<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>

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 in vivo. 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 in vitro, it is not required in vivo. Therefore, even though FcγRIIB can inhibit antibody responses, other mechanisms (such as epitope masking and enhanced antigen clearance) play a more dominant role in vivo. 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. 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 in vivo. 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 in vivo. Taken together, these results show that Fc receptor-mediated antigen presentation is a major mechanism underlying antibody feedback enhancement.

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 &gt;1000-fold enhancement or &gt;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 in vivo and in vitro 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 in vitro 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>Circulating immune complexes play an important role in the modulation of antibody responses and in the pathogenesis of immune diseases. This thesis deals with the <i>in vivo </i>regulatory properties of antibodies and their specific Fc receptors.</p><p>The immunosuppressive function of IgG is used clinically, to prevent rhesus-negative women from becoming sensitized to rhesus-positive erythrocytes from the fetus. The mechanism behind this regulation is poorly understood but involvement of a receptor for IgG, FcγRII, has been suggested. It is shown in this thesis that IgG and also IgE induce immunosuppression against sheep erythrocytes to a similar extent both in mice lacking all the known Fc receptors as in wild-type animals. These findings imply that antibody-mediated suppression of humoral responses against particulate antigens is Fc-independent and that the major operating mechanism is masking of epitopes.</p><p>Immunization with soluble antigens in complex with specific IgG leads to an augmentation of antibody production. The cellular mechanism behind this control is examined here and it is found that the capture of IgG2a immune complexes by a bone marrow-derived cell expressing FcγRI (and FcγRIII) is essential. An analysis of the ability of IgG3 to mediate this regulation indicated that, in contrast, this subclass of IgG augments antibody responses independently of FcγRI (and FcγRIII). These findings suggest that distinct mechanisms mediate the enhancing effect of different subclasses of antibodies.</p><p>Finally, the contribution of FcγRIII was studied in the development of collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis in humans. It was discovered that while DBA/1 wild-type control mice frequently developed severe CIA, with high incidence, FcγRIII-deficient mice were almost completely protected, indicating a crucial role for FcγRIII in CIA.</p><p>The results presented here help to understand how immune complexes regulate immune responses <i>in vivo</i> and show that Fc receptors for IgG, if involved, could be new targets for the treatment of immune complex-related disorders.</p>

Circulating immune complexes play an important role in the modulation of antibody responses and in the pathogenesis of immune diseases. This thesis deals with the in vivo regulatory properties of antibodies and their specific Fc receptors. The immunosuppressive function of IgG is used clinically, to prevent rhesus-negative women from becoming sensitized to rhesus-positive erythrocytes from the fetus. The mechanism behind this regulation is poorly understood but involvement of a receptor for IgG, FcγRII, has been suggested. It is shown in this thesis that IgG and also IgE induce immunosuppression against sheep erythrocytes to a similar extent both in mice lacking all the known Fc receptors as in wild-type animals. These findings imply that antibody-mediated suppression of humoral responses against particulate antigens is Fc-independent and that the major operating mechanism is masking of epitopes. Immunization with soluble antigens in complex with specific IgG leads to an augmentation of antibody production. The cellular mechanism behind this control is examined here and it is found that the capture of IgG2a immune complexes by a bone marrow-derived cell expressing FcγRI (and FcγRIII) is essential. An analysis of the ability of IgG3 to mediate this regulation indicated that, in contrast, this subclass of IgG augments antibody responses independently of FcγRI (and FcγRIII). These findings suggest that distinct mechanisms mediate the enhancing effect of different subclasses of antibodies. Finally, the contribution of FcγRIII was studied in the development of collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis in humans. It was discovered that while DBA/1 wild-type control mice frequently developed severe CIA, with high incidence, FcγRIII-deficient mice were almost completely protected, indicating a crucial role for FcγRIII in CIA. The results presented here help to understand how immune complexes regulate immune responses in vivo and show that Fc receptors for IgG, if involved, could be new targets for the treatment of immune complex-related disorders.

