La molécule HLA-DR du complexe majeur d'histocompatibilité de classe II (CMH-II) est fortement incorporée dans l'enveloppe virale du virus d'immunodéficience humaine de type 1 (VIH-1). L'expression à la membrane cytoplasmique de cette même molécule est modulée à la baisse par la protéine virale Nef. D'autre part, Nef augmente l'expression de la chaîne invariante CD74, une autre protéine du CMH-II associée avec HLA-DR dans un complexe protéique immature. D'autres protéines accessoires virales, telles Vpu, interagissent avec le CMH-II. Vpu diminue l'expression de HLA-DR et interagit avec la portion intracellulaire de CD74. Cette étude a pour but de vérifier si les complexes immatures de CMH-II sont incorporés dans l'enveloppe virale du VIH-1. Elle veut aussi vérifier l'impact de la présence de Nef et de Vpu sur l'incorporation du CMH-II. La modulation à la baisse de HLA-DR et l'augmentation d'expression de CD74 induite par Nef ont été confirmées à la surface de lymphocytes T CD4+. La molécule CD74 représentant un complexe de CMH-II immature ne semble pas être incorporée dans l'enveloppe virale, peu importe la présence ou l'absence de Nef et de Vpu lors de l'infection. La présence de Nef ne semble pas influencer l'incorporation de HLA-DR qui demeure toujours fortement incorporée. Vpu semble important pour l'incorporation du HLA-DR, puisqu'un virus sans Vpu incorpore nettement moins de HLA-DR. Un mutant déficient en phosphorylation sur deux serines de Vpu, pour sa part, semble permettre une incorporation partielle de cette même molécule. Ces résultats semblent indiquer que HLA-DR est incorporée selon son stade de maturation et que Vpu est essentielle à l'incorporation de HLA-DR.

Apesar de uma diversidade de estudos científicos, em que dezenas de milhares de pacientes foram analisados e tratados, a sepse continua sendo um grande desafio para a medicina contemporânea. Investigações bem conduzidas levaram a uma reavaliação do modelo clássico da sepse, tradicionalmente vista como um processo descontrolado de hiperinflamação sistêmica, uma vez que se observou a existência de uma atividade antiinflamatória ao longo do seu curso evolutivo. Nesse contexto, o comportamento do sistema imune inato se assemelha ao de indivíduos idosos submetidos ao fenômeno da imunossenescência, interseção ainda mais relevante ao considerarmos o crescente incremento na faixa etária média dos pacientes internados em UTI. O presente estudo visou a estabelecer a epidemiologia da sepse em um hospital público de um país de renda media, como é o caso do Brasil. Ademais, através de citometria de fluxo, buscamos definir a cinética da expressão monocitária de moléculas HLA-DR e CD64 ao longo do processo de envelhecimento humano. Comparamos essas observações com o comprometimento do sistema imune inato visto na sepse visando discriminar as alterações da senescência do sistema imune associada ao envelhecimento daquelas associadas ao fenômeno da imunoparalisia da sepse. Na investigação epidemiológica, nós encontramos uma taxa de ocorrência de 5,9 casos de sepse por 100 pacientes e uma densidade de incidência de 6,4 casos por 1000 pacientes-dia. Documentamos ainda sua associação com uma elevada incidência de sepse e documentamos sua associação com uma elevada taxa de comordidades crônicas. A sepse foi diagnosticada tardiamente (72% dos casos após 12 horas de evolução) e em estágio avançado como atestado pelos elevados escores de gravidade de doença e de disfunção orgânica. O presente estudo identificou vários obstáculos à efetiva implementação das recomendações da Surviving Sepsis Campaign. No segundo estudo, observamos correlações negativas significativas entre idade e intensidade de expressão dos biomarcadores avaliados. Durante a sepse, a expressão de mHLA-DR foi ainda mais reduzida, enquanto a expressão de CD64 foi majorada. Para melhor discriminar imunossenescência de imunoparalisia, valores de cutoff de 9.700 m/c para mCD64 e de 7.000 m/c para mHLA-DR forneceram as melhores combinações de sensibilidade e especificidade. / Despite a multiplicity of trials enrolling several thousands of treated patients, sepsis still represents a major challenge to contemporary medicine. Since several well-conducted investigations have consistently indicated the existence of an anti-inflammatory activity during sepsis evolution, there has been a reappraisal of the classic sepsis model in which the