An individual's Human Leukocyte Antigen (HLA) type is an essential immunogenetic parameter, influencing susceptibility to a variety of autoimmune and infectious diseases, to certain types of cancer and the likelihood of adverse drug reactions. I present and evaluate two models for the accurate statistical determination of HLA types for single-population and multi-population studies, based on SNP genotypes. Importantly, SNP genotypes are already available for many studies, so that the application of the statistical methods presented here does not incur any extra cost besides computing time. HLA*IMP:01 is based on a parallelized and modified version of LDMhc (Leslie et al., 2008), enabling the processing of large reference panels and improving call rates. In a homogeneous single-population imputation scenario on a mainly British dataset, it achieves accuracies (posterior predictive values) and call rates >=88% at all classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DRB1) at 4-digit HLA type resolution. HLA*IMP:02 is specifically designed to deal with multi-population heterogeneous reference panels and based on a new algorithm to construct haplotype graph models that takes into account haplotype estimate uncertainty, allows for missing data and enables the inclusion of prior knowledge on linkage disequilibrium. It works as well as HLA*IMP:01 on homogeneous panels and substantially outperforms it in more heterogeneous scenarios. In a cross-European validation experiment, even without setting a call threshold, HLA*IMP:02 achieves an average accuracy of 96% at 4-digit resolution (>=91% for all loci, which is achieved at HLA-DRB1). HLA*IMP:02 can accurately predict structural variation (DRB paralogs), can (to an extent) detect errors in the reference panel and is highly tolerant of missing data. I demonstrate that a good match between imputation and reference panels in terms of principal components and reference panel size are essential determinants of high imputation accuracy under HLA*IMP:02.

Le système ubiquitine-protéasome est le principal mécanisme par lequel les protéines intracellulaires sont dégradées. Le protéasome dit constitutif (PC) est donc essentiel à l’homéostasie mais aussi à la régulation de la majorité des processus cellulaires importants. La découverte d’un deuxième type de protéasome, appelé immunoprotéasome (IP), soulève toutefois de nouvelles questions. Pourquoi existe-t-il plus d’un type de protéasome ? L’IP a-t-il des rôles redondants ou complémentaires avec le PC ? L’IP étant présent principalement dans les cellules immunitaires ou stimulées par des cytokines, plusieurs groupes ont tenté de définir son rôle dans la réponse immunitaire. Or, l’implication de son homologue constitutif dans un éventail de processus non spécifiquement immunitaires nous laisse croire que l’IP pourrait lui aussi avoir un impact beaucoup plus large. L’objectif de cette thèse était donc de caractériser certains rôles cellulaires de l’IP dans les cellules dendritiques. Nous avons d’abord étudié l’impact global de l’IP sur la présentation antigénique de classe I. Ce faisant, nous avons pu déterminer ses deux contributions principales, soit l’augmentation drastique du nombre et de la diversité des peptides présentés sur les complexes majeurs d’histocompatibilité de classe I. Les différences de clivage entre le PC et l’IP pourraient expliquer en partie cette diversité du répertoire peptidique, notamment par l’affinité apparente de l’IP pour les régions protéiques non structurées. Dans un deuxième temps, nous avons dévoilé un nouveau rôle de l’IP sur un processus dépassant le cadre immunitaire : la transcription. Nous avons découvert que l’IP modifie l’abondance des ARNm en agissant principalement au niveau de leur synthèse. L’impact de l’IP sur le transcriptome est majeur et serait dû en partie à une dégradation différente de facteurs de transcription des familles IRF, STAT et NF-kB. Les cellules dendritiques IP-déficientes activent moins efficacement les lymphocytes T CD8+ et nous croyons que cette défaillance est causée (du moins en partie) par la perturbation transcriptomique provoquée par l’absence d’IP. Il importe donc de comprendre les différents rôles moléculaires de l’IP afin de mieux définir sa contribution globale au fonctionnement de la cellule et comprendre l’avantage évolutif, au niveau de l’organisme, procuré par une telle plasticité du système ubiquitine-protéasome. / The