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

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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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13/02/2024

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06/03/2025

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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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https://twitter.com/user/status/1767374835200438645
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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.
 
Some interesting things I didn't know before (links to S4ME threads for references):
Autoimmune disease has been reported to be significantly more prevalent in first-degree relatives of ME/CFS patients (OR=5.30; 95%CI: 1.83-15.38; p=0.001), although this finding has yet to be validated in a large cohort (Moslehi, A. Kumar, and Dzutsev 2022).
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Severe ME/CFS patients had distinct serum antibody epitope repertoires targeting Lachnospiracaea bacterial flagellins and other gut microbiome antigens (Vogl et al. 2022).
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The same HLA alleles increased risk of ME/CFS and predicted greater efficacy of cyclophosphamide:
a GWAS on 5000 individuals found both Class I and Class II HLA associations (HLA-C*07:04 and HLA-DQB1*03:03) in 10% of ME/CFS patients and estimated that these were associated with a 1.5-2 fold increased risk of developing ME/CFS (Lande et al. 2020). An open-label clinical trial of cyclophosphamide treatment in ME/CFS has demonstrated modest efficacy, particularly in twelve individuals with HLA DQB1*03:03 and/or HLA-C*07:04 who had an 83% response rate, compared to the remaining cohort (24 patients) who had a 43% response rate (Ingrid G. Rekeland et al. 2020).
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One mild/moderate ME/CFS sample was excluded because they possibly had lymphoma. I wonder if they were able to inform this person:
Additionally sample SBL146 was excluded because it was evident from the B cell counts 4×107CD19+ cells) that this sample had a B cell malignancy. Repertoires from patients with B cell malignancies are heavily skewed regarding clonality and V gene usage and would be a significant outlier in all repertoire metrics. Upon examination of the repertoire for this individual, 96% of their BCRs used IGHV4-34 (Table 2.3) and more than 90% of the repertoire was made up of a single IgG CDR3: "CARVGAHYYYYYMDVW" (Table 2.4). The patient belonged to the mild/moderate ME group and could represent a lymphoma patient mis-diagnosed with ME/CFS or an ME/CFS patient who developed B cell lymphoma in addition to their ME/CFS. [...]

It is interesting that this was observed since ME/CFS patients are reported to have a higher risk of developing non-Hodgkin’s lymphoma (Chang, Warren, and Engels 2012).
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Suggested future directions:
In future it could be valuable to identify whether IGHV3-30 gene usage is associated with a particular cell population in ME/CFS. This could be achieved by sorting B cell populations prior to repertoire sequencing. Analysing BCR repertoires from sorted B cell populations would substantially improve the power to detect and interpret potential BCR signatures, as many BCR signatures reported in infection and autoimmunity are restricted to particular B cell subsets.
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More future directions from the final paper:
Studies in which IGHV3-30 heavy chains and their cognate light chains from ME/CFS patients are expressed as recombinant antibodies and their specificity tested against panels of target antigens [51] could reveal whether these BCRs have a common antigenic target.
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Given the emerging evidence of altered serum antibody repertoires targeting microbiome antigens [56]) in ME/CFS patients, future studies should also consider sequencing IgA, as well as IgM and IgG isotypes.
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Future repertoire sequencing studies in ME/CFS where repertoires from patients and controls are sampled prior to and following vaccination could shed light on whether B-cell responses to antigenic challenge are impaired in ME/CFS.
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B-cell responses mostly take place in lymphoid tissues and sampling peripheral blood provides a very limited picture of adaptive immune responses.
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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.
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Samples were obtained from a study examining T cell receptor repertoires in ME/CFS performed by Joshua Dibble under supervision of Prof. Chris Ponting at the University of Edinburgh. The aim of the TCR work was to replicate preliminary findings presented by Dr Mark Davis regarding clonal expansion in Lyme disease, MS and ME/CFS. We established a collaboration with the Ponting lab to allow us to examine the BCRs from the same ME and MS patients and healthy controls used in their TCR study.
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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)
Nevertheless, with experimentally-determined data the method detected no statistically significant differences between TCR clonotype diversities of people with ME and others with MS or healthy control individuals, or between the TCR clonotypes of severely affected versus mildly- or moderately-affected people with ME (MEsa and MEmm).
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Utsikt said:
I have no idea what any of this means.
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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.
 
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forestglip said:
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 invades 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. So it's unclear what this means. But it's shown up two or three times, so it seems promising.
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Really appreciate this comment. Thanks. I understood far more more reading it than 15 minutes spent on search engines trying to decypher the jargon.
 
forestglip said:
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. So it's unclear what this means. But it's shown up two or three times, so it seems promising.
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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.
 
Kitty said:
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.
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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:
Very few findings have been replicated in ME/CFS and therefore the observation of increased usage of IGHV3-30 in two BCR repertoire studies and one plasma proteomics paper is noteworthy. It may reflect a common antigen exposure, or a propensity to form auto-reactive B cells.
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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):
This could reflect a shared foreign or self antigen exposure, such as EBV infectious mononucleosis, although I did not observe other signals associated with recent or ongoing infection or an active B cell response such as clonal expansion or somatic hypermutation.

IGHV3-30 gene usage was variable across the severe ME/CFS patients, but it would be valuable to repeat this analysis with the severe patients stratified by age since severe ME/CFS patients tended to belong to the younger age groups.

Mild-moderate ME/CFS patients also had a greater proportion of IgM BCRs that could reflect a more naive B cell population, consistent with findings from a previous study (A. S. Bradley, Ford, and Bansal 2013).

It would be valuable to investigate BCR repertoires in Long Covid patients who meet ME/CFS diagnostic criteria, given that the infectious trigger is known in these patients. Sorting antigen-specific B cells against SARS-CoV2 as well as characterising the BCR repertoire of their naive, memory and atypical B cell subsets could reveal whether this disease phenotype is associated with B cell dysfunction.

Furthermore, characterising EBV-specific B and T cells in terms of repertoire and, phenotype by flow cytometry, could be insightful given that EBV-infection has been proposed as a potential trigger for ME/CFS, EBV-reactivation can occur upon SARS-CoV2 infection (Gold et al. 2021; Bernal and Whitehurst 2023) and serological evidence of recent EBV reactivation has been associated with fatigue in Long Covid patients (Peluso et al. 2023).
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With so little known about the pathophysiology of ME/CFS it is difficult to speculate on the biological underpinnings of the observations made in our study.

A large-scale Genome Wide Association Study on ME/CFS due to be published in 2024 will hopefully begin to shed light on what future research priorities should be pursued in this disease.

Nonetheless, the finding that BCR repertoires are not suitable as a general diagnostic of ME/CFS is valuable.
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forestglip said:
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. So it's unclear what this means. But it's shown up two or three times, so it seems promising.
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Very nice explanation. You're an asset to this forum, thanks for all your contributions!
 
This could reflect a shared foreign or self antigen exposure
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Thanks for highlighting that, it's helped clarify the meaning a bit more!

Nonetheless, the finding that BCR repertoires are not suitable as a general diagnostic of ME/CFS is valuable.
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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.
 
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forestglip said:
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.
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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?
 
Utsikt said:
So we have higher than normal IGHV3-30 usage that isn’t caused by any of the usual suspects?
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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.

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

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