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The body’s humoral immune response plays a larger role in the body’s defenses beyond screening for invading pathogens. Modulation of this response is also vital for tissue regeneration, drug delivery, and vaccine development. The immune system operates within a complicated feedback loop and as such, altering the strength of the immune response can be approached from an engineering perspective. While a strong initial input can direct the response to either a pro- or anti-inflammatory bias, extreme responses can be deleterious, as in the case of allergic reactions or sepsis. Therefore, the objective of this thesis was to develop a novel biomaterials platform that can be used to alter the immune response in a tunable manner. Antibodies are not only the workhorses of the adaptive immune response but are also powerful immunomodulators through their Fc (constant fragment) regions. By coating microparticles with Fc ligands in variable surface densities, we were able to utilize the sensitivity of multivalent signaling to tune the response of the immune response. Microparticle size was also varied to decouple the effects of physical versus biochemical signaling. The goal of this thesis was to analyze the effects of Fc coated particles on two major components of the humoral immune responses: macrophages and the complement system. We first looked at the mechanical response of macrophages through phagocytosis and found that both Fc density and microparticle size had significant impacts on macrophage phagocytosis. These results also provide a particle delivery “toolbox” for future applications. We then analyzed the downstream effects of Fc particles on macrophage phenotype and on phenotype plasticity. This showed that the addition of Fc particles lead to increased production of TNFα and IL-12 and inverted the response of LPS treated macrophages. Finally, we applied our particles to activate the complement system, an often overlooked cascade of serum protein activation that results in bacterial cell lysis. Cleaved components of the complement system are also powerful chemokines and can act as a vaccine adjuvant. Fc density on particles played a large role in complement system activation, both through the classical and alternative pathway, as it lead to a binary response for smaller particles and a tunable response for larger particles. We then applied these results to create a novel form of antibiotic by using Fc particles to direct complement-mediated bacterial cytotoxicity. The use of immune activation by Fc particles was also applied to better understand and improve the tuberculosis vaccine. Our findings are significant to the biomaterials and immunology fields as we showed that Fc microparticles can generally be used to alter the immune response in a tunable manner for a broad range of applications, as well answering fundamental immunology questions.

Dissertação apresentada para obtenção do grau de Mestre emMétodos Computacionais em Ciência e Engenharia, na Faculdade de Ciências da Universidade do Porto

PhD / The aims of this study were to test the efficacy of immunoglobulin and its Fab and Fc fragment in the treatment of experimental autoimmune neuritis (EAN) in Lewis rats, to investigate which portion of immunoglobulin is operative in the effect of IVIg, and to clarify the possible mechanisms by which immunoglobulin exerts its action in the treatment of rats EAN. EAN was induced by immunization with whole bovine peripheral nerve myelin. The immunized rats were randomized into groups, assessed clinically, electrophysiologically, and histologically, and intravenously injected with normal saline, albumin, human IVIg preparation, purified Fab or Fc fragments. The treatment efficacy was compared between normal saline and albumin groups, albumin and IVIg groups, albumin and Fab groups, albumin and Fc groups, Fab and Fc groups, Fab and IVIg groups, and Fc and IVIg groups. Methods of myelin isolation, antibody purification, and Western blot techniques were also applied. The results revealed that treatment with Fc fragment and IVIg at the onset of signs of disease effectively prevented further progression of disease, shortened disease duration, and facilitating recovery from illness as shown in clinical, electrophysiological and histological parameters. In the study which the efficacy of albumin and IVIg was compared, 5 out of 17 rats (29%) in the albumin group and 12 out of 17 (71%) in the IVIg group completely recovered from the clinical disease by day 30. The animals receiving IVIg treatment exhibited lower clinical scores, less prolongation of S wave latencies, better maintained S wave amplitudes, less reduction of distal motor NCVs, better maintained distal and proximal CMAP amplitudes, and lower histological grades. In the study which the efficacy of albumin, Fab fragment, Fc fragment, and IVIg was compared, 0 out of 8 (0%) in the albumin group, 1 out of 8 (13%) in the Fab group, 4 out of 8 (50%) in the Fc group, and 6 out of 9 (67%) rats in the IgG group completely recovered from the clinical disease by day 30. The animals receiving Fc fragment and IVIg treatment exhibited lower clinical scores, less prominent weight loss, less prolongation of S wave latencies, better maintained S wave amplitudes, less reduction of distal motor NCVs, better maintained distal and proximal CMAP amplitudes, and lower histological grades.

