Thesis Sequencing B cell receptor repertoires in human disease: applications in ME/CFS and in experimental malaria infection, 2024, Ryback

https://era.ed.ac.uk/handle/1842/41600

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

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Date
13/02/2024

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Restricted Access

Embargo end date
06/03/2025

Author
Ryback, Audrey

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The human adaptive immune systemi 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 antigen-binders 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 the 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 which potentially underlie the pathology of ME/CFS.

We compared the 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.

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https://hdl.handle.net/1842/41600

http://dx.doi.org/10.7488/era/4332
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• Biological Sciences thesis and dissertation collection

 

https://www.actionforme.org.uk/research-and-campaigns/our-research-work/our-projects-and-phds/

The first Clare Francis Research fellowship was awarded to Audrey Ryback. Audrey will be affiliated to the Genetics Centre of Excellence at the University of Edinburgh, with Professor Chris Ponting supervising the 2-year fellowship. Addressing research priorities 3 (accurate and reliable diagnostic test) and 10 (understanding the impact of ME/CFS on mitochondria) as identified by The ME/CFS Priority Setting Partnership, Audrey’s research aims to identify serum factors possibly causing changes to mitochondria in those with ME/CFS which could, in turn, be developed into a diagnostic test.

 

The embargo is finished and the full thesis can now be accessed here:
https://era.ed.ac.uk/handle/1842/41600

 

Some interesting things I didn't know before (links to S4ME threads for references):

The same HLA alleles increased risk of ME/CFS and predicted greater efficacy of cyclophosphamide:

One mild/moderate ME/CFS sample was excluded because they possibly had lymphoma. I wonder if they were able to inform this person:

Suggested future directions:

More future directions from the final paper:

One thing I wonder is about how IGHV3-30 is one of the most common V genes even in healthy people (proportion relative to other V genes shown in the figure from the study), which might mean it has some important function in everyone, and maybe this function is more needed in ME/CFS, so it seems like it might be useful to figure out the reason that it's higher in healthy people.

 

Rybak’s orher work and IGHV3-30 has been discussed here as well: https://www.s4me.info/threads/deep-...nic-fatigue-syndrome-2025-ryback-et-al.42142/

I have no idea what any of this means.

 

That paper has the same data as this thesis.

Edit: But this thesis has a couple other B cell repertoire related investigations, one in malaria.

 

So these B cells are from the same participants whose T cells were studied here, where no differences were found:

S4ME link: Comparison of T-cell Receptor Diversity of people with Myalgic Encephalomyelitis versus controls (2024, BMC Research Notes)

 

I don't really understand much (OK, any!) of this. But I do think Audrey's summation of ME/CFS is very neat indeed.

 

I can try to give you the absolute basics, as I understand them, at least. B cells have B cell receptors (BCRs) sticking out of their surface. Each B cell randomly generates a BCR from a collection of V, D, and J genes. (Each B cell has many identical BCRs.) There are a huge number of possible combinations of these genes, because we're meant to be able to make B cells that match pretty much any possible antigen that could invade the body.

When BCRs bind to an antigen (like a bacteria or virus), the B cell is activated, proliferates so that lots of matching/very similar B cells are created, and the activated B cells release antibodies which also match the structure of the BCRs from the original B cell, so they will attach to that same antigen to tag it for destruction or to interfere with its functioning.

What Ryback et al and other studies found was increased numbers of B cells in ME/CFS that use a specific V gene, IGHV3-30, out of all the possible V genes that could be used for BCRs. Sato et al speculated that it might be because there's an antigen in people with ME/CFS that BCRs/antibodies with IGHV3-30 bind to, so there would be more of these B cells to counter the antigen. But Ryback et al didn't find any other signs that would be expected if B cells were responding to an antigen (such as matching B cells proliferating). So it's unclear what this means. But it's shown up two or three times, so it seems promising.

 

Really appreciate this comment. Thanks. I understood far more more reading it than 15 minutes spent on search engines trying to decypher the jargon.

 

This was the bit I was struggling with, so thanks for that! I guess I don't need to understand anything more than that it's interesting at the very least, and possibly even promising.

 

Yeah, I mainly wanted to read the thesis to see if it included any more speculation about the implication of this finding, because I don't know what to make of it other than "interesting" either, but it basically only says:

 

The thing about common antigen exposure's intriguing, isn't it, specially when the chances are that we've been exposed to exactly the same antigens as all the people around us at the time (none of whom got ME/CFS). Yet the people who did get it tend to show this pattern.

 

Oh there's a final discussion at the end of the thesis with a bit more of where to go from here, but still basically says that it's unclear what it means (line breaks added):

 

Very nice explanation. You're an asset to this forum, thanks for all your contributions!

 

Oh thank you, that's really nice.

 

Thanks for highlighting that, it's helped clarify the meaning a bit more!

It is, and if she hadn't looked at it she wouldn't have found more evidence of that possible V gene link. It may not mean much in isolation, but it could be a bit of the background in the jigsaw picture.

 

Thank you!!

So we have higher than normal IGHV3-30 usage that isn’t caused by any of the usual suspects?

And it isn’t know if increased IGHV3-30 has any downstream consequences, and if it’s a benign consequence if something upstream?

Essentially, IGHV3-30 could have some kind of connection to some of the more unique characteristics of ME/CFS, and researchers are trying to work backwards and find out how and if it all fits together?

 

Well, she still says it could be caused by an antigen. But the other expected signs are missing, so maybe a weird response to an antigen, or maybe something yet unknown.

I haven't been able to find much else about this gene other than being increased in response to certain antigens (like malaria, rabies, influenza, SARS-CoV-2), which is the main purpose of these genes, and some associations with certains cancers (like hairy cell leukemia and chronic lymphocytic leukemia) but I'm still looking for any paper that speculates about reasons for an increase other than because of antigens.

Also, this paper about an association with Wiskott-Aldrich Syndrome that I haven't looked at yet.

Yeah, as Kitty said, another puzzle piece that might eventually fit with other puzzle pieces or might guide where to look next.