Two anti-DNA autoantibody-secreting hybridomas, derived from murine models of the disease systemic lupus erythematosus (SLE), were provided. MAb-32, an IgG1, kappa, bound both single- (ss) and double-stranded (ds) DNA while mAb F-423, an IgG3, kappa, bound only ssDNA. F-423 also reacted with RNA and poly (dA) and its binding to ssDNA was found to be greatly amplified by histones. The VH genes of both antibodies and the VK gene of mAb F-423 were isolated by PCR, cloned, and sequenced. The two VH genes were also cloned into the pSW1.Fab expression vector and bacterial clones which showed DNA-binding were isolated. Continued induction of these clones led to the loss of their DNA-binding activity, due to a recombination event within the expression vector leading to the loss of its heavy chain-encoding genes. This event, seen only in anti-DNA clones, was proposed to be due to toxicity of the anti-DNA Fab fragments, leading to selection of recombinant bacteria. A number of experiments were carried out to improve the stability of the anti-DNA vector in E.coli. E.coli XL1-Blue was found to be preferable to E.coli TG1 as host strain both in terms of its lower frequency of recombination and tighter control of expression, while maximal production of Fab fragments was shown to be achieved after only 1-3 hours of induction. Furthermore, antigen binding of antibody fragments produced at 25[Special character omitted]C was found to be considerably higher than that of fragments produced at 37[Special character omitted]C. Mixing of CDRs from the two anti-DNA autoantibodies was carried out by CDR grafting.

Dans le cadre du diagnostic biologique des maladies auto-immunes, les autoanticorpssont généralement recherchés par des méthodes qui utilisent des antigènes(ou peptides) présélectionnés. De telles techniques ont des limites qui pourraient êtrecontournées par de nouvelles approches. Dans le premier temps de ce travail, nousrapportons l'intérêt d’évaluer les réactivités d'auto-anticorps vis-à-vis de ciblesmodifiées par des processus post traductionnels, comme la citrullination dans lecadre diagnostique de la polyarthrite rhumatoïde. Cette approche ne peut toutefoispas être transposée à toutes les pathologies auto-immunes, et notamment lasclérose en plaques. Dans un deuxième temps, notre démarche s’est élargie àl’analyse globale de la réactivité des immunoglobulines G sériques dirigée contredifférents extraits d’antigènes tissulaires, notamment issus du cerveau. Malgré lagrande hétérogénéité des réponses interindividuelles, il a été possible d’observer desprofils de réactivités distinguant les sujets sains de patients souffrant de différentespathologies à composantes auto-immunes. Cette signature sérologique permetégalement de distinguer les profils auto-immuns associés à différentes maladies etmême de distinguer leurs formes cliniques. Dans le modèle expérimental développéau laboratoire, nous avons observé que des modifications de ces profils apparaissentde façon précoce et peuvent être liés à une évolution péjorative ou favorable de lamaladie auto-immune. Ces résultats ont été confirmés dans la sclérose en plaques,où l’étude menée chez des patients ne présentant qu’un syndrome clinique isolé, amontré que les profils de réactivité sérique des IgG sont déjà marqués de l’empreintede la maladie qui se révèlera cliniquement en moyenne trois ans plus tard. De façonintéressante, d'autres travaux ont montré que le répertoire des IgM pouvaitégalement être perturbé au cours d’un processus auto-immun dont la pathogénierepose sur un dysfonctionnement des lymphocytes T (syndrome APECED). Cesrésultats suggèrent que des mécanismes T dépendants mais aussi T indépendantspèsent sur la constitution et l’entretien du répertoire auto-immun pathologique.Pour identifier les cibles des réactivités discriminantes révélées par cette premièreapproche, nous avons développé une technique de caractérisation moléculairefaisant appel à la technique immunoprotéomique. Alors que les pathologieshumaines et les modèles animaux étudiés sont principalement des pathologiesspécifiques d’organe, les antigènes tissulaires qui ont été identifiés comme cibles desréactivités spécifiquement associées à ces pathologies sont des antigènesubiquitaires, et non des antigènes spécifiques d’organe. Ces résultats posent laquestion de l’implication réelle de ces cibles dans la physiopathologie des maladiesauto-immunes. Ils illustrent également l’impérative nécessité de connaître les limitesdes résultats apportés par les méthodes d’immunoprotéomique.La caractérisation de la signature sérologique d’un processus pathologique, autravers de l’analyse des perturbations globales des réactivités sériques qui lui sontassociées, offre des perspectives intéressantes tant en termes de prise en charge dupatient qu’en termes de compréhension physiopathologique des maladies autoimmunes.Elle pourrait aboutir à d’utiles débouchés thérapeutiques. Ces attentesjustifient pleinement l’investissement qui a été mis en place par notre laboratoiredans le cadre de sa validation méthodologique. / In autoimmune diseases, specific autoantibodies detected in patients’ sera areusually investigated by techniques using purified self-antigens and/or relevantpeptides from preselected targets. Such a restrictive view may be overcome by usingnew biological techniques to improve the diagnostic procedure. In a first step, weevaluated the impact of slight changes in target self-antigens related to posttranslationalmodifications, such as citrullination. In view of the weak resultsobtained, we further focused on some properties of the humoral response. Westudied the global self-reactive IgG antibody patterns against a large panel ofantigens derived from different target tissue extracts, especially brain antigens.Despite inter-individual differences, some reactivities allowed us to discriminatebetween the immune profiles of healthy individuals and those of patients. The selfreactivefootprints can also differentiate distinct autoimmune diseases and theirclinical forms. When we induced experimental autoimmune diseases, dynamicchanges occurred at the early phases with significant patterns related to pathogenicor protective events. A pathological distortion of the self-reactive antibodyrepertoire was also found in clinically isolated syndromes predictive of multiplesclerosis. Despite the predominant organ-specific symptoms in the clinical andexperimental situations studied, discriminant self-IgG reactivities mostly involvedubiquitous antigens rather than organ specific targets. Interestingly, discriminantIgM reactivities targeting both tissue-specific and ubiquitous antigens were alsospecifically observed in a T-dependent autoimmune disease (autoimmunepolyendocrinopathy syndrome), suggesting that T-cell-dependent but also T-cellindependentmechanisms might be involved in pathological changes in the selfreactiverepertoire. Although these footprints have allowed the identification ofuseful new biomarkers, their pathophysiological relevance remains to be defined.The molecular characterization of specific antigenic targets in autoimmune disease isa critical step towards understanding the pathological mechanisms and developinguseful diagnostic and therapeutic tools. In this perspective, we emphasize the needfor accurate methodological approaches. Our analysis of self-reactive footprintshighlights the potent role of complementary events related to putative dysfunctionin the innate/natural immune response in autoimmune diseases.