<p>IgG, IgM and IgE in complex with antigen have the capacity to regulate specific immune responses. In this investigation, the role of Fc receptors for IgG (FcγRI, FcγRII and FcγRIII) and complement receptors 1 and 2 (CR1/2) for antibody-mediated enhancement of antibody responses are investigated.</p><p>IgM is known to efficiently activate complement and thereby enhance specific antibody responses but it is not known if this involves binding to CR1/2. Using CR1/2 deficient mice, immunized with sheep erythrocytes alone or together with specific IgM, we present evidence that IgM-mediated enhancement is completely dependent on CR1/2 expression, whereas IgG or IgE in complex with bovine serum albumin (BSA) induce strong antibody responses in CR1/2-deficient mice. Enhancement by IgE is mediated via the low affinity receptor for IgE (FcεRII, CD23). However, the receptors which are involved in IgG-mediated enhancement are not known. We find that γ-chain-deficient mice (lacking FcγRI and FcγRIII) have impaired antibody responses to IgG/BSA complexes. In contrast, FcγRIII deficient mice have normal responses, suggesting that FcγRI mediates the effect. Furthermore, IgG/BSA complexes induce up to 189-fold stronger antibody responses in FcγRIIB-deficient mice than in wild-type mice. The threshold dose of IgG/BSA required was lower, the response was sustained for longer and initiated earlier in FcγRIIB-deficient than in wild-type animals. The findings suggest that FcγRIIB acts as a "safety-valve" preventing excessive antibody production during an immune response. We show for the first time that IgG3/BSA complexes can mediate enhancement of specific antibody responses. Their effect does not involve known Fcγ receptors.</p>