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

Les anticorps monoclonaux ont révolutionné le traitement de nombreuses pathologies. Cependant, leur pharmacocinétique (PK) et l’influence de leur concentration sur la réponse clinique restent mal connues. Nous avons étudié les sources de variabilité interindividuelle de la PK du cetuximab, un anticorps anti- EGFR, ainsi que l’influence de l’exposition à cet anticorps sur la réponse. Nous avons validé une méthode ELISA de dosage du cetuximab. Dans un modèle murin, nous avons étudié l’absorption pulmonaire du cetuximab. Nous avons étudié la PK du cetuximab chez un patient hémodialysé. Nous avons décrit la PK du cetuximab chez des patients traités pour cancer colorectal métastatique, à l’aide d’un modèle combinant des éliminations d’ordre 0 et 1. Enfin, nous avons identifié la clairance globale du cetuximab, paramètre pouvant être estimé précocement par la concentration résiduelle à J14, comme un facteur influençant la survie sans progression des patients. Nos travaux montrent qu’une description de la PK d’un anticorps par approche compartimentale permet d’identifier les sources de variabilité et d’étudier l’impact de la PK sur la réponse clinique. / Monoclonal antibodies have profoundly modified the treatment of many diseases. However, their pharmacokinetics (PK) and the influence of their concentrations on the clinical response are poorly known. We studied the sources of the interindividual variability of PK of cetuximab, an anti-EGFR, and the influence of the exposure to this antibody on the response. We validated an ELISA technique to measure cetuximab concentrations. We studied the pulmonary absorption of cetuximab in a murine model. We studied cetuximab PK in a hemodialysed patient. In metastatic colorectal cancer patients, we described cetuximab PK with the help of a model combining zero- and first-order eliminations. Finally, we identified the global clearance of cetuximab, a parameter which can be estimated by residual concentration on day 14, as a factor influencing progression-free survival of the patients. Our work shows that the description of the PK of an antibody by compartmental approach allows to identify sources of variability and to study the impact of PK on the clinical response.

No description available.

