A imunodeficiência combinada grave (SCID) é uma doença caracterizada por profunda deficiência de células T, que afeta as imunidades celular e humoral e gera anormalidades graves no desenvolvimento e funções do sistema imune. Recém-nascidos com SCID apresentam a doença nos primeiros meses de vida e tem grande susceptibilidade a infecções. Sem tratamento, essas condições são invariavelmente fatais, porém se reconhecidas precocemente, há a possibilidade da realização do transplante de células-tronco hematopoiéticas, o tratamento curativo, o que torna a SCID uma emergência pediátrica. A investigação do defeito genético é uma prerrogativa para o condicionamento adequado do transplante, a terapia gênica, o aconselhamento genético e o diagnóstico pré-natal. No Brasil, o conhecimento sobre SCID é incipiente e não existem dados moleculares sobre pacientes com a doença. Sendo assim, este estudo teve por objetivo investigar defeitos genético-moleculares de pacientes brasileiros com SCID. Foram incluídos 13 pacientes, todos com início precoce dos sintomas e manifestações clínicas esperadas em SCID (principalmente infecções respiratórias, de pele, diarreia crônica e atraso de crescimento). Os patógenos isolados foram vírus, bactérias e fungos oportunistas comumente encontrados em pacientes SCID. A partir da quantificação de TRECS e KRECs e imunofenotipagem de linfócitos, foi montado o perfil imunológico de cada paciente, que guiou o sequenciamento direto de Sangerdos genes mais frequentemente mutados em cada imunofenótipo de SCID. Mutações em 3 pacientes foram identificadas por Sanger e, posteriormente, 8 pacientes cujas mutações não foram encontradas no Sanger foram encaminhados para o sequenciamento completo de exoma, que resultou na identificação do gene afetado em 62,5% dos casos. Ao todo, foram identificadas mutações patogênicas em 8 dos 13 pacientes. Os resultados revelaram 6 alterações em 5 genes de SCID clássica (IL7R, RAG2, DCLRE1C, JAK3, IL2RG), 1 mutação no gene CD3G e 2 alterações em CECR1. Das 9 mutações encontradas, 5 não possuíam registro na literatura. O estudo genético de SCID em nosso país é problemático, principalmente porque ainda hoje, a esmagadora maioria dos pacientes não é diagnosticada. A implementação da quantificação de TRECs e KRECs como triagem neonatal para linfopenias graves é uma ferramenta fundamental para que os pacientes SCID possam ser identificados, investigados e tratados adequadamente. / Primary immunodeficiencies are a heterogeneous group of genetic diseases that lead to increased susceptibility to infections and affect mostly children. Severe Combined Immunodeficiency (SCID) is the most severe of all these diseases and is characterized by profound T cell deficiency, which affects cellular and humoral immunities and leads to severe abnormalities in the development and function of the immune system. Newborns with SCID present the disease in the first months of life and are highly susceptible to infections. Without treatment, these conditions are invariably fatal, but if recognized early, there is the possibility of hematopoietic stem cell transplantation, the curative treatment, which makes SCID a pediatric emergency. Identifying the genetic defect of SCID patients is a prerequisite for proper transplant conditioning, gene therapy, genetic counseling and prenatal diagnosis. Knowledge about SCID is still incipient in Brazil, and there are virtually no molecular data on patients with the disease. Therefore, this study aimed to investigate genetic-molecular defects of Brazilian patients with SCID. Thirteen patients were recruited, all with early onset of symptoms and clinical manifestations expected of classic SCIDs (mainly respiratory and skin infections, chronic diarrhea and failure to thrive). The pathogens isolated were opportunistic viruses, bacteria and fungi often reported in SCID patients. The immunological profile from each patient was defined by the quantification of TRECS and KRECs and lymphocyte immunophenotyping, which was meant to guide direct sequencing by Sanger of the most frequently mutated genes of each SCID immunophenotype. Mutations in 3 patients were identified by Sanger and, subsequently, 8 patients whose mutations were not identified by Sanger were referred for whole exome sequencing, which resulted in the identification of the affected gene in 62,5% of cases. Pathogenic mutations were identified in 8 of the 13 patients. The results revealed 6 mutations in 5 genes associated to classical SCID genes (IL7R, RAG2, DCLRE1C, JAK3, IL2RG), 1 mutation in the CD3G gene, and 2 mutations in CECR1. Five of the 9 mutations found had no record in the literature. SCID genetic investigation in our country is troublesome, mainly because even nowadays, the vast majority of patients are not diagnosed properly. Newborn screening for SCID and other severe lymphopenias by the quantification of TRECs and KRECs is key for the identification, investigation and proper treatment of SCID patients.