Der SC Motor/FC Carl Zeiss Jena war seit Ende der 50-er Jahre bis in die 80-er Jahre hinein ein vom DFV der DDR und vom DTSB immer wieder benannter und bestätigter Schwerpunktclub innerhalb der sogenannten zivilen Clubs. Der SC Turbine/FC Rot-Weiß Erfurt konnte diesen Status innerhalb des Fußballverbands dagegen nie erreichen. Die zentrale Frage dieser Dissertation nach den spezifischen Bedingungsgefügen des zivilen Schwerpunktclubs FC Carl Zeiss Jena (und Vorgänger) und des zivilen Nichtschwerpunktclubs FC Rot-Weiß Erfurt (und Vorgänger) im DDR-Fußballsystem ergab sich aus dieser unterschiedlichen Privilegierung und den ungleichen Erfolgsbilanzen dieser beiden Clubs. Die Hypothese der komparativ angelegten Fallstudie vermutete einen unmittelbaren Zusammenhang zwischen diesen deutlich sichtbaren Erfolgsunterschieden der beiden Mannschaften in der DDR und den erfolgten Schwerpunktfestlegungen. Zusätzlich konnte vermutet werden, dass ein beträchtlicher Anteil an den Jenaer Erfolgen auf die besonders starke Unterstützung des wirtschaftlich mächtigen VEB Carl Zeiss Jena zurückzuführen war. Um diesen Zusammenhängen nachzugehen, fragte der Autor nach den konkreten Bevorzugungen des Jenaer Schwerpunktclubs und den Benachteiligungen des Erfurter Nichtschwerpunktclubs und nach den spezifischen Bedingungen und Handlungsspielräumen der beiden Thüringer Mannschaften in der DDR. Daraus ergaben sich eine Reihe von detaillierten, auf einen Vergleich der verschiedenen Bedingungen in Erfurt und in Jena hin orientierte, Fragen, welche in der vorliegenden Untersuchung detailliert beantwortet werden: Wie sah die besondere Förderung des DFV bzw. des DTSB für einen Schwerpunktclub wie Jena überhaupt aus? Wer nahm Einfluss auf die Clubs, von wem waren diese abhängig, wer förderte sie durch welche Leistungen? Wie wurden diese Beschlüsse vor Ort umgesetzt? Wer waren die Trägerbetriebe und in welchem Maße und wodurch engagierten sich diese für den Fußball in Erfurt und Jena? Wie kamen die häufigen Wechsel der besten Spieler Erfurts nach Jena zustande? Warum war die Richtung dieser Wechsel insgesamt einseitig in Richtung Jena? Welche finanziellen, materiellen und sozialen Bedingungen konnten den Spielern in Jena und Erfurt geboten werden? Die vorliegenden Ergebnisse dieser erstmals für die zivilen Clubs auf der Mikroperspektive angelegten systematischen Untersuchung bestätigen das bereits von Hans Joachim Teichler als grundlegend für den DDR-Fußball beschriebene Konfliktmuster des „Fußball-Lokalpatriotismus versus Parteiräson“. Eigenmächtige Handlungen vieler Betriebsleiter und zahlreicher Partei- und Gewerkschaftsfunktionäre in den Trägerbetrieben konnten beispielsweise in Erfurt bei der eigenmächtigen Erhöhung der Aufnahmezahlen von Fußballern an die KJS Erfurt oder in Jena bei der Anstellung der Fußballer im Zeisswerk nachgewiesen werden. Das am sowjetischen Vorbild orientierte Sportsystem der DDR mit seinen engen Bindungen an die Trägerbetriebe provozierte geradezu verdeckte Zuwendungen der Betriebe, die über die Clubs an die Spieler weitergereicht wurden. Für die zentralen Instanzen des DDR-Fußballs war das ein Dauerproblem, weil sich damit ein Großteil der Vorgänge vor Ort der Steuerung entzog. Wie in der vorliegenden Arbeit beschrieben wird, war genau dies jedoch der Schlüssel für den Erfolg des SC Motor/FC Carl Zeiss Jena vom Ende der 50-er bis in den Anfang der 80-er Jahre bzw. für den vergleichsweisen Misserfolg des SC Turbine/FC Rot-Weiß Erfurt im gleichen Zeitraum. Dass letztlich die finanziellen, materiellen und sozialen Möglichkeiten die entscheidende Gründe für die Spieler waren, zu einem anderen Club oder einer BSG zu wechseln, mithin demnach Marktmechanismen, und hier in erster Linie der Grund für die Stärke des SC Motor/FC Carl Zeiss Jena zu suchen ist, ist eine zentrale Erkenntnis dieser Arbeit. / The SC Motor/FC Carl Zeiss Jena was in the late 50's to the 80's one of the DFV der DDR and of the DTSB repeatedly nominated and confirmed priority club within the so-called civilian clubs. The SC Turbine/FC Rot-Weiss Erfurt could never reach this status within the Football Association. The central question of this thesis to the specific structure of conditions of the civilian priority club FC Carl Zeiss Jena (and earlier) and of the civilian non-focal clubs FC Rot-Weiss Erfurt (and earlier) in the East German football system resulted from these different privileges, and the uneven track records of these two clubs. The hypothesis of the comparative case study to suspected a direct relationship between these highly visible success differences between the two teams in the East and made the key requirements. Additionally it was suggested that a significant proportion of the Jena successes to the particularly strong support of the economically powerful VEB Carl Zeiss Jena was due. To investigate these relationships, the author asked about the specific preferences of the Jena focal club and the disadvantages of the Erfurt non-focal clubs and on the specific conditions and scope for action of the two Thuringian teams in the GDR. This resulted in a series of detailed, based on a comparison of the different conditions in Erfurt and Jena out questions that are answered in detail in this study: What was the specific support of the DFV or the DTSB for a priority club like Jena at all from? Who took effect on the club, by whom they were dependent on who they supported by what is included? How these decisions were implemented on site? Who were the carrier companies and to what extent and how dedicated these for football in Erfurt and Jena? How did the frequent change of the best players reached Erfurt in Jena? Why was the overall direction of these changes unilaterally in the direction of Jena? What financial, material and social conditions were the players in Jena and Erfurt are offered? The present results of this first time to the civilian clubs at the micro-perspective scale systematic study to confirm the already by Hans Joachim Teichler as fundamental to the East German football described patterns of conflict of "Football local patriotism versus party argue." Unauthorized actions of many managers and many party and union officials in the support operations for example, could be detected in Erfurt at the arbitrary increase in the numbers of pictures of football players at the KJS Erfurt in Jena or to the appointing of the footballers in the Zeiss factory. The Soviet model based on the GDR sports system provoked by his close ties to the carrier companies almost hidden benefits of companies that have been passed down through the clubs to the players. For the central authorities of the East German football was a constant problem because it deprived a large part of the operations of local control. As described in the present work, exactly that was, however, the key to the success of the SC Motor/FC Carl Zeiss Jena of the late 50's until the early 80's and for the comparative failure of the SC Turbine/FC Rot-Weiss Erfurt in the same period. That were the financial, physical and social opportunities the key reason for the players, ultimately, to move to another club or BSG, consequently, therefore market mechanisms, and this is primarily the reason for the strength of the SC Motor/FC Carl Zeiss Jena looking for is is a central finding of this study.

