Para atestar a imunogenicidade de vacinas contra raiva, os testes de titulação e de soroneutralização de vírus rábico em células BHK-21 foram validados quanto à linearidade, precisão, exatidão e robustez. Cinco esquemas de imunização com diferentes concentrações, vias de inoculação e intervalos entre as doses foram avaliados em camundongos com vacina contra raiva em células Vero. O grupo II que recebeu um esquema de 2 doses e intervalos maiores (0 e 60 dias) apresentou títulos de anticorpos neutralizantes [ACN] mais elevados (46,1 UI/mL) do que o Grupo I (19,4 UI/mL), de 3 doses e intervalos menores (0,7 e 28 dias). O grupo III, que recebeu 2 doses, sendo a 1ª diluída 1/10, apresentou ACN semelhantes ao grupo II (39,2 UI/mL), sendo igualmente eficaz. Nenhum animal do grupo IV, imunizado por via oral (VO) apresentou ACN, indicando supressão e todos os do grupo V, imunizados VO com antígeno adsorvido/encapsulado em sílica nanoestruturada SBA-15, apresentaram títulos de ACN detectáveis, mostrando que o antígeno foi protegido da degradação no trato gastrointestinal. / To assure the reliability of the results of immunogenicity of rabies vaccines, tests of virus titration and neutralization in BHK-21 cells were validated for linearity, precision, accuracy and robustness. Five schemes of immunization with different concentrations, routes of inoculation and intervals between doses were evaluated in mice with rabies vaccine in Vero cells. Group II, immunized with 2 doses and enlarged interval (0 and 60 days) presented higher levels of neutralizing antibody (NAs) (46,1 IU/mL) than group I (19,4 IU/mL), with 3 doses and shorter intervals (0,7 and 28 days). Group III, which received 2 doses, the 1st diluted to 1/10, presented results similar to group II (39,2 IU/mL). None mouse of Group IV, immunized by oral route, presented NAs, indicating suppression and all mice of group V, immunized by oral route with vaccine adsorbed/encapsulated in nanostructured SBA-15 silica presented detectable NAs titers, showing that the SBA-15 silica prevented the antigen degradation in the gastrointestinal tract.

A deficiência da proteína C5 do sistema complemento humano é rara com 38 casos relatados na literatura e freqüentemente associada a severas infecções provocadas por bactérias Neisseria. O objetivo do trabalho é caracterizar imunológica e geneticamente esta deficiência encontrada pela primeira vez em brasileiros. Por imunodifusão dupla obtivemos níveis expressivos de C3, C4, C6, C7, C8, C9, Fator B, Fator H e Fator I em todos os membros desta família, a proteína C5 não foi detectada no soro de três irmãos: II:9, II:4 e II:5. Por ELISA a concentração de C5 nestes indivíduos foi (0,9; 1,0; 1,3 µg/ml, 45- 190 µg/ml). Soros destes probandos não apresentam atividade hemolítica mediada pelo sistema complemento. O cDNA de C5 dos indivíduos I:1, I:2, II:4 e II:9 apresenta a deleção do éxon 30. Causada pela substituição de GAG4028 por GAA4028 no último nucleotídeo deste éxon que leva a um erro no splicing. Este defeito provavelmente produz uma proteína incompleta e destinada à degradação. / The deficiency of the C5 component of the complement system is rare with 38 described cases in the literature. This deficiency is frequently associated with severe infections, especially caused by Neisseria. Our objective is to characterize immunologically and genetically this deficiency, the first of its type described in the Brazilian population.We noted that C3, C4, C6, C7, C8, C9, Factor B, Factor H and Factor I have expressive levels in all the individuals sera of this family. C5 was absent in individuals II:4, II:5 and II:9. By ELISA a C5 concentration in this individuals were 0,9; 1,0; 1,3 µg/ml (normal: 45 - 190 µg/ml). Their serum doesn´t present hemolytic activity mediated by complement system. The C5 cDNA from individuals I:1, I:2, II:4 and II:9 has éxon 30 deleted. Caused by the substitution of GAG4028 for a GAA4028 in the last codon of exon 30. This defect was responsible for the deficiency of C5 in this family and this deletion would probably produce an unstable protein destined for degradation.

