Le système immunitaire joue un double rôle dans le cancer : il peut non seulement supprimer la croissance tumorale en détruisant les cellules cancéreuses, mais aussi promouvoir la progression de la tumeur en sélectionnant les cellules tumorales ou en créant un microenvironnement tumoral immunosuppresseur. Notre idée principale est de développer des stratégies pour mieux comprendre l'immunologie du cancer colique.Au cours de ma thèse, je me suis tout d'abord intéressée à une population du système immunitaire : les MDSC (Myeloïd Derived Suppressor Cells). Nous avons exploré des stratégies pour réduire le nombre de ces cellules au cours de la croissance tumorale. Nous avons pu découvrir que de petites doses de 5 fluorouracil sont capables d'induire spécifiquement une mort par apoptose des cellules myéloïdes suppressives. Nous avons ainsi caractérisé un effet immunologique positif nouveau du 5-fluorouracil. Cet effet immunologique contribue à l'effet antitumoral du 5-fluorouracil chez la souris. Dans une deuxième partie nous avons étudié le rôle pronostic des infiltrats immunitaires dans une série de patients présentant un cancer du côlon avec des métastases hépatiques. Nous avons étudié le rôle pronostic des infiltrats en cellules CD8, CD45R0 et Foxp3. Nous avons mis en évidence que la présence d'un fort infiltrat en cellules CD45RO et Foxp3 est un facteur de bon pronostic. L'association des 2 marqueurs permet de définir 3 groupes pronostics et ainsi d'individualiser un groupe de mauvais pronostic ne bénéficiant probablement pas de la chirurgie hépatique.

Psoriasiform diseases are a group of cutaneous disorders that are characterized by impaired keratinocyte maturation leading to epidermal hyperplasia and thickening of skin. This group of disorders includes psoriasis, seborrheic dermatitis (SD) and mycosis fungoides (MF). Psoriasis has been recently shown to be mediated by the pro-inflammatory T helper cell subset, namely Th17 cells, whereas the pathogenesis of SD and MF are still poorly understood. SD is characterized by inflamed skin that primarily manifests on areas populated with sebaceous glands. Interestingly, SD is very common amongst immunosuppressed patients such as those with HIV-AIDS, suggesting the importance of an immune response in the development of SD. Because SD shares common clinical and histopathological features with psoriasis, a disease in which Th17 axis cytokines is known to be involved, and given that Th17 cells and their related cytokines have been implicated in the pathogenesis of a wide range of autoimmune and inflammatory disorders, it is possible that Th17 axis cytokines play a role in the pathogenesis of SD. We explored the involvement of Th17 axis cytokines in a D2C mouse model of psoriasiform disease that shows a high degree homology to the clinicopathological characteristics of human seborrheic dermatitis. IL-6 and IL-23, which are important for the differentiation of Th17 cells, and IL-17 and IL-22, which are the Th17 effector molecules, were measured at both protein and mRNA levels in sera and lesional skin from D2C mice. An immunohistochemical analysis was also performed to detect the presence of IL-17 in D2C lesional skin relative to normal skin from DBA/2 controls. Our data demonstrated significantly elevated levels of IL-6, IL-17 and IL-22 in sera from diseased D2C mice compared to controls and/or convalescent mice. There were no significant differences in IL-23 protein levels in sera from D2C mice compared to those from wild type mice or convalescent D2C mice. RT-PCR revealed a significant increase in IL-23 and IL-17 gene expression in D2C lesional skin relative to normal skin. Gene expression levels of IL-22, but not IL-6, were statistically significant elevated in D2C skin lesions compared to controls, by real time PCR. Our IHC study of IL-17 expression showed an abundance of positively stained mononuclear cells in D2C lesional skin relative to DBA/2 normal skin. Altogether, our data demonstrate that Th17 axis cytokines are elevated locally at mRNA levels for IL-23, IL-17, and IL-22 and systematically at protein levels for IL-6, IL-17, and IL-22. This data lay the foundation for further studies investigating a role for Th17 axis cytokines in the cutaneous inflammatory disease seen in our mouse model of SD and, ultimately, in the development of human SD. Mycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma (CTCL). The etiology of MF is unknown, but there is substantial evidence suggesting a potential role for a yet unidentified infectious agent in the pathogenesis of MF. Many studies have claimed that there is an association between MF and the Human T cell Lymphotorpic Virus Type 1 (HTLV-I); however, the involvement of this virus in the etiology of MF is a controversial topic. In our study, we used nested PCR to explore the association between HTLV-I infection and MF by screening genomic DNA from 114 skin biopsies for the presence of HTLV-I provirus. We also utilized a ViroChip and high-throughput sequencing (HTS), as a case study, to attempt to detect novel virus-specific oligonucleotides that may be associated with CTCL. Our data showed no evidence for HTLV-I proviral integration in the 114 MF samples that were screened using nested-PCR. The ViroChip and HTS results also did not reveal any signature sequence for known or unknown infectious agent in the CTCL case study. Collectively, this data argue against the involvement of HTLV-I provirus in the pathogenesis of MF.