condition was traditionally seen as an uncontrolled systemic hyper-inflammatory process. During sepsis, the innate immune system behavior has some similarities to the immunosenescence process experienced by elderly people; something that is even more relevant when considering the progressive increase of the mean age of the patients admitted to ICUs along the last decades. This study investigated the epidemiology of sepsis in a public university hospital from a middle-income country, as is the case of Brazil. Also, applying a flow cytometry methodology, we tried to establish the kinetics of the molecular expression of HLA-DR and CD64 on monocytes during human aging. To put it in context, we compared such findings with those obtained from septic patients aiming at the discrimination between changes related to the normal aging process from those of the immunoparalysis phenomenon seen in sepsis. In the epidemiological arm of this study, we found an occurrence rate of 5.9 cases of sepsis per 100 patients, and an incidence density of 6.4 cases per 1000 patient-days. We documented the association of sepsis with an elevated rate of chronic comorbid conditions. Sepsis was detected late (72% evolving for more than 12 hours) and in advanced stages as indicated by severity and organ failure scores. The study detected several obstacles to the effective implementation of the Surviving Sepsis Campaign recommendations. In the second study, we observed significant negative correlations between age and the levels of HLA-DR and of CD64 expression on monocytes. During sepsis, HLA-DR expression decreased further while CD64 expression was upregulated. To better discriminate immunosenescence from immunoparalysis, cutoff values of 9,700 m/c for mCD64 and of 7,000 m/c for HLA-DR provided the best combination of sensitivity and specificity.

Apesar de uma diversidade de estudos científicos, em que dezenas de milhares de pacientes foram analisados e tratados, a sepse continua sendo um grande desafio para a medicina contemporânea. Investigações bem conduzidas levaram a uma reavaliação do modelo clássico da sepse, tradicionalmente vista como um processo descontrolado de hiperinflamação sistêmica, uma vez que se observou a existência de uma atividade antiinflamatória ao longo do seu curso evolutivo. Nesse contexto, o comportamento do sistema imune inato se assemelha ao de indivíduos idosos submetidos ao fenômeno da imunossenescência, interseção ainda mais relevante ao considerarmos o crescente incremento na faixa etária média dos pacientes internados em UTI. O presente estudo visou a estabelecer a epidemiologia da sepse em um hospital público de um país de renda media, como é o caso do Brasil. Ademais, através de citometria de fluxo, buscamos definir a cinética da expressão monocitária de moléculas HLA-DR e CD64 ao longo do processo de envelhecimento humano. Comparamos essas observações com o comprometimento do sistema imune inato visto na sepse visando discriminar as alterações da senescência do sistema imune associada ao envelhecimento daquelas associadas ao fenômeno da imunoparalisia da sepse. Na investigação epidemiológica, nós encontramos uma taxa de ocorrência de 5,9 casos de sepse por 100 pacientes e uma densidade de incidência de 6,4 casos por 1000 pacientes-dia. Documentamos ainda sua associação com uma elevada incidência de sepse e documentamos sua associação com uma elevada taxa de comordidades crônicas. A sepse foi diagnosticada tardiamente (72% dos casos após 12 horas de evolução) e em estágio avançado como atestado pelos elevados escores de gravidade de doença e de disfunção orgânica. O presente estudo identificou vários obstáculos à efetiva implementação das recomendações da Surviving Sepsis Campaign. No segundo estudo, observamos correlações negativas significativas entre idade e intensidade de expressão dos biomarcadores avaliados. Durante a sepse, a expressão de mHLA-DR foi ainda mais reduzida, enquanto a expressão de CD64 foi majorada. Para melhor discriminar imunossenescência de imunoparalisia, valores de cutoff de 9.700 m/c para mCD64 e de 7.000 m/c para mHLA-DR forneceram as melhores combinações de sensibilidade e especificidade. / Despite a multiplicity of trials enrolling several thousands of treated patients, sepsis still represents a major challenge to contemporary medicine. Since several well-conducted investigations have consistently indicated the