published_or_final_version / Pathology / Master / Master of Philosophy

<p>Southern Africa is facing one of the most serious HIV epidemics. This project contributes to the HIVNET, Network for Prevention Trials cohort for vaccine development. HIV’s biology and rapid mutation rate have made vaccine design difficult. We examined HIV-1 subtype C diversity and how it relates to CTL epitope location along viral gag sequences. We found a negative correlation between codon sites under positive selection and epitope regions / suggesting epitope regions are evolutionarily conserved. It is possible that epitopes exist in non-conserved regions, yet fail to be detected due to the reference strain diverging from the circulating viral population. To test if CTL clustering is an artifact of the reference strain, we calculated differences between the gag codons and the reference strain. We found a weak negative correlation, suggesting epitopes in less conserved regions maybe evading detection. Locating conserved and optimal epitopes that can be recognized by CTLs is essential for the design of vaccine reagents.</p>

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The aim of this study was to design a resolution typing system for the HLA-B gene. This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this technique to serological analysis can improve serology detection which is currently hindered by antibody cross-reactivity and the unavailability of useful typing reagents.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Des études récentes ont relevés que les CSMn pouvaient être in vivo des cellules périvasculaires avec des caractéristiques des péricytes. Pour évaluer cette hypothèse, nous avons cultiver des CSMn issues de MO dans des conditions spécifiques pour l'expansion des péricytes puis nous avons testé leur potentiel de souchitude. De plus, nous les avons comparées avec des CSMs cultivées dans des conditions standards en maintenant les même donneurs. Des échantillons de MO ont été cultivés dans un milieu pro-Pericytaires (milieu EGM2) ou dans un milieu standard. Après culture, les cellules ont été caractérisées. Les cellules de caractère péricytaire avaient exprimé une upregulation des marqueurs de souchitude d’OCT4, NANOG et SOX2 pour un potentiel neuronal. Ces cellules ont démontré un grand potentiel in vivo. / Native mesenchymal stem cells were found tore perivascular cells with pericyte features. This suggests that pericyte phenotype is crucial for the stenness of MSC. We cultured MSC from bone marrow upon in vitro conditions (EGM2 versus standard mediums). They all express MSC, markers and character. Cells cultivated into ECM2 were found to be more immature than cells obtained from standard conditions (expressed OCT4, NANOG and SOX2), with high neuronal and engraftment potential

Die Regulation von Effektor-T-Zellen ist ein wichtiger Mechanismus zur Kontrolle organspezifischer Entzündungen. Dabei sind regulatorische T-Zellen (Treg) maßgeblich an der Aufrechterhaltung peripherer Immuntoleranz und parenchymaler Immunhomöostase beteiligt. Eine neue Population von humanen, natürlich vorkommenden Treg Zellen wurde durch ihre konstitutive Expression des immuntolerogenen Moleküls HLA-G identifiziert. Im ersten Teil dieser Arbeit wurden die Mechanismen, durch die CD4+ HLA-Gpos Treg Zellen ihre Zielzellen (autologe HLA-Gneg T-Zellen) modulieren, aufgeklärt. Unter Verwendung eines Suppressionsansatzes in Abwesenheit von antigenpräsentierenden Zellen (APC) wurden T-T-Zell-Interaktionen, die die Proliferation von HLA-Gneg T-Zellen hemmen, demonstriert. Diese Suppression, die durch die Stimulierung des T-Zell-Rezeptors auf HLA-Gpos Treg Zellen verstärkt wurde, war unabhängig vom Zell-Zell-Kontakt. Die HLA-Gneg T-Zellen erlangten nach Entfernung der HLA-Gpos Treg Zellen und einer erneuten Stimulierung ihrer T-Zell- Rezeptoren ihre Fähigkeit zur Proliferation wieder. Dies wies auf die Umkehrbarkeit dieser Suppression hin. Darüber hinaus war die HLA-Gpos Treg-vermittelte Suppression entscheidend von der IL-10- Sekretion, nicht jedoch von TGF-&#946; abhängig. Zusammengefasst beschreibt dieser Teil der Arbeit eine detaillierte