In der humanen Haut spielen antimikrobielle Peptide (AP) bei Entzündungsgeschehen bakteriellen und nicht-bakteriellen Ursprungs eine bedeutende Rolle. Neben einer konstitutiven Expression AP können Zytokine deren vermehrte oder abgeschwächte Expression bewirken. In dieser Arbeit wurden die AP humanes β-Defensin (HBD) -2, HBD-3 und Psoriasin (PSO) in Bezug auf deren Expression in gesunder Haut und deren Modulation durch Zytokine in vivo anhand des xenogenen NOD-SCID-Maus-Transplantationsmodells untersucht. Nach erfolgreicher Transplantation von humaner Haut auf NOD-SCID Mäuse wurden die Zytokine TNF-α, IFN-γ und IL-13 in unterschiedlicher Dosierung einzeln und in Kombination intradermal appliziert. Für TNF-α konnte eine erhöhte Expression von HBD-2, HBD-3 und PSO auf RNA-Ebene mittels in-situ-Hybridisierung und Protein-Ebene mittels immunhistochemischer Nachweismethoden gezeigt werden. Eine erhöhte Expression nach Injektion von IFN-γ ließ sich für HBD-3 auf RNA-Ebene und Protein-Ebene und für HBD-2 auf RNA-Ebene erfolgreich belegen. PSO zeigte auf Protein-Ebene nach Modulation mit IFN-γ eine bei höherer Dosierung leicht abnehmende Expression. Eine Änderung der Expression durch IL-13 ließ sich nicht eindeutig belegen. In dieser Arbeit konnte die in der Literatur in vitro beschriebene Modulationsfähigkeit der untersuchten AP durch die verwendeten Zytokine in vivo belegt werden.