The Major Histocompatibility Complex (MHC) encodes proteins critical to the vertebrate immune response; therefore MHC diversity is an indicator of population health. I have (1) Isolated exon 2 of the class II gene DQA in Tursiops truncatus in the North Indian River Lagoon (IRL) (n=17), South IRL (n=29) and adjacent Atlantic waters (n=20), (2) assessed genetic variability between groups, (3) developed a method to genotype individuals, (4) typed 11 unique alleles in 66 individuals, (5) detected geographic patterns of diversity between estuarine and coastal individuals (FST=0.1255, p<0.05), (6) found evidence of positive selection centered in the binding pockets P1, P6 and P9 of the peptide binding region (w=2.08), (7) found that patterns of polymorphism did not closely match patterns of diversity in neutral markers, (8) performed a pilot study with Orcinus orca. The initial findings highlight the need for further comparative work and suggest that silent mutations are not neutral. / by Tatiana Ferrer. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram clinicamente relevantes. Desde então, procuramos observar as diferenças entre melhor ou pior prognóstico de acordo com a imunogenética que mostrou diferenciação entre os grupos R e NR ao tratamento em relação ao prognóstico da resposta terapêutica. Quando as diferenças entre as sequências da NS5B e a resposta ao tratamento foram consideradas verificou-se que associada a mutação R254K, estava a C316N que poderia conduzir a uma não resposta à terapia no genótipo 1b. Os nossos dados também suportaram forte associação de IL-28B rs12979860, com elevada probabilidade de resposta à terapia de IFN + RBV. Nossos dados evidenciam a presença de pacientes virgens de tratamento que abrigam mutações de resistência previamente descritas na literatura. A análise dos fatores preditores de resposta virológica mostrou que a predição de boa resposta ou não ao tratamento e ainda da progressão da doença é dependente de uma importante interação entre a genética viral e a do hospedeiro. Fato este importante para que no momento de avaliação de diagnóstico e conduta terapêutica, o médico possa tomar medidas apropriadas para o tratamento de cada paciente individualmente independentemente do genótipo do HCV em questão. / It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid fragment of NS5B was examined in 69 Hepatitis C Virus drug naïve (HCV)-infected individuals in Rio de Janeiro, Brazil. In the second phase, the mutations revealed clinically relevant from the gene in question. Since then, we seek to observe the differences between better or worse prognosis according to immunogenetic showed that differentiation between the immunogenetics of the groups R and NR to treatment in relation to prognosis of therapeutic response. When the differences between the NS5B sequences at baseline and the treatment response were considered we found that R254K associated with C316N mutations could lead to a non-response to IFN-RBV therapy in genotype 1b. Our data also strong support the association of rs12979860 IL-28B polymorphism with high probability of response to IFN + RBV therapy. Our data highlight the presence of HCV genotypes from drug naïve patients harboring resistance mutations previously described in literature. The analysis of predictors virologic response demonstrated that the prediction of better or worse therapy response and further the disease progression is dependent of a significant interaction between viral and host genetics. This fact is important for diagnosis evaluation and clinical therapeutic, the medico can take appropriate measures to treat each individual patient irrespective of the genotype of HCV in question.