existence of an anti-inflammatory activity during sepsis evolution, there has been a reappraisal of the classic sepsis model in which the condition was traditionally seen as an uncontrolled systemic hyper-inflammatory process. During sepsis, the innate immune system behavior has some similarities to the immunosenescence process experienced by elderly people; something that is even more relevant when considering the progressive increase of the mean age of the patients admitted to ICUs along the last decades. This study investigated the epidemiology of sepsis in a public university hospital from a middle-income country, as is the case of Brazil. Also, applying a flow cytometry methodology, we tried to establish the kinetics of the molecular expression of HLA-DR and CD64 on monocytes during human aging. To put it in context, we compared such findings with those obtained from septic patients aiming at the discrimination between changes related to the normal aging process from those of the immunoparalysis phenomenon seen in sepsis. In the epidemiological arm of this study, we found an occurrence rate of 5.9 cases of sepsis per 100 patients, and an incidence density of 6.4 cases per 1000 patient-days. We documented the association of sepsis with an elevated rate of chronic comorbid conditions. Sepsis was detected late (72% evolving for more than 12 hours) and in advanced stages as indicated by severity and organ failure scores. The study detected several obstacles to the effective implementation of the Surviving Sepsis Campaign recommendations. In the second study, we observed significant negative correlations between age and the levels of HLA-DR and of CD64 expression on monocytes. During sepsis, HLA-DR expression decreased further while CD64 expression was upregulated. To better discriminate immunosenescence from immunoparalysis, cutoff values of 9,700 m/c for mCD64 and of 7,000 m/c for HLA-DR provided the best combination of sensitivity and specificity.

La polyarthrite rhumatoïde (PR) est le rhumatisme inflammatoire chronique le plus fréquent avec une prévalence mondiale qui varie selon les pays mais se situe aux alentours de 0,5% dans le monde. La PR est caractérisée par une atteinte articulaire souvent bilatérale et symétrique, évoluant par poussées inflammatoires, une production d'auto-anticorps, une destruction du cartilage et de l'os entrainant des déformations. La PR peut survenir à tous les âges mais apparaît le plus souvent entre 40 et 60 ans, avec une forte prédominance féminine (3 : 1). Il existe des variations géographiques au sein d'un même continent ou d'un même pays en raison de facteurs environnementaux, immunologiques mais aussi génétiques. Depuis bientôt 40 ans, l’implication du gène HLA-DRB1 est connue. Les études à grande échelle sur tout le génome ont permis d’identifier 110 nouveaux polymorphismes qui n’expliquent qu’une partie de la composante génétique de la PR. Ces études ont été principalement menées dans les populations d’Amérique du Nord, d’Asie ou d’Europe du Nord. L’objectif de ma thèse était donc d’étudier des facteurs génétiques de prédisposition à la PR 1) codant pour des microARNs ou mARNs, 2) dans deux populations jusqu’ici peu ou pas étudiées, et 3) portés par le chromosome X.Pour cela, j’ai travaillé sur deux ethnies différentes (Tunisienne et Française) afin d’effectuer une étude d’association « cas-contrôle » via une approche « gènes candidats». J’ai genotypé 3 polymorphismes (SNPs) sur le locus Xq28, et 2 SNPs sur le gène REL et quantifié le niveau d’expression de 13 micro-ARNs (miR-363, miR-106a, miR-20b, miR-188, miR-92a, miR-532, miR-652, miR-221, miR-222, miR-223, miR-98, let-7f miR-718 et miR-3202) situés sur le chromosome X. J’ai également évalué les composantes HLA et l’épitope partagé (EP) dans ces deux populations. J’ai effectué une analyse des haplotypes et du degré de déséquilibre de liaison (LD) pour chaque locus. Enfin j’ai validé mes résultats grâce à des méta-analyses.Mes résultats sur un échantillon cas/témoins de 995 individus montrent que les locus Xq28 et REL sont fortement associés à la PR chez les femmes Tunisiennes et Françaises, avec des différences entre ces deux populations. Les résultats des allèles du complexe HLA-DRB1 pourraient expliquer ces différences puisque ce ne sont pas les mêmes allèles HLA-DRB1 qui prédominent dans les deux populations. Parmi les 11 micro-ARNs étudiés, deux