Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 6, 2008). Includes bibliographical references.

CD4 T cells and B cells are cells associated with the adaptive immune system. The adaptive immune system is designed to mount a rapid antigen-specific response to pathogens by way of clonal expansions of T and B cells bearing discrete antigen-specific receptors. During viral infection, interactions between CD4 T cells and B cells occur in a dynamic process, where B cells that bind to the virus internalize and degrade virus particles. The B cells then present viral antigens to virus-specific CD4 T cells that activate the B cells and cause them to proliferate and differentiate into virus-specific antibody-secreting cells. Yet, non-specific hypergammaglobulinemia and the production of self-reactive antibodies occur during many viral infections, and studies have suggested that viral antigen-presenting B cells may become polyclonally activated by CD4 T cells in vivo in the absence of viral engagement of the B cell receptor. This presumed polyclonal B cell activation associated with virus infection is of great medical interest because it may be involved in the initiation of autoimmunity or contribute to the long-term maintenance of B cell memory. In order to directly examine the interactions that occur between T cells and B cells, I asked what would happen to a polyclonal population of B cells that are presenting viral antigens, if they were transferred into virus-infected hosts. I performed these studies in mice using the well-characterized lymphocytic choriomeningitis virus (LCMV) model of infection. I found that the transferred population of antigen-presenting B cells had two fates. Some antigen-expressing B cells were killed in vivo by CD4 T cells in the first day after transfer into LCMV-infected hosts. However, B cells that survived the cytotoxicity underwent a dynamic polyclonal activation manifested by proliferation, changes in phenotype, and antibody production. The specific elimination of antigen-presenting B cells following adoptive transfer into LCMV-infected hosts is the first evidence that MHC class II-restricted killing can occur in vivo during viral infection. This killing was specific, because only cells expressing specific viral peptides were eliminated, and they were only eliminated in LCMV-infected mice. In addition to peptide specificity, killing was restricted to MHC class II high cells that expressed the B cell markers B220 and CD19. Mice depleted of CD4 T cells prior to adoptive transfer did not eliminate virus-specific targets, suggesting that CD4 T cells are required for this killing. I found that CD4 T cell-dependent cytotoxicity cannot be solely explained by one mechanism, but Fas-FasL interactions and perforin are mechanisms used to induce lysis. Polyclonal B cell activation, hypothesized to be the cause of virus-induced hypergammaglobulinemia, has never been formally described in vivo. Based on previous studies of virus-induced hypergammaglobulinemia, which showed that CD4 T cells were required and that hypergammaglobulinemia was more likely to occur when virus grows to high titer in vivo, it was proposed that the B cells responsible for hypergammaglobulinemia may be expressing viral antigens to virus-specific CD4 T cells in vivo. CD4 T cells would then activate the B cells. However, because the antibodies produced during hypergammaglobulinemia are predominantly not virus-specific, nonvirus-specific B cells must be presenting viral antigens in vivo. In my studies, the adoptively transferred B cells that survived the MHC class II-restricted cytotoxicity became polyclonally activated in LCMV-infected mice. Most of the surviving naïve B cells presenting class II MHC peptides underwent an extensive differentiation process involving both proliferation and secretion of antibodies. Both events required CD4 cells and CD40/CD40L interactions to occur but B cell division did not require MyD88-dependent signaling, type I interferon signaling, or interferon γ signaling within B cells. No division or activation of B cells was detected at all in virus-infected hosts in the absence of cognate CD4 T cells and class II antigen. B cells taken from immunologically tolerant donor LCMV carrier mice with high LCMV antigen load became activated following adoptive transfer into LCMV-infected hosts, suggesting that B cells can present sufficient antigen for this process during a viral infection. A transgenic population of B cells presenting viral antigens was also stimulated to undergo polyclonal activation in LCMV-infected mice. Due to the high proportion of B cells stimulated by virus infection and the fact that transgenic B cells can be activated in this manner, I conclude that virus-induced polyclonal B cell activation is independent of B cell receptor specificity. This approach, therefore, formally demonstrates and quantifies a virus-induced polyclonal proliferation and differentiation of B cells which can occur in a B cell receptor-independent manner. By examining the fate of antigen-presenting B cells following adoptive transfer into LCMV-infected mice, I have been able to observe dynamic interactions between virus-specific CD4 T cells and B cells during viral infection. Adoptive transfer of antigen-presenting B cells results in CD4 T cell-mediated killing and polyclonal activation of B cells during LCMV infection. Studies showing requirements for CD4 T cells or MHC class II to control viral infections must now take MHC class II-restricted cytotoxicity into account. Polyclonal B cell activation after viral infection has the potential to enhance the maintenance of B cell memory or lead to the onset of autoimmune disease.