A deficiência de CD40 Ligante (CD40L) ou síndrome de Hiper-IgM ligada ao X (X-HIGM) é considerada uma imunodeficiência primária combinada de células T e B. O CD40L é expresso na superfície de linfócitos T ativados e interage com o CD40 expresso na superfície de linfócitos B, macrófagos, células dendríticas, células endoteliais e neutrófilos. A interação CD40L-CD40 transmite sinais que induzem ativação, diferenciação e proliferação celular. Nosso objetivo foi analisar as alterações genético-moleculares da molécula CD40L que acometeram indivíduos de 5 famílias brasileiras, ocasionando X-HIGM. Genotipamos 25 indivíduos, sendo 6 pacientes com X-HIGM, 13 parentes relacionados heterozigotos e 6 homozigotos sadios. Dentre os pacientes com X-HIGM dois eram de origem caucasóide e 4 eram mestiços. A idade dos pacientes variou de 2 a 20 anos e o quadro clínico de infecções de repetição teve início em média nos primeiros 4 meses de vida. As principais infecções recorrentes manifestadas pelos pacientes foram pneumonia e otite. O paciente TB apresentou blastomicose, observação original nesta imunodeficiência. A análise genético-molecular foi heterogênea. No paciente TB foi detectado um defeito de splicing levando a deleção do exon 3 (r.345_402del do gene CD40L (CD40LG) no paciente FS uma nova substituição missense g.11856 G>C (c.476 G>C, pW140C), no paciente KC uma substituição nonsense g.11855 G>A (c.475G>A, p. W140X), e nos pacientes CH, FE e VIC uma deleção g. 3074_3077delTAGA, levando a alteração no processamento do RNA. A fenotipagem dos leucócitos demonstrou que a contagem de linfócitos T auxiliares (CD3+CD4+), linfócitos citotóxicos (CD3+CD8+), linfócitos B (CD19+CD40+) e linfócitos T ativados (CD3+CD69+) dos pacientes foram similares aos controles sadios. Contudo, foi observada uma redução significativa nos níveis de expressão de CD40L na superfície de linfócitos CD3+ e CD4+ dos pacientes. A análise dos linfócitos T por microscopia confocal revelou que as células dos homozigotos com expressão residual do CD40L em sua superfície também apresentam redução na densidade da expressão da molécula CD3, sugerindo a necessidade da integridade molecular do CD40L para a expressão normal do CD3. Concluímos que mutações no CD40L que levam à síndrome de X-HIGM são heterogêneas e a análise genético-molecular permitiu um diagnóstico preciso tornando possível o aconselhamento genético e a triagem dos recém-nascidos das famílias avaliadas. / CD40-Ligand (CD40L) deficiency or X linked Hyper-IgM syndrome (X-HIGM) is considered a T and B cell combined primary immunodeficiency. CD40L is expressed on the cell surface of activated T lymphocytes and interacts with CD40, expressed on the surface of B lymphocytes, macrophages, dendritic cells, endothelial cells, and neutrophils. The CD40L-CD40 interaction induces activation, differentiation, and cell proliferation. Our aim was to analyze the molecular-genetic alterations of CD40L molecule affecting individuals of 5 brazilian families, leading to X-HIGM. We genotyped 25 individuals, whom 6 were X-HIGM patients, 13 were heterozygote related patients, and 6 were healthy homozygotes. Within the patients with X-HIGM, two of them were of caucasoid origin and four were mestiços. The patients age ranged from 2 to 20 years and their recurrent infections started in average during their first 4 months of life. The main recurrent infections were pneumonia and otitis. The patient TB presented blastomycosis, a unique observation in this immunodeficiency. The molecular-genetic analysis revealed heterogeneity. TB patient presented a splicing defect causing a deletion of exon 3 (r.345_402del) of CD40L gene (CD40LG). Patient FS presented a new missense mutation g.11856 G>C (c.476 G>C, pW140C). Patient KC presented a nonsense substitution g.11855 G>A (c.475G>A, p. W140X). Patients CH, VIC, and FE presented a deletion g. 3074_3077delTAGA, causing an alteration on RNA processing. The leukocytes fenotyping demonstrated that T helper lymphocytes (CD3+CD4+), T cytotoxic lymphocytes (CD3+CD8+), B lymphocytes (CD19+CD40+), and T activated (CD3+CD69+) cell counts of patients were similar to healthy controls. However it was observed a significant reduction of CD40L expression on cell surface patients CD3+ and CD4+ lymphocytes. The T lymphocyte confocal microscopy analysis revealed that homozygotes with residual expression of CD40L in their surface also presented a reduction on the density of CD3 molecule expression, suggesting the need of molecular integrity of CD40L for normal CD3 expression. We conclude that mutations on CD40L leading to X-HIGM syndrome are heterogeneous and the molecular-genetic analysis allowed a precise diagnosis making possible the genetic counseling and newborn screening of the involved families.