Charakterisierung der Mechanismen, wie HLA-Gpos Treg HLA-Gneg TZellen supprimieren. Das tiefere Verständnis der Wirkmechanismen von HLA-Gpos Treg könnte in therapeutischen Strategien verwendet werden, in denen die regulatorische Funktion der T-Zell-Suppression verstärkt oder moduliert werden soll. Im zweiten Teil dieser Arbeit wurde die potenzielle Rolle von HLA-Gpos Treg bei der Multiplen Sklerose (MS) untersucht, einer klassischen Autoimmunerkrankung des Zentralnervensystems (ZNS). Im Gegensatz zu Vergleichspatienten mit nicht-entzündlichen Erkrankungen konnte im Liquor von MS Patienten eine erhöhte Anzahl von HLA-Gpos Treg gefunden werden. Diese aus dem Liquor isolierten HLA-Gpos Treg wiesen phänotypische Merkmale von zentralen Gedächtnis-T-Zellen (CD45RA- CD27+) auf, exprimierten den Aktivierungsmarker ICOS sowie deutlich höhere Level des Chemokinrezeptors (CCR) CCR5 und agierten als starke Suppressoren der autologen CD4+ T-Zellproliferation. Durch Verwendung eines in vitro Modells der humanen Bluthirnschranke konnte demonstriert werden, dass HLA-Gpos Treg eine starke Neigung zur Migration haben, die durch die CCR5- Liganden MIP1&#945; und RANTES, nicht jedoch durch MIP3&#946; (Ligand von CCR7) unterstützt wird. Diese Chemokin-induzierte Migration von HLA-Gpos Treg war auch mit einer Steigerung der suppressiven Kapazität nach Zelltransmigration assoziiert. Im Gegensatz zu CD4+CD25+, FoxP3-exprimierenden Treg zeigten HLA-Gpos Treg von MS-Patienten keine beeinträchtigte Funktionalität. Dies deutet auf eine selektive Rekrutierung von HLA-Gpos Treg zu Entzündungsherden im ZNS und ihre Beteiligung an der Bekämpfung der destruktiven Entzündung hin. Die Ergebnisse dieser Studien tragen zum weitergehenden Verständnis der Rolle und Funktion HLA-Gpos Treg Zellen bei und stellen somit ein wichtiges pathophysiologisches Beispiel „gutartiger“ T-Zell-Entzündung während der ZNS Autoimmunität dar, das sowohl aus pathophysiologischer als auch therapeutischer Sicht interessant ist. / Regulation of effector T cells is an important mechanism to control organ-specific inflammation. Thereby regulatory T cells (Treg cells) are essential for maintaining peripheral immune tolerance and for establishing parenchyma immune homeostasis. A novel population of natural human Treg characterized by the constitutive expression of the immune-tolerogenic human HLA-G molecule has been identified. In the first part of the study, we elucidated the mechanism(s) by which CD4+ HLA-Gpos Treg modulates their cellular targets namely autologous HLA-G negative responder T cells (HLAGneg Tresp). Using a suppression system free of antigen-presenting cells (APC), we demonstrate a T-T cell interaction resulting in suppression of HLA-Gneg Tresp. We could also show that this suppression was independent of cell-cell contact. Importantly, stimulus of T cell receptor (TCR) on HLA-Gpos Treg facilitated their suppressive capacity. We also observed that removal of HLA-Gpos Treg from the established co-cultures could restore the ability of HLA-Gneg Tresp to proliferate upon TCR re-stimulation, indicating that the suppression was reversible. Further, HLA-Gpos Treg–mediated suppression was critically depending on the secretion of IL-10 but not TGF-&#946;. Taken together, this part of the work provides an in-depth characterization of the mechanisms of how HLA-Gpos Treg suppresses T responder cells in direct T-T interactions. Understanding the suppressive mechanism used by HLA-Gpos Treg may help to develop therapeutic strategies to modulate regulatory arms of T-cell suppression. In the second part of this study, the potential role of HLA-Gpos Treg in the pathophysiological process of Multiple Sclerosis (MS), a prototypic autoimmune inflammatory central nervous system (CNS), has been investigated. We found that HLA-Gpos Treg are enriched in the cerebrospinal fluid (CSF) from MS patients, but not in non-inflammatory controls. CSFderived HLA-Gpos Treg showed predominance of central memory (CD45RA-CD27+) phenotype, exhibited markers of activation (ICOS), and had significantly higher expression of the inflammatory chemokine receptor CCR5. Importantly, these cells