Antigen presented to CD4+ T cells by major histocompatibility complex class II molecules (MHCII) plays a key role in adaptive immunity. Antigen presentation is initiated by the proteolytic cleavage of pathogenic or self proteins and loading of resultant peptides to MHCII. The loading and exchange of peptides to MHCII is catalyzed by a nonclassical MHCII molecule, HLA-DM (DM). It is well established that DM promotes peptide exchange in vitro and in vivo. However, the mechanism of DM-catalyzed peptide association and dissociation, and how this would affect epitope selection in human responses to infectious disease remain unclear. The work presented in this thesis was directed towards the understanding of mechanism of DM-mediated peptide exchange and its role in epitope selection. In Chapter II, I measured the binding affinity, intrinsic dissociation half-life and DM-mediated dissociation half-life for a large set of peptides derived from vaccinia virus and compared these properties to the peptide-specific CD4+ T cell responses. These data indicated that DM shapes the peptide repertoire during epitope selection by favoring the presentation of peptides with greater DM-mediated kinetic stability, and DM-susceptibility is a strong and independent factor governing peptide immunogenicity. In Chapter III, I computationally simulated peptide binding competition reactions and found that DM influences the IC50 (50% inhibition concentration) of peptides based on their susceptibility to DM, which was confirmed by experimental data. Therefore, I developed a novel fluorescence polarization-based method to measure DM-susceptibility, reported as a IC50 (change in IC50 in the absence and presence of DM). Traditional assays to measure DM-susceptibility based on differential peptide dissociation rates are cumbersome because each test peptide has to be individually labeled and multiple time point samples have to be collected. However, in this method developed here only single probe peptide has to be labeled and only single reading have to be done, which allows for fast and high throughput measure of DM-susceptibility for a large set of peptides. In Chapter IV, we generated a series of peptide and MHCII mutants, and investigated their interactions with DM. We found that peptides with non-optimal P1 pocket residues exhibit low MHCII affinity, low kinetic stability and high DM-susceptibility. These changes were accompanied with conformational alterations detected by surface plasmon resonance, gel filtration, dynamic light scattering, small-angle X-ray light scattering, antibody-binding, and nuclear magnetic resonance assays. Surprisingly, all these kinetic and conformational changes could be reversed by reconstitution with a more optimal P9 pocket residue. Taken together, our data demonstrated that conformation of MHCII-peptide complex constrained by interactions throughout the peptide binding groove is a key determinant of DM-susceptibility. B cells recognizing cognate antigen on the virion can internalize and process the whole virion for antigen presentation to CD4+ T cells specific for an epitope from any of the virion proteins. In turn, the epitope-specific CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to B cells to generate antibody responses against any protein from the whole virion. This viral intermolecular help model in which CD4+ T cells provide help to B cells with different protein specificities was established in small size influenza virus, hepatitis B virus and viral particle systems. For large and complex pathogens such as vaccinia virus and bacteria, the CD4+ T cell-B cell interaction model may be complicated because B cells might not be able to internalize the large whole pathogen. Recently, a study in mice observed that CD4+ T cell help is preferentially provided to B cells with the same protein specificity to generate antibody responses against vaccinia virus. However, for larger pathogens such as vaccinia virus and bacteria the CD4+ T cell-B cell interaction model has yet to be tested in humans. In Chapter V, I measured in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors the CD4+ T cell responses and antibody responses against four vaccinia viral proteins (A27L, A33R, B5R and L1R) known to be strongly targeted by cellular and humoral responses. We found that there is no direct linkage between antibody and CD4+ T cell responses against each protein. However, the presence of immune responses against these four proteins is linked together within donors. Taken together, our data indicated that individual viral proteins are not the primary recognition unit and CD4+ T cells provide intermolecular help to B cells to generate robust antibody responses against large and complicated vaccinia virus in humans.

INTRODUÇÃO: O penfigoide bolhoso é uma doença autoimune, vesicobolhosa com incidência de 0,2 a 1,4 por 100.000 hab. Sua fisiopatologia caracteriza-se pela ação de autoanticorpos na junção dermoepidérmica dos hemidesmossomos, promovendo a formação de bolhas subepidérmicas na pele e mucosas. Estudos vêm sendo publicados demonstrando a associação de penfigoide com alelos do sistema HLA classe II em diferentes populações do mundo, entretanto não há dados sobre a população brasileira, uma das mais heterogêneas do mundo. PACIENTES E MÉTODOS: O grupo de estudo incluiu 17 pacientes brasileiros com diagnóstico confirmado de PB de um hospital na cidade de São Paulo, sudeste do Brasil. O DNA foi extraído a partir de sangue periférico utilizando kits Qiagen (QIAamp DNA Mini Kit®) e a tipagem HLA loci A, B, C, DR e DQ foi realizada por meio de PCR e a amplificação utilizando o oligonucleótido de sequência específica (SSO) contido nos kits LABType®. O