Les lymphocytes T sont caractérisés par l’expression d’un récepteur à l’antigène des cellules T (TCR), soit le preTCR, soit le γδ TCR et le αβ TCR clonotypique, associé à un complexe de transduction formé du CD3 et de la chaîne ζ. La signalisation du complexe TCR:CD3:ζ est cruciale pour le développement des cellules T et pour leur activation spécifique par l'antigène. Ces signaux déclenchent la prolifération, la différentation, les fonctions effectrices et l’apoptose des cellules T. Les évènements proximaux de la signalisation du TCR:CD3:ζ impliquent des protéines tyrosine kinases (PTK) des familles CSK, SRC, SYK et TEC dont les acteurs principaux sont CSK (C-terminal SRC kinase), LCK (lymphocyte-specific protein tyrosine kinase), ZAP-70 (ζ chain-associated protein tyrosine kinase of 70 kDa) et ITK (interleukin-2-inducible T cell kinase). Après stimulation du complexe TCR:CD3:ζ, les PTKs sont activées et déclenchent une cascade de phosphorylation sur tyrosine dont la phosphorylation des motifs activateurs ITAM (immunoreceptor tyrosine-based activation motif) du CD3 et de la chaîne ζ, et la phosphorylation des protéines adaptrices. Celles-ci conduisent à l’assemblage du signalosome LAT:SLP-76, lequel contrôle en grande partie la diversité des signaux associés au complex TCR:CD3:ζ, dont le flux calcique, l’activation de la cascade des MAP kinases, l’activation des facteurs de transcription NF-κB, NFAT et AP-1 ainsi que la réorganisation du cytosquellette d’actine, l’adhésion cellulaire et la motilité. Pendant les cinq dernières décennies, le système immunitaire a été analysé extensivement in vitro et à l’aide de modèles animaux comme la souris. Par ailleurs, l’étude des déficits immunitaires héréditaires chez l’homme a permis aussi des avancées importantes et novatrices dans la compréhension du système immunitaire humain que les modèles animaux ne permettaient pas d’appréhender. Dans ma thèse de doctorat je rapporte le premier cas de déficit humain en LCK de transmission autosomique récessive et l’identification de nouvelles mutations autosomiques récessives provoquant un défaut humain de ZAP-70 et un défaut humain d’ITK. Je rends compte des phénotypes cliniques et immunologiques associés à ces immunodéficiences et je caractérise les défauts biochimiques de la signalisation TCR:CD3:ζ associés à ces déficits. Enfin, je compare mes observations avec les modèles murins déficients Lck-/-, Zap-70-/- et Itk-/- déjà bien établis. / T lymphocytes express either a preTCR, or a clonotypic γδ TCR or αβ TCR together with the CD3-complex and the associated ζ-chain. TCR:CD3:ζ-signalling is crucial for T cell development and antigen-specific activation including proliferation, differentiation, effector functions and apoptosis of mature T cells. Protein tyrosine kinase (PTK) cascades lie at the heart of proximal TCR:CD3:ζ-signalling. The CSK-, SRC-, SYK- and TEC-family members C-terminal SRC kinase (CSK), lymphocyte-specific protein tyrosine kinase (LCK), ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP-70) and interleukin-2-inducible T cell kinase (ITK), respectively, are the major T cell players. After TCR:CD3:ζ-complex triggering, activation of PTKs results in tyrosine phosphorylation signals. These include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chains, and adaptor proteins that nucleate the proximal LAT:SLP-76-signalosome controlling almost all TCR:CD3:ζ-induced signalling events. These events initiate Ca2+-flux, activation of mitogen-activated protein kinases (MAPKs), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), activation of nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) as well as actin reorganization, cell-adhesion and motility. Throughout the last five decades, the immune system has been extensively investigated in vitro and in animal models such as the murine system. Additionally, studying and taking care of human primary immunodeficiency diseases (PIDs) has been seminal for our understanding of the human immune system as animal models not always recapitulate the subtleties found in men. In my doctoral thesis I report the first case of autosomal recessive human LCK-deficiency, a novel autosomal recessive mutation leading to human ZAP-70-deficiency and a novel autosomal recessive mutation leading to human ITK-deficiency. I provide detailed clinical, immunological and biochemical analyses especially of TCR:CD3:ζ-signalling and compare my findings to the well-established Lck-/-, Zap-70-/- and Itk-/- murine models.

<p>T lymphocytes are primarily involved in adaptive, cell-mediated, immune reactions. In this thesis T cells were studied regarding central and peripheral differentiation and homeostatic mechanisms for maintanance of the immune repertoire.</p><p>The influence by mature T cells on thymic development was studied in C.B-17 <i>scid/scid</i> (SCID) mice, devoid of mature T and B cells, and whose thymocyte development is arrested at the early pro-T cell stage. When mature syngeneic T cells were injected the developmental block was overcome and there was an accumulation of CD4⁺CD8⁺ thymocytes. This event was accompanied by the maturation of medullary epithelial cells in thymus which seemed to be driven by CD8⁺ T cells. In the periphery there was initially a spontaneous T-cell proliferation and later, the majority of the donor T lymphocytes showed a memory phenotype with high expression of CD44 and with an early onset of proliferation and cytokine production upon stimulation. This stable pool of memory type of cells sustained for more than a year following treatment.</p><p>Treating SCID mice with allogeneic T cells results in graft-versus-host disease (GVHD). Severe GVHD was dependent on the MHC-haplotype of the donor cells and was accompanied by profound alterations of the TCR-Vβ repertoire and with high production of IFN-γ.</p><p>Kappa light chain (κ)-deficient mice have only half the number of B cells as their normal counterparts but normal levels of immunoglobulins. When T cells from κ-deficient mice were stimulated <i>in vitro</i> there was a bias towards production of B-cell stimulatory type 2 cytokines. This is proposed as a mechanism for the homeostatic control of serum immunoglobulin levels in κ-deficient mice.</p>

