In autoimmune diseases such as rheumatoid arthritis (RA), self-reactive antibodies are present at high levels, which contributes to disease pathogenesis. The antibodies mediate their effect predominantly by binding to Fc gamma receptors (FcγR) on various leukocytes, such as monocytes, macrophages and mast cells, where FcγR ligation leads to cell activation. In this thesis the role of FcγR in RA was investigated. We could, for the first time, demonstrate an increased expression of the inhibitory FcγRIIb in RA synovial tissue, while this receptor as well as FcγRI were almost absent in healthy synovial tissue. The enhanced FcγRI expression in RA synovia was reduced by one intraarticular injection of glucocorticoids, indicating that FcγRI participates in the joint inflammation. Interestingly, RA patients with an ongoing joint inflammation exhibited blood monocytes with immune compromised features, such as decreased FcγR binding of IgG1-IC and reduced TNF production. These effects were associated with high levels of auto-antibodies in the patients, implying that the monocyte FcγR are saturated with IgG. In order to investigate whether soluble FcγR could be used as a therapy in arthritis, we injected human soluble FcγR into mice with collagen-induced arthritis (CIA). The soluble FcγR reduced the levels of pathogenic IgG anti-collagen type II (CII) antibodies, arthritis severity and pro-inflammatory cytokines. Thus, suggesting that soluble FcγR may represent a novel therapeutic agent in RA. We also studied the disease-aggravating role of mast cells in arthritis by investigating mouse mast cell protease-4 (mMCP-4) in CIA. We found that mMCP-4 deficient mice displayed a reduced IgG anti-CII response and reduced arthritis severity. This indicates a role for mMCP-4 in adaptive immunity. In conclusion, these data demonstrate that IgG occupancy of FcγR and mast cell secretion of mMCP-4 play vital roles in the development of autoimmune arthritis.

Les antigènes exprimés sur les cellules cancéreuses peuvent être ciblées par des anticorps monoclonaux thérapeutiques (mAbs) capables, d'induire une réduction de la masse tumorale. Dans la plupart des cas, la fixation des mAbs à la surface des cellules tumorales induit, par la portion Fc, le recrutement de cellules phagocytaires et cytotoxiques qui expriment des récepteurs pour la pour la portion Fc des IgG (RFcy). Chez l'homme et chez la souris plusieurs RFcy existent et ont différentes fonctions et expression à la surface des cellules hématopoïétiques. En revanche, lequel parmi les multiples RFcy est responsable pour l'activité thérapeutique des anticorps monoclonaux n'est pas connu, ni quelle population cellulaire qui les exprime est responsable de la destruction des cellules tumorales. Au cours de ma thèse j'ai développé deux modèles d'immunothérapie à base d'anticorps monoclonaux chez la souris : un modèle syngenique (Melanome B16) et un modèle xenogenique (Cancer du sein humain BT474). Dans ces modèles, l'injection de l'anticorps murin TA99 qui reconnaît la protéine gp75 exprimée par les cellules tumorales B16, d'une part, et l'injection de l'anticorps humanisé Trastuzumab qui reconnaît le récepteur HER2 exprimé par les cellules BT474, d'autre part, induit une réduction de la masse tumorale. En utilisant ces deux modèles j'ai identifié des récepteurs murins (RFcyI/RFcyIII) et humains (RFcyI/RFcyIIA) responsables de l'activité anti tumorale de ces anticorps. De plus, j'ai identifié les neutrophiles comme population cellulaire responsable de la destruction des cellules tumorales en présence des anticorps thérapeutiques.