grupo controle foi composto por um banco de dados de 297 doadores falecidos da cidade de São Paulo. Este banco de dados é parte do Sistema de Transplantes da Secretaria de Saúde do Governo do Estado de São Paulo. RESULTADOS: Os resultados mostram que os alelos HLA C*17, DQB1*03:01, DQA1*01:03 e DQA1*05:05 estão associados com o aparecimento da doença na população brasileira, com risco relativo de 8,31 (2,46 a 28,16), 3,76 (1,81 a 7,79), 3,57 (1,53 a 8,33) e 4,02 (1,87 a 8,64), respectivamente (p < 0,005). O nível de significância estatística foi ajustado utilizando a correção de Bonferroni, dependendo das frequências fenotípicas avaliadas para HLA de classe I (A, B e C) e classe II (DRB1, DQB1 e DQA1). DISCUSSÃO: Os dados indicam que pacientes brasileiros com PB apresentam a mesma predisposição genética ligada ao HLA-DQB1*03:01 relatado anteriormente em caucasianos e indivíduos iranianos e o estudo apresenta três novos alelos (C *17, DQA1*01:03 e DQA1* 05:05) envolvidos na fisiopatologia da PB. CONCLUSÕES: Os resultados mostram que os alelos HLA C*17, DQB1*03:01, DQA1*01:03 e DQA1*05:05 estão associados com o aparecimento da doença na população brasileira / BACKGROUND: Bullous pemphigoid (BP) is an autoimmune disease with bullous vesicles and an incidence of 0.2 to 1.4 per 100,000 inhabitants. Its pathophysiology is characterized by the action of autoantibodies on hemidesmosomes at the dermalepidermal junction, promoting subepidermal blister formation in the skin and mucous membranes. Many studies have been published demonstrating the association of pemphigoid with HLA class II system alleles in different populations, however there are no data on the Brazilian population, one of the most heterogeneous in the world. PATIENTS AND METHODS: The study group included 17 Brazilian patients with a confirmed diagnosis of BP from a hospital in Sao Paulo city, southeast Brazil. DNA was extracted from peripheral blood using Qiagen kits (QIAamp DNA Mini Kit®) and HLA A, B, C, DR and DQ typing was performed using PCR and amplification using Sequence-Specific Oligonucleotide (SSO) contained in LABType® kits. The control group was composed of a database of 297 deceased donors from the city of São Paulo. This database is part of the Transplants State System of the Government\'s Health Secretary from the State of Sao Paulo. RESULTS: Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population, with relative risks of 8.31 (2.46 to 28.16), 3.76 (1.81 to 7.79), 3.57 (1.53 to 8.33), and 4.02 (1.87 to 8.64), respectively (p < 0.005). The statistical significance level was adjusted using the Bonferroni correction depending on the phenotypic frequencies evaluated for HLA class I (A, B and C) and class II (DRB1, DQB1 and DQA1). DISCUSSION: Our data indicate that Brazilian patients with BP present the same genetic predisposition linked to HLA-DQB1*03:01 previously reported in Caucasian and Iranian individuals and our study introduces three new alleles (C*17, DQA1*01:03 and DQA1*05:05) involved in the pathophysiology of BP. CONCLUSIONS: Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population

INTRODUÇÃO: O penfigoide bolhoso é uma doença autoimune, vesicobolhosa com incidência de 0,2 a 1,4 por 100.000 hab. Sua fisiopatologia caracteriza-se pela ação de autoanticorpos na junção dermoepidérmica dos hemidesmossomos, promovendo a formação de bolhas subepidérmicas na pele e mucosas. Estudos vêm sendo publicados demonstrando a associação de penfigoide com alelos do sistema HLA classe II em diferentes populações do mundo, entretanto não há dados sobre a população brasileira, uma das mais heterogêneas do mundo. PACIENTES E MÉTODOS: O grupo de estudo incluiu 17 pacientes brasileiros com diagnóstico confirmado de PB de um hospital na cidade de São Paulo, sudeste do Brasil. O DNA foi extraído a partir de sangue periférico utilizando kits Qiagen (QIAamp DNA Mini Kit®) e a tipagem HLA loci A, B, C, DR e DQ foi realizada por meio de PCR e a amplificação utilizando o oligonucleótido de sequência específica (SSO) contido nos kits LABType®. O grupo controle foi composto por um banco de dados de 297 doadores falecidos da cidade de São Paulo. Este banco de dados é parte do Sistema de Transplantes da Secretaria de Saúde do Governo do Estado de São Paulo. RESULTADOS: Os resultados mostram que os alelos HLA C*17, DQB1*03:01, DQA1*01:03 e DQA1*05:05 estão associados com o aparecimento da doença na população brasileira, com risco relativo de 8,31 (2,46 a 28,16), 3,76 (1,81 a 7,79), 3,57 (1,53 a 8,33) e 4,02 (1,87 a 8,64), respectivamente (p < 0,005). O nível de significância estatística foi ajustado utilizando a correção de Bonferroni, dependendo das frequências fenotípicas avaliadas para HLA de classe I (A, B e C) e classe II (DRB1, DQB1 e DQA1). DISCUSSÃO: Os dados indicam que pacientes brasileiros com PB apresentam a mesma predisposição genética ligada ao HLA-DQB1*03:01 relatado anteriormente em caucasianos e indivíduos iranianos e o estudo apresenta três novos alelos (C *17, DQA1*01:03 e DQA1* 05:05) envolvidos na fisiopatologia da PB. CONCLUSÕES: Os resultados mostram que os alelos HLA C*17, DQB1*03:01, DQA1*01:03 e DQA1*05:05 estão associados com o aparecimento da doença na população brasileira / BACKGROUND: Bullous pemphigoid (BP) is an autoimmune disease with bullous vesicles and an incidence of 0.2 to 1.4 per 100,000 inhabitants. Its pathophysiology is characterized by the action of autoantibodies on hemidesmosomes at the dermalepidermal junction, promoting subepidermal blister formation in the skin and mucous membranes. Many studies have been published demonstrating the association of pemphigoid with HLA class II system alleles in different populations, however there