The nonobese diabetic mouse (NOD) is an excellent animal model to study type 1 diabetes. As with some humans, disease in the NOD mouse is effected by a combination of genetic and environmental factors. At least 20 insulin dependent diabetes (Idd) susceptibility loci have been identified so far, both in humans and in the NOD mouse. In this thesis, the overall aim has been to understand the genetic basis of diabetes in the NOD mouse by assessing immunogically-related phenotypes. As lymphocytes are the main players in the onset and progression to overt diabetes, we searched for physiological abnormalities in T and B cells, which could contribute to the breakdown of tolerance to pancreatic antigens. Ultimately, we postulate that abnormalities in the T or B cell compartments, under the genetic control of a previously defined diabetes susceptibility regions (Idds) could unravel the biological mechanisms underlying diabetes susceptibility and facilitate the identification of etiological polymorphisms involved in the disease. NOD T cells are defective in upregulating CTLA-4 upon in vitro activation. Previous studies have shown that this defect is, at least in part, controlled by gene(s) in the Idd5 region on chromosome 1. In paper I, we provide evidence that defective upregulation of the CTLA-4 in NOD T cells is not controlled by the Idd5.1 and Idd5.2 regions, but rather by genes linked to the telomeric region of chromosome 1 and to the Idd3 locus, for which the prime candidate gene is Il-2. Interestingly, we could restore some of the defective CTLA-4 expression in NOD T cells by the addition of exogenous IL-2 during T cell activation in vitro. In paper II, we show that NOD thymocytes are resistant to superantigen-mediated negative selection and that this trait is under control of the Idd5.2 region. Interestingly, it appears to operate in a T cell non-autonomous manner. In paper III, we describe a competitive advantage of NOD thymocytes to mature when they co-develop with B6 thymocytes in embryo aggregation chimeras. These results imply that defects exist in the positive/negative selection mechanisms in the NOD thymus. Apart from T cells, B cells also play an important role in the initiation of diabetes in NOD mice, probably as antigen presenting cells. In paper IV, we report that the genetic basis of an enlarged marginal zone (MZ) B cell population observed in the NOD mice is linked to the Idd9/Idd11 region. Together, these findings contribute to the dissection of the molecular mechanisms underlying diabetes pathogenesis, and shed light on the contribution of central and peripheral tolerance mechanisms to this process.