IgG, IgM and IgE in complex with antigen have the capacity to regulate specific immune responses. In this investigation, the role of Fc receptors for IgG (FcγRI, FcγRII and FcγRIII) and complement receptors 1 and 2 (CR1/2) for antibody-mediated enhancement of antibody responses are investigated. IgM is known to efficiently activate complement and thereby enhance specific antibody responses but it is not known if this involves binding to CR1/2. Using CR1/2 deficient mice, immunized with sheep erythrocytes alone or together with specific IgM, we present evidence that IgM-mediated enhancement is completely dependent on CR1/2 expression, whereas IgG or IgE in complex with bovine serum albumin (BSA) induce strong antibody responses in CR1/2-deficient mice. Enhancement by IgE is mediated via the low affinity receptor for IgE (FcεRII, CD23). However, the receptors which are involved in IgG-mediated enhancement are not known. We find that γ-chain-deficient mice (lacking FcγRI and FcγRIII) have impaired antibody responses to IgG/BSA complexes. In contrast, FcγRIII deficient mice have normal responses, suggesting that FcγRI mediates the effect. Furthermore, IgG/BSA complexes induce up to 189-fold stronger antibody responses in FcγRIIB-deficient mice than in wild-type mice. The threshold dose of IgG/BSA required was lower, the response was sustained for longer and initiated earlier in FcγRIIB-deficient than in wild-type animals. The findings suggest that FcγRIIB acts as a "safety-valve" preventing excessive antibody production during an immune response. We show for the first time that IgG3/BSA complexes can mediate enhancement of specific antibody responses. Their effect does not involve known Fcγ receptors.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases. / May 2005

Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions. We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women.

The antibody Fc region is critical for the therapeutic potency by virtue of its role in recruiting and activating the cytotoxic pathways of immune cells, complement activation and its role in antibody homeostasis (a process mediated by the pH dependent binding to the neonatal receptor FcRn). Bacterially produced antibodies lack of glycosylation at Asn297 and therefore do not bind to the surface Fc[gamma]Rs on effector innate immune cells, nor can they activate complement. This dissertation describes the engineering of aglycosylated bacterially expressed antibodies for binding to a specific Fc[gamma]R and therefore eliciting therapeutically relevant effector functions. Aglycosylated Fc mutants that bind to desired Fc binding ligands were isolated by a new E. coli homodimeric Fc display system coupled with high throughput flow cytometry. Two amino acids mutation in the CH3 domain (Fc5) conferred selectively high binding affinity of aglycosylated Fc domains to the Fc[gamma]RI receptor. Flow cytometry screening from a randomized Fc5 library resulted in the isolation of Fc mutants exhibiting higher affinity binding to Fc[gamma]RI receptor than the Fc5. Aglycosylated Fc[gamma]RI specific IgG containing the variable regions of the clinically important anti-Her2 antibody trastuzumab elicited dendritic cell-mediated ADCC in sharp contrast to the clinical grade trastuzumab (Herceptin) or the glycosylated coutreparts of the engineered antibodies, neither of which could potentiate target cell lysis with dendritic cells as effectors. In separate studies, a system was developed for the screening of periplasmically anchored E. coli libraries and the isolation of clones expressing antibodies that are specific to insoluble antigens or to cell surface markers. Following three rounds of flow cytometric screening, spheroplasts expressing specific scFvs were enriched 950-fold from a large excess (1,000x) of spheroplasts expressing nonspecific antibodies. / text