are no data on the Brazilian population, one of the most heterogeneous in the world. PATIENTS AND METHODS: The study group included 17 Brazilian patients with a confirmed diagnosis of BP from a hospital in Sao Paulo city, southeast Brazil. DNA was extracted from peripheral blood using Qiagen kits (QIAamp DNA Mini Kit®) and HLA A, B, C, DR and DQ typing was performed using PCR and amplification using Sequence-Specific Oligonucleotide (SSO) contained in LABType® kits. The control group was composed of a database of 297 deceased donors from the city of São Paulo. This database is part of the Transplants State System of the Government\'s Health Secretary from the State of Sao Paulo. RESULTS: Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population, with relative risks of 8.31 (2.46 to 28.16), 3.76 (1.81 to 7.79), 3.57 (1.53 to 8.33), and 4.02 (1.87 to 8.64), respectively (p < 0.005). The statistical significance level was adjusted using the Bonferroni correction depending on the phenotypic frequencies evaluated for HLA class I (A, B and C) and class II (DRB1, DQB1 and DQA1). DISCUSSION: Our data indicate that Brazilian patients with BP present the same genetic predisposition linked to HLA-DQB1*03:01 previously reported in Caucasian and Iranian individuals and our study introduces three new alleles (C*17, DQA1*01:03 and DQA1*05:05) involved in the pathophysiology of BP. CONCLUSIONS: Our findings show that alleles HLA C*17, DQB1*03:01, DQA1*01:03 and DQA1*05:05 are associated with the onset of the disease in the Brazilian population

This thesis is comprised of studies of proteins involved in class I and class II major histocompatibility complex (MHC) antigen procressing. In class I MHC processing, structural and functional studies were conducted of an aminopeptidase, ERAP1, that mediates the final step in antigen processing to understand how it is particularly suitable for cleavage of antigenic peptides for class I MHC presentation. In the class II MHC antigen presentation pathway, structural studies were conducted to characterize a fluorogenic peptide that can be used to understand peptide loading events in vivo and in real time. Also structural studies of class II MHC and peptide complexes were conducted to understand the nature of an unique C-terminal secondary structure element exhibited by an HIV derived peptide in the peptide binding groove of class II MHC. The studies discussed in this thesis provide insights into the proteins involved in the class I and class II MHC antigen presentation pathway. The endoplasmic reticulum (ER) aminopeptidase, ERAP1, is a 941 amino acid member of the M1 family of zinc metalloaminopeptidases. Unlike other aminopeptidases, ERAP1 has a length and C-terminal preference for its substrates. Interestingly, ERAP1 has been shown to trim antigenic peptides to lengths of 8 or 9 amino acids long. This length matches the length required to bind into the peptide binding groove of class I MHC molecules. In addition, ERAP1 is upregulated in the ER of cells treated with interferon gamma (IFN-γ). Knock-down of ERAP1 by siRNA results in less overall antigenic presentation during IFN-γ treatment, although the knock-down does not affect all class I MHC epitopes equally. Knock-out studies show that ERAP1 effects the antigen repertoire at the cell surface. These and other data implicate ERAP1 as an important player in class I MHC antigen presentation. A chapter of this thesis will describe the crystallographic work describing the structures of ERAP1 with an aminopeptidase inhibitor, bestatin, and ERAP1 without an inhibitor that suggest possible peptide binding site in ERAP1 that will allow it to generate suitable substrates for a subset of class I MHC alleles. Class II MHC plays a key role in the immune response by presenting antigenic peptides on CD4+ cytotoxic cell surfaces for T-cell response. The binding of peptides onto the MHC is an important step in creating an immune response. Structures of peptide bound MHC class II show conserved side chain binding pockets within the overall peptide-binding groove. In HLA-DR1, a common human class II MHC, the P1 pocket shows a preference for large hydrophobic side chains. Development of environmentally sensitive peptide analogs, that can bind into the class II MHC the same way as native peptides, can assist in visualizing the antigen binding process. A chapter in this thesis describes the crystallographic work showing that (4-DAPA)-HA can be used to study antigen-presenting processes in a cell by visualizing the changes in fluorescence of the synthesized peptide upon antigen loading. Crystallographic analysis of MHC class II, HLA-DR1, in complex with HIV gag-derived peptide, GagP16(PEVIPMFSALSEGATP), and superantigen, SEC3- 3B2, reveals the conventional polyproline conformation up to MHC binding pocket residue, P9, while the C-terminus of GagP16 adopts an unusual β- hairpin loop structure. Additionally, interactions between the leucine at P8 (LeuP8) and other residues on the loop such as ThrP16 and AlaP14 of the hairpin loop, was observed. Importantly, GagP16 requires the last 4 amino acids (P13-P16), which is part of the hairpin loop, for T-cell recognition. Understanding what dictates the C-terminal hairpin loop and the interaction motif of HLA-DR1/GagP16 complex with its TCR will provide insights on why it is important for T cell activation. A chapter in this thesis discusses the structural investigation conducted to understand the determinants of the loop at the C-terminus of GagP16 using designed peptides. It will also discuss work involving HLA-DR1 with the T cell receptor, AC25, that was cloned from T cells that are specific to HLA-DR1 in complex with the GagP16 peptide.