Natural autoantibodies are defined as self-reactive antibodies present in healthy individuals in the absence of purposeful immunization or any known antigenic stimulation. While most NAAbs are of the IgM isotype, T cell dependent isotypes such as IgG and IgA NAAbs have been reported. Natural antibodies are conserved through evolution; they have been detected in sharks and humans, the phylogenetic limits of vertebrates capable of producing antibodies. The phylogenetic conservation of NAAbs suggests that they play an important, but overlooked, role in the maintenance of physiological homeostasis of the immune system.My project involved the characterization of two NAAbs. The first part of my dissertation elucidates the production and functional characterization of an anti-T Cell Receptor NAAb, OR3. OR3 is an IgM heterohybridoma made from the peripheral blood B cells of a patient with Rheumatoid Arthritis. The results show that OR3 specifically binds to the Complementarity Determining Region 1 segment of TCRV b8 regions, is specific for a subset of T cells and blocks the antigen activation of these T cells. Importantly, OR3 does not induce apoptosis or necrosis of T cells. My results support our hypothesis that anti-TCR NAAbs are immunomodulatory and indicate that autoantibodies present in humans may have significant functional activity in the regulation of T cell responses.The second part of the dissertation describes the phenomenon of NAAbs to human delta opioid receptor. I affinity purified IgG antibodies to human DOR from intravenous immunoglobulin, a therapeutic blood product that contains purified IgG isolated from the plasma of thousands of healthy donors. The anti-DOR NAAbs bind to DOR protein, and initiate an activating signaling cascade, in a manner similar to a receptor specific agonist. Interestingly, there is a significant difference in the agonistic activity of the autoantibodies compared to the synthetic DOR agonist DPDPE. Unlike DPDPE, these autoantibodies display significant immunomodulatory activity as evinced by changes in CCR5 and CXCR4 chemokine receptor expression. The presence of functional autoantibodies in IVIG shows that they are a part of the normal healthy immune repertoire in humans. This work also presents data supporting the interconnecting network between the neuroendocrine and immune systems.

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