Holdermann, Sebastian. Antibody mediated autoimmunity: characterizing the pathomechanism of autoantibodies derived from myasthenia gravis patients and healthy donors. 2024, Doctoral Thesis, University of Basel, Faculty of Science.
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Abstract
Background and rationale: Myasthenia gravis (MG) represents a prototypic autoimmune disease in which hypermutated, autoreactive B cells produce antibodies against proteins located at the post-synaptic membrane of the neuromuscular junction. Despite decades of prolific research that determined the acetylcholine receptor (AChR) as most prevalent autoantibody target and described several processes that mediate the clinical symptoms, little is known about the fundamental pathomechanisms of AChR-specific autoantibodies and the B cells producing them. We established a method to isolate B cells from the peripheral blood of patients and healthy donors that recognize a specific membrane-antigen, which provided the exceptional opportunity to characterize AChR-specific, monoclonal antibodies and B cells.
Results: We produced six monoclonal MG patient-derived IgG antibodies that bound specifically to the human AChR, of which none exhibited pathogenic properties when tested in vitro and in vivo. However, combinations of autoantibodies with distinct AChR-subunit specificities generated an increased complement activation even if the individual antibodies were incapable of activating complement. Similarly, an antibody combination with synergistic subunit-specificities proved pathogenic when tested in live rats. With a subsequent animal study, which included the pathogenic antibody combination and additionally a C5 complement inhibitor we demonstrated the causal relationship between increased complement activation and myasthenic symptoms. To explain the observation of enhanced activation by antibody combinations we constructed three different hypotheses: the density model, the antigen array model, and the hexamer model. All three competing hypotheses postulated distinct structural patterns for the antigen-antibody interaction. The opposing predictions for complement activation depending on specified antibody characteristics allowed us to experimentally test the three models. We used anti-AChR IgG antibodies with different properties and even included somatically hypermutated, AChR-specific IgM isolated from MG patients and healthy donors. At present, the results of the antibodies tested individually and in combination suggest that the antigen array hypothesis is the most accurate. Further evaluation of the AChR-targeting IgM antibodies revealed that neither the relative binding strength nor the number of hypermutations had a positive impact on complement activation and a class switch to IgG caused a loss of binding capacity.
Conclusion: The antibody repertoire is a determining factor for the pathology in MG, as single, seemingly non-pathogenic antibodies can induce disease symptoms if included in a combination with synergistic AChR-subunit specificities. Even antibodies incapable of complement activation due to an inept IgG subclass can participate in complement-driven pathogenicity by cross-linking receptors and stabilizing an advantageous antigen arrangement. A hexameric arrangement according to the prevalent model might not be necessary nor sufficient to activate the complement cascade, as indicated by IgM antibodies with strong specific binding to the AChR but without the ability to activate the complement system. These hypermutated IgM B cells that lose their binding when switched to IgG do not constitute a pathogenic precursor of the autoimmune disease. Finally, the population of somatically hypermutated, AChR-specific IgM B cells encountered in MG patients and healthy donors alike, probably reflect a regulatory B cell phenotype.
Results: We produced six monoclonal MG patient-derived IgG antibodies that bound specifically to the human AChR, of which none exhibited pathogenic properties when tested in vitro and in vivo. However, combinations of autoantibodies with distinct AChR-subunit specificities generated an increased complement activation even if the individual antibodies were incapable of activating complement. Similarly, an antibody combination with synergistic subunit-specificities proved pathogenic when tested in live rats. With a subsequent animal study, which included the pathogenic antibody combination and additionally a C5 complement inhibitor we demonstrated the causal relationship between increased complement activation and myasthenic symptoms. To explain the observation of enhanced activation by antibody combinations we constructed three different hypotheses: the density model, the antigen array model, and the hexamer model. All three competing hypotheses postulated distinct structural patterns for the antigen-antibody interaction. The opposing predictions for complement activation depending on specified antibody characteristics allowed us to experimentally test the three models. We used anti-AChR IgG antibodies with different properties and even included somatically hypermutated, AChR-specific IgM isolated from MG patients and healthy donors. At present, the results of the antibodies tested individually and in combination suggest that the antigen array hypothesis is the most accurate. Further evaluation of the AChR-targeting IgM antibodies revealed that neither the relative binding strength nor the number of hypermutations had a positive impact on complement activation and a class switch to IgG caused a loss of binding capacity.
Conclusion: The antibody repertoire is a determining factor for the pathology in MG, as single, seemingly non-pathogenic antibodies can induce disease symptoms if included in a combination with synergistic AChR-subunit specificities. Even antibodies incapable of complement activation due to an inept IgG subclass can participate in complement-driven pathogenicity by cross-linking receptors and stabilizing an advantageous antigen arrangement. A hexameric arrangement according to the prevalent model might not be necessary nor sufficient to activate the complement cascade, as indicated by IgM antibodies with strong specific binding to the AChR but without the ability to activate the complement system. These hypermutated IgM B cells that lose their binding when switched to IgG do not constitute a pathogenic precursor of the autoimmune disease. Finally, the population of somatically hypermutated, AChR-specific IgM B cells encountered in MG patients and healthy donors alike, probably reflect a regulatory B cell phenotype.
Advisors: | Derfuss, Tobias Johannes |
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Committee Members: | Rüegg, Markus A. and Schreiner, Bettina |
Faculties and Departments: | 03 Faculty of Medicine > Bereich Medizinische Fächer (Klinik) > Neurologie > Molekulare Neuroimmunologie (Derfuss) 03 Faculty of Medicine > Departement Klinische Forschung > Bereich Medizinische Fächer (Klinik) > Neurologie > Molekulare Neuroimmunologie (Derfuss) 05 Faculty of Science |
UniBasel Contributors: | Derfuss, Tobias Johannes and Rüegg, Markus A. |
Item Type: | Thesis |
Thesis Subtype: | Doctoral Thesis |
Thesis no: | 15457 |
Thesis status: | Complete |
Number of Pages: | 109 |
Language: | English |
Identification Number: |
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edoc DOI: | |
Last Modified: | 14 Sep 2024 04:30 |
Deposited On: | 13 Sep 2024 14:29 |
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