The feasibility of the Alb-uPA/SCID/Bg chimeric mouse as a model for Hepatitis C Virus (HCV) infection was assessed experimentally by (1) the infection and treatment with another hepatotropic virus, Hepatitis B Virus (HBV) and (2) the infection of the model with HCV and the subsequent treatment of that infection with a pro-apoptotic factor (BID) targeted to infected hepatocytes. In the former, the infected mouse responded favorably, and in the manner of human patients, to a standard imunoglobulin therapy. In the latter, HCV-infected hepatocytes were successfully targeted for cell death, with repeated doses of Adenovirus-delivered BID being the most effective at inhibiting virus spread. Efficacy and toxic side-effects of BID treatment could be reconciled by modulating the timing between doses, the most effective tested being three doses of BID at 7-day intervals. Analyses of chimeric model production were undertaken to improve the quality of human hepatocyte engraftment (typically only 25-35% of mice receiving grafts are currently used experimentally). Minor variations in success rates were experienced with respect to donor age or health status, or the age of recipient mice within an operational window of 5 to 13 days from birth. The greatest obstacle to useful engraftment (aside from technical challenges) was deemed to be the genetic/cellular integrity of the recipient mouse. This conclusion was based on variable engraftment success with ‘healthy’ donor cell preparations and a consideration of variability in immune deficiency arising in mice within a SCID/Bg mouse colony. / Experimental Surgery

Bone marrow (BM) mesenchymal stem cells (MSC) support proliferation and differentiation of hematopoietic progenitor cells (HPC) in vitro. Since they represent a rare subset of BM cells, MSC preparations for clinical purposes involves a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on MSC supportive activity. MSC were expanded for up to 10 passages. MSC and CD34+ cells were seeded in cytokinefree co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed. Early passage MSC supported HPC expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage MSC did not support HPC and myeloid cell outgrowth but maintained B cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for one week in contact with MSC was effective until the fourth MSC passage and declined afterwards. CD34+ cells achieved higher levels of engraftment in NOD/SCID mice when co-injected with early passage MSC; however MSC expanded beyond 9 passages were ineffective in promoting CD34+ cell engraftment. Non-contact cultures indicated that MSC supportive activity involved diffusible factors. Among these, interleukin (IL)-6 and IL-8 contributed to the supportive activity of early passage MSC but not of late passage MSC. MSC phenotype as well as fat, bone and cartilage differentiation capacity did not change during MSC culture. Extended MSC culture alters their supportive ability toward HPC without concomitant changes in phenotype and differentiation capacity.

T lymphocytes are primarily involved in adaptive, cell-mediated, immune reactions. In this thesis T cells were studied regarding central and peripheral differentiation and homeostatic mechanisms for maintanance of the immune repertoire. The influence by mature T cells on thymic development was studied in C.B-17 scid/scid (SCID) mice, devoid of mature T and B cells, and whose thymocyte development is arrested at the early pro-T cell stage. When mature syngeneic T cells were injected the developmental block was overcome and there was an accumulation of CD4+CD8+ thymocytes. This event was accompanied by the maturation of medullary epithelial cells in thymus which seemed to be driven by CD8+ T cells. In the periphery there was initially a spontaneous T-cell proliferation and later, the majority of the donor T lymphocytes showed a memory phenotype with high expression of CD44 and with an early onset of proliferation and cytokine production upon stimulation. This stable pool of memory type of cells sustained for more than a year following treatment. Treating SCID mice with allogeneic T cells results in graft-versus-host disease (GVHD). Severe GVHD was dependent on the MHC-haplotype of the donor cells and was accompanied by profound alterations of the TCR-Vβ repertoire and with high production of IFN-γ. Kappa light chain (κ)-deficient mice have only half the number of B cells as their normal counterparts but normal levels of immunoglobulins. When T cells from κ-deficient mice were stimulated in vitro there was a bias towards production of B-cell stimulatory type 2 cytokines. This is proposed as a mechanism for the homeostatic control of serum immunoglobulin levels in κ-deficient mice.