ubiquitin-proteasome system is the major mechanism by which intracellular proteins get degraded. Constitutive proteasomes (CPs) are thus essential for cellular homeostasis but also to regulate the majority of important cellular processes. However, the discovery of a second type of proteasome, named immunoproteasome (IP), raises new questions. Why are there more than one type of proteasome? Does the IP perform redundant or complementary roles with the CP? The IP is predominantly expressed in immune or cytokine-stimulated cells and several groups worked at defining its role during the immune response. Yet, the implication of its constitutive homolog in a variety of processes suggests that the IP may also have a much broader impact. The objective was to characterize cellular roles of the IP in dendritic cells. We first studied the global impact of the IP on class I antigen presentation. We discovered that the IP drastically increases the number and the diversity of peptide presented by class I major histocompatibility complexes. Cleavage differences between the CP and the IP are likely part of the explanation for this peptide repertoire diversity, notably due to IP’s apparent affinity for unstructured protein regions. Second, we discovered a new role for the IP in a process unrestricted to the immune system: transcription. We found that the IP affects transcript abundance mostly at the level of mRNA synthesis. The impact of IPs on the transcriptome is major and would be partly based on a different degradation of IRF, STAT and NF-kB transcription factor family members by the two types of proteasomes. IP-deficient dendritic cells are less potent activators of CD8+ T cells and we believe that this defect is at least partly caused by the transcriptome alterations induced by the absence of IPs. It is therefore important to understand the different molecular roles of the IP in order to better define its global contribution to cellular functions and to understand the evolutionary advantage, at the level of the organism, brought by such plasticity of the ubiquitin- proteasome system.

L’étude des interactions hôtes-parasites est devenue un thème de recherche incontournable pour les sciences de l’évolution. Cette coévolution complexe dépend de nombreux compromis évolutifs et peut être grandement influencée par les facteurs environnementaux. Nous nous proposons ici d’étudier les interactions hôtes-parasites à plusieurs échelles, à travers des approches expérimentales et des études en populations naturelles, en étudiant les parasites de la malaria aviaire. Dans un premier temps, nous nous sommes intéressés à l’influence des caractéristiques de l’hôte et notamment au système immunitaire. Le système immunitaire est bénéfique pour l’hôte dans sa lutte contre le parasite, mais peut également engendrer des coûts immunopathologiques. Des traits d’histoire de vie, comme l’âge ou le statut social peuvent modifier la parasitémie au sein des hôtes, sans toutefois avoir d’effet sur la prévalence. Dans un second temps, l’effet de certains facteurs environnementaux a été évalué au sein des interactions hôtes-parasites. La température et la contamination en métaux lourds ont un effet sur la prévalence dans les populations, mais n’affectent pas la parasitémie. Au cours de cette thèse, nous avons également montré l’influence directe des parasites sanguins sur la structure génétique des populations hôtes, notamment au niveau des gènes du CMH. / Host-parasite interactions are one of the main topics in evolutionary sciences. This complex coevolution depends on several trade-offs and can be influenced by environmental factors. Here, we propose to study host-parasite interactions with a multi-level approach, using experimental and natural population studies, focusing on avian malaria parasites. First, we studied the effect of host characteristics, and more precisely the immune system. The immune system confers benefits in terms of protection against the parasite, but can also generated immunopathological costs. Life history traits, like age or social status, appear to modify parasitemia but not prevalence. In a second part, we evaluated the effect of environmental factors on host-parasite interactions. We found that temperature and heavy metal contamination had an effect on population prevalence, but not on host parasitemia. We also showed the direct parasite influence on host population genetic structure, and more precisely on MHC genes.

