relevant model of GVHD by transplanting allogeneic bone marrow across minor histocompatibility antigen (HA) barriers in the rat. To enhance alloreactive T-cell stimulation, bone marrow (BM) was co-administered with a fraction of CD8[superscript]+ cells magnetically selected from spleen to induce GVHD. Bone marrow tissue was isolated from a donor rat Fischer 344 (F344, RT1lv) and transplanted into lethally irradiated (10 Gray) Lewis rat (LEW, RT1l). Once GVHD was induced, MSCs derived from umbilical cord WJCs were either co-transplanted at day 0 with bone marrow, or given on day 2 post-BMT intravenously. The prophylactic potential of WJCs in an in vivo GVHD model was assessed as survival time, clinical symptomatology occurrence, and histopathology injuries in target tissues. Results indicate that while co-administration of WJCs with hematopoietic cells on day 0 failed to alleviate GVHD associated symptomatology and mortality. WJCs administered on day 2 post-induction ameliorated GVHD-associated symptomatology, improved engraftment and survival.

Thesis (doctoral)--Katholieke Universiteit te Nijmegen.

Orientador: Ana Gabriela Sálvio / Resumo: A Doença Enxerto Contra o Hospedeiro (DECH) é uma das principais complicações em pacientes pós transplante de células tronco hematopoiéticas (TCTH), onde as células T do doador agridem os tecidos do receptor resultando em uma das principais causas de mortalidade e morbidade. Ocorre em cerca de metade dos pacientes que realizaram transplante de células tronco hematopoiéticas (TCTH). A pele é um dos órgãos mais acometidos. A DECH é dividida da forma aguda (DECH-a) e crônica (DECH-c), de acordo com o tempo de aparecimento dos achados clínicos e histopatológicos. A DECH de pele é classificada em graus variados, de I a IV, de acordo com o comprometimento. O presente estudo teve como objetivo avaliar a incidência de DECH-a cutânea entre os pacientes submetidos ao TCTH alogênico, aparentado, no Serviço de Transplante de Medula Óssea do Hospital Amaral Carvalho em Jaú/SP. Também foi objetivo do estudo a análise do perfil destes pacientes, a incidência e o impacto da doença na mortalidade precoce nos 100 primeiros dias pós TCTH. Os procedimentos metodológicos envolveram análise retrospectiva de dados retirados de planilhas do setor administrativo do Serviço de Transplante de Medula Óssea do Hospital Amaral Carvalho em Jaú/SP, contendo informações de 1113 pacientes que estiveram internados para a realização de transplante de medula óssea alogênico, aparentado, no período de agosto de 1996 a dezembro de 2013. Foram incluídos 582 pacientes cujas as doenças eram de linhagem mielóide - as ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre