Sequencing B cell receptor repertoires in human disease: applications in myalgic encephalomyelitis/chronic fatigue syndrome and in experimental malaria infection

Abstract

The human adaptive immune system has the capacity to respond to any potential pathogen, to fine-tune the specificity of this response upon encountering an antigen, and commit the effective B or T cells to immune memory. This specificity relies on selecting antigenbinders from a vastly diverse pool of B cell receptors (BCRs) produced by VDJ gene segment recombination and junctional diversification during B cell development, and affinity maturation upon encounter with a cognate antigen. Adaptive Immune Receptor Repertoire sequencing (AIRRseq) enables us to characterise features of B cell populations by sequencing BCRs. In this thesis AIRRseq was used to investigate properties of the human BCR repertoire in two different disease settings. We also attempted to improve on existing methods for BCR-antigen mapping, which would address a major limitation of current AIRRseq analyses.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a common chronic illness with unknown aetiology and characterised uniquely by the exacerbation of symptoms following exertion. Chronic infection and autoimmunity have been proposed as two mechanisms that potentially underlie the pathology of ME/CFS. We compared the BCR repertoires of 25 patients with mild-moderate ME, 36 patients with severe ME, 21 healthy controls and 28 patients with Multiple Sclerosis to see if we could find signatures of infection or autoimmune responses. ME patients did not display increased clonality or differential somatic hypermutation compared to healthy controls and patients with Multiple Sclerosis. One of two V genes reported to be differentially used in ME patients in a previous study, was replicated in patients with mild/moderate disease. There were no obvious differences in affinity maturation in the ME cohort, but we observed skewing of the ratio of IgM to IgG BCRs in a majority of ME patients.

The second chapter explores a cohort of seven volunteers undergoing a first and second homologous challenge with Plasmodium falciparum. The BCR repertoires of volunteers infected with malaria displayed clonal expansion and somatic hypermutation of repertoires in a primary challenge but, upon re-challenge, we did not observe any signatures of clonal expansion or recurrence of clones expanded in the first challenge. Twenty-eight days post challenge, volunteers showed a trend towards an enrichment of unmutated IgG B cell receptors in their repertoires and this signature was enhanced in the second infection. This was an unexpected finding that warrants further investigation.

Finally, we attempted optimisation of a protocol to pair native B cell receptor heavy and light chains as expression-ready scFv libraries for phage display at high throughput in a user-friendly microfluidics system. While significant progress was made with improving on existing protocols and developing the method, including making a low-cost alternative to a commercially available droplet generator to generate uniform and stable emulsions at high throughput, the full reactions to pair native heavy and light chains in single cell reactions were not achieved. The work described here provides a basis for future lab members to fully optimise the reactions and will allow the lab to interrogate the antigen specificity of sequenced BCR repertoires in future. Taken together, these three chapters explore the uses and limitations of state-of-the-art BCR repertoire sequencing, and generated and analysed two high-quality BCR repertoire datasets.