Les cellules neurales expriment des molécules dites immunes qui peuvent exercer des rôles différents de ceux exercés dans le système immunitaire. Les molécules du CMH-I classiques présentent des peptides représentatifs du contenu protéique de chaque cellule aux sentinelles du système immunitaire. Cependant, il est documenté que ces molécules ont aussi des fonctions « non immunes ». En effet, les molécules du CMH-I classiques jouent un rôle dans l'établissement et la plasticité des synapses. Sur divers types cellulaires, elles peuvent aussi interagir avec des récepteurs membranaires en cis, moduler leur stabilité à la membrane et en conséquence leur activité. RAE-1 est un membre de la famille des molécules du CMH-I, décrite initialement dans le système nerveux central embryonnaire. Pour le système immunitaire, RAE-1 est un ligand du récepteur activateur NKG2D, exprimé par les cellules NK, NKT, les lymphocytes T γδ et CD8+. RAE-1 est peu ou pas exprimé dans la plupart des tissus adultes. Son expression est induite par le stress génotoxique, la transformation tumorale ou l'infection virale ce qui permet au système immunitaire d'éliminer les cellules « malades » grâce à l'activation des cellules cytotoxiques exprimant NKG2D. Je décris l'expression de RAE-1 par les cellules neurales progénitrices et le rôle non immun de cette molécule dans la prolifération cellulaire. L'expression de RAE-1 est fortement corrélée au niveau de prolifération cellulaire et est dépendante du facteur de croissance EGF. / Neural cells express immune molecules which roles differ from those in the immune system. Classical MHC-I molecules present peptides originated from the proteic content of each cell to patrolling immune cells. However, these molecules can also have nonimmune roles. Indeed, classical MHC-I molecules participate in the establishment of synapses and synaptic plasticity. They can also interact in cis with different membrane receptors on different cell types, and modulate the receptors' membrane stability and activity. RAE-1, a member of MHC-I family, was initially described in the embryonic central nervous system. In the immune system, RAE-1 is a ligand of the activating receptor NKG2D, expressed by NK cells and by NKT, γδT and some CD8+ T lymphocytes. RAE-1 is weakly or not expressed in most adult tissues. Its expression is induced by genotoxic stress, tumoral transformation or viral infection and triggers the elimination of transformed cells by the cytotoxic immune cells which express NKG2D. I describe here the expression of RAE-1 by neural progenitor cells and its role in cell proliferation. RAE-1 expression level is highly correlated with the rate of cell proliferation and depends on the presence of epidermal growth factor (EGF). Exposition to EGF induces the colocalization of RAE-1 and phosphorylated EGF-receptor (EGFR) inside lipid rafts and endocytosed vesicles, which supports a role of RAE-1 as a partner of EGFR. RAE-1 expression is also induced in the nervous tissue in different models of CNS pathologies. In these conditions, RAE-1 could be expressed by proliferating microglia under the control of M-CSF.

A hepatite auto-imune (HAI) é uma doença inflamatória crônica do fígado, de etiologia desconhecida, que acomete preferencialmente mulheres, com destruição progressiva do parênquima hepático e que, sem tratamento imunossupressor, evolui freqüentemente para cirrose. É uma doença rara na infância, com menos de 10% dos pacientes com doença hepática crônica, porém de alta mortalidade. Caracteriza-se pela presença de hipergamaglobulinemia, auto-anticorpos não órgãos-específicos e infiltrado inflamatório portal linfoplasmocitário. Cerca da metade dos pacientes atendidos no Instituto da Criança, apresenta também níveis elevados de IgE, sem causas aparentes como parasitoses ou atopia. A suscetibilidade genética à doença está principalmente associada a genes que codificam as moléculas de histocompatibilidade (HLA). A presença do HLA-DR é importante, mas não suficiente para o desenvolvimento dessa doença rara, fazendo inclusive supor um forte componente externo/ambiental no desencadeamento da doença. Genes recém descritos, localizados na região de classe III do Complexo Principal de Histocompatibilidade (CPH) e ligados ao controle da resposta imune, em especial, alguns próximos à junção com a região de classe I, têm sido investigados como \"loci\" secundários para o desenvolvimento de doenças auto-imunes. A forte