Les molécules classiques du complexe majeur d’histocompatibilité de classe II (CMHII) sont des glycoprotéines de surface spécialisées dans la présentation de peptides, principalement dérivés de pathogènes extracellulaires, aux récepteurs des lymphocytes T CD4+ afin d’initier la réponse immunitaire adaptative. Elles sont encodées, avec celles du CMH de classe I, par les gènes les plus polymorphiques identifiés jusqu’à maintenant, avec plusieurs loci et une grande diversité allélique à chacun d’eux. De plus, le polymorphisme des gènes du CMHII n’est pas limité qu’aux séquences codantes. Il est également observé dans les promoteurs où on a démontré ses effets sur le niveau d’expression des gènes. La variation de la régulation d’un gène est considérée comme un facteur important et pour laquelle des modifications morphologiques, physiologiques et comportementales sont observées chez tous les organismes. Des séquences d’ADN répétées impliquées dans cette régulation ont été identifiées dans les régions non-codantes des génomes. D’un autre côté, la sélection par les pathogènes permettrait l’évolution et le maintien du polymorphisme des gènes du CMH chez les vertébrés. À ce sujet, plusieurs études ont montré l’implication de différents allèles du CMH dans la résistance ou la susceptibilité aux maladies. Cette étude avait pour objectifs de caractériser le polymorphisme du gène MHIIb chez l’omble de fontaine (Salvelinus fontinalis) et de documenter ses effets au niveau de la survie conférée par des allèles et/ou génotypes particuliers lors d’une infection, ainsi que sur la variation du niveau d’expression du gène dans différentes conditions. Dans une première partie, nous avons identifié un total de 6 allèles du gène MHIIb, désignés Safo-DAB*0101 à Safo-DAB*0601, qui montrent une grande similarité avec les séquences codantes provenant de poissons téléostéens et de l’humain. L’analyse des séquences du domaine b1 a permis de détecter l’effet d’une pression sélective positive pour maintenir le polymorphisme dans cette région de la molécule. Quatre de ces allèles ont été testés lors d’une expérience d’infection avec le pathogène Aeromonas salmonicida afin d’évaluer l’effet qu’ils pouvaient avoir sur la survie des poissons. Nous avons trouvé que l’allèle DAB*0101 était significativement associé à la résistance à la furonculose. En plus d’avoir été identifié chez les individus homozygotes pour cet allèle, l’effet a également été remarqué au niveau de la survie les poissons de génotype DAB*0101/*0201. À l’opposé, les facteurs de risque élevé obtenus pour les génotypes DAB*0201/*0301 et DAB*0301/*0401 suggèrent plutôt une association à la susceptibilité. Étant donné la faible fréquence à laquelle l’allèle DAB*0101 a été retrouvé dans la population, le modèle de la sélection dépendante de la fréquence pourrait expliquer l’avantage conféré par ce dernier et souligne l’importance de ce mécanisme pour le maintien du polymorphisme du gène MHIIb chez l’omble de fontaine. Dans une seconde partie, nous avons rapporté la présence d’un minisatellite polymorphique formé d’un motif de 32 nucléotides dans le second intron du gène MHIIb, et pour lequel un nombre exclusif de répétitions du motif a été associé à chaque allèle (69, 27, 20, 40, 19 et 25 répétitions pour les allèles DAB*0101 à DAB*0601 respectivement). L’expression relative de quatre allèles a été évaluée dans des poissons hétérozygotes aux températures de 6 ºC et 18 ºC. Les résultats indiquent que les allèles possédant un long minisatellite montrent une réduction de l’expression du gène d’un facteur 1,67 à 2,56 par rapport aux allèles qui en contiennent un court. De même, des allèles qui incluent des minisatellites de tailles similaires n’affichent pas de différence significative au niveau de l’abondance du transcrit aux deux températures. De plus, l’effet répressif associé aux longs minisatellites est amplifié à la température de 18 ºC dans des poissons de trois génotypes différents. Nous avons finalement observé une augmentation significative par un facteur 2,08 de l’expression totale du gène MHIIb à la température de 6 ºC. Ces résultats appuient l’implication des séquences d’ADN répétées dans la régulation de l’activité transcriptionnelle d’un gène et suggèrent qu’un minisatellite sensible aux différences de températures pourrait être soumis aux forces sélectives et jouer un rôle important dans l’expression de gènes et l’évolution des organismes poïkilothermes. / Classical major histocompatibility complex class II (MHCII) molecules are cell-surface glycoproteins specialized in the presentation of peptides, mainly derived from extracellular pathogens, to the antigen receptors of CD4+ T cells in the adaptive immune system. They are encoded, with those of the MHC class I, by the most polymorphic genes known to date, with multiple loci and high allelic diversity at each one. Moreover, the polymorphism within MHCII genes is not restricted to coding sequences. It has also been observed in promoters where it was shown to affect the expression level of the genes. Variation in gene regulation is believed to be an important factor from which modification in morphology, physiology or behaviour can be observed in all organisms. Repeated DNA sequences with functional roles in this regulation have been identified within the non-coding parts of the genomes. On the other hand, pathogen-driven selection is also believed to be important in the evolution and maintenance of the polymorphism of the MHC genes in