Antigen-specific human B cells in viral and autoimmune conditions

Background and rationale: The low frequency of the antigen specific B cells in the peripheral blood and the complexity of the epitopes they recognize are some of the features of the human immune response that make the isolation and characterization of B cells with a given specificity challenging. While studies of serum and plasma have shown that antibodies that bind to the acetylcholine receptor (AChR) are the main driver of pathogenicity in patients with AChR-myasthenia gravis (MG), little is known about the B cells that secrete these antibodies which hampers the development of more targeted therapeutics.
Results: In the first part of this thesis, we established a novel method for the isolation of B cells recognizing membrane-expressed antigens using influenza haemagglutinin (HA) as a model antigen. We demonstrated that B cells can extract HA together with its intracellular GFP-tag from the plasma membrane of HA-expressing cells and subsequently become activated as evidenced by upregulation of surface-expressed CD69. Most of the HA-specific peripheral B cells after influenza vaccination were somatically hypermutated and their CD20+CD27+CD71+CD21low phenotype matched that of antigen-experienced activated B cells.
In the second part of this thesis, we then adapted the membrane-antigen capture model to isolate B cells that recognize acetylcholine receptor from patients with myasthenia gravis and from healthy controls (HC). From 3 patient with MG, we isolated 6 IgG+ AChR-specific B cells: 3 IgG1, 1 IgG3, and 2 clonally related IgG4 B cells. We provided evidence that somatically hypermutated and clonally expanded AChR-specific IgM B cells circulate not only in patients with MG, but also in HCs. AChR-specific IgG B cells were monoreactive, whereas the IgM compartment contained both polyreactive and monoreactive clones.
Conclusion: We have developed a sensitive approach for the isolation of human B cells with a rare antigen specificity that retains conformational epitopes even of large and complex antigens such as multiprotein receptor units. We generated a diverse dataset of AChR-specific BCR sequences from MG patients and HCs that is currently unparalleled. The presence of somatically hypermutated, expanded AChR-specific IgM clones in HCs raises questions on the antigen-experience of these B cells, the pathogenicity of the expressed antibodies, and the presence of an unknown post-germinal center tolerance checkpoint.