associação da HAI com genes na região do MHC, bastante comuns na população, aliada a incidência muito baixa da doença, leva à questão da presença de genes adicionais de suscetibilidade, que, junto com o HLA-DR, seriam responsáveis pela suscetibilidade genética observada. Dessa forma, o HLADRB1* 13, além de um fator por si, também pode ser um marcador da região cromossômica, ou seja, de um haplótipo específico e que, portanto, carrega mais de um gene de suscetibilidade. Estudamos polimorfismos, tipo SNP, de xx genes próximos ao HLA-DRB1, como TNFA, LTA, NFKBIL1 e BAT1, buscando haplótipos de susceptibilidade à doença, em pacientes HAI-1 (n=105) e controles sadios (n=227). O haplótipo ancestral 8.1 que inclui HLA-DRB1*03 e o alelo raro na posição -308 do gene TNFA estava aumentado (p=0.0005). Já o alelo HLA-DRB1*13, presente na maioria dos pacientes, não mostrou haplótipo específico associado. Também avaliamos genes de citocinas envolvidas na produção de IgE, elevado em parte dos pacientes HAI-1. Na comparação com controles, a freqüência dos SNPs IL- 4+33 e IL13+110, localizados em genes vizinhos, apresentou aumento estatisticamente significante nos pacientes, sugerindo haver um grupo gênico adicional no cromossomo 5q31 envolvido na susceptibilidade à HAI / Autoimmune hepatitis (AIH) is an inflammatory chronic liver disease of unknown etiology found predominantly in females, leading when untreated, to cirrhosis. It is a rare disease in the childhood, corresponding to about 10% of patients with chronic hepatitis, but exhibits high mortality. It is characterized by hipergammaglobulinemia, organ nonspecific circulating autoantibodies, and an inflammatory liver-infiltrating lymphocytes and plasma cells. Almost half of patients investigated at Instituto da Criança had increased plasma IgE levels, without any apparent cause such as parasite infestation or atopy. Genetic predisposition to AIH has been mainly linked to genes coding for HLA class II molecules. HLA-DR is important but not sufficient to explain this rare disease, suggesting there is an external/environmental component triggering the disease. Recently, several genes in the class III region of MHC, linked to immune responses, especially near the junction with the MHC class I region have been investigated as secondary loci for autoimmune disease susceptibility. The strong association of AIH with genes in the MHC region, common in the population, but a disease with the very low incidence, suggests additional genes linked with HLA-DR could add to the disease susceptibility. So, HLA-DR*13 besides being a factor by itself, could also be a chromosomal region marker, taking part of a specific haplotype carrying more than one susceptibility gene. We studied single nucleotide polymorphisms (SNP) in genes near HLA-DRB1, like TNFA, LTA, NFKBIL1 and BAT1 searching for disease susceptibility haplotypes, in HAI-1 patients (n=105) compared to healthy controls (n=227). The ancestral haplotype 8.1, which includes HLA-DRB1*03 and the rare TNFA allele at position -308 was increased (p=0.0005). However, HLA-DRB1*13, though present in the majority of the patients, did not show any specific haplotype associated to it. xxii We also analyzed cytokine genes involved in IgE production, which is increased in a part of the AIH type 1 patients. In comparison with controls, the frequency of the SNPs IL-4+33 and IL13+110 was significantly increased, suggesting the existence of an additional gene cluster in chromosome 5q31 involved in the susceptibility to AIH

The immune system is essential for keeping the integrity of multicellular organisms. We were able to make a step forward in studying the complex immune reactions in mammals in vivo and/ or in situ using the major histocompatibility complex (MHC) class II/ enhanced green fluorescent protein (EGFP) knock-in mouse model. Due to the EGFP visualization of MHC II expressing cells we were able to observe antigen presenting cells, which are essential for the onset of immune responses, in their natural environment. Thus, we report some original features of the immune system. We have identified MHC II+ cell clusters with unknown, probably unique function, in the intestine. We have also described MHC II+ cell migration to the lactating mammary gland and tested few hypotheses about the role of this phenomenon for the development of the mammary gland, milk secretion or infant immune system establishment. Lastly, we observed residential macrophages in the cornea. The presence of APCs in the cornea is a very contradictory issue due to the fact that cornea is an immunologically privileged tissue and therefore harbors special immune features. key words: antigen presenting cells (APC), major histocompatibility complex class II (MHC II), enhanced green fluorescent protein (EGFP), immune system, knock-in mouse model