ne sont pas détectables (miR-718 et miR-3202) dans les PBMCs des patients atteints de PR, cinq (miR-221, miR-222, miR-223, miR-106a, miR-98) montrent une différence statistique d’expression entre les femmes contrôles et les femmes PR, et 6 (let-7f, miR-188, miR-652, miR-20b, miR-363, miR-92a) ne montrent aucune différences entre les deux groupes. Le cluster miR-221-222 montre une corrélation avec le génotype (AA+AG) de SNP rs3761548 et (AG+GG) versus (AA) et rs2223356 du gène FoxP3 chez les patients atteints de PR seulement avec une stratification en fonction du sexe.En conclusion, cette étude de 3 types de facteurs génétiques de prédisposition à la PR apporte un éclairage nouveau aux précédentes études Asiatiques ou d’Europe et d’Amérique du Nord, mettant en évidence des différences ethniques et géographiques. Des études de génomique fonctionnelle et sur de larges cohortes masculines seront nécessaires pour une meilleure compréhension de la physiopathologie de la PR et de l’importance du chromosome X dans cette maladie. Par ailleurs, le rôle des micro-ARNs en tant que facteurs génétiques de prédisposition de la PR reste peu étudié et mérite de futures explorations. / Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease with a worldwide prevalence that varies by country but is around 0.5% worldwide. RA is characterized by a bilateral and symmetrical joint disease, relapsing inflammation, production of auto-antibodies, cartilage destruction and bone causing deformations. RA can occur at any age, however, it appears most often between 40 and 60 years, with a strong female predominance (3: 1). There are geographical variations within the same continent or in the same country because of environmental factors, immunological but also genetical reasons. For nearly 40 years, the involvement of the HLA-DRB1 gene is known. Large-scale studies of the genome have identified 110 new polymorphisms (SNPs) that explain only a part of the genetic component of RA. These studies were mainly conducted in North American, Asian and North European populations. The aim of my thesis was to study genetic factors of susceptibility to RA 1) encoding microRNAs and mRNAs, 2) in two unstudied populations, and 3) with a specific focus on the X chromosome.For that, I worked on two different ethnicities (Tunisian and French) to conduct a "case-control" study of association via a "candidate gene" approach. I have genotyped 3 SNPs on the Xq28 locus (rs1059702, rs1059703, rs13397) and two SNPs on the REL gene , and quantified the expression level of 11 micro-RNAs (has_Mir-223, has_Mir-363, has_Mir-106a, 20b-has_Mir, has_Mir-188, has_Mir-92a, has_Mir-532, has_Mir-652, has_Mir-221, has_Mir -222, has_Mir-223, has_Mir-98 and let-7f) located within the X chromosome. I also assessed the HLA components and the shared epitope (SE) in these two populations. I performed a haplotype analysis and a linkage disequilibrium (LD) study for each locus. Finally I validated my results through a Meta-analysis.My results on a case/control sample of 995 individuals show that the Xq28 locus and REL locus are strongly associated with RA in both Tunisian and French women, with however differences between the two populations. The results of the HLA-DRB1 alleles might explain these differences since different HLA-DRB1 alleles predominate in each population. In overall our analysis showed that among the 11 studied X-linked miRNAs, two are not detectable (miR-718 and miR-3202) in PBMCs of RA patients, five (miR-221, miR-222, miR-223, miR-106a, miR-98) show a statistical difference between controls and RA women, and 6 (let-7f, miR-188, miR-652, miR-20b, miR-363, miR-92a) show no differences between controls and RA women. MiR-222 and miR-221 was statically correlation with (AA+AC) versus (CC) FoxP3 SNP rs3761548 and (AG+GG) versus (AA) FoxP3 SNP rs2223356 in RA patients only with gender stratification.In conclusion, this study focusing on three types of genetic predisposition to RA sheds new light on the previous studies from Asia, Northern Europe and America, highlighting ethnical and geographical differences. Functional genomic studies and large male cohorts will be needed for a better understanding of the pathophysiology of RA and to study the importance of the X chromosome in this disease. Moreover, the role of micro-RNAs as genetic factors of RA remains under-studied and needs further exploration.