vertebrates. Studies have shown the implication of different MHC alleles in disease resistance or susceptibility. In this study, our aims were to characterize the polymorphism of the MHIIb gene in brook charr (Salvelinus fontinalis), to document its effects on the survival conferred by specific alleles and/or genotypes following an infection and on the variation of the expression level of the gene in different environmental conditions. In a first part, we identified a total of 6 MHIIb alleles, designated Safo-DAB*0101 to Safo-DAB*0601, showing a high similarity to coding sequences from teleost fish and human. Analysis of the b1 domain sequences indicates the effect of a positive selection pressure to select polymorphic mutations in that region of the molecule. Four of these alleles were tested in a challenge experiment against the pathogen Aeromonas salmonicida to evaluate their effect on fish survival. We found that one allele, DAB*0101, was significantly associated with resistance to furonculosis. In addition to homozygotes for this allele, its resistance effect was also detected in the heterozygote individuals of the DAB*0101/*0201 genotype. In contrast, other allelic combinations, namely heterozygous genotypes DAB*0201/*0301 and DAB*0301/*0401 were significantly associated with increased susceptibility. Given that its frequency was relatively low in the population, the negative frequency dependant selection hypothesis could explain the advantage associated with the allele DAB*0101 over the other alleles and highlight the importance of this mechanism to sustain variation at the MHC in brook charr. In a second part, we reported the identification of a polymorphic minisatellite formed of a 32 nucleotides motif in the second intron of MHIIb gene, and for which distinctive repeat numbers of the motif were associated to each alleles (69, 27, 20, 40, 19 and 25 repeats for the DAB*0101 to DAB*0601 alleles respectively). Relative expression levels of four alleles were determined in heterozygous fish at temperature of 18 ºC and 6 ºC. Results indicate that alleles carrying the longest minisatellite showed a 1.67 to 2.56-fold reduction in the transcript expression relatively to the shortest one. In contrast, no significant differences were seen in the expression levels between alleles with comparable minisatellite length at both temperatures. Furthermore, the repressive activity associated to the longest minisatellite was more effective at temperature of 18 ºC in fish from three different genotypes. We finally observed a significant 2.08-fold up-regulation of the total MHII transcript amount at 6 ºC. The results support the implication of repeated DNA sequences in the regulation of the gene transcriptional activity and suggest that a temperature-sensitive minisatellite could potentially be submitted to selective forces and therefore play an important role in gene expression and evolution in ectothermic organisms.

INTRODUÇÃO. A proteína Fator Inibitório da Migração de Macrófagos (MIF) é frequentemente observada com expressão elevada em tecidos tumorais quando comparados aos tecidos equivalentes normais e estudos têm sugerido seu papel como marcador prognóstico de neoplasias malignas, incluindo carcinomas hepatocelular, de ovário, de esôfago e também de cabeça e pescoço. Adicionalmente, alguns de seus mecanismos de ação já demonstrados, como a indução da proliferação e migração celular permitem implicar essa expressão diferencial no desenvolvimento tumoral e, consequentemente, no prognóstico das neoplasias malignas. OBJETIVO. Esse estudo objetivou avaliar o papel diagnóstico e prognóstico da proteína MIF em carcinoma epidermóide da cavidade bucal. METODOLOGIA. O estudo foi composto por 50 pacientes com carcinoma epidermoide da cavidade bucal coletados prospectivamente e 57 casos retrospectivos admitidos nos Serviços de Cirurgia de Cabeça e Pescoço do Hospital Heliópolis e da Faculdade de Medicina da Fundação ABC. As análises foram feitas por meio de imunoistoquímica dos tecidos tumorais e margens epiteliais normais e de ELISA das amostras de soro e saliva, coletadas pré e pós-tratamento cirúrgico, dos pacientes participantes. Os resultados foram correlacionados aos achados clínicos e histopatológicos. RESULTADOS. A expressão da proteína MIF e seu receptor CD74 mostrou-se elevada em tecido tumoral quando comparado ao tecido epitelial livre de neoplasia (p < 0,0001). Associação entre a alta expressão da MIF no tumor e infiltração vascular linfática foi observada (p=0,005) e alta expressão da MIF no epitélio livre de tumor apresentou correlação marginalmente significativa com ocorrência de segundo tumor primário (p=0,072). A expressão positiva do receptor CD74 não apresentou associação com variáveis clínicas ou histopatológicas. A concentração sorológica da proteína MIF apresentou associação inversa com metástase linfonodal (p=0,018) e estádios patológicos avançados (p=0,040) e foi significativamente reduzida após a ressecção do tumor (p=0,001). A concentração da MIF na saliva não apresentou redução significativa após o tratamento cirúrgico, mas foi associada aos estágios pT3 e pT4 (p=0,001) e a estádios patológicos avançados (p=0,032). CONCLUSÕES. A redução significante da concentração da MIF observada no soro dos pacientes após a ressecção cirúrgica do tumor permite sugerir papel potencial dessa proteína como biomarcador para a detecção precoce de recorrência do carcinoma epidermoide da cavidade bucal. A expressão tecidual da proteína MIF e seu