demonstrated as potent suppressors to autologous CD4+ T-cell proliferation. Using an in vitro model of human blood brain barrier, we showed that HLA-Gpos Treg have a strong propensity to migrate, which could be facilitated by MIP1&#945; and RANTES (ligands of CCR5) but not MIP3&#946; (a ligand of CCR7). The HLA-Gpos Treg migration triggered by chemokines was also associated with a gain of suppressive capacity upon cellular transmigration. In contrast to CD4+CD25+ naturally occurring FoxP3-expressing Treg, HLA-Gpos Treg from patients with MS did not exhibit impaired function, suggesting that HLA-Gpos Treg are selectively recruited to the sites of CNS inflammation in an effort to combat destructive inflammation during MS. Our results contribute to the understanding of the role and function of HLA-Gpos Treg and provide an important example of “beneficial” T-cell inflammation in CNS autoimmunity- interesting both from a patho/-physiological and a therapeutically point of view.

Orientador: Erick da Cruz Castelli / Resumo: O gene KIR2DL4 codifica um importante receptor de células Natural Killer (NK). O único ligante de KIR2DL4 conhecido é a molécula HLA-G, expressa principalmente na placenta, modulando a ação das células NK durante a gestação. O gene KIR2DL4 parece ser bastante variável, quando considerado os bancos de dados que armazenam suas sequências conhecidas, porém não está claro o nível de diversidade deste gene em populações reais e heterogêneas. Polimorfismos presentes no gene KIR2DL4 poderiam influenciar a interação entre KIR2DL4 e HLA-G, modificando a ação das células NK. Neste estudo exploramos a variabilidade genética de KIR2DL4 em 157 indivíduos oriundos do Estado de São Paulo/Brasil. Devido à alta similaridade de sequências entre os genes KIR, erros de genotipagem são esperados quando se utiliza sequenciamento de segunda geração. Por este motivo, desenvolvemos uma abordagem para classificar cada leitura com base em sequências KIR conhecidas, endereçando-as ao gene mais provável. Também utilizamos o painel SNPforID 34-plex para avaliar a ancestralidade dessas amostras. Considerando o segmento completo desse gene, indo da região 5’URR até a 3’UTR, com aproximadamente 13kb, o gene KIR2DL4 se mostrou pouco polimórfico, com 152 pontos de variações identificados (MAF 1%). Esses pontos de variação estão organizados em 32 haplótipos estendidos que codificam 13 proteínas diferentes. Foram encontrados 11 haplótipos na região promotora, sendo que 8 possuem MAF maior que 1%. Na região codif... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: KIR2DL4 is the most unusual Killer Cell Immunoglobulin-Like receptor (KIR) family member in terms of structure, expression, and signaling properties. The only known KIR2DL4 ligand is HLA-G, and polymorphisms might disrupt this interaction. KIR2DL4 variability is not well explored in admixed populations. Here we explored KIR2DL4 exon variability in 157 individuals from the State of São Paulo/Brazil. Because of sequence similarity with other KIR genes, it is expected genotyping errors when using secondgeneration sequencing. We developed an approach to score each read based on known KIR sequences, addressing them to the most likely locus. We evaluated the SNPforID 34-plex panel to assess ancestry. The methodology was applied to survey the variability of a very admixed population, such as Brazilian, counting with 157 samples of São Paulo State. Considering a segment of about 13-kb, KIR2DL4 gene was conserved with few different and frequent sequences. Overall, 152 variable sites were detected, arranged in 32 haplotypes codifying 13 protein. We found 11 promoter haplotypes, 8 with a frequency greater than 1%. In the coding region we detected 70 haplotypes, four of which correspond to 50% of the coding sequences (KIR2DL4 * 0080204, * 008105, * 001, * 005). In the 3'UTR region, 14 haplotypes were identified with MAF greater than 1%. The KIR2DL4 coding region was the most variable segment. We observed that KIR2DL4 variability is strongly influenced by the sample ancestry background. K... (Complete abstract click electronic access below) / Mestre