According to the Joint United Nations Programme against HIV/AIDS 2009 AIDS epidemic update, there were a total of 33.3 million (31.4 million–35.3 million) people living with HIV worldwide in 2009. The majority of the epidemic occurs in Sub-Saharan Africa. Of the 33.3 million people living with HIV worldwide in 2009, a vast majority of 22.5 million (20.9 million-24.2 million) were from Sub-Saharan Africa. There were 1.8 million (1.6 million-2.0 million) new infections and 1.3 million (1.1 million-1.5 million) AIDS-related deaths in Sub-Saharan Africa in 2009 (UNAIDS, 2009). Statistical models and analysis are required in order to further understand the dynamics of HIV/AIDS and in the design of intervention and control strategies. Despite the prevalence of this disease, its pathogenesis is still poorly understood. A thorough understanding of HIV and factors that influence progression of the disease is required in order to prevent the further spread of the virus. Modelling provides us with a means to understand and predict the progression of the disease better. Certain genetic factors play a key role in the way the disease progresses in a human body. For example HLA-B types and IL-10 genotypes are some of the genetic factors that have been independently associated with the control of HIV infection. Both HLA-B and IL-10 may influence the quality and magnitude of immune responses and IL-10 has also been shown to down regulate the expression of certain HLA molecules. Studies are therefore required to investigate how HLA-B types and IL-10 genotypes may interact to affect HIV infection outcomes. This dissertation uses the Sinikithemba study data from the HIV Pathogenesis Programme (HPP) at the Medical School, University of KwaZulu-Natal involving 450 HIV positive and treatment naive individuals to model how certain outcome biomarkers (CD4+ counts and viral loads) are associated with immuno genetic parameters (HLA-B types and IL-10 genotypes). The work also seeks to exploit novel longitudinal data methods in Statistics in order to efficiently model longitudinally measured HIV outcome data. Statistical techniques such as linear mixed models and generalized estimating equations were used to model this data. The findings from the current work agree quite closely with what is expected from the biological understanding of the disease. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

HIV-1 specific CD4+ T cell activity in clade C infected subjects has not been studied. CD4+ T cells play a vital role in controlling infectious diseases and there is a need to augment our knowledge of HIV immunology to aid vaccine design. We therefore embarked on a study to characterize HIV-1 specific CD4+ T cell activity in both adults and infants; assess the relationship between CD4+ and CD8+ immune responses; and the relationship between CD4+ T cell activity and markers of disease progression (viral loads and CD4 counts). Our study revealed that the magnitude of CD8+ T cell responses correlated significantly with CD4+ T cell responses, but that the percentage of CD8+ T cells directed against HIV-1 was always greater than that of CD4+ T cells. Gag was the frequently targeted HIV-1 protein by CD4+ T cells and had the highest density of epitopes targeted by CD4+ T cells. Patients with either a dominant CD4 or CD8 T cell response against Gag had significantly lower viral loads than patients in whom non-Gag proteins were the main target (p< 0.0001 for CD4 activity and p= 0.007 for CD8 responses). Single IFN- producing CD4+ T cells were present in significantly higher numbers than cells producing both IFN- and IL-2 simultaneously (p=0.009). Gag also dominated the CD4+ T cell response in acutely infected infants with IFN- production detected more frequently than IL-2 or TNF- . Longitudinal analysis of infants receiving early ARV treatment and then ceasing after 12 months revealed that early treatment conferred no protection against increasing viremia and disease progression. CD4+ T cell responses were detected sporadically in untreated infants indicating a dysfunctional immune response in the face of constant exposure to high levels of viremia. Taken together, the data reveal that a vaccine inducing Gag specific CD4+ T cell responses has the potential to confer some degree of protection, but other immunological parameters need to be investigated especially in infants. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2011.

L’idée qu’une cellule puisse effectuer la cytolyse de cellules transformées, comme une cellule Natural Killer (NK), tout en ayant la capacité de présenter des antigènes, comme une cellule dendritique (DC), peut sembler fantaisiste. Cependant, de telles cellules furent bel et bien identifiées chez la souris en 2006. Ces cellules, nommées Interferon-producing Killer Dendritic Cells (IKDC), furent l’objet d’une caractérisation extensive qui révéla leur énorme potentiel immunologique. La combinaison de fonctions associées à des cellules NK et à des DC a doté les IKDC d’un pouvoir antitumoral remarquable. D’ailleurs, il a été démontré que les IKDC sont plus efficaces que les cellules NK pour limiter la croissance tumorale. Ainsi, suite à leur découverte, les IKDC ont suscité beaucoup d’intérêt. Cependant, une controverse