Epstein-Barr virus (EBV) infects almost all humans and establishes lifelong latency in B cells. Posttransplant lymphoproliferative disorder (PTLD) is a rare but serious complication after transplantation triggered by immunosuppression and often related to EBV infection. The aim of this thesis was to study the role of EBV in relation to clinical and histological features of PTLD, regulatory T cells (Tregs), and donor or recipient origin of PTLD. EBV surveillance after allogeneic hematopoietic stem cell transplantation (allo-HSCT) showed that EBV reactivations were common, but that symptomatic EBV disease (including PTLD) only occurred in the high-risk group (unrelated or mismatched related grafts, reduced-intensity conditioning). A threshold of 1000 copies/ml plasma distinguished EBV disease from asymptomatic reactivations. In a population-based cohort of 135 PTLDs/lymphomas after solid organ transplantation (SOT) almost half were EBV–. EBV+ PTLDs were associated with B cell phenotype, non-germinal center subtype of diffuse large B cell lymphoma (DLBCL), early-onset, graft involvement, antithymocyte globulin treatment, and younger age. EBV– PTLDs were associated with T cell phenotype, bone marrow involvement, and hepatitis C. Most PTLDs displayed few or no intratumoral Tregs with the marker FoxP3, possibly due to heavy immuno­suppres­sion. Half of both FoxP3+ and FoxP3– PTLDs were EBV+. FoxP3+ PTLDs were associated with B cell phenotype and hepatitis C. All PTLDs for which tumor origin could be determined were recipient-derived and half of them were EBV+. Eight of twelve recipient-derived graft PTLDs were disseminated outside the graft. T cell PTLD and hepatitis C were independently associated with inferior overall survival, whereas subtype of DLBCL, FoxP3-expression, and EBV-status did not influence survival. In conclusion, monitoring of EBV DNAemia in high-risk patients after allo-HSCT and pre-emptive therapy is valuable for prevention of PTLD. Use of anti­thymocyte globulin increases the risk for EBV+ PTLDs after allo-HSCT and SOT. With long follow-up time, a large proportion of PLTDs after SOT are EBV– with a different clinical presentation. Tregs are rare in PTLD and do not affect survival. The vast majority of PTLDs after SOT is of recipient origin. Graft PTLDs are more likely recipient-derived if disseminated. EBV-status is not associated with intratumoral Tregs or PTLD of recipient origin.

Head and neck squamous cell carcinomas (HNSCC) remain a significant cause of morbidity worldwide, with approximately 550,000 new cases diagnosed each year. The main etiological factors include smoking and alcohol consumption. The incidence of non-oropharyngeal HNSCC is gradually decreasing while the incidence of squamous cell oropharyngeal carcinoma (OPSCC) is still on the rise. This increasing incidence can be most likely attributed to an increasing prevalence of human papillomavirus (HPV) infection. From the clinical point of view the most significant fact is that patients with HPV positive OPSCC have better prognosis. HNSCC is linked to an alteration in the immune system. Only a limited number of studies have correlated both the immunological parameters and HPV status with patient prognosis. Therefore, we focused on the research of the immunological profile of patients with HNSCC of viral and non-viral etiology. In our study, 110 patients with HNSCC were enrolled. They were divided into HPV-positive and HPV-negative groups based on the expression of HPV 16 E6 mRNA detected in the tumor tissue. Basic lymphocyte subpopulations (CD3+, CD4+ CD25+ Treg, CD4+ CD25+ FoxP3 Treg, CD4+, CD8+, CD19, and CD3- CD16+ CD56+ cells) were determined by flow cytometry in the peripheral blood (PB). We observed...