A hepatite auto-imune (HAI) é uma doença inflamatória crônica do fígado, de etiologia desconhecida, que acomete preferencialmente mulheres, com destruição progressiva do parênquima hepático e que, sem tratamento imunossupressor, evolui freqüentemente para cirrose. É uma doença rara na infância, com menos de 10% dos pacientes com doença hepática crônica, porém de alta mortalidade. Caracteriza-se pela presença de hipergamaglobulinemia, auto-anticorpos não órgãos-específicos e infiltrado inflamatório portal linfoplasmocitário. Cerca da metade dos pacientes atendidos no Instituto da Criança, apresenta também níveis elevados de IgE, sem causas aparentes como parasitoses ou atopia. A suscetibilidade genética à doença está principalmente associada a genes que codificam as moléculas de histocompatibilidade (HLA). A presença do HLA-DR é importante, mas não suficiente para o desenvolvimento dessa doença rara, fazendo inclusive supor um forte componente externo/ambiental no desencadeamento da doença. Genes recém descritos, localizados na região de classe III do Complexo Principal de Histocompatibilidade (CPH) e ligados ao controle da resposta imune, em especial, alguns próximos à junção com a região de classe I, têm sido investigados como \"loci\" secundários para o desenvolvimento de doenças auto-imunes. A forte associação da HAI com genes na região do MHC, bastante comuns na população, aliada a incidência muito baixa da doença, leva à questão da presença de genes adicionais de suscetibilidade, que, junto com o HLA-DR, seriam responsáveis pela suscetibilidade genética observada. Dessa forma, o HLADRB1* 13, além de um fator por si, também pode ser um marcador da região cromossômica, ou seja, de um haplótipo específico e que, portanto, carrega mais de um gene de suscetibilidade. Estudamos polimorfismos, tipo SNP, de xx genes próximos ao HLA-DRB1, como TNFA, LTA, NFKBIL1 e BAT1, buscando haplótipos de susceptibilidade à doença, em pacientes HAI-1 (n=105) e controles sadios (n=227). O haplótipo ancestral 8.1 que inclui HLA-DRB1*03 e o alelo raro na posição -308 do gene TNFA estava aumentado (p=0.0005). Já o alelo HLA-DRB1*13, presente na maioria dos pacientes, não mostrou haplótipo específico associado. Também avaliamos genes de citocinas envolvidas na produção de IgE, elevado em parte dos pacientes HAI-1. Na comparação com controles, a freqüência dos SNPs IL- 4+33 e IL13+110, localizados em genes vizinhos, apresentou aumento estatisticamente significante nos pacientes, sugerindo haver um grupo gênico adicional no cromossomo 5q31 envolvido na susceptibilidade à HAI / Autoimmune hepatitis (AIH) is an inflammatory chronic liver disease of unknown etiology found predominantly in females, leading when untreated, to cirrhosis. It is a rare disease in the childhood, corresponding to about 10% of patients with chronic hepatitis, but exhibits high mortality. It is characterized by hipergammaglobulinemia, organ nonspecific circulating autoantibodies, and an inflammatory liver-infiltrating lymphocytes and plasma cells. Almost half of patients investigated at Instituto da Criança had increased plasma IgE levels, without any apparent cause such as parasite infestation or atopy. Genetic predisposition to AIH has been mainly linked to genes coding for HLA class II molecules. HLA-DR is important but not sufficient to explain this rare disease, suggesting there is an external/environmental component triggering the disease. Recently, several genes in the class III region of MHC, linked to immune responses, especially near the junction with the MHC class I region have been investigated as secondary loci for autoimmune disease susceptibility. The strong association of AIH with genes in the MHC region, common in the population, but a disease with the very low incidence, suggests additional genes linked with HLA-DR could add to the disease susceptibility. So, HLA-DR*13 besides being a factor by itself, could also be a chromosomal region marker, taking part of a specific haplotype carrying more than one susceptibility gene. We studied single nucleotide polymorphisms (SNP) in genes near HLA-DRB1, like TNFA, LTA, NFKBIL1 and BAT1 searching for disease susceptibility haplotypes, in HAI-1 patients (n=105) compared to healthy controls (n=227). The ancestral haplotype 8.1, which includes HLA-DRB1*03 and the rare TNFA allele at position -308 was increased (p=0.0005). However, HLA-DRB1*13, though present in the majority of the patients, did not show any specific haplotype associated to it. xxii We also analyzed cytokine genes involved in IgE production, which is increased in a part of the AIH type 1 patients. In comparison with controls, the frequency of the SNPs IL-4+33 and IL13+110 was significantly increased, suggesting the existence of an additional gene cluster in chromosome 5q31 involved in the susceptibility to AIH

Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in Asia. JEV causes serious inflammation of the brain, which may lead to permanent brain damage and has a high mortality rate. Almost 3 billion people live in JE endemic areas and JEV causes an estimated 20,000 cases of disease and 6000 deaths per year. JEV is a positive stranded RNA virus belonging to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. JEV transmission occurs through a zoonotic cycle involving mosquitoes and vertebrate amplifying hosts, chiefly pigs and ardeid birds. Humans are infected when bitten by an infected mosquito and are dead end hosts. The role of humoral and cell mediated immune responses during JEV infection have been studied by several groups. While the humoral responses play a central role in protection against JEV, the cell mediated immune responses contributing to this end are not fully understood. The MHC molecules have been known to play predominant roles in host responses to viral infections and the consequences of virus infection on the expression of MHC molecules are varied. The expression of MHC-I molecules is known to decrease upon infection with many viruses such as HIV, MCMV, HCMV, Adv, and EBV. In contrast, infection with flavivirus such as West Nile Virus (WNV) has been shown to increase the cell surface expression of both MHC-I and MHC-II molecules. It has been reported previously that WNV infection increases the cell surface expression of adhesion molecules such as ICAM-1, VCAM-1 as well as E-Selectin and these changes were mediated directly by WNV and not by soluble cytokines. In contrast to classical MHC-I molecules, the nonclassical MHC-I molecules do not belong to a single group of structurally and functionally homologous proteins and normally have lower cell surface expression. Earlier studies have shown that the expression of nonclassical MHC-I molecules were induced during infection with JHM strain of mouse hepatitis virus (MHV). However, the functional significance of this induction is unclear. Expression of nonclassical MHC-I molecules upon flaviviral infection is not very well understood. In this thesis, evidence is presented that JEV infection induces the expression of both classical and nonclassical MHC-I molecules on primary mouse brain astrocytes, mouse embryonic fibroblasts (MEFs) and H6 (hepatoma cell). The levels of adhesion molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results clearly demonstrate that JEV infection induces their expression on astrocytes, MEFs and H6. The role of NF-κB and type-I IFNs in the induction of classical and nonclassical MHC-I molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results demonstrated that type-I IFN mediated signaling is responsible for the induction of these molecules during JEV infection. Chapter 1 discusses the innate and adaptive immune system, the role of classical and nonclassical MHC molecules in the initiation of immune response and diverse strategies adapted by different viruses to evade the immune response. It also includes a detailed discussion about the IFN and NF-κB signaling pathways and their modulation by viral infection. Finally, the genome organization, epidemiology, transmission cycle, pathogenesis and pathology, clinical features, humoral as well as cell mediated immune response to JEV infection and the current vaccine status to JEV infection are briefly discussed. Chapter 2 describes the general materials and methods used in this study. It includes the details of the reagents and cell lines used in the experiments. It also discusses the various techniques such as RT-PCR, FACS analysis, EMSA and ELISA. Chapter 3 focusses on the validation of different