receptor CD74 apresentou papel controverso, mas a concentração salivar da proteína MIF parece relacionar-se a um possível papel pró-tumoral em carcinoma epidermoide da cavidade bucal / INTRODUCTION. The Macrophage Migration Inhibitory Factor (MIF) overexpression is frequently observed in tumor tissues compared to normal tissues and some previous studies have suggested its role as a prognostic marker of malignancies, including hepatocellular, ovarian, esophageal and also head and neck carcinoma. Additionally, some of its mechanisms of action, as migration and cell proliferation induction, have been demonstrated, which allow imply a differential expression in tumor progression and therefore in the prognosis of malignant neoplasms. OBJECTIVES. This study aimed to evaluate the role of MIF protein and its receptor CD74 in prognosis and diagnostic of oral squamous cell carcinoma. METHODS. The study consisted of 50 patients with oral squamous cell carcinoma prospectively collected and 57 patients retrospectively collected admitted at the Head and Neck Surgery Service from Heliópolis Hospital and ABC Medical School. The analysis were performed by Imunohistochemistry of tumor and normal tissues and by ELISA of serum and saliva samples collected pre and post-surgical treatment. Results were correlated to clinical and histopathological data. RESULTS. The expression of MIF protein and of its receptor CD74 was higher in OSCC than in normal epithelium (p < 0,0001). Association between overexpression of MIF in tumor tissue and lymphatic vessel invasion was observed (p=0,005) and higher concentration of MIF in normal epithelium showed correlation of marginal significance with second primary tumor occurrence (p=0,072). The positive expression of the receptor CD74 did not presented association with clinical or histopathological variables. Serum MIF concentration presented inverse association with lymph node metastasis (p=0,018) and advanced pathological stage (p=0,040) and it was significantly reduced after the surgery (p=0,001). The salivary MIF concentration was not significantly reduced after the surgery, but it was associated with pT3 and pT4 stages (p=0,001) and advanced pathological findings (p=0,032). CONCLUSIONS. The results showing significant reduction of MIF concentration in post-surgical serum of patients suggest its potential role as a biomarker to early detection of oral squamous cell carcinoma recurrence. The MIF and CD74 expression presented controversial role, but the salivary concentration of MIF seems to develop a possible pro-tumoral role

Les molécules classiques du complexe majeur d’histocompatibilité de classe II (CMHII) sont des glycoprotéines de surface spécialisées dans la présentation de peptides, principalement dérivés de pathogènes extracellulaires, aux récepteurs des lymphocytes T CD4+ afin d’initier la réponse immunitaire adaptative. Elles sont encodées, avec celles du CMH de classe I, par les gènes les plus polymorphiques identifiés jusqu’à maintenant, avec plusieurs loci et une grande diversité allélique à chacun d’eux. De plus, le polymorphisme des gènes du CMHII n’est pas limité qu’aux séquences codantes. Il est également observé dans les promoteurs où on a démontré ses effets sur le niveau d’expression des gènes. La variation de la régulation d’un gène est considérée comme un facteur important et pour laquelle des modifications morphologiques, physiologiques et comportementales sont observées chez tous les organismes. Des séquences d’ADN répétées impliquées dans cette régulation ont été identifiées dans les régions non-codantes des génomes. D’un autre côté, la sélection par les pathogènes permettrait l’évolution et le maintien du polymorphisme des gènes du CMH chez les vertébrés. À ce sujet, plusieurs études ont montré l’implication de différents allèles du CMH dans la résistance ou la susceptibilité aux maladies. Cette étude avait pour objectifs de caractériser le polymorphisme du gène MHIIb chez l’omble de fontaine (Salvelinus fontinalis) et de documenter ses effets au niveau de la survie conférée par des allèles et/ou génotypes particuliers lors d’une infection, ainsi que sur la variation du niveau d’expression du gène dans différentes conditions. Dans une première partie, nous avons identifié un total de 6 allèles du gène MHIIb, désignés Safo-DAB*0101 à Safo-DAB*0601, qui montrent une grande similarité avec les séquences codantes provenant de poissons téléostéens et de l’humain. L’analyse des séquences du domaine b1 a permis de détecter l’effet d’une pression sélective positive pour maintenir le polymorphisme dans cette région de la molécule. Quatre de ces allèles ont été testés lors d’une expérience d’infection avec le pathogène Aeromonas salmonicida afin d’évaluer l’effet qu’ils pouvaient avoir sur la survie des poissons. Nous avons trouvé que l’allèle DAB*0101 était significativement associé à la résistance à la furonculose. En plus d’avoir été identifié chez les individus homozygotes pour cet allèle, l’effet a également été remarqué au niveau de la survie les poissons de génotype DAB*0101/*0201. À l’opposé, les facteurs de risque élevé obtenus pour les génotypes DAB*0201/*0301 et DAB*0301/*0401 suggèrent plutôt une association à la susceptibilité. Étant donné la faible fréquence à laquelle l’allèle DAB*0101 a été retrouvé dans la population, le modèle de la sélection dépendante de la fréquence pourrait expliquer l’avantage conféré par ce dernier et souligne l’importance de ce mécanisme pour le maintien du