A Artrite Reumatóide (AR) é uma artropatia autoimune multifatorial com etiologia desconhecida que afeta aproximadamente 1% da população adulta. A estratégia padrão para o tratamento da AR consiste na administração de baixas doses de Metotrexato (MTX), cujo efeito anti-inflamatório está relacionado com a manutenção dos níveis elevados de adenosina (ADO) extracelular. No entanto, uma parte considerável dos pacientes com AR é refratária ao tratamento com MTX e o mecanismo pelo qual este fenômeno ocorre ainda não está totalmente esclarecido. Neste contexto, o presente estudo descreveu que a eficácia terapêutica ao MTX está associada com a expressão em células Tregs da ectoenzima CD39, cuja função biológica é a geração de ADO extracelular via metabolização do ATP. Especificamente, através da realização de um estudo longitudinal, observamos que pacientes respondedores ao MTX (R-MTX) apresentam uma expansão de células Tregs circulantes expressando CD39 após o tratamento com MTX. Por outro lado, identificamos que pacientes não respondedores ao MTX (UR-MTX) possuem uma redução da expressão de CD39 em células Tregs, o que culmina em um comprometimento das suas funções supressoras. Ainda, demonstramos que a expressão de CD39 em células Tregs é um biomarcador apto em predizer a resposta terapêutica ao MTX, visto que pacientes UR-MTX apresentam uma expressão reduzida de CD39 em Tregs mesmo antes do início do tratamento com MTX. Posteriormente, nós investigamos as bases moleculares que acarretam na expressão reduzida de CD39 observada em células Tregs de pacientes URMTX. Demonstramos que a estimulação com TGF-? tanto em células Tregs isoladas quanto diferenciadas in vitro aumenta a expressão de CD39 através da ativação sequencial da seguinte plataforma molecular: receptores de TGF-? (TGFBRII e TGFBRI), transdutor de sinal SMAD2, fator de transcrição CREB, de modo dependente da atividade de p38. Uma vez identificada a via envolvida com a indução da expressão de CD39, demonstramos que células Tregs diferenciadas de indivíduos que apresentam uma expressão reduzida de CD39 são incapazes de induzir a expressão desta ectoenzima através da estimulação com TGF-?. Por fim, transpondo nossos achados para pacientes com AR, observamos que pacientes UR-MTX apresentam uma redução nos níveis de RNAm para TGFBRII e CREB bem como também uma redução das proteínas fosforiladas SMAD2 e CREB em células CD4+ e Tregs, sugerindo que o comprometimento na cascata de sinalização de TGF-?, envolvida com a indução da expressão de CD39 em células Tregs, está associado com a resistência ao MTX. / Rheumatoid arthritis (RA) is an autoimmune multifactorial arthropathy with unknown etiology that affects approximately 1% of the adult population. The standard strategy for RA treatment comprises the administration of low doses of methotrexate (MTX), whose antiinflammatory effects are associated with maintenance of high levels of extracellular adenosine (ADO). However, a considerable proportion of RA patients is resistant to MTX treatment and the mechanisms underlying this phenomenon occurs is poorly understood. Within this context, the present study showed that therapeutic efficacy of MTX is associated with expression on Treg cells of the ectoenzyme CD39, whose function is related to the generation of extracellular ADO by ATP metabolism. Specifically, we conducted a longitudinal study and observed that responsive patients to MTX (R-MTX) exhibit an increase in the frequency of circulating Treg cells expressing CD39 after MTX treatment. On the other hand, we found that non-responsive patients to MTX (UR-MTX) have a reduction of CD39 expression on Treg cells, which culminates in an impairment of Treg function. Furthermore, these findings indicate that CD39 expression on Treg cells is a biomarker for therapeutic response to MTX, since UR-MTX patients had a depressed CD39 expression on Treg cells even before MTX treatment. Subsequently, the present study investigated the molecular mechanisms that would cause the reduction of CD39 expression on Treg cells from UR-MTX patients. For this, we demonstrated that TGF-? stimulation increases CD39 expression in isolated and in vitro differentiated Treg cells through participation/activation of the following molecules: receptors of TGF-?, TGFBRII and TGFBRI, signal transducer SMAD2 and transcription factor CREB, through p38 activity dependent-manner. Once identified these molecules involved with CD39 induction, we demonstrated that differentiated Treg cells from healthy individuals with an intrinsic reduction of CD39 expression on circulating Treg cells are unable to increase CD39 expression by TGF-? stimulation. Transposing our findings to RA patients, we found that UR-MTX patients exhibit a reduction of mRNA for TGFBRII and CREB as well as reduction on levels of phospho-SMAD2 and phospho-CREB in CD4+ and Treg cells, suggesting that an impairment in TGF-? signaling pathway, related to induction of CD39 expression on Treg cells, is associated with MTX resistance.