knockout MEFs used in the study as well as confirming the purity of primary astrocyte cultures established from pub brains. The susceptibility of various cells to JEV infection has also been investigated. Our results confirmed the authenticity of all the cells and the purity of primary astrocyte cultures used in the study. Our results also indicated that all the cells used in the study are susceptible to JEV infection. Chapter 4 discusses the expression of MHC and related genes involved in immune response upon JEV infection of primary mouse brain astrocytes, MEFs and H6. Chapter 4 demonstrates for the first time that JEV infection induces the expression of nonclassical MHC-I or class Ib molecules namely Qa-1, Qb1 and T10 in addition to the induction of classical MHC-I molecules. In contrast to WNV, there was no increase in the cell surface expression of MHC-II molecules upon JEV infection of primary mouse brain astrocytes. JEV infection also induces the expression of adhesion molecules as well as molecules involved in antigen processing and presentation namely Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10. Chapter 5 demonstrates that JEV infection induces NF-κB activation in astrocytes and MEFs. Studies using MEFs deficient in classical and alternate pathways of NF-κB activation indicate that JEV activates the classical pathway of NF-κB activation and is dependent on canonical lKKβ/IKK2 activity. JEV infection of astrocytes, MEFs and H6 induces the production of type-I IFNs. To determine the mechanism of type-I IFN induction during JEV infection, MEFs deficient in NF-κB signaling and IFN signaling were used. Results indicate that type-I IFN production in MEFs occurs by both NF-κB dependent and independent mechanisms. In contrast, the production of IFN-α was completely abrogated in IFNAR-\- MEFs whereas IFN-β production was greatly reduced. Production of type-I IFNs in IFNGR-\- MEFs is also reduced upon JEV infection but the reason for this is unclear. Chapter 6 demonstrates that JEV induced expression of classical MHC-I molecules occurs by type-I IFN mediated signaling. This result is in contrast to WNV infection, in which both NF-κB and type-I IFNs are involved in the induction of classical MHC-I molecules. Type-I IFNs were also shown to be involved in the induction of nonclassical MHC molecules namely, Qa-1 and Qb1 during JEV infection. In contrast, the expression of T10, another nonclassical MHC molecule occurs independent of type-I IFN signaling. The expression of molecules involved in antigen processing and presentation namely, Tap1, Tap2, Lmp2 and Lmp7 was type-I IFN-mediated, whereas the expression of Tapasin and Lmp10 was mediated by both type-I IFN dependent and independent mechanisms. The expression of VCAM-1 was dependent on NF-κB mediated signaling. Chapter 7 precisely describes the underlying mechanism of induction of MHC and various other related molecules and their significance during JEV infection. In addition, it also includes a working model for the induction of these molecules during JEV infection. In summary, this is the first study in which the mechanism of JEV mediated induction of classical as well as nonclassical MHC molecules has been studied in detail. This study clearly demonstrated that type-I IFNs are involved in the induction of classical and nonclassical MHC-I molecules during JEV infection. The functional significance of this JEV mediated induction of classical MHC-I molecules is unclear, but it has been proposed that this is to escape from the action of NK cells. The absence of MHC-II induction during JEV infection could be important because it may lead to the initiation of an immune response which is different from that induced during other viral infections which induce the expression of MHC-II molecules. In contrast to classical MHC-I molecules, the functional and biological significance of nonclassical MHC-I molecules are poorly studied. Nonclassical MHC-I molecules play an important role in bridging adaptive and innate immune response. So the nonclassical MHC molecules induced during JEV infection may play an important role in the initiation of immune response during JEV infection. The role these nonclassical MHC-I molecules in antigen presentation during JEV infection is not known. These nonclassical antigens are also recognized by NK and γδT cells, thus the expression of nonclassical MHC-I molecules during JEV infection might also confer a protective role.