Antibodies neutralize and eliminate pathogens, malignancies, and toxins by acting either alone or in association with Fc receptors which, once engaged, activate the elimination mechanisms of phagocytic cells. Based on structural differences, antibodies are divided into functionally distinct classes (IgM, IgD, IgG, IgE and IgA). Structure-function relationships within these classes are not well characterized. In addition, animal models for the assessment of potential therapeutic strategies for the modulation of the interaction between antibodies and Fc receptors are not established. Nonhuman primates are widely used to model human diseases and, represent excellent in vivo systems for this assessment. Therefore, we have studied nonhuman primate IgD as well as IgG and IgA specific Fc receptors in rhesus macaques, cynomolgus macaques, baboons and sooty mangabeys. IgD genes had not been identified in nonhuman primates nor the IgD receptors characterized in any species. We characterized IgD genes of the four monkey species, as well as chimpanzees and dogs. In contrast to other antibody classes, the IgD hinge regions are highly conserved between human and nonhuman primates, thus indicating a role in Fc receptor binding. In humans, Fc receptors CD16a (natural killer cells) and CD16b (neutrophils) bind IgG1 and IgG3, and CD89 (myeloid cells) binds IgA. To assess ligand binding and glycosylation properties of nonhuman primate CD16a, CD16b, and CD89, we sequenced, cloned, and generated recombinant molecules in a mammalian expression system. Our results verify the presence of CD16a, but not CD16b in nonhuman primates. CD16a is expressed on monocytes and a subpopulation of lymphocytes. In sooty mangabeys, CD16 is also expressed on neutrophils. Recombinant sooty mangabey/baboon CD16a binds to human IgG1 and IgG2, but not IgG3 and IgG4. Monkey CD89 has the same peripheral blood leukocyte expression profiles as humans, and binds human and recombinant macaque IgA. Blocking of N-glycans inhibited expression of CD89, but only marginally CD16a expression. Although extensive similarities of antibody/Fc receptor interactions exist between human and nonhuman primates, several differences must be considered when evaluating therapeutic strategies. However, these differences can be exploited to further characterize the structure-function relationships existing within antibody molecules and respective receptors.

Host defense against infection and disease relies on the reciprocal communication between the immune and neuroendocrine systems where sex hormones exert negative and positive feedback actions on immune functions. Indeed, sex hormones have been implicated in gender dimorphic immune response and in the potentiation of immune-related disorders. The female hormone estrogen plays a role as an immunomodulator and may exert immunosuppressive and immunostimulatory effects. Though many studies focus on estrogen’s role in immunity within the female reproductive tract and autoimmunity, the modulatory effects of estrogen on vaccine responses are largely unexplored. The insufficient efficacy of some vaccines in certain target populations, as for example the elderly population, is well recognized. Hormones fluctuate throughout an individual’s life, and females in particular undergo several necessary reproductive (pregnancy and menopause) and lifestyle (oral contraceptive use) changes which involve sex hormones. Vaccine efficacy might be influenced by endogenous estrogen levels or by exogenous estrogen administration. Therefore, in the pursuit of improved vaccine efficacy, it is necessary to consider such hormonal factors and their contribution to immune status. We have studied estrogen’s role in modulation of vaccine responses using a mouse ovariectomy model where exogenous estrogen delivery can be controlled. Our studies included two different types of vaccines, a bacterial toxoid formulation and a bacterial secreted protein formulation. Results from these studies indicate that estrogen enhances vaccine-specific antibody production by likely supporting a general TH2 pathway and also modulates expression of genes encoding molecules critical in innate immune signaling and required for development of proper adaptive immune responses and antigen clearance through antibody-mediated mechanisms. The level at which estrogen modulates antibody responses appears to be dependent on the route of vaccine administration. The enhancement of specific humoral responses may involve mechanisms involving TLR2 and antibody Fc receptor expression on macrophages, cells that link innate and adaptive immune responses. Advances in our understanding of the relationship between sex hormones and the immune system may provide new insights into the mechanisms by which hormones act and thus may be exploited to guide the design of future vaccine strategies.

Los receptores Fc-gamma (Fc-gR) median muchas de las funciones inmunes de la IgG. Están presentes en numerosas células del sistema inmune y su activación (tras la unión al fragmento Fc de la IgG) permite el desarrollo de funciones tales como la fagocitosis, la citotoxicidad dependiente de anticuerpos y la liberación de enzimas proteolíticas. Existen polimorfismos genéticos que modifican la afinidad de los Fc-gR y por tanto su capacidad funcional. Estos polimorfismos se han relacionado con enfermedades autoinmunes, infecciosas y con la eficacia de agentes monoclonales. El penfigoide ampolloso (PA) es una patología ampollosa autoinmune caracterizada por el deposito de autoanticuerpos en la membrana basal de la piel. Estos anticuerpos activan a neutrófilos vía el receptor Fc-gR, los cuales liberan enzimas proteolíticas que degradan la membrana basal causando el daño tisular. Los agentes monoclonales han revolucionado el tratamiento de las formas moderadas y graves de psoriasis. Sin embargo, en torno a un 30% de los pacientes no presentarán una respuesta adecuada. En la actualidad no existen factores farmacogenéticos definidos que puedan predecir la respuesta a biológicos en la psoriasis. Teniendo en cuenta el contrastado papel del los Fc-gR en modelos animales de PA, los polimorfismos genéticos de Fc-gR podrían relacionarse a nivel clínico con el PA, tanto como marcadores como modificadores de esta enfermedad. Por otra parte, los polimorfismos de Fc-gR también podrían predecir la respuesta a biológicos en la psoriasis, ya que los agentes empleados (etanercept, infliximab y adalimumab) contienen el fragmento Fc en su estructura. Esta influencia podría venir dada por un aumento en las capacidades citotóxicas de estos agentes, o por una alteración de los mecanismos de eliminación de estos fármacos dependientes del sistema reticuloendotelial. El objetivo de esta tesis fue estudiar la influencia de los polimorfismos de Fc-gR en 2 patologías cutáneas inmunomediadas mediante el estudio genético de grupos de pacientes y controles. Por un lado, se pretendía detectar si los polimorfismos de Fc-gR se asocian al PA o si modifican el pronostico de esta enfermedad, y por otro, si dichos polimorfismos son marcadores farmacogenéticos de respuesta clínica en aquellos pacientes psoriásicos tratados con agentes biológicos. Se realizó estudio genético de los polimorfismos Fc-gRIIA-H131R, Fc-gRIIB-I187T y Fc-gRIIIA-V158F en un grupo de 41 pacientes con PA y un grupo control de 115 individuos sanos y de Fc-gRIIA-H131R y Fc-gRIIIA-V158F en un grupo de 70 pacientes con psoriasis tratados agentes anti-TNF-alfa. Se realizó un análisis retrospectivo de los datos clínicos de los pacientes con PA y una valoración clínica retrospectiva de la respuesta a biológicos en pacientes con psoriasis. En el caso del PA observamos, en un modelo multivariante, una tendencia a la asociación del alelo F de Fc-gRIIIA-V158F con las formas más graves de PA, definidas como la necesidad de añadir tratamiento inmunosupresor. Por otra parte, los pacientes psoriáticos con alelos de alta afinidad (aislados o en combinación) de los polimorfismos a estudio presentaron, de forma independiente, una respuesta terapéutica más rápida a terapia biológica en forma de un porcentaje de superficie corporal afecta menor a las 6-8 semanas de tratamiento, probablemente en relación con una mayor eliminación de células patogénicas con TNF-alfa en membrana. Nuestros resultados tienen implicación clínica ya que, por un lado, los pacientes con PA y presencia del alelo F, podría beneficiarse de la introducción temprana de inmunosupresores y, por otro, la presencia de alelos de alta afinidad en los polimorfismos de Fc-gR podría predecir una respuesta clínica mas precoz en pacientes con psoriasis tratados con biológicos, lo cual puede ser de especial importancia en casos graves. / Fc gamma receptors (Fc-gammaR) mediate most of the immune functions of IgG. There are single-nucleotide polimorphims affecting Fc-gammaR genes that influence affinity and thus functions of Fc-gammaR. These polymorphisms have been linked clinically with infectious and autoimmune disease but also with the degree of response to monoclonal antibodies containing the Fc fragment. Bullous pemphigoid (BP) is an autoimmune disease where pathogenic antibodies bind antigens in the basal membrane of the skin. Such antibodies activate neutrophils through Fc-gammaR, which make these cells liberate proteolytic enzymes that cause tissue injury and blisters in the skin. Monoclonal agents have improved greatly the outcome of patients with psoriasis. However up to 30% does not achieve a significant response. There are not currently pharmacogenetic markers that could predict the outcome of biological therapy in psoriasis. The main objective of this thesis was to study the influence of Fc-gammaR polymorphisms in 2 immune-mediated skin diseases: a) a possible influence, in terms of susceptibility or disease modification, of Fc-gammaR polymorphisms in BP; and b) the potential role of Fc-gammaR polymorphisms as pharmacogenetic markers in patients with psoriasis treated with anti-TNF-alpha therapy. We performed the determination of the genotype of Fc-gammaRIIA-H131R, Fc-gammaRIIB-I187T and Fc-gammaRIIIA-V158F in 41 patients with BP and 115 controls and Fc-gammaRIIA-H131R and Fc-gammaRIIIA-V158F genotypes in 70 patients with psoriasis that underwent anti-TNF-alpha treatment. Clinical charts were reviewed in order to establish correlations. In the BP study, we observed an association between the presence of Fc-gammaRIIIA-158F with the most severe forms of BP, defined as the need for immunosuppressants. With regard to the psoriasis study, we detected that patients with high affinity alleles (alone or in combination) presented a quick response to biologics, measured as a lower body surface area affected in the week 6-8. Our results present clinical implications, since for example, patients with BP harboring the F allele may benefit from an early introduction of immunosuppressants. On the other hand, the presence of Fc-gammaR high affinity alleles may predict an early response to biologics in psoriasis, which may be critical especially in severe cases.

