MHCII molecules expose a weave of antigens, which send survival or activation signals to T lymphocytes. The ongoing process of peptide binding to the MHC class II groove implicates three accessory molecules: the invariant chain, DM and DO. The invariant chain folds and directs the MHCII molecules to the endosomal pathway. Then, DM exchanges the CLIP peptide, which is a remnant of the degraded invariant chain, for peptides of better affinity. Expressed in highly specialized antigen presenting cells, DO competes with MHCII molecules for DM binding and favors the presentation of receptor-internalized antigens. Altogether, these molecules exhibit potential immunomodulatory properties that can be exploited to increase the potency of peptide vaccines. DO requires DM for maturation and to exit the ER. Interestingly, it is possible to monitor this interaction through a conformation change on DOβ that is recognized by the Mags.DO5 monoclonal antibody. Using Mags.DO5, we showed that DM stabilizes the interactions between the DO α1 and β1 chains and that DM influences DO folding in the ER. Thus, the Mags.DO5+ conformation correlates with DO egress from the ER. To further evaluate this conformation change, directed evolution was applied to DO. Of the 41 unique mutants obtained, 25% were localized at the DM-DO binding interface and 12% are at the solvent-exposed β1 domain, which is thought to be the Mags.DO5 epitope. In addition, I used the library to test the ability of HLA-DO to inhibit HLA-DM and sorted for the amount of CLIP. Interestingly, most of the mutants showed a decrease inhibitory effect, supporting the notion that the intrinsic instability of DO is a required for its function. Finally, these results support the model in which DO competes against classical MHCII molecules by sequestering DM chaperone’s function. MHCII molecules are also characterized by their ability to present superantigens, a group of bacterial or viral toxins that coerces MHCII-TCR binding in a less promiscuous fashion than what is observed in a canonical setting. While the mechanism of how bacterial superantigens form trimeric complexes with TCR and MHCII is well understood, the mouse mammary tumor virus superantigens (vSAG) are poorly defined. In the absence of a crystal structure, I chose a functional approach to examine the relation between vSAG, MHCII and TCR with the goal of uncovering the overall trimolecular architecture. I showed that TCR concomitantly binds both the MHCII α chain and the vSAG and that TCR-MHCII docking is almost canonical when coerced by vSAGs. Because many peptides may be tolerated in the MHCII groove, the pressure exerted by vSAG seems to tweak conventional TCR-MHCII interactions. Furthermore, my results demonstrate that vSAG binding to MHCII molecules is conformation-dependent and abrogated by the CLIP amino-terminal residues extending outside the peptide-binding groove. In addition, they also suggest that vSAGs cross-link adjacent MHCIIs and activate T cells via a TGXY motif. / Les molécules du CMH présentent une panoplie d'antigènes qui, lorsque reconnus par un lymphocyte T spécifique, indique à ce dernier de survivre ou de s'activer. Le processus menant à la liaison d'un peptide à la poche du CMH de classe II, implique trois molécules accessoires, soit la chaine invariante, DM et DO. La chaine invariante replie et dirige les molécules du CMHII jusqu'à la voie endosomale. Ensuite, DM échange CLIP, peptide découlant de la dégradation de la chaine invariante, pour d'autres ayant une meilleure affinité. Exprimé seulement chez certaines cellules présentatrices, DO compétitionne avec les molécules du CMHII pour la liaison à DM et ainsi favorise la présentation d'antigènes internalisés par des récepteurs membranaires. Ensemble, ces protéines ont un potentiel immunomodulatoire pouvant être exploité afin d'augmenter l'efficacité de vaccin peptidique. DO requiert DM pour arriver à maturité et sortir du RE. Cette interaction, qui induit un changement de conformation dans la chaine β de DO, peut être sondée à l'aide de l'anticorps monoclonal Mags.DO5. En utilisant cet anticorps, nous avons montré que DM stabilise l'interaction entre les domaines α1 et β1 de DO et influence son repliement dans le RE. Donc, la conformation qui révèle l’épitope Mags.DO5 corrèle