Introdução: Os receptores Fc são proteínas de importância crucial no processo saúde-doença. São responsáveis pela ativação de mecanismos efetores e modulam a resposta imune e inflamatória. Têm papel central na patogênese de doenças autoimunes, sepse e doenças neoplásicas. O lúpus, protótipo das doenças autoimunes e a sepse, infecção grave que causa disfunção orgânica, são doenças inflamatórias nas quais o papel dos receptores Fc vêm sendo desvendados. Essas patologias têm alta morbidade e mortalidade, impondo enormes custos para sociedade. A descoberta que a E. coli se liga ao receptor CD16 (FcGamaRIII) para evadir-se do sistema imune, através da ligação com a proteína wzxe presente em sua membrana, torna esse receptor um alvo terapêutico interessante. O CD16 é um FcGamaR com ITAM que classicamente tem função ativadora e gera respostas inflamatórias ao se ligar a imunocomplexos. Porém, na sepse, a ligação direta com a E. coli induz uma ativação ITAMi, que bloqueia a produção de ROS e inibe a fagocitose e a morte desta bactéria. A manipulação dessa ativação inibitória (ITAMi), aparentemente anti-inflamatória, pode ser uma estratégia efetiva para o tratamento de doenças inflamatórias como a sepse e o lúpus. Inicialmente visamos avaliar a importância fisiológica e o papel terapêutico do peptídeo ligante do CD16, em modelo de sepse e em modelo de lúpus induzido por pristane, respectivamente. De maneira similar ao CD16, o CD89 (FcAlfaRI) é capaz de mediar uma sinalização dual, ativatória ou inibitória, que depende da forma como se dá sua ligação às imunoglobulinas e imunocomplexos. Por isso, num segundo momento, averiguamos se o FcAlfaRI (CD89) poderia se ligar à bactérias de maneira direta, na ausência de ligantes cognatos e mediar respostas pro ou anti-inflamatórias, protegendo ou não o hospedeiro. Metodologia: Camundongos C57Bl/6, WT e CD16KO com lúpus induzido por pristane, foram tratados com o peptídeo CYWGGTEGAC(IRG Bioscience,USA). A expressão gênica e protéica de diversas citocinas, assim como genes associados a assinaturas de interferon foram avaliados nos pulmões desses animais. Utilizamos também um modelo de sepse através da injeção intra-peritoneal de E. coli WT e E. coli mutante wzxe -/-, no qual avaliamos mortalidade e produção de citocinas. Realizamos experimentos in vitro com BMM e BMDC murinos, fagócitos humanos e bactérias. A expressão de CD89 e de receptores cognatos foi avaliada através de citometria de fluxo. Empregamos a citometria de fluxo com imagem para análise da fagocitose. Foram realizados também, experimentos in vivo com camundongos WT e transgênicos: CD89tg, CD89R209Ltg CD89tgCD16KO, CD16KO e PCRKO. Comparamos mortalidade, produção de citocinas, quantidade de bactérias e lesão tecidual em modelos de CLP e de pneumonia por administração nasal de S. pneumoniae. Produção de ROS pelos BMM foi avaliada por microscopia confocal e, nos PMN, por quimioluminescência. Imunoprecipitação e immunoblotting foram utilizados para avaliar recrutamento de syk e SHP-1. Utilizamos ELISA para ensaios de ligação de bactérias com CD89 e para quantificar TNF-Alfa, IL-1 e IL-6. Resultados: Camundongos injetados com a bactéria mutante wzxe-/- sobreviveram mais e produziram quantidade menor de citocinas reforçando o papel chave da proteína wzxe, no mecanismo de evasão imune da E. coli. Camundongos WT e CD16KO com lupus induzido por pristane, tratados ou não com peptídeo CYWGGTEGAC, não apresentaram diferenças na expressão gênica nem protéica de citocinas nem em genes associados a assinaturas de interferon em seus pulmões. O CD89 interage diretamente com bactérias gram-positivas e gram-negativas. A interação bactéria-CD89 em macrófagos murinos induz ativação celular, fagocitose e morte bacteriana, que são dependentes da cadeia FcRGama. Essa mesma interação protege contra a mortalidade em dois modelos de sepse (CLP e pneumonia) e é dependente da cadeia FcRGama e indepedente de PCR e IgA anti-bactéria. Conclusões: O CD16 e o CD89 são FcRs com ITAM que apresentam uma dualidade na forma de ativação através do ITAM, que em algumas situações pode ser inibitória (ITAMi). Nessa tese reforçamos o papel chave da proteína wzxe, ligante do CD16, como responsável pela evasão bacteriana da E. coli através de sinalização ITAMi. Por outro lado, falhamos em demostrar diferenças após o tratamento de camundongos com lúpus induzido por pristane usando o peptídeo CYWGGTEGAC. Acreditamos que isso tenha ocorrido devido a dose inadequada do peptídeo ou proteólise por enzimas endógenas do camundongo, assim que o peptídeo é injetado. Novas doses ou a manipulação da estrutura do peptídeo são perspectivas futuras para este projeto. Quanto ao CD89, provamos seu papel extremamente importante na imunidade inata. Esse receptor, à semelhança do CD16, foi capaz de ligar-se diretamente a bactérias, na ausência de opsoninas e ligantes cognatos. O CD89 foi protetor tanto para infecção por gram-positivo quanto por gram-negativo enquanto o CD16 foi protetor apenas em modelo de pneumonia por gram-positivo / Introduction: Fc receptors are proteins of crucial importance in the health-disease process. They are responsible for the activation of effector mechanisms and modulate the immune and inflammatory responses. They play a central role in the pathogenesis of autoimmune diseases, sepsis and neoplastic diseases. Lupus, a prototype of autoimmune diseases and sepsis, a serious infection that causes organ dysfunction, are inflammatory diseases in which the role of Fc receptors has been unraveled. These pathologies have high morbidity and mortality, imposing enormous costs for society. E. coli has been found to bind directly to the FcGammaRIII(CD16) receptor to evade the immune system. This is due to the binding to the wzxe protein present in its membrane, making this receptor an interesting therapeutic target. CD16 is an FcGammaR with ITAM that classically has an activating function and generates inflammatory responses when binding to immunocomplexes. However, in sepsis, direct binding with E. coli induces an ITAMi activation, which blocks ROS production and inhibits phagocytosis and death of this bacterium. The manipulation of this apparently anti-inflammatory inhibitory signaling (ITAMi) may be an effective strategy for the treatment of inflammatory diseases such as sepsis and lupus. Initially we aimed to evaluate the physiological importance and therapeutic role of the CD16 binding peptide, in a sepsis model and pristane-induced lupus model, respectively. Similarly to CD16, CD89 (FcAlphaRI) is capable of mediating dual, activating or inhibitory signaling, which depends on how it binds to immunoglobulins and immunocomplexes. Therefore, we assessed whether FcAlphaRI (CD89) could bind to bacteria directly, in the absence of cognate ligands and mediate pro or anti-inflammatory responses, protecting or not the host. Methods: C57Bl/6 mice, WT and CD16KO with pristane-induced lupus were treated with the peptide CYWGGTEGAC (IRG Bioscience, USA). Gene and protein expression of cytokines, as well as genes associated to interferon signatures were evaluated in the lungs of these animals. We also used a sepsis model through the intra-peritoneal injection of E. coli WT and E. coli mutant wzxe-/-, in which we evaluated mortality and production of cytokines. We performed in vitro experiments with murine BMM and BMDC, human phagocytes and bacteria. Expression of CD89 and cognate receptors was assessed by flow cytometry. Flow cytometry with imaging was employed for phagocytosis analysis. In vivo experiments were also performed on WT and transgenic mice: CD89tg, CD89R209Ltg CD89tgCD16KO, CD16KO and CRPKO. We compared the mortality, cytokine production, amount of bacteria and tissue injury in CLP and pneumonia by nasal administration of S. pneumoniae. ROS production by BMM was evaluated with confocal microscopy and, in PMN, by chemiluminescence. Immunoprecipitation and immunoblotting were used to evaluate recruitment of syk and SHP-1. We used ELISA for binding assays with CD89 and bacteria and quantification of TNF-Aphla, IL-1 and IL-6. Results: Mice injected with wzxe-/- mutant E. coli survived more and produced smaller amounts of cytokines, reinforcing the key role of the wzxe protein in the mechanism of immune evasion of E. coli. WT and CD16KO pristane induced lupus mice, treated or not with the peptide CYWGGTEGAC didn\'t show differences in gene or protein expression of cytokines nor in interferon signature genes in their lungs. The bacterial-CD89 interaction in murine macrophages induces cellular activation, phagocytosis and bacterial death, which are dependent on the FcRGamma chain. This same interaction protects against mortality in two models of sepsis (CLP and pneumonia) and is dependent on the FcRGamma chain and independent of PCR and IgA anti-bacterium. Conclusions: CD16 and CD89 are ITAM-bearing FcRs that present a duality in the form of activation through ITAM, which in some situations may be inhibitory (ITAMi). In this thesis we reinforce the key role of wzxe protein, a CD16 ligand, as responsible for the bacterial evasion of E. coli through ITAMi signaling. On the other hand, we failed to demonstrate differences after treatment of pristane-induced lupus mice using the CYWGGTEGAC peptide. We believe that this was due to inadequate dose of the peptide or proteolysis by endogenous mouse enzymes, so the peptide is injected. New doses or manipulation of the peptide structure are future prospects for this project. As to CD89, we proved the extremely important role of CD89 in innate immunity. That receptor, similarly to CD16, was able to bind directly to bacteria, in the absence of opsonins and cognate ligands. CD89 was protective for both gram-positive and gram-negative infection while the CD16 was protective only in a model of gram-positive pneumonia