émergea sur la nature des IKDC. Plusieurs groupes indépendants tentèrent de reproduire les expériences attestant les fonctions de DC des IKDC, sans y parvenir. De plus, des études additionnelles révélèrent que les IKDC possèdent des similitudes très importantes avec les cellules NK. Ces observations ont mené la communauté scientifique à suggérer que les IKDC sont des cellules NK en état d’activation (aNK). Malgré cette controverse, les caractéristiques antitumorales des IKDC sont si uniques et considérables qu’il est primordial de poursuivre l’étude de ces cellules. Pour y arriver, il est essentiel de déterminer la nature des IKDC et de mettre fin à ce débat. Par la suite, il sera important d’identifier des façons de cibler spécifiquement les IKDC pour permettre leur usage dans le cadre de thérapies antitumorales. Ainsi, l’objectif de cette thèse est de définir l’identité des IKDC, puis de déterminer les facteurs génétiques responsables de la régulation de ces cellules. Nous avons démontré que les IKDC ne sont pas des cellules aNK, contrairement à ce qui avait été suggéré. Nous avons constaté que les IKDC prolifèrent activement et possèdent un phénotype unique, des caractéristiques associées à des cellules NK très immatures. Afin de déterminer si les IKDC peuvent acquérir un phénotype mature, nous avons effectué des expériences de transfert adoptif. Suite à leur injection in vivo, les IKDC acquièrent un phénotype de cellules matures, mais étonnamment, elles se différencient aussi en cellules NK. Ainsi, nous avons révélé que les IKDC sont un intermédiaire dans la différenciation des cellules NK. En parallèle, nous avons démontré que la proportion d’IKDC varie grandement entre des souris de fond génétique différent, indiquant que des facteurs génétiques sont impliqués dans la régulation de ces cellules. Nous avons alors effectué une analyse génétique qui a révélé que les IKDC sont régulées par des facteurs génétiques compris dans une région distale du chromosome 7. Les résultats présentés dans cette thèse constituent une avancée importante pour la recherche sur les IKDC. Ils ont permis de définir la nature des IKDC et d’identifier un intervalle génétique impliqué dans la régulation de ces cellules. Ces découvertes sont des connaissances précieuses pour l’identification des IKDC chez l’Homme et la création de nouvelles thérapies dans la lutte contre le cancer. / The idea that a cell could kill transformed cells, like a Natural Killer (NK) cell, all the while exhibiting also the capacity to present antigens to T cells, like a Dendritic Cell (DC), may seem farfetched. However, in mice, a cell presenting these specific properties was identified in 2006. These cells were named Interferon-producing Killer Dendritic Cells (IKDC) and extensive studies revealed that they were endowed with an important immunological potential. Indeed, the fact that IKDCs exhibit properties of both DC and NK cells conferred them with an exceptional anti-tumor potential. Notably, on a per cell basis, the in vivo anti-tumor activity of IKDCs is more efficient than NK cells. Therefore, following their identification, IKDCs showed great therapeutic promise. However, a debate on the cell lineage origin of IKDCs emerged. Several independent groups could not replicate the finding that IKDCs showed functional antigen-presentation properties similar to DCs. Also, additional studies revealed that IKDCs are very similar to NK cells. These and other observations led the scientific community to believe that IKDCs were activated NK cells. Despite this controversy, IKDCs clearly exhibit a unique and outstanding anti-tumor potential, highlighting the relevance to further explore these cells. We must first close the debate regarding the lineage origin of IKDCs. We subsequently need to identify a means to specifically target IKDCs to facilitate their use in novel anti-tumor therapies. Thus, the objective of my thesis is first, to define the identity of IKDCs and second, to determine the genetic factors implicated in the regulation of these cells. For the first objective, we demonstrated that IKDCs do not represent activated NK cells, as previously suggested. We show that IKDCs are highly proliferative and exhibit a unique phenotype associated with very immature NK cells. In an attempt to verify if IKDCs could acquire a mature phenotype, we conducted an adoptive transfer experiment. We found that, after adoptive transfer, IKDCs adopt a mature phenotype, but also surprisingly differentiate into NK cells. These findings indicate that IKDCs represent an intermediate in NK-cell differentiation. For the second objective, we demonstrated that the IKDC proportion was highly variable between strains of different background origins, indicating that these cells are regulated by genetic factors. A genetic study revealed that genetic factors in distal arm of chromosome 7 associate with the proportion of IKDCs. The results presented in this thesis represent an important breakthrough for the research on IKDCs. They allowed to define the cell lineage origin of IKDCs and to identify a genetic region involved in the regulation of this cell type. These discoveries are valuable knowledge for the identification of human IKDCs and the development of novel anti-tumor therapies.