avec la migration de DO hors du RE. Afin d'étudier plus précisément ce changement de conformation, DO fut contraint à une ronde d’évolution dirigée. Des 41 mutants obtenus, 25% se retrouvent à l'interface DO- DM et 12% se retrouvent à la surface exposée au solvant du domain β1, région hypothétique de l'épitope Mags.DO5. De plus, la bibliothèque de mutants a été testée pour son habileté à inhiber l'activité de DM. La plupart des mutants montre une activité inhibitrice diminuée, ce qui supporte le model où DO compétionne les molécules du CMHII en séquestrant le rôle chaperon de DM. Les molécules du CMHII ont l'unique habileté de présenter les superantigènes, une famille de toxines virales et bactériennes qui force l'interaction CMHII-TCR de façon beaucoup moins spécifique qu'en contexte canonique. Alors que la façon dont les superantigènes bactériens s'assemblent avec le CMHII et le TCR soit bien comprise, la nature du complexe trimoléculaire découlant des superantigènes du virus de la tumeur mammaire de la souris (vSAG) reste mal définie. En l'absence d'une structure cristalline, une approche fonctionnelle a été choisie pour examiner la relation des vSAGs avec le CMHII et le TCR avec le but de dévoiler l'architecture de ce multi-complexe protéique. Je montre que le TCR lie parallèlement la chaine α du CMHII et vSAG, ce qui résulte en une interaction presque canonique. Puisque différents peptides peuvent être tolérés lors de cet ancrage, il semble que vSAG ajuste les interactions CMHII-TCR conventionnelles. En outre, mes résultats montrent que vSAG reconnait un épitope conformationnel et que cette liaison peut être abrogée par l'extension amino-terminale du peptide CLIP, laquelle s'étend en deçà de la niche. Finalement, mes résultats suggèrent que vSAG peut réticuler plusieurs CMHII adjacents et active les cellules T via son motif TGXY.

Antibody responses can be up- or down-regulated by passive administration of specific antibody together with antigen. Depending on the structure of the antigen and the antibody isotype, responses can be completely suppressed or enhanced up to a 1000-fold of what is seen in animals immunized with antigen alone. IgG suppresses primary antibody responses against erythrocytes. Suppression works well in mice lacking Fc-receptors for IgG, C1q, C3, or complement receptor 1 and 2 (CR1/2). Here, we demonstrate that IgG anti-NP given to mice together with NP-conjugated sheep erythrocytes, suppresses the generation of NP-specific extra-follicular antibody-secreting cells, NP-specific germinal center B cells, induction of memory and long-lived plasma cells. IgG increases antigen clearance but this does not explain the suppressed antibody response. It is demonstrated that IgG-mediated suppression of IgG responses is epitope specific, suggesting that epitope masking is the dominant explanation for IgG-mediated suppression of antibody responses. Both IgE and IgG3 can enhance antibody responses against soluble antigens. IgE-antigen complexes bind to recirculating B cells expressing CD23, an Fc-receptor for IgE.  Thirty minutes after intravenous administration, IgE-antigen is found in splenic follicles. Subsequently, germinal center responses, antigen-specific T cell proliferation, and antibody responses are enhanced. We show that also antigen conjugated to anti-CD23 can bind to CD23+ B cells and be transported to splenic follicles. CD11+ spleen cells, rather than CD23+ B cells, present IgE-antigen complexes to T cells. Here, it is demonstrated that CD8α− conventional dendritic cells is the CD11c+ cell population presenting IgE-antigen to T cells. IgG3-mediated enhancement is dependent on CR1/2. We find that IgG3-antigen complexes, administered intravenously to mice, bind to marginal zone B cells via CR1/2. These cells then transport IgG3-antigen into splenic follicles and deposit antigen onto follicular dendritic cells. Mice treated with FTY720, a drug which dislocates marginal zone B cells from the marginal zone, impairs this transport. Studies in bone marrow chimeric mice show that CR1/2 on both B cells and follicular dendritic cells are crucial for IgG3-mediated enhancement. In summary, these observations suggest that antibodies can feedback regulate antibody responses by modulating the access of antigen to the immune system.