Head and neck squamous cell carcinomas (HNSCC) remain a significant cause of morbidity worldwide, with approximately 550,000 new cases diagnosed each year. The main etiological factors include smoking and alcohol consumption. The incidence of non-oropharyngeal HNSCC is gradually decreasing while the incidence of squamous cell oropharyngeal carcinoma (OPSCC) is still on the rise. This increasing incidence can be most likely attributed to an increasing prevalence of human papillomavirus (HPV) infection. From the clinical point of view the most significant fact is that patients with HPV positive OPSCC have better prognosis. HNSCC is linked to an alteration in the immune system. Only a limited number of studies have correlated both the immunological parameters and HPV status with patient prognosis. Therefore, we focused on the research of the immunological profile of patients with HNSCC of viral and non-viral etiology. In our study, 110 patients with HNSCC were enrolled. They were divided into HPV-positive and HPV-negative groups based on the expression of HPV 16 E6 mRNA detected in the tumor tissue. Basic lymphocyte subpopulations (CD3+, CD4+ CD25+ Treg, CD4+ CD25+ FoxP3 Treg, CD4+, CD8+, CD19, and CD3- CD16+ CD56+ cells) were determined by flow cytometry in the peripheral blood (PB). We observed...

Due to high incidence, medical and socioeconomic burden and impact on individual quality of life and productivity, allergic disorders are a crucial issue for 21st century immunology. Much still remains to be elucidated, particularly regarding the very early processes in allergy development. In order to introduce timely, effective preventive measures, novel, more reliable predictive factors of allergy risk also need to be established. Dysregulation of proper balance between the branches of immune response, particularly unwarranted dominance of Th2, is the underlying cause of allergy. After birth, new immune balance needs to be established to prepare the neonate for adequate reactivity towards newly encountered environmental stimuli. Regulatory T cells (Treg) play a central role in finely setting this balance and inducing tolerance towards harmless environmental antigens, including allergens. Interactions with external factors, most importantly microbiota, modulate this process during the early postnatal "window of opportunity." Analysis of cord blood Treg of children of allergic mothers uncovered decreased presence of function-associated surface markers and lower production of IL-10. Furthermore, decreased proportion of Helios- induced Treg was observed in children with higher risk of allergy....

<p>Inflammatory Bowel Disease (IBD) is a chronic intestinal inflammation that has caused many challenges for healthcare providers in treating the disease and also altered the quality of life of the patients. The cure for IBD is still symptomatic-based; the causes mechanism and pathogenesis of IBD are to be further investigated. Currently, IBD has been considered as an excessive immune response to commensal flora that in normal condition is tolerable to the host. Antigen presenting cells (APCs) play an important role in the pathogenesis of IBD. Macrophage is one of the professional APCs that present antigen information to T cells and induce the T cell subtype proliferation. Aside from this role, macrophages also phagocytose pathogens and clean cell debris in thebody.</p> <p>T cell immunoglobulin and mucin domain (TIM)-4 is a glycoprotein expressed on the surface of macrophage, which recognizes phosphatidylserine (PS) that is expressed mainly on the surface of the early apoptotic cell phospholipid membrane; the latter is a negatively charged molecule that can bind to the TIM-4 to enhance the phagocytosing activity. In IBD, the loss of intestinal epithelial cells (IECs) due to apoptosis is prominent in the site of inflammation especially in ulcerative colitis (UC).</p> <p>The aim of this study is to elucidate whether there is an increase of TIM-4 expression in colitis mice model after exposure to excessive number of apoptotic IECs and whether TIM-4 plays a role in the development of colitis in mice.</p> <p>The expression of TIM-4 is measured with several tests; including flow cytometry, immunohistochemistry, western blotting and real time RT-PCR. In the first step, we tried to see if there is a difference in the TIM-4 expression in colitis mice and ethanol control mice. After the association was established, we further observed the role of TIM-4 in the pathogenesis of IBD by injecting TIM-4+ macrophages into the mice prior to inducing a mild colitis in the mice and finally injected neutralizing anti TIM-4 antibodies to block the available TIM-4 receptors.</p> <p>We found that TIM-4 expression was higher in a colitis mouse model compared to the control. Also by injecting TIM-4+ macrophages into the mice, the frequency of intestinal T regulatory (Treg) cells was decreased significantly. Finally in the group treated with anti-TIM-4 neutralizing antibodies prior to colitis induction, the frequency of intestinal Treg cells increased significantly and the inflammation response was less severe than the colitis control group. This study revealed, for the first time in the world, that TIM-4 expression in the colon of colitis mice was significantly increased, which suppressed Tregs and promoted T effector cells.</p> / Master of Science in Medical Sciences (MSMS)