L'hépatite C reste un problème majeur de santé publique. Malgré la mise au point d'un modèle de réplication du virus de l'hépatite C (VHC) in vitro, les mécanismes conduisant à l'infection restent encore mal connus. Le VHC est sécrété et circule dans le sérum associé à des lipoprotéines. L'importance des lipoprotéines pour le cycle viral nous a conduits à étudier le rôle de la lipoprotéine lipase (LPL), une enzyme lipolytique, dans l'infection de la cellule par le VHC. Nous avons montré que la LPL potentialise l'attachement et l'internalisation du virus par un mécanisme similaire à la clearance hépatique des lipoprotéines. La LPL dimérique forme un pont entre les lipoprotéines associées au virus et les HSPG à la surface des cellules. Néanmoins son action conduit à une inhibition de l'infection par les souches virales JFH-1 et J6/JFH-1 produites en culture cellulaire et dans les hépatocytes humains greffés à des souris chimériques uPA-SCID. L'analyse par ultracentrifugation en gradient d'iodixanol des virus produits in vitro et in vivo a montré la présence de deux populations virales : la première, de densité très faible, est beaucoup plus infectieuse que la seconde, de densité plus élevée. L'infection in vitro par ces deux populations virales est inhibée par la LPL. La LPL représente donc un nouvel inhibiteur de l'infection par le VHC. Nous avons également démontré que la présence d'un réseau de microtubules intact et dynamique est cruciale pour l'entrée du VHC et les étapes post-fusion qui mènent à l'infection. Enfin, nous avons mis en évidence une interaction de la protéine de capside avec les tubulines α et β, conduisant à une augmentation de la polymérisation des microtubules. Ces observations suggèrent que le VHC pourrait utiliser les mécanismes de polymérisation des microtubules pour établir l'infection, et la protéine de capside jouer un rôle essentiel dans ce processus. Les nouvelles approches antivirales pourraient donc cibler les éléments du cytosquelette et/ou des lipoprotéines associées aux particules virales.

Human T cells may be genetically modified to express targeted chimeric antigen receptors (CARs). We have previously demonstrated that T cells modified to express a CAR specific to the B cell tumor antigen CD19, termed 19-28z, successfully eradicate systemic human CD19+ tumors in SCID-Beige mice. While these results are encouraging, this xenogeneic tumor model fails to address potential limitations of this therapeutic approach in the clinical setting wherein these modified T cells encounter a hostile tumor microenvironment. Specifically, these models fail to address potential effector T cell inhibition mediated by endogenous regulatory T cells (Tregs). To investigate the role of inhibitory Tregs, we initially assessed the in vitro function of CAR-modified T cells in the context of Tregs. We found that CD19-targeted T cell proliferation and cytotoxicity were inhibited by purified natural Tregs. To further assess the role of these Tregs in vivo, we isolated and genetically modified Tregs to express the CD19-targeted 19z1 CAR. We verified specific trafficking of targeted Tregs to CD19+ tumors in vivo, and demonstrate that 19z1 Tregs wholly inhibit anti-tumor function of subsequently injected 19-28z effector T cells even at low Treg to effector T cell ratios (1:8). In order to overcome this limitation, we assessed whether the addition of a pro-inflammatory cytokine in vitro could overcome Treg inhibition. Indeed, the addition of exogenous IL-12 mediated resistance of 19-28z T cells to Treg inhibition. In light of this data we generated a bicistronic retroviral vector containing both the 19-28z CAR as well as the murine IL-12 fusion gene (19-28z IRES IL-12). Significantly, we found that 19-28z/IL-12+ T cells when compared to 19-28z+ T cells exhibited enhanced proliferation in vitro as well as resistance to Treg mediated inhibition. Finally, we demonstrate that 19-28z/IL-12+ T cells overcome Treg inhibition in vivo in our SCID-Beige Treg tumor model. In conclusion, tumor targeted T cells modified to express IL-12 demonstrate significantly enhanced in vivo anti-tumor efficacy in the presence of Tregs that are similarly targeted to the site of tumor. These results validate utilization of IL-12 secreting tumor targeted T cells in future clinical trials.

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