In einer Vielzahl von Arbeiten konnten anti-inflammatorische Eigenschaften von Ethylpyruvat (EP) aufgezeigt werden. An verschiedenen Modellen der Sepsis, des hämorrhagischen Schocks, von Verbrennungsschäden, des Apoplex oder der Ischämie und Reperfusion wurde bei der Behandlung mit EP ein protektiver Effekt sowie eine verminderte Produktion von pro-inflammatorischen Zytokinen nachgewiesen. Als biochemische Grundlage wurde die Interaktion von EP mit dem Transkriptionsfaktor NF-κB identifiziert, die spezifischen Regulationsmechanismen konnten bisher allerdings nicht zufriedenstellend aufgeklärt werden. In dieser Arbeit wurde als eine neue mögliche Erklärung für die anti-inflammatorischen Eigenschaften des EP und weiterer α-oxo-Karbonsäureester die Inhibierung der Glyoxalase I (Glo-I) aufgezeigt. In vitro-Experimente zur Enzymaktivität belegten die Hemmung der Glo-I durch EP, während α-Hydroxy-Karbonsäureester wie L-Ethyllaktat (EL) keine inhibierenden Eigenschaften aufwiesen. Dennoch waren sowohl EP als auch EL und weitere Laktatester in der Lage, die LPS-induzierte Produktion von pro-inflammatorischen Zytokinen wie IL-1β, IL-6, IL-8 und TNF-α von humanen immunkompetenten Zellen zu supprimieren und die Expression von Immunrezeptoren wie HLA-DR, CD14 und CD91 zu modulieren. Somit konnten erstmals anti-inflammatorische Eigenschaften von Laktatestern nachgewiesen sowie eine Verbindung zwischen den Glyoxalase-Enzymen und dem Immunsystem etabliert werden. Diese und weitere Ergebnisse zur Einflussnahme der Karbonsäureester auf die Zellvitalität präsentieren das Glyoxalasesystem als mögliches Ziel neuer Therapiekonzepte für die Immunsuppression und bestätigen dessen Bedeutung für die Entwicklung von Anti-Tumor-Agenzien.

Abstract The etiology of juvenile idiopathic arthritis (JIA) is still unknown but genetic and enviromental factors play role in the pathogenesis. The aim of the study was to detect endoscopic and immunohistological changes in the gut in JIA compared with the controls and potential correlation of mucosal immunological activation with clinical activity of JIA. JIA patients (n=26) and negative controls (n=71) suffering from gastrointestinal symptoms without significant gastrointestinal disease were recruited for the study. Positive controls were patients with cows milk protein sensitive enteropathy (n=24). The intraepithelial lymphocytes counts, cytotoxic (granzyme A, B) and gamma/delta T-cell count and HLA-DR antigens were evaluated by using immunohistochemistry and messenger RNA expression levels of important immune mediators were assessed with real time PCR (RT-PCR) from fresh frozen intestinal mucosal samples. In JIA compared with negative controls, there was increased presence of lymphonodular hyperplasia and expression of HLA-DR antigens in abnormal mucosal cites, in crypts of the ileum. These changes were correlating with activity of JIA. In JIA compared with negative controls, there were found elevated granzyme B but decreased cytoprotective heat shock protein expression. The mRNA expression levels of anti- inflammatory mediators like TGFβ, IL10 and transcriptor factor of regulatory T-cells FOXP3, inversely correlated with activity of JIA. In conclusion, patients with JIA suffering from gastrointestinal symptoms display evidence of intestinal mucosal immune activation and there is an association between levels of mucosal immune alteration and clinical activity of JIA. These findings support the hypothesis that there is a link between the intestinal immune system and pathogenesis of juvenile idiopathic arthritis. In order to confirm these findings, more extensive series of JIA patients without gastrointestinal symptoms needs to be examined. / Tiivistelmä Lastenreuman tautimekanismi on tuntematon. Geneettiset ominaisuudet ja ympäristötekijät ovat yhteydessä taudin syntyyn. Tutkimuksen tavoitteena oli selvittää, onko suolen limakalvolla endoskooppisia tai immunohistologisia muutoksia enemmän lastenreumassa kuin kontrolleilla, ja että liittyvätkö muutokset niveltaudin aktiivisuuteen. Tutkimukseen otettiin 26 suolioireista lastenreumapotilasta, 76 verrokkia joilla ei ollut autoimmuunisairautta sekä 24 viivästynyttä maitoallergiaa sairastavaa lasta, joille tehtiin suolen tähystystutkimus. Ohutsuolinäytteistä arvioitiin immunohistologisesti solunsisäisten lymfosyyttien, gamma/delta-positiivisten lymfosyyttien sekä sytotoksisten (grantsyymi-A ja -B) lymfosyyttien määrä. Lisäksi määritettiin immunohistologisesti ohutsuolen limakalvon epiteelisolujen HLA-DR- antigeenien ja epiteelisolua