As células dendríticas (DCs) são as principais células apresentadoras de antígeno do sistema imune. No entanto, trabalhos têm demonstrado seu envolvimento na manutenção da tolerância imunológica. As células T CD4+CD25+Foxp3+ possuem a capacidade de suprimir respostas imunes. Neste estudo avaliamos as alterações ocorridas na população de células T CD4+CD25+Foxp3+ após co-cultura de células de linfonodo com DCs. Nossos resultados demonstram que após a co-cultura há um aumento da população de células CD4+CD25+Foxp3+ de maneira independente do estado de ativação das DCs ou da presença de antígenos exógenos. No entanto, o aumento observado é maior quando DCs imaturas são incubadas com antígenos exógenos. Notamos ainda que há presença de TGF-ß em todas as condições experimentais em que observamos aumento da população de células CD4+CD25+Foxp3+. Nossos dados sugerem ainda que este aumento se deve à proliferação das células T CD4+CD25+Foxp3+. / Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system. However, DCs have also been implicated in maintaining immunologic tolerance. CD4+CD25+Foxp3+ T lymphocytes are known as cells with regulatory properties. In this study we evaluated the changes in the CD4+CD25+Foxp3+ T cell population after co-culture of lymph-node cells with DCs. Our results show an increase in the CD4+CD25+Foxp3+ T cell population after co-culture and occurs regardless of the activation state of DCs and the presence of exogenous antigens; however it is greater when immature DCs are previously pulsed with exogenous antigen. We also noticed that TGF-? is present in all cultures conditions in which the CD4+CD25+Foxp3+ T cell population increases. Our data also suggests that the increase of the CD4+CD25+Foxp3+ T cell population may be due to the proliferation of these cells.

Les cellules iNKT (de l’anglais invariant Natural Killer T) constituent un sous-type particulier de lymphocytes T caractérisé par un profil de type inné. Ces cellules répondent rapidement à des antigènes lipidiques et glycolipidique présentés par le CD1d, une glycoprotéine exprimée par les différentes cellules présentatrices d'antigène. Suite à l’activation, les cellules iNKT sont capables de produire de grandes quantités de cytokines anti-inflammatoires et pro-inflammatoires et elles sont impliquées dans différentes maladies, telles que l'allergie, l'auto-immunité, le cancer et les infections, parmi lesquelles la leishmaniose. Les parasites protozoaires de les espèces Leishmania sont les agents causaux de la leishmaniose, une maladie tropicale négligée dont la manifestation la plus grave affecte les organes viscéraux et qui peut être mortelle si elle n'est pas traitée. Le succès de l'infection dépend de la capacité du parasite à maitriser la réponse immunitaire de l'hôte. Récemment, quelques groupes, y compris le nôtre, ont observé que les parasites Leishmania libèrent des vésicules extracellulaires (VE). Les VE sont formées par une bicouche membranaire lipidique, contenant des lipides, des protéines et du matériel génétique et elles peuvent transmettre des molécules dérivées des pathogènes aux cellules hôte sans contact direct entre les cellules. Les VE produites par les parasites Leishmania et aussi par d’autres protozoaires ont été associés à des effets pro-parasite car elles favorisent un environnement plus permissif à l'établissement de l'infection. Dans cette thèse, nous avons étudié l'effet du matériel extracellulaire (ME), correspondant aux VE et aux molécules non-associées aux VE, libéré par les promastigotes de L. infantum sur les cellules iNKT. Dans le début de ce travail, il a été observé que le ME de L. infantum empêche l'expansion ex-vivo des cellules iNKT humaines à partir de cellules mononucléaires du sang périphérique. Cela a mis en évidence la communication entre les cellules iNKT et le ME de L. infantum, ce qui a été exploré par la suite. Le ME de L. infantum module la capacité très importante des cellules iNKT à produire des cytokines. En effet, le ME de L. infantum empêche la production des différentes cytokines par les cellules iNKT, comme par exemple IL-4 et IFNγ. De plus, nous avons aussi démontré que le ME de L. infantum compète avec l’α-GalCer, un agoniste très puissant des cellules iNKT, pour la liaison à la molécule CD1d, ce qui justifie l’effet inhibiteur dans l'activation des cellules iNKT. Nous avons aussi montré que les lipides