Cowan, Graeme

Gray, Mohini

Simpson, Ian

Sutherland, Catherine

2023-12-21T14:28:08Z

2023-12-21T14:28:08Z

2023-12-21

Adaptive Immune Receptor Repertoire Sequencing (AIRR-seq) uses high throughput sequencing to characterise the state and dynamics of B and T cell receptor (BCR and TCR) repertoires. In this thesis, I utilise this technique both in bulk and at the single cell level to explore adaptive immune cell populations. I also document development of a simulation tool that facilitates benchmarking of AIRR-seq analysis pipelines. Firstly, TCR repertoire sequencing was used to characterise Epstein-Barr Virus (EBV)-specific T cell products from the Scottish National Blood Transfusion Service. These cells are used to treat reactivation of latent EBV in immunosuppressed individuals, and a new manufacturing method is under development. Both next generation and current products were sequenced and found to be oligoclonal. There was substantial variation across all samples, with some being highly enriched for TCR sequences previously determined to be EBV-specific. No significant effect of manufacturing technique was found. Products targeted against SARS-CoV2, generated in the same manner, showed less restricted diversity and less enrichment for previously identified virus-specific sequences than the EBV samples. This work provides detail on these clinically important T cells, indicates that the repertoires of cells in the next generation EBV bank are not dissimilar to those currently used in treatment, and shows that virus specific products targeting different viruses have altered repertoire characteristics. In the second chapter, I establish the source of a previously identified “hypomutated” IgG signature in rheumatoid arthritis (RA) by integrating single cell whole transcriptome and BCR sequencing. I found that no individual blood B cell population was enriched for sequences with few mutations in RA. However, differential gene expression analysis revealed altered expression of a small number of genes in cells with hypomutated BCR sequences. A similar gene expression signature was observed in the RA synovium, where BCRs with low levels of mutation were also found across all identified B cell subsets. This suggests a widespread phenomenon of reduced somatic hypermutation in B cells from RA patients, rather than the presence of a single pathogenic hypomutated population that could be targeted for treatment. Finally, I developed AIRRSHIP, a flexible and fast Python package that produces synthetic human B cell receptor sequences. Work to assess the reliability of the numerous tools developed to analyse the complex data produced by AIRR-seq experiments is sorely needed. Benchmarking of this kind is dependent on the ability to produce high quality simulated datasets with known ground truth. AIRRSHIP produces such datasets by using a comprehensive set of reference data to replicate key mechanisms in the immunoglobulin recombination process. The resulting repertoires are highly similar to experimental data and can be used to determine the accuracy of tools that process BCR sequences. By enabling thorough, systematic assessment of their performance, AIRRSHIP will allow more informed decisions on tool use to be made, and for error rates to be accounted for in analysis. Overall, this thesis uses AIRR-seq to aid understanding of the adaptive immune response in differing situations and illustrates its potential to inform treatment of human disease. It also details a simulation tool that can be used to improve and advance methodologies for AIRR-seq analyses.

https://hdl.handle.net/1842/41308

http://dx.doi.org/10.7488/era/4043

en

The University of Edinburgh

Bioinformatics: Catherine Sutherland and Graeme J M Cowan, AIRRSHIP: simulating human B cell receptor repertoire sequences, Bioinformatics, Volume 39, Issue 6, June 2023, btad365, https://doi.org/10.1093/bioinformatics/btad365

2027-01-14

AIRR-seq

Repertoire sequencing

Epstein-Barr virus

T cells

clonotypes

B cells

rheumatoid arthritis

synovium

AIRRSHIP

synthetic human BCR sequences

Understanding adaptive immunity using immune receptor repertoire sequencing

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy

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