Les objectifs de cette thèse étaient d'explorer les mécanismes moléculaires et les bases génétiques impliquées dans la survie des huîtres Crassostrea gigas à des maladies infectieuses, en considérant deux souches de Vibrio pathogènes pour l'huître (V. splendidus LGP32 et V. aestuarianus LPi 02/41) qui ont été associées aux phénomènes de mortalités massives d'huîtres en France. Par l'approche transcriptomique de « Digital Gene Expression », nous avons identifié des composants génétiques d'une réponse efficace à des infections par des Vibrio virulents. La capacité de survie des huîtres se traduit par l'expression basale d'une combinaison de 14 gènes hémocytaires, une signature de survie, et par l'induction de différentes fonctions cellulaires au cours de la réponse immunitaire. Une analyse transcriptomique détaillée au niveau individuel a révélé un extraordinaire polymorphisme d'expression basale des gènes, incluant des cas où chez certaines huîtres des transcrits sont absents. Afin de comprendre ce polymorphisme, nous nous sommes intéressés à la caractérisation d'une nouvelle famille de peptides antimicrobiens (PAMs), les big défensines (Cg-BigDef). Nous avons montré que Cg-BigDef est une famille de PAMs composée de trois membres et diversifiée en termes de séquences, d'organisation génomique et de régulation de l'expression des gènes. Les Cg-BigDefs sont codées par des gènes distincts dont l'expression est régulée suivant différents modes en réponse à une infection. Chose intéressante, certaines huîtres n'expriment pas simultanément les trois formes de Cg-BigDef ou dans certains cas, n'en expriment aucune. Nous avons démontré que l'absence d'expression basale de Cg-BigDef est liée à l'absence de gène correspondant dans le génome des huîtres. C'est la première mise en évidence chez un invertébré de variation de présence/absence (PAV) de gènes, un phénomène qui pourrait contribuer à une susceptibilité accrue aux maladies infectieuses. / The objectives of this thesis were to explore the molecular mechanisms and genetic bases involved in Crassostrea gigas oyster survival to infectious diseases, considering two Vibrio strains (V. splendidus LGP32 and V. aestuarianus LPi 02/41) pathogenic for oysters which have been shown to be involved in C. gigas mass mortalities in France. By the Digital Gene Expression transcriptomic approach, we have identified some genetic components implicated in a successful response and survival to virulent Vibrio infections. Oyster survival capacity is reflected by the basal expression of a selected combination of hemocyte genes, a 14-gene survival signature, and by the induction of some cellular functions during the oyster immune response. A detailed transcriptomic analysis at individual level revealed an extraordinary interindividual polymorphism in basal gene expression, including cases where some transcripts are fully absent. In order to understand this striking variability in gene expression, we have focused on the characterization of a novel family of antimicrobial peptides (AMP) in C. gigas oysters, the big defensins (Cg-BigDef). We have shown that Cg-BigDef is an AMP family, composed of three members, and diversified in terms of sequences but also in terms of genomic organization and regulation of gene expression. Each Cg-BigDef form is encoded by a distinct gene that follows different patterns of gene regulation upon Vibrio infection. Interestingly, some oysters were shown do not express simultaneously the three Cg-BigDef forms or any Cg-BigDef. We demonstrated that the absence of Cg-BigDef basal gene expression is likely due to the absence of the Cg-bigdef gene in oyster genome. This is the first evidence in an invertebrate of a presence/absence variation (PAV) of genes, a phenomenon that could be associated to a susceptibility to infectious diseases.