qui sont présents dans chaque fraction du ME de L. infantum ont un rôle très important dans l’inhibition de l'activation et l'expansion des cellules iNKT. Ainsi, le ME de L. infantum, par ces lipides, peut participer à l’altération de l’activation des cellules iNKT dépendante du CD1d. Cela ajoute une nouvelle évidence de la contribution du ME de L. infantum dans la subversion de la réponse immunitaire de l’hôte. La communication entre le ME libéré par un pathogène et les cellules iNKT a été étudié pour la première fois, ce qui a suggéré un mécanisme de modulation de ces cellules qui n’avait jamais été décrit. Ce travail ouvre des perspectives pour l'étude de l'interaction de ME libéré par d'autres pathogènes avec des cellules iNKT. De plus, l'analyse des lipides contenus dans le ME de L. infantum pourra aboutir à la découverte de nouvelles molécules spécifiques pour inhiber les cellules iNKT. Cela apporterait des avantages significatifs dans les approches cliniques ciblant la modulation de l'activation des cellules iNKT. / The invariant natural killer T (iNKT) cells constitute a particular subset of T lymphocytes characterized by an innate-like profile. These cells rapidly respond to lipid and glycolipid antigens bound by the glycoprotein CD1d expressed by different antigen presenting cells. Once activated, they release large amounts of anti- and proinflammatory cytokines. Thus, iNKT cells are endowed with a remarkable immunomodulatory potential and they have been implicated in different disorders, such as allergy, autoimmunity, cancer and infection, among which is leishmaniasis. Leishmania spp. are a group of protozoan parasites that includes the causative agents of leishmaniasis. This is a neglected tropical disease in which the most severe form of manifestation affects visceral organs and could be fatal if left untreated. Importantly, the success of Leishmania infection relies on the capacity of the parasite to subvert host immune responses. Recently, a few groups, including ours, observed that Leishmania parasites release extracellular vesicles (EVs). EVs are vesicles formed by a lipid bilayer membrane, containing other lipids, proteins and genetic material on their surface as well as in their lumen. Due to their potential to transmit messages between pathogens and host cells without a direct cell contact, they have been a focus of great interest regarding infection. EVs derived from Leishmania and other protozoan parasites have been associated with pro-parasite effects, by creating a more permissive environment to the establishment of the infection. Herein, we studied the effect of the extracellular material (ExM), which encloses both EVs and vesicle-depleted material, released by L. infantum promastigotes in iNKT cells. In the first steps of this work, it was observed that L. infantum ExM is capable of impairing the expansion of human iNKT cells ex vivo from peripheral blood mononuclear cells. This evidenced the cross-talk between iNKT cells and L. infantum ExM that we explored further. L. infantum ExM also modulates the important capacity of iNKT cells to release cytokines, impairing the production of different cytokines, such as IL-4 and IFNγ by these cells. Furthermore, we also show that L. infantum ExM competes with α-GalCer, a potent iNKT cell agonist, for CD1d binding, which justifies its effect in the impairment of iNKT cell activation. Additionally, we also proved the lipids present in each fraction of L. infantum ExM take an important role in the inhibition of iNKT cell activation and expansion. Thus, L. infantum ExM, through their lipids, is suggested to participate in the impairment of CD1d-mediated activation of iNKT cells, adding a new evidence regarding the contribution of the parasite ExM to subvert host immune responses. To the best of our knowledge, this is the first time that the cross-talk between the ExM released by a microbe and iNKT cells was assessed, shedding light on a mechanism of iNKT cell modulation that remained unexplored so far. This opens new perspectives regarding the study of the interaction of the ExM released by other pathogens with iNKT cells. Moreover, a further analysis of the lipid content of L. infantum ExM might allow the finding of new inhibitory molecules specific to iNKT cells, which can bring significant advantages in clinical approaches targeting the modulation of iNKT cell activation.