Antibodies are the defining characteristic of the humoral immune response. Their functions are diverse, including direct neutralization of pathogens and recruitment of other immune molecules or cells. While most successful vaccines induce protective neutralizing antibody responses, effective vaccine-elicited neutralizing antibodies against some pathogens, including HIV, HCV, malaria, and TB, remain elusive. Thus, researchers have begun to focus on how vaccines can elicit strong non-neutralizing antibody functions, including recruitment of innate immune factors for antibody-dependent cellular cytotoxicity, complement deposition, and anti\-body-dependent phagocytosis. The antibody's constant region (Fc) mediates most effector functions through isotype and subclass selection or alteration of the structure of the Fc-attached N-glycan, which controls function with exquisite specificity. Glycan modifications are naturally induced during inflammatory conditions such as autoimmune disease and natural infection however, the specific signals that regulate Fc-glycosylation remain unknown. This dissertation sought to understand how antibody glycosylation is regulated and how it can be programmed through vaccination. To do this, we first developed a technique to analyze antibody glycan structures both of bulk Fc and antigen-specific antibodies. Using this technique, we observed significant modulation of antibody glycans during viral infection as well as in vaccine-elicited antibodies. To identify specific signals important for altering the antibody glycan, we transcriptionally profiled stimulated B cells and identified a set of innate and adaptive stimuli that regulate the genes responsible for antibody glycosylation. The results described in this dissertation begin to define the specific mechanism(s) by which infection and vaccination modulate antibody glycosylation to elicit functional antibodies that can ultimately provide effective and sustained protection from infection.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

Fcγ receptors (FcγR) which recognize the Fc fraction of IgG play key roles in the modulation of a range of cellular responses as part of the host defense against foreign microbes and antigens. An important function of FcγR is to mediate internalization of soluble IgG-containing immune complexes via endocytosis. The mechanisms of internalization and intracellular transport of FcγR after internalization are less clear. In this thesis, I investigated the trafficking behaviours of human FcγRIIA and FcγRIIB2 upon clustering with immune complexes. In Chapter 3, I demonstrate FcγRIIA, when engaged with multivalent heat aggregated IgG (agIgG), is delivered along with its ligand to lysosomal compartments for degradation, whereas FcγRIIB2 becomes dissociated from the ligand and routed separately into a recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase (SFK) activity or receptor ubiquitylation. Upon co-engagement, these two receptors are sorted independently to distinct final fates after dissociating from their co-clustering ligand. In Chapter 4, I show that while the ubiquitin-conjugating system is required for FcγRIIA-mediated endocytosis, it is not required for FcγRIIB2 endocytosis. FcγRIIB2 internalizes immune complexes at a faster rate than FcγRIIA and accelerates the endocytosis of FcγRIIA upon receptor co-engagement. Taken together, these results reveal fundamental differences in the trafficking behaviour of FcγRIIA and FcγRIIB2 both during the initial induction of endocytosis as well as during subsequent intracellular sorting.

Fcγ receptors (FcγR) which recognize the Fc fraction of IgG play key roles in the modulation of a range of cellular responses as part of the host defense against foreign microbes and antigens. An important function of FcγR is to mediate internalization of soluble IgG-containing immune complexes via endocytosis. The mechanisms of internalization and intracellular transport of FcγR after internalization are less clear. In this thesis, I investigated the trafficking behaviours of human FcγRIIA and FcγRIIB2 upon clustering with immune complexes. In Chapter 3, I demonstrate FcγRIIA, when engaged with multivalent heat aggregated IgG (agIgG), is delivered along with its ligand to lysosomal compartments for degradation, whereas FcγRIIB2 becomes dissociated from the ligand and routed separately into a recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase (SFK) activity or receptor ubiquitylation. Upon co-engagement, these two receptors are sorted independently to distinct final fates after dissociating from their co-clustering ligand. In Chapter 4, I show that while the ubiquitin-conjugating system is required for FcγRIIA-mediated endocytosis, it is not required for FcγRIIB2 endocytosis. FcγRIIB2 internalizes immune complexes at a faster rate than FcγRIIA and accelerates the endocytosis of FcγRIIA upon receptor co-engagement. Taken together, these results reveal fundamental differences in the trafficking behaviour of FcγRIIA and FcγRIIB2 both during the initial induction of endocytosis as well as during subsequent intracellular sorting.