Introdução e Objetivos: A maior prevalência de doença celíaca (DC) em indivíduos com diabetes mellitus tipo I (DM1) já é reconhecida. Ambas as doenças tem causa autoimune, em que os genes HLA classe 2 representam o principal fator genético de risco. Porém, existe uma considerável parcela da população que não manifesta tais doenças e são portadores desses genes. Estudo de associação genômica (GWAS) identificaram polimorfismos de susceptibilidade às duas doenças em genes diferentes do sistema HLA, que poderão auxiliar na compreensão da causa e das suas variabilidades clínicas. Os objetivos desse estudo foram avaliar as frequências dos polimorfismos HLA e não HLA em pessoas como DM1 e com DC e relacionar esses dados com a ocorrência de sintomas gastrointestinais, com a idade do diagnóstico da DM1 e com história alimentar. Métodos: Delineamento transversal, com avaliações retrospectivas e prospectivas, em pessoas com DM1 com e sem DC. Foram realizadas entrevista e revisão de prontuário dos pessoas, seguido de coleta de sangue ou saliva. A pesquisa dos genes RGS1, IL2-IL21, BACH2, TLR7/TLR8 e IL18RAP foi realizada por PCR Real-Time. Os alelos DQA1* 0501 e DQB1* 0201 para DQ2.5 e o alelo DQB1*0302 para DQ8 foram identificados a partir da técnica de genotipagem de HLA Tag-single-nuleotide polymorphism (Tag SNP). Resultados: As frequências alélicas e genotípicas entre 273 pessoas com DM1 sem DC e 39 pessoas com DM1 e DC não apresentaram diferença significativa. A presença de sintoma gastrointestinal foi mais frequente nos portadores dos polimorfismos dos genes RGS1 e IL18RAP. O tempo de aleitamento materno, a idade de introdução do glúten e a idade do diagnóstico da DM1 foram semelhantes entre os grupos. A comparação dos cinco polimorfismos com a combinação dos haplótipos para DQ2.5 e DQ8 não apresentou diferença significativa. Nos 312 indivíduos, com DM1 com e sem DC e nos 66 indivíduos portadores de DC sem DM1 foi identificado alelos DQ2.5 e ou DQ8 em 97% dos casos, enquanto que nos indivíduos com DC sem DM1 identificou-se em 76% dos casos. DQ2.5 foi mais frequente entre pessoascom DC e DQ8 foi mais frequentes entre pessoas com DM1. Conclusões: A presença dos polimorfismos dos genes estudados não modificou a chance do indivíduo com DM1 ter ou não DC. Houve associação dos genes RGS1 e IL18RAP com sintomas gastrointestinais. A pesquisa dos alelos DQ2.5 e DQ8, pela técnica Tag-SNP, permitiu determinar um alto valor preditivo negativo no diagnóstico de DC na população com DM1 e com DC, semelhante ao descrito na literatura com a técnica convencional. / Introduction and Objectives: The higher prevalence of celiac disease (CD) in individuals with diabetes mellitus type I (T1D) is already recognized. Both diseases have autoimmune cause, where HLA genes class 2 represent the major genetic risk factor. However, there is a considerable portion of the population that does not manifest such diseases and are carriers of these genes. Genome-wide association studies (GWAS) have identified susceptibility polymorphisms to both diseases in different genes of the HLA system that may assist in understanding the etiology and in its clinical variabilities. The objectives of this study were to evaluate the frequencies of HLA and non-HLA polymorphisms in patients with T1D and CD, related to the occurrence of gastrointestinal symptoms, the age of diagnosis of T1D and food history. Methods: Mixed design with retrospective and prospective evaluations in patients with T1D with and without DC. They were conducted interview and review of medical records of patients, followed by collecting blood or saliva. The search for genes RGS1, IL21-IL2, BACH2, TLR7 / TLR8 and IL18RAP was performed by Real-Time PCR. The alleles DQA1 * 0501 and DQB1 * 0201 for DQ2.5 and DQB1 * 0302 for DQ8 were identified from the Tag-single-nucleotide polymorphism (tag SNP) genotyping HLA technique Results: The allelic and genotypic frequencies between 273 T1D patients without CD and 39 patients with T1D and CD showed no significant difference. The presence of gastrointestinal symptoms were more frequent in patients with polymorphisms of genes RGS1 and IL18RAP. The duration of breastfeeding, the age of introduction