Characterization of GAD-reaktive B cells and their B-cell receptor repertoire in the periphery and central nervous system

Biljecki, Michelle (2022): Characterization of GAD-reaktive B cells and their B-cell receptor repertoire in the periphery and central nervous system. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)

Abstract

Antibodies (abs) against the intracellular cytoplasmic protein glutamic acid decarboxylase 65 (GAD65) occur in different neurological disorders. Hereby, patients with anti-GAD65 ab associated autoimmune neurological disorders (GAD-AIND) exhibit high levels of anti-GAD65 abs in the serum as well as in the cerebrospinal fluid (CSF). However, the sites of immunological dysregulation and the triggers for the production of auto-abs against GAD65 are unclear. Furthermore, investigations on the pathogenic relevance of anti-GAD65 abs and their producing cells have yielded conflicting results. In consequence, the treatment of patients with GAD-AIND remains challenging. Therefore, investigating the functional properties of GAD-reactive B cells and anti-GAD65 abs could help to gain insights into the pathophysiology of GAD-AIND and assist the development of new treatment strategies. To this end, we analyzed GAD-reactive B cells in peripheral blood of patients with GAD-AIND as well as bone marrow (bm) cells of one patient. As a next step, we generated monoclonal patient-derived abs from peripheral blood (PB) and CSF cells and characterized their reactivity to GAD in different assays. Moreover, we analyzed the auto-abs for sequence specificities, e.g., subclass distribution, and somatic hypermutations (SHMs). In the first study, we could detect anti-GAD65 ab producing cells derived from GAD-reactive B cells in the peripheral blood (PB) of fifteen patients with GAD-AIND. Compared to our healthy control group consisting of nineteen donors, cells producing anti-GAD65 abs were highly elevated. Furthermore, we could identify bm plasma cells as an additional source of anti-GAD65 abs. The frequency of GAD-reactive B cells was comparable to B cells reactive for common recall antigens (ags) e.g. tetanus toxoid. In a second study, we included six patients with GAD-AIND and were able to generate 30 monoclonal abs (mabs) with 25 mabs derived from CSF cells and 5 mabs derived from PB cells. Mabs could be generated in patients with a short disease duration at the time of sampling. Thereby, we identified 10 GAD65-reactive mabs (CSF: 6/25; PB: 4/5) with enzyme-linked-immunosorbent-assay (ELISA). Furthermore, we could demonstrate that the intrathecal anti-GAD65 response is polyclonal and that the mabs had undergone affinity maturation when being compared to non-GAD65-reactive mabs. By reverting the SHMs of three GAD-reactive mabs to their germline sequences, we could show that affinity maturation is essential for the reactivity towards GAD65. Taken together, our data characterize the B cell response in the CSF of patients with GAD-AIND, showing that the anti-GAD65 response is polyclonal and affinity maturation is necessary for the recognition of GAD65. Additionally, we could for the first time proof that anti-GAD65-ab-producing cells are present in the central nervous system. In addition, our data support the clinical observation that a B-cell depleting therapy might not be suitable for the treatment of long-standing GAD-AIND.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: GAD, Autoantibodies, B cells
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Graduate School of Systemic Neurosciences (GSN)
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 22. Juli 2022
• 1. Bericht­erstatter:in: Thaler, Franziska