polymorphisme du gène MHIIb chez l’omble de fontaine. Dans une seconde partie, nous avons rapporté la présence d’un minisatellite polymorphique formé d’un motif de 32 nucléotides dans le second intron du gène MHIIb, et pour lequel un nombre exclusif de répétitions du motif a été associé à chaque allèle (69, 27, 20, 40, 19 et 25 répétitions pour les allèles DAB*0101 à DAB*0601 respectivement). L’expression relative de quatre allèles a été évaluée dans des poissons hétérozygotes aux températures de 6 ºC et 18 ºC. Les résultats indiquent que les allèles possédant un long minisatellite montrent une réduction de l’expression du gène d’un facteur 1,67 à 2,56 par rapport aux allèles qui en contiennent un court. De même, des allèles qui incluent des minisatellites de tailles similaires n’affichent pas de différence significative au niveau de l’abondance du transcrit aux deux températures. De plus, l’effet répressif associé aux longs minisatellites est amplifié à la température de 18 ºC dans des poissons de trois génotypes différents. Nous avons finalement observé une augmentation significative par un facteur 2,08 de l’expression totale du gène MHIIb à la température de 6 ºC. Ces résultats appuient l’implication des séquences d’ADN répétées dans la régulation de l’activité transcriptionnelle d’un gène et suggèrent qu’un minisatellite sensible aux différences de températures pourrait être soumis aux forces sélectives et jouer un rôle important dans l’expression de gènes et l’évolution des organismes poïkilothermes. / Classical major histocompatibility complex class II (MHCII) molecules are cell-surface glycoproteins specialized in the presentation of peptides, mainly derived from extracellular pathogens, to the antigen receptors of CD4+ T cells in the adaptive immune system. They are encoded, with those of the MHC class I, by the most polymorphic genes known to date, with multiple loci and high allelic diversity at each one. Moreover, the polymorphism within MHCII genes is not restricted to coding sequences. It has also been observed in promoters where it was shown to affect the expression level of the genes. Variation in gene regulation is believed to be an important factor from which modification in morphology, physiology or behaviour can be observed in all organisms. Repeated DNA sequences with functional roles in this regulation have been identified within the non-coding parts of the genomes. On the other hand, pathogen-driven selection is also believed to be important in the evolution and maintenance of the polymorphism of the MHC genes in vertebrates. Studies have shown the implication of different MHC alleles in disease resistance or susceptibility. In this study, our aims were to characterize the polymorphism of the MHIIb gene in brook charr (Salvelinus fontinalis), to document its effects on the survival conferred by specific alleles and/or genotypes following an infection and on the variation of the expression level of the gene in different environmental conditions. In a first part, we identified a total of 6 MHIIb alleles, designated Safo-DAB*0101 to Safo-DAB*0601, showing a high similarity to coding sequences from teleost fish and human. Analysis of the b1 domain sequences indicates the effect of a positive selection pressure to select polymorphic mutations in that region of the molecule. Four of these alleles were tested in a challenge experiment against the pathogen Aeromonas salmonicida to evaluate their effect on fish survival. We found that one allele, DAB*0101, was significantly associated with resistance to furonculosis. In addition to homozygotes for this allele, its resistance effect was also detected in the heterozygote individuals of the DAB*0101/*0201 genotype. In contrast, other allelic combinations, namely heterozygous genotypes DAB*0201/*0301 and DAB*0301/*0401 were significantly associated with increased susceptibility. Given that its frequency was relatively low in the population, the negative frequency dependant selection hypothesis could explain the advantage associated with the allele DAB*0101 over the other alleles and highlight the importance of this mechanism to sustain variation at the MHC in brook charr. In a second part, we reported the identification of a polymorphic minisatellite formed of a 32 nucleotides motif in the second intron of MHIIb gene, and for which distinctive repeat numbers of the motif were associated to each alleles (69, 27, 20, 40, 19 and 25 repeats for the DAB*0101 to DAB*0601 alleles respectively). Relative expression levels of four alleles were determined in heterozygous fish at temperature of 18 ºC and 6 ºC. Results indicate that alleles carrying the longest minisatellite showed a 1.67 to 2.56-fold reduction in the transcript expression relatively to the shortest one. In contrast, no significant differences were seen in the expression levels between alleles with comparable minisatellite length at both temperatures. Furthermore, the repressive activity associated to the longest minisatellite was more effective at temperature of 18 ºC in fish from three different genotypes. We finally observed a significant 2.08-fold up-regulation of the total MHII transcript amount at 6 ºC. The results support the implication of repeated DNA sequences in the regulation of the gene transcriptional activity and suggest that a temperature-sensitive minisatellite could potentially be submitted to selective forces and therefore play an important role in gene expression and evolution in ectothermic organisms.