suojaavien lämpöshokkiproteiinien ilmenemistä sekä käänteis-PCR-menetelmällä keskeisten välttäjäaineiden lähetti-RNA-tasoja. Tutkimuksessa lastenreumaa sairastavilla esiintyi enemmän suolen imukudoskertymää (lymfonodulaarinen hyperplasia) negatiiviseen verrokkiryhmään nähden sekä HLA-DR antigeenejä epätyypillisellä alueella ohutsuolen loppuosan limakalvon kryptassa. Nämä löydökset olivat yhteydessä lastenreuman aktiivisuuteen. Lastenreumassa oli verrokkeja enemmän sytotoksisia lymfosyyttejä ja vähemmän lämpöshokkiproteiineja. Tulehdusta suojaavat lähetti- RNA-tasot korreloivat käänteisesti lastenreumataudin aktiivisuuteen. Väitöstutkimuksen suolioireisilla lastenreumapotilailla oli suolen limakalvolla muutoksia, jotka sopivat poikkeavaan antigeenien käsittelyyn. Nämä löydökset tukevat hypoteesia, että lastenreumassa suolen limakalvon immunologinen aktivaatio on yhteydessä taudin puhkeamiseen. Jotta tulokset voisi yleistää, tarvittaisiin jatkotutkimus, joka on tehty suolioireettomilla lastenreumapotilailla ja riittävällä otoskoolla.

L’histoire naturelle du cancer implique des interactions entre la tumeur et les mécanismes de défense de l’hôte, tout particulièrement avec le système immunitaire adaptatif. Ainsi la transformation de cellules normales en cellules malignes peut engendrer l’expression d’antigènes tumoraux reconnus par les lymphocytes T. Plusieurs sous-populations de lymphocytes T (LT) CD4 contrôlent les réponses antitumorales, parmi elles, les LT CD4 helper de type-1 (Th1) jouent un rôle activateur majeur de l’immunité à médiation cellulaire antitumorale. Ils deviennent actifs grâce à la reconnaissance des peptides de 15 à 20 acides aminés dérivés d’antigènes tumoraux et présentés par les molécules HLA de classe II. Ils sont nécessaires à l’induction et la fonction des cellules effectrices dirigées contre les tumeurs notamment les lymphocytes T CD8 cytotoxiques (CTL). De plus la présence de lymphocytes CD4 Th1 infiltrant les tumeurs est souvent associée à un bon pronostic chez les patients. A l’aide d’un modèle in vitro chez l’homme et in vivo chez des souris transgéniques HLA, nous avons identifié quatre nouveaux peptides CD4 dérivés de la télomérase (TERT) un antigène de tumeur exprimé dans la majorité des cancers humains. Ces peptides appelés «Universal Cancer Peptide, UCP» se lient à la majorité des allèles HLA-DR et sont capables d’activer spécifiquement les LT CD4 de type-1. Des LT CD4 circulants spécifiques des UCP sont naturellement détectables dans plusieurs cancers humains mais absents chez des individus sains. Des clones T CD4 spécifiques des UCP générés à partir des lymphocytes de patients, produisent de forts taux d’IFN, TNF, et d’IL-2, cytokines associées à la polarisation Th1. L’analyse par ELISPOT IFN, de LT CD4 anti-UCP circulants au sein d’une cohorte de 84 patients atteints de cancers bronchiques métastatiques a montré la présence naturelle de ces lymphocytes chez 38 % des patients. De plus un effet bénéfique de la présence de cette réponse sur la survie globale a été observé chez les patients ayant une réponse clinique objective après chimiothérapie (13 vs 10 mois, P< 003). In vivo, l’immunisation de souris transgéniques HLA-A2/HLA-DR1 (Tg A2/DR1) avec les peptides UCP stimule des réponses T CD4 spécifiques caractérisées par une polarisation Th1. Nous avons montré que la présence in vivo de LT CD4 anti-UCP est nécessaire pour l’induction de réponses CTL antitumorales efficaces. Ainsi chez des souris co-immunisées en présence d’un peptide UCP, on observe un accroissement en nombre et de la qualité des réponses CTL proportionnellement à l’aide délivrée par les LT CD4 anti-UCP. L’induction de LT CD4 anti-UCP s’accompagne également d’une activation des cellules dendritiques in vivo via un mécanisme impliquant CD40L, IFN et GM-CSF. Dans un modèle de mélanome transplantable chez les souris Tg A2/DR1 nos résultats ont montré qu’une vaccination thérapeutique comportant un peptide UCP favorise un meilleur recrutement de CTL fonctionnels dans les tumeurs et améliore ainsi l’efficacité antitumorale du vaccin. Ces résultats confirment le rôle antitumoral majeur des lymphocytes CD4 Th1 et soulignent l’intérêt clinique de stimuler des réponses T CD4 spécifiques d’antigènes tumoraux de relevance clinique comme TERT. / Recent advances in immunology have now validated the concept of cancer immunosurveillance and the leading role of adaptative T cell immunity. Until a few years ago, antitumor CD8 T cell responses have been the most studied due to their direct cytotoxic activity on tumor