No / Vitiligo is characterized by a mostly progressive loss of the inherited skin color. The cause of the disease is still unknown, despite accumulating in vivo and in vitro evidence of massive oxidative stress via hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) in the skin of affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Since depletion of the essential amino acid l-tryptophan (Trp) severely affects various immune responses, we here looked at Trp metabolism and signaling in these patients. Our in vivo and in vitro data revealed total absence of epidermal Trp hydroxylase activities and the presence of H2O2/ONOO− deactivated indoleamine 2,3-dioxygenase. Aryl hydrocarbon receptor signaling is severely impaired despite the ligand (Trp dimer) being formed, as shown by mass spectrometry. Loss of this signal is supported by the absence of downstream signals (COX-2 and CYP1A1) as well as regulatory T-lymphocytes and by computer modeling. In vivo Fourier transform Raman spectroscopy confirmed the presence of Trp metabolites together with H2O2 supporting deprivation of the epidermal Trp pool by Fenton chemistry. Taken together, our data support a long-expressed role for in loco redox balance and a distinct immune response. These insights could open novel treatment strategies for this disease.—Schallreuter, K. U., Salem, M. A. E. L., Gibbons, N. C. J., Maitland, D. J., Marsch, E., Elwary, S., Healey, A. R. Blunted epidermal l-tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 2: epidermal H2O2/ONOO−-mediated stress in vitiligo hampers indoleamine 2,3-dioxygenase and aryl hydrocarbon receptor-mediated immune response signaling.

Dans le contexte de l'étude pan-génomique de données d'expression des gènes (transcriptome), différents outils existent déjà. Parmi eux, les techniques de réduction de dimensionnalité cherchent les formes remarquables et les composants importants du système qui peuvent aider à résumer les données. Au cours de ma thèse, j'ai étudié en profondeur les différentes techniques existantes dans ce domaine. Nous avons ensuite développé notre propre approche basée sur la combinaison de la décomposition en valeurs singulières (Singular Value Decomposition) et le Multidimensional Scaling. Nous avons prouvé son utilité et sa précision. En plus des outils d'analyse de données spécifiques à l'étude de l'expression des gènes, nous avons développé un logiciel qui permet de correler l'expression des gènes à des réseaux d'interactions protéine-protéine. Et ceci afin de lier l'information sur l'expression des gènes à celle des interactions entre protéines (protéome) qui ont lieu au sein de la cellule. Tous les outils venant d'être décrits et de nombreux autres ont été utilisés afin d'analyser différent types de données biologiques. La première application a été de corréler l'expression d'auto-anticorps et de cytokines dans le corps humain lors d'une infection au paludisme. Nous avons déterminé des marqueurs spécifiques du paludisme cérébral, permetant à termes de prévenir et détecter plus tôt la maladie. La plus grande analyse que nous avons réalisé visait à définir le profil du transcriptome des cellules T régulatrices (Treg). Ces cellules sont détruites au cours d'une infection par le VIH, une bonne caractérisation moléculaire de celles-ci permettrait par exemple de mieux suivre l'évolution des Treg au cours des traitements pour le SIDA. Parmi les nouveaux marqueurs moléculaires de Treg que nous avons étudié, un nouveau facteur de transcription FOXLF a été découvert, qui pourrait jouer un rôle important dans l'apparition du caractère de "regulation" chez les Treg.