Les protéines solubles sont généralement faiblement immunogènes, ce qui constitue unelimite pour le développement de vaccins sous unitaires à base de protéines. Mes travaux de thèseont eu pour objectif de décrypter certains mécanismes moléculaires et cellulaires qui contribuent àl’immunogénicité et d’en tirer partie pour développer des approches originales permettantd’améliorer la capacité des protéines à déclencher la réponse immunitaire. Pour cela, j’aiprincipalement utilisé le transactivateur transcriptionnel (Tat) du VIH-1. J’ai montré quel’oligomérisation de Tat permet à un mécanisme de collaboration B-TH-2 d’induire la réponseimmunitaire en absence d’adjuvant. J’ai identifié le déterminant minimal responsable de l’effet etmontré qu’il confère la propriété adjuvante à d’autres antigènes. J’ai ensuite montré que laprésentation aux cellules T restreinte aux CMH I et CMH II est accrue lorsque les protéines sontdotées de la capacité à lier des sucres sulfatés d’expression ubiquitaire: les héparanes sulfate. Cestravaux ont permis de définir de nouvelles approches pour améliorer l’immunogénicité de protéinessusceptibles d’être intégrées dans des préparations vaccinales. / Soluble proteins are usually poorly immunogenic, which is a limit to the development ofsubunit vaccines based on proteins. My thesis work aimed to decipher some molecular and cellularmechanisms that contribute to the immunogenicity and to exploit them to develop innovativeapproaches to improve the ability of proteins to trigger the immune response. For this purpose, Imainly used the transcriptional transactivator (Tat) of HIV-1. I showed that the oligomerization of Tatenables a B-TH-2 collaborative mechanism to induce the immune response in the absence ofadjuvant. I identified the minimum region determining the effect and showed that it confers the selfadjuvantingproperty to other antigens. In the second part of my work, I showed that the MHC I andMHC II restricted presentation to T cells is increased when the proteins are endowed with the abilityto bind ubiquitous sulfated polysaccharides: heparan sulfates. This work helped to define newapproaches to improve the immunogenicity of proteins that are likely to be included in vaccinepreparations.

Nos travaux décrivent l’identification et la caractérisation d’une cellule immunitaire : l’IKDC pour Interferon Producing killer dendritic cell. Cette population cellulaire, découverte à la faveur de la combinaison Imatinib mesilate et Interleukine 2 dans le traitement des métastases pulmonaires de mélanome B16F10 chez la souris, combine des caractéristiques de cellule dendritique (présentation antigénique) et de cellule natural killer (lyse sans apprentissage). Le phénotype des IKDC est le suivant :CD3-, CD19-, CD11c+,B220+, NK1.1+. Nous avons établi une stratégie de culture des IKDC ex vivo grâce à la trans-présentation de l’IL-15 et montré que ces cellules sont capables de présenter des antigènes aux lymphocytes T et de vacciner une souris contre une tumeur. Ce projet se poursuit par la mise en place d’un essai clinique de phase I utilisant Imatinib mesilate et l’Interleukine 2. / In this manuscript, we describe the isolation and the characterisation of a novel immune cell: the IKDC for Interferon Producing killer dendritic cell. This cell population, discovered thanks to the combination Imatinib mesilate and Interleukine 2 for the treatment of lung metastases of B16F10 murine melanoma, shares dendritic cell (antigen presentation) and natural killer cell (non specific lysis) properties. IKDC phenotype is the following: CD3-, CD19-, CD11c+, B220+, NK1.1+. We established a culture strategy for the IKDC ex vivo thanks to the trans-presentation of IL-15 and demonstrated that these cells once cultivated are able to prime lymphocytes and to protect mice from tumor development. This project has led to a phase I clinical trial testing the antitumor effect of the combination Imatinib mesilate and Interleukin 2.

Les molécules du Complexe Majeur d'Histocompatibilité de classe I (CMH I) contrôlent la plasticité synaptique dans le système nerveux central (SNC) et plusieurs travaux expérimentaux suggèrent des interactions antigène-dépendantes entre des neurones infectés par des virus et les lymphocytes T CD8. Cependant, le rôle de la présentation des antigènes par le CMH I des neurones sur la physiopathologie de l'infection par Toxoplasma gondii (T. gondii) n'a pas encore été clarifié. Après la dissémination aigue sous forme de tachyzoites, T. gondii se convertit en bradyzoites, forme persistante dans les neurones du SNC. Chez les individus immunocompétents, la toxoplasmose latente est associée à des variations des fonctions cognitives ainsi qu'à des pathologies neuropsychiatriques. Les sujets dont le système immunitaire est dysfonctionnel peuvent développer une encéphalite létale causée par T. gondii, qui est caractérisée par une réplication du parasite, une infiltration massive et des agrégats leucocytaires et l'activation des cellules gliales. Les lymphocytes T (LT) CD8 et le CMH I sont des facteurs-clés contrôlant la résistance à l'encéphalite. Utiliser les LT CD8 pour éliminer les kystes chez des sujets à risque est une piste thérapeutique intéressante en raison de l'absence d'approches pharmacologiques ciblant les bradyzoites. A ce jour, les mécanismes et la pertinence fonctionnelle de la présentation des antigènes dérivés de T. gondii par les neurones restent à déterminer, ainsi que la contribution des différents stades parasitaires au contrôle de l'infection. L'utilisation de nouveaux parasites exprimant un antigène immunodominant uniquement au stade tachyzoite a permis de montrer que la reconnaissance par les LT CD8 d'antigènes issus des tachyzoites est suffisante pour une protection efficace contre l'encéphalite.