of gluten and the age of diagnosis of T1D were similar between the groups. The comparison of the five polymorphisms with the combination of haplotypes for DQ2.5 and DQ8 showed no significant difference. In 312 individuals with DM1 with and without CD and 66 individuals with CD without T1D was identified alleles DQ2.5 and/or DQ8 in 97% of cases, whereas in individuals with CD without T1D was identified in 76% of cases . DQ2.5 was more frequent among patients with CD and DQ8 was more frequent among patients with T1D Conclusions: The presence of polymorphisms of genes studied did not modify the chance of T1D whether or not DC. There was an association of RGS1 and IL18RAP genes with gastrointestinal symptoms. The survey of DQ2.5 and DQ8 alleles by Tag-SNP technique allowed determining a high negative predictive value in the diagnosis of CD in the population of patients with T1D and DC, similar to that described in the literature with the conventional technique.

The haematopoietic phosphatase PTPN22 is a key regulator in balancing immune responses between self-reactivity and tolerance. PTPN22 downregulates T cell signaling and harbors the non-HLA genetic variation most strongly associated with autoimmune disease in humans, the single nucleotide polymorphism R620W. The effect of this mutation is currently controversial due to confounding results in mouse and human models. The polymorphism is linked to increased susceptibility to autoimmunity in both human and mouse models, although the latter does depend on genetic background. However, mouse data clearly shows that the polymorphism has a loss-of-function effect on T cell signalling, whereas studies in human models largely demonstrate a gain-of-function effect for R620W. A confounding issue in human studies is that they depend on comparison of T cells from distinct individuals, on protein over-expression, or on RNA interference, techniques for which it is difficult to control for genetic and environmental variables, changes in stoichiometry, and off-target effects or incomplete knockdown, respectively. We aimed to create isogenic human cell lines with mutations in PTPN22 at the genomic level to alleviate the complications inherent in analysing human data. In addition to autoimmune pathogenesis, we are interested in the role of PTPN22 in a cancer setting. Because PTPN22 has a strong suppressive effect on T cell responses to weak affinity antigen, which encompass most tumour antigens, we postulated that knocking out PTPN22 may better enable T cells to kill tumour cells. Furthermore, we have shown that PTPN22 knockout (KO) leads to increased IL-2 expression in mouse T cells, and that this effect is protective against TGF-β mediated suppression, a common driver of T cell inhibition in the tumour microenvironment. T cell transfer experiments in mice showed that PTPN22 KO T cells are indeed more effective at reducing tumour size. Based on these findings, we aim to determine whether PTPN22 KO in human cells confers a similar effect on signaling. To investigate the effects of PTPN22 KO on human T cell signaling, we used CRISPR gene-editing to target PTPN22 in a Jurkat cell line. By combining this technique with lentiviral transduction of a specific T cell receptor, we generated human cell lines which are genetically identical, save for specific alterations to PTPN22, and which can be stimulated with strong or weak cognate antigen. We found that PTPN22 KO Jurkat cells develop an enhanced activation phenotype upon stimulation, including increased IL-2 expression. Additionally, PTPN22 KO Jurkat cells show enhanced Erk signalling following stimulation with weak affinity antigen, but this difference is lost as stimulus strength increases. CRISPR technology has presented the opportunity to create novel models of PTPN22 signalling in the context of human T cell lines. The data from these lines suggests that, unlike the R620W mutation, complete loss of PTPN22 has a comparable effect in human and mouse T cells. In conjunction with our previous findings, these results suggest that knocking out PTPN22 may lead to signalling alterations that improve adoptive T cell cancer therapy.