Introdução: Sepse é uma síndrome complexa definida por resposta inflamatória sistêmica, de origem infecciosa e caracterizada por manifestações múltiplas que podem determinar disfunção ou falência de um ou mais órgãos ou sistemas. É a principal causa de morte em unidades de terapia intensiva em pacientes críticos e tem representado uma fonte constante de preocupação para os sistemas de saúde em todo o mundo, devido, principalmente, às taxas elevadas de morbimortalidade. O tratamento da sepse é um desafio e continua a ser uma tarefa difícil devido a inúmeros fatores interferentes. Um estudo do nosso grupo demonstrou que a Escherichia coli (E. coli) é capaz de se ligar CD16 de um modo independente de opsonina, levando a um aumento na resposta inflamatória e a inibição da sua própria fagocitose, por conseguinte, procurou-se identificar os peptídeos no proteoma da E. coli envolvidos neste cenário. Metodologia: Utilizando a metodologia de Phage Display, que consiste numa técnica de clonagem, que permite a expressão de diversas sequências de peptídeos na superfície de bacteriófagos, nós identificamos 2 peptídeos que obtiveram interação com CD16. Após a seleção dos peptídeos identificamos uma proteína de membrana de E.coli que possui alta similaridade com um de nossos peptídeos selecionados. Nós acreditamos que esta proteína de membrana possa estar envolvida no processo de evasão imune desenvolvida pela E.coli e parece ser um forte candidato como uma nova opção terapêutica para controlar infecções por E. coli. Conclusão: A identificação de proteínas capazes de induzir inibição de fagocitose, através do receptor CD16, pode ser usada como uma nova forma de tratamento da sepse, assim como explorada no tratamento de doenças autoimunes / Introduction: Sepsis is a complex syndrome defined by a systemic inflammatory response of infectious origin and characterized by multiple manifestations that can determine dysfunction/failure of one or more organs and systems. It is the leading cause of death in intensive care units and represents a major health problem around the world, mainly due to its high mortality and morbidity rates. The treatment of sepsis is challenging and remains a difficult task due to numerous interfering factors. A study from our group demonstrated that Escherichia coli (E. coli) is able to bind CD16 in an opsoninindependent manner, leading to an increase in the inflammatory response and inhibition of its own phagocytosis, therefore we sought to identify the peptides in the E. coli proteome involved in this scenario. Methods and Results: Using the Phage Display technique, which is a cloning technique that allows the expression of various peptide sequences on the surface of bacteriophages (phages) and selecting these on the basis of affinity for a target molecule, we identified two peptides that interact with CD16. Next, using bioinformatic tools, we found an E. coli membrane protein that has high similarity with one of our selected peptides. We believe this membrane protein is involved in the process of immune evasion developed by E. coli and it is a strong candidate as a new therapeutic option to control E. coli infections. Conclusion: The identification of proteins capable of inducing inhibition of phagocytosis through the CD16 receptor, can be used as a new treatment of sepsis, as well as exploited in the treatment of autoimmune diseases