cells. On the other hand, study of antitumor CD4 T cell responses are even more challenging because of the heterogeneity and plasticity of the various CD4 T cells subpopulations described. Among them, CD4 T helper type-1 cells (Th1), mainly characterized by the production of IFN, control the activation of antitumor cellular immunity. Thus, stimulation of specific CD4 Th1 cells may have a major interest for the development of anticancer immunotherapies. During this research thesis, we characterized novel HLA class II epitopes derived from a relevant tumor antigen, telomerase (TERT), and studied their capacities to stimulate specific CD4 Th1 cell responses. Using a method based on predictive immunology, we identified 4 peptides derived from TERT, referred as « Universal Cancer Peptides » (UCPs), enable to bind the most commonly found HLA-DR alleles in human. Using HLA-A2/HLA-DR1 transgenic mouse model, we first evaluated the in vivo immunogenicity of these peptides. Immunization of mice with UCPs induces high avidity specific CD4 T cells. The study of their polarization showed that UCP-specific CD4 T cells do not produce IL-4, -5, -10 or -17 cytokines, excluding a Th1, Treg or Th17 differentiation. In contrast, we measured high amount of IFN and IL-2 which characterize a Th1 pattern. The study of helper role allow us to demonstrate that CD8 peptide-based vaccinations in presence of UCPs enhance the efficacy of tumor specific CTL responses. Indeed, the intensity of these responses is strongly correlated with that of UCP-specific CD4 T cells induced in vivo. Furthermore, the stimulation of UCP-specific CD4 T cells promotes activation and IL-12 release by dendritic cells through a mechanism that involves IFN, GM-CSF and CD40L. We also demonstrated the antitumor efficacy of UCPs during a therapeutic vaccination in mice, as well as their capacity to foster the recruitment of specific CD8 T cells at the tumor site. In addition, the presence of naturally occurring UCP-specific CD4 T cell responses was found in different types of cancers such as leukemia, lung, colorectal or renal cancers. A study conducted in a cohort of 84 metastatic lung cancer patients revealed a synergistic effect of spontaneous UCP-specific CD4 Th1 and chemotherapy-treatment. Altogether, this study provides further evidences that stimulation of antitumor CD4 Th1 cells is a powerful method to improve cancer vaccines and also highlights the interest of TERT-derived UCPs for the innovative monitoring of antitumor CD4 T cell responses

Monocytic human leukocyte antigen-D related (mHLA-DR) expression determined by flow cytometry has been suggested as a biomarker of sepsisinduced immunosuppression. In order to facilitate use of HLA-DR in clinical practice, a quantitative real-time PCR technique measuring HLA-DR at the transcription level was developed and evalutated. Levels of HLA-DR mRNA correlated to mHLADR expression and were robustly measured, with high reproducibility, during the course of infection. Dynamics of mHLA-DR expression was studied during the first weeks of bloodstream infection (BSI) and was found to be dependent on the bacterial etiology of BSI. Moreover, mHLA-DR was shown to be inversely related to markers of inflammation. In patients with unfavourable outcome, sustained high C-reactive protein level and high neutrophil count were demonstrated along with low mHLA-DR expression and low lymphocyte count. This supports the theory of sustained inflammation in sepsis-induced immunosuppression. The association between mHLA-DR and bacterial etiology may be linked to the clinical trajectory via differences in ability to cause intractable infection. Staphylococcus aureus was the dominating etiology among cases with unfavourable outcome. With focus on patients with S. aureus BSI, those with complicated S. aureus BSI were found to have lower HLA-DR mRNA expression during the first week than those with uncomplicated S. aureus BSI. If these results can be confirmed in a larger cohort, HLA-DR measurement could possibly become an additional tool for early identification of patients who require further investigation to clear infectious foci and achieve source control. In conclusion, PCR-based measurement of HLA-DR is a promising method for measurements of the immune state in BSI, but needs further evaluation in the intensive care unit setting to define the predictive and prognostic value for deleterious immunosuppression. The etiology of infection should be taken into consideration in future studies of translational immunology in sepsis.