To be able to make informed descicions regarding the research of new drug molecules (ligands), it is crucial to have access to information regarding the chemical interaction between the drug and its biological target (protein). Computer-based methods have a given role in drug research today and, by using methods such as molecular docking, it is possible to investigate the way in which ligands and proteins interact. Despite the acceleration in computer power experienced in the last decades many problems persist in modelling these complicated interactions. The main objective of this thesis was to investigate and improve molecular modelling methods aimed to estimate protein-ligand binding. In order to do so, we have utilised chemometric tools, e.g. design of experiments (DoE) and principal component analysis (PCA), in the field of molecular modelling. More specifically, molecular docking was investigated as a tool for reproduction of ligand poses in protein 3D structures and for virtual screening. Adjustable parameters in two docking software were varied using DoE and parameter settings were identified which lead to improved results. In an additional study, we explored the nature of ligand-binding cavities in proteins since they are important factors in protein-ligand interactions, especially in the prediction of the function of newly found proteins. We developed a strategy, comprising a new set of descriptors and PCA, to map proteins based on their cavity physicochemical properties. Finally, we applied our developed strategies to design a set of glycopeptides which were used to study autoimmune arthritis. A combination of docking and statistical molecular design, synthesis and biological evaluation led to new binders for two different class II MHC proteins and recognition by a panel of T-cell hybridomas. New and interesting SAR conclusions could be drawn and the results will serve as a basis for selection of peptides to include in in vivo studies.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that leads to degradation of cartilage and bone mainly in peripheral joints. In collagen-induced arthritis (CIA), a mouse model for RA, activation of autoimmune CD4+ T cells depends on a molecular recognition system where T-cell receptors (TCRs) recognize a complex between the class II MHC Aq protein and CII259-273, a glycopeptide epitope from type II collagen (CII). Interestingly, vaccination with the Aq/CII259-273 complex can relieve symptoms and cause disease regression in mice. This thesis describes the use of modified glycopeptides to explore interactions important for binding to the Aq protein and recognition by autoimmune T-cell hybridomas obtained from mice with CIA. The CII259-273 glycopeptide was modified by replacement of backbone amides with different amide bond isosteres, as well as substitution of two residues that anchor the glycopeptide in prominent pockets in the Aq binding site. A three-dimensional structure of the Aq/glycopeptide complex was modeled to provide a structural basis for interpretation of the modified glycopeptide’s immunological activities. Overall, it was found that the amide bond isosteres affected Aq binding more than could be explained by the static model of the Aq/glycopeptide complex. Molecular dynamics (MD) simulations, however, revealed that the introduced amide bond isosteres substantially altered the hydrogen-bonding network formed between the N-terminal 259-265 backbone sequence of CII259-273 and Aq. These results indicated that the N-terminal hydrogen-bonding interactions follow a cooperative model, where the strength and presence of individual hydrogen bonds depended on the neighboring interactions. The two important anchor residues Ile260 and Phe263 were investigated using a designed library of CII259-273 based glycopeptides with substitutions by different (non-)natural amino acids at positions 260 and 263. Evaluation of binding to the Aq protein showed that there was scope for improvement in position 263 while Ile was preferred in position 260. The obtained SAR understanding provided a valuable basis for future development of modified glycopeptides with improved Aq binding. Furthermore, the modified glycopeptides elicited varying T-cell responses that generally could be correlated to their ability to bind to Aq. However, in several cases, there was a lack of correlation between Aq binding and T-cell recognition, which indicated that the interactions with the TCRs were determined by other factors, such as presentation of altered epitopes and changes in the kinetics of the TCR’s interaction with the Aq/glycopeptide complex. Several of the modified glycopeptides were also found to bind well to the human RA-associated DR4 protein and elicit strong responses with T-cell hybridomas obtained from transgenic mice expressing DR4 and the human CD4 co-receptor. This encourages future investigations of modified glycopeptides that can be used to further probe the MHC/glycopeptide/TCR recognition system and that also constitute potential therapeutic vaccines for treatment of RA. As a step towards this goal, three modified glycopeptides presented in this thesis have been identified as candidates for vaccination studies using the CIA mouse model.