[...] / In the Central Nervous System (CNS), Major Histocompatibility Complex class I (MHC I) molecules regulate synaptic plasticity and evidence suggests antigen-specific interactions between virus-infected neurons and CD8 T cells. Yet, little is known about the impact of neuronal MHC I presentation on the pathophysiology of infection by the neurotropic Toxoplasma gondii (T. gondii) parasite. Following acute dissemination as tachyzoites, T. gondii converts into bradyzoites that persist inside cysts within neurons of the CNS. In immunocompetent hosts, latent toxoplasmosis is associated with cognitive changes and neuropsychiatric disorders. Hosts with sub-optimal immune responses may develop a lethal T. gondii Encephalitis (TE), characterized by parasite replication, granuloma-like structures with massive immune cell influx and glial cell activation. CD8 T cells and MHC I are key determinants of TE resistance. Harnessing CD8 T cells in at-risk individuals may turn helpful in the future as we are currently lacking an effective pharmacological approach to eradicate bradyzoites. Yet the mechanisms and functional relevance of neuronal MHC I presentation of T. gondii remain unexplored, as well as which stage of the parasite contributes to efficient control of the infection. Using new T. gondii parasites with restricted expression of the immunodominant antigen at the tachyzoite stage, this work showed that CD8 T cell recognition of tachyzoite antigens at early stages of brain invasion is enough to protect from TE. Interestingly, by comparing situations of toxoplasmosis with varying TE severity and by pioneering antigen presentation assays with T. gondii-infected primary neurons, we revealed that TE susceptibility may be underlied by sub-optimal MHC I presentation of tachyzoites antigens by neurons. At last, we describe a mouse model that allows conditional deletion of a MHC I allele that is essential for TE resistance (H-2Ld). [...]

Mice infected with neurotropic variants of the murine coronavirus, mouse hepatitis virus, (strains JHMV or J2.2–V–1) develop acute and chronic CNS infections, and provide a model system to study the pathogenesis of virus–induced neuroinflammation, mechanisms of virus persistence, and anti–viral immune responses in the CNS. Using the J2.2–V–1 model of CNS infection, we addressed the role of sustained CCL2 production during viral infection using mice in which CCL2 was expressed transgenically in oligodendrocytes. Tonic CCL2 expression in the CNS resulted in delayed kinetics of virus clearance, and converted what is typically a mild, nonlethal disease to acutely lethal encephalitis, with the majority of mice succumbing to the infection. CCL2 induced a rapid and dysregulated inflammatory response that was no longer protective and was unable to efficiently clear virus from the CNS. Infected CCL2 Tg mice had increased numbers of Foxp3–expressing CD4 T cells (Tregs) and of macrophages and microglia expressing elevated levels of YM–1, a marker for alternatively activated macrophages, and nitric oxide. Our results showed that CCL2 has effects beyond serving as a chemoattractant for leukocytes, and has effects on the composition and function of inflammatory cells at sites of infection. In a separate set of experiments, I identified and characterized two additional heteroclitic variants of the JHMV epitope S598 that induced CD8 T cells with greater antigen sensitivity to the native S598 determinant relative to the cells primed by the native epitope. One of these heteroclitic epitopes elicited a T cell response with nearly complete cross–reactivity towards the native peptide. The structural data show that these heteroclitic epitopes induced modest conformational changes in the local environment of the peptide–MHCI complex. I also provide data to support the notion that heteroclitic determinants augment functional avidity by increasing surface epitope density. Collectively, these data will help guide the design of heteroclitic epitopes in the setting of vaccine development. Lastly, I examined the consequences of oxidative stress induced by viral infection on antigen presentation. The brains of JHMV–infected mice were found to have signs of oxidative stress, with significantly decreased ratios of reduced (GSH) to oxidized (GSSG) glutathione, suggesting that there is an environment that is conducive for cysteine modification with oxidized glutathione. We found that virus–induced oxidative stress resulted in the presentation of both native and S–glutathionylated forms of the JHMV epitope S510 by infected cells. A subset of the S510–specific CD8 T cells failed to recognize the modified form of the epitope, suggesting that GSH–modification of a cysteine–containing viral epitope might interfere with T cell recognition. Further, GSH-modified peptides were identified in stressed human cells, including herpes virus–transformed B cells, suggesting that the modification is not limited to mouse cells. Collectively these findings have implications for both anti–viral immunity and anti–tumor immunity, where oxidative stress has been shown to play a role during infection and tumorgenesis.