The vector-borne protozoan Leishmania spp. cause the spectrum of disease known as leishmaniasis in human and animal hosts. The most common manifestations of leishmaniasis are the chronic, ulcerative skin disease cutaneous leishmaniasis (CL), and the more serious visceral leishmaniasis (VL) in which parasites take up residence in internal organs, causing death if not treated. The role of neutrophils (PMNs) in the immune response to CL and VL is unclear. It is s generally thought that PMNs are only a short-lived effector cell, and have been disregarded as playing a role in chronic Leishmania spp. infection. As both CL and VL are diseases characterized by increased inflammatory immune mediators, we hypothesized that PMNs from human or animal models of chronic leishmaniasis would display different properties from PMNs from healthy controls. We found in a subset of CL and VL patients circulating PMNs expressing HLA-DR, the human form of MHC class II, a molecule thought to be restricted primarily to professional antigen cells. When we examined PMNs recruited to CL skin lesions in human patients, or similar lesions in experimental murine model of CL, we found significantly increased MHC class II+ PMNs. Circulating HLA-DR+ PMNs also expressed the co-stimulatory molecules CD80, CD86 and CD40. While this suggested an antigen-presenting cell-like phenotype by these HLA-DR+ PMNs, compared to conventional HLA-DR- PMNs, HLA-DR+ PMNs showed not only a neutrophil-like appearance and function, but in fact increased activation, degranulation, intracellular MPO and phagocytosis of parasites and zymosan particles. Incubation of healthy control whole blood with inflammatory cytokines resulted in increased HLA-DR+ PMNs and the presence of hladrb1 mRNA, suggesting a connection between neutrophil “priming” and upregulation of HLA-DR. In addition to HLA-DR+ PMNs in CL patients, we also identified the presence of so-called “low-density” neutrophils (LD-PMNs). These neutrophils, which are enriched in low-density fractions following centrifugation of blood over a density gradient, are reported in numerous disease states, including cancer, HIV, and systemic lupus erythematosus. In some disease states, LD-PMN are reported to be immunosuppressive toward T cell activation and proliferation. However, LD-PMNs from leishmaniasis patients showed no evidence of immunosuppression. Additionally, we found that LD-PMNs show significantly increased surface expression of MHC class II, suggesting a heretofore unappreciated connection between these atypical neutrophil phenotypes. We also investigated the in vitro interactions with different Leishmania infantum life-stages, both those that cause acute infection (promastigotes) and amastigotes, which are found during chronic stages of the disease. We found that PMNs are readily infected by all L. infantum life-stages, but that amastigotes may have different methods of interacting with PMN surface receptors and are better equipped to avoid PMN anti-microbial responses. These data suggest that circulating PMNs in chronic leishmaniasis may have unique phenotypes and interact differently with the Leishmania spp. life-cycle present during chronic infection. Further investigation of the role of PMNs and atypical PMN phenotypes in chronic disease may help identify new immunomodulatory roles for this cell type.

Diss. (sammanfattning) Stockholm : Karol inst., 2002. / Härtill 6 uppsatser.