<p>Antibodies have the ability to influence the antibody response against the very antigen they are specific for, in a process called antibody feedback regulation. Depending on the nature of the antigen, the antibody response can be either enhanced or almost completely inhibited. This thesis focuses on the underlying mechanisms of antibody feedback regulation <i>in vivo</i>. </p><p>Antigen-specific IgG can inhibit the antibody response to a particulate antigen. Based on its ability to inhibit B cell activation, the inhibitory FcγRIIB (low affinity receptor for IgG) has been suggested to be involved. Here we show that although FcγRIIB is required for efficient suppression<i> in vitro, </i>it is not required <i>in vivo</i>. Therefore, even though FcγRIIB can inhibit antibody responses, other mechanisms (such as epitope masking and enhanced antigen clearance) play a more dominant role<i> in vivo</i>.</p><p>The antibody response to soluble antigen is greatly enhanced when it is introduced to the immune system in complex with antigen-specific IgG or IgE. We found that FcγRIIB attenuates the magnitude of IgG-mediated enhancement. In mice lacking FcγRIIB, IgG enhanced the antibody response much more efficiently than in normal mice.</p><p>Since B cells require CD4⁺ T cell help in order to become antibody-producing cells, we examined the CD4⁺ T cell response to immune complexes <i>in vivo</i>. Using an adoptive transfer strategy with transgenic ovalbumin (OVA)-specific CD4⁺T cells, we could show that the enhanced OVA-specific IgG response to IgG2a/OVA and IgE/OVA complexes was preceded by a potent OVA-specific CD4⁺ T cell response. IgG2a-mediated enhancement was dependent on activating Fcγ receptors, whereas IgE-mediated enhancement was dependent on CD23, the low affinity receptor for IgE. We identified CD23⁺ B cells as the responsible effector cells for IgE-mediated enhancement<i> in vivo</i>. Taken together, these results show that Fc receptor-mediated antigen presentation is a major mechanism underlying antibody feedback enhancement. </p>

<p>Antibodies, passively administered or actively produced, regulate immune responses to the antigen they recognize. This phenomenon is called antibody-mediated feedback regulation. Feedback regulation can be positive or negative, resulting in >1000-fold enhancement or >99% suppression of the specific antibody response. The outcome depends on size, structure, dose, and route of administration of the antigen as well as on class and subclass of the regulating antibody. This thesis investigates the role of T cells in antibody-mediated feedback enhancement, using both<i> in vivo</i> and <i>in vitro</i> approaches. IgE-antibodies enhance antibody responses to small soluble proteins. This effect is entirely dependent on the low-affinity receptor for IgE, CD23, and most likely depends on increased antigen presentation by CD23⁺ B cells. Strengthening this hypothesis, we show that IgE-mediated CD4⁺ T cell proliferation<i> in vitro</i> required the presence of CD19⁺ CD43^- CD23⁺ B cells. CD23 has also been shown to negatively regulate immune responses. Transgenic mice overexpressing CD23 are known to have impaired responses to antigens in alum. We here demonstrate that they are normal regarding IgE-mediated enhancement. IgG3 enhances antibody responses, and previous data suggested involvement of complement. We found that IgG3-mediated enhancement works well in mice lacking the only Fc-receptor known to bind IgG3, CD64. Although IgG3 could enhance antibody responses it had no major effect on T cell responses. Complement-receptors 1/2 (CR1/2) are required for the initiation of normal antibody responses. Although mice lacking CR1/2 had impaired antibody responses after immunization with sheep erythrocytes, their specific T cell responses were unaffected. The presented data do not support the idea that increased complement-mediated antigen presentation is a major mechanism behind the involvement of complement in antibody responses. They support the hypothesis that antigens forming complement-containing immune complexes may activate specific B cells by co-crosslinking BCR and CR1/2.</p>