Deep Sequencing of BCR Heavy Chain Repertoires in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, 2025, Ryback et al

Nightsong

Nightsong

Senior Member (Voting Rights)
Now published: see here

Abstract:
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a common and debilitating chronic illness of unknown aetiology. Chronic infection and autoimmune responses have been proposed as two mechanisms that potentially underlie the pathogenesis of ME/CFS. To explore these disease hypotheses, we characterised the antigen-specific receptors of B cells using adaptive immune receptor repertoire sequencing.

We compared the B cell receptor (BCR) repertoires of 25 patients with mild-moderate ME/CFS, 36 patients with severe ME/CFS, 21 healthy controls and 28 patients with Multiple Sclerosis (MS) to identify signatures of infection or autoimmune responses. ME/CFS patients did not display increased clonality or differential somatic hypermutation compared to healthy controls and patients with MS.

One of two Immunoglobulin Heavy Variable (IGHV) genes, IGHV3-30, reported to be increased in ME/CFS patients in a previous study, was replicated in patients with mild/moderate disease in our cohort. However, there was no evidence of ongoing adaptive responses in IGHV3-30 repertoires from mild-moderate ME/CFS patients with increased IGHV3-30 usage. There were no detectable repertoire signatures associated with infection or autoimmunity in repertoires from ME/CFS patients, but we observed skewing of the ratio of IgM to IgG BCRs in patients with mild/moderate ME/CFS, a preliminary finding that presents an opportunity for follow-up work.

Link (Front. Immunol., abstract only ahead of publication)
 
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Nice to see this out.

The finding of skewing to usage of a particular antibody heavy chain variable region gene may seem obscure but it is the sort of finding that I think makes sense. As a replication it is really intriguing.

This sort of skewing suggests to me that the immune system is being 'leant on' by some general regulatory signal rather than some specific antibody response to a particular microbe. They did not find any indication of specific responses it seems. Regulatory signals could be cytokines or complement pathways.
 
Yann04 said:
From what I understand, and this is confusing to me, this refers to a specific part of an antibody that is different across different antibodies to adapt to fighting different antigens.
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To give an analogy: say antibodies are a bit like Yale style keys and foreign antigens like locks.

When a B cell chooses to make a specific antibody species it picks from a range of options for which genes it splices together for making antibody variable regions - in this case the variable bit of the heavy chain. VH3-30 is one option. You can think of it as a bit like choosing an Ingersoll key rather than a Yale or a dozen other types. In the key case that determines how many grooves there are along the key and which type of lock the key will slot in to. It does not determine whether it will open the door of number 45 or number 57 house.

So we cannot actually say anything about what antigen any of these antibodies using VH3-30 bind to, just that they were made in a particular factory (if you like).

The antibody situation isn't quite as simple as this because the VH gene chosen does contribute to the binding - there isn't a clear separation between grooves and teeth like in keys. But in practice this is roughly the significance.
 
@Jonathan Edwards Just trying to make sure I understand what the abstract is saying. There were two different, but potentially related, findings. In ME/CFS, antibodies were more often made with the IGHV3-30 gene. And there was a higher ratio of IgG vs IgM antibodies compared to controls.

Do you know how these findings could potentially be related?

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Looking for the study they say they replicated. Maybe one of these:

Plasma proteomic profiling suggests an association between antigen driven clonal B cell expansion and ME/CFS, 2020, Lipkin et al
IGHV3-23/30 and immunoglobulin kappa variable region 3–11 were significantly associated with ME/CFS without sr-IBS.
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Skewing of the B cell receptor repertoire in myalgic encephalomyelitis/chronic fatigue syndrome, 2021, Sato et al
By applying a next-generation sequencing to determine the clone-based IGHV/IGHD/IGHJ repertoires, we revealed a biased usage of several IGHV genes in peripheral blood B cells from ME/CFS patients. Results of receiver operating characteristic (ROC) analysis further indicated a possibility of distinguishing patients from healthy controls, based on the skewed B cell repertoire. Meanwhile, B cell clones using IGHV3-30 and IGHV3-30-3 genes were more frequent in patients with an obvious infection-related episode at onset, and correlated to expression levels of interferon response genes in plasmablasts. Collectively, these results imply that B cell responses in ME/CFS are directed against an infectious agents or priming antigens induced before disease onset.
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forestglip said:
Do you know how these findings could potentially be related?
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Not specifically. Jo Cambridge might know more but we may need someone with detailed knowledge of VH3-30 regulation - which is a niche topic. In broad terms I think it could easily relate to signals that regulate the way B cell clones are expanded and antibodies modified as an immune response matures.

For instance, IgM is made first and tends to be 'rough and ready'. IgG takes longer and is more specific and focused on an antigen. We also know that some VH genes are also used early more than later in an immune response. For instance VH4-34 carrying antibodies are quite often made but almost never used for producing fine-tuned antibodies through affinity maturation. Complement is relevant here because it is essential to the way B cells talk to each other and swap bits of antigen to encourage each other to make better and better antibodies.

Most VH gene usage is probably fairly random and evenly represented but there are a few of these genes, like VH4-34 that seem to be a bit rogue, as if they have some special function in early responses. VH4-34 antibodies naturally bind to some red cell antigens and that might be of some value in picking up antigens early, for other genes to be used later.
 
forestglip said:
Looking for the study they say they replicated. Maybe one of these:
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I was about to ask the same thing. So this paper is the second replication of the IGHV3-30 finding, with different methods being used.

This is a slide from an Ian Lipkin presentation regarding his teams work
https://neuroscienceblueprint.nih.gov/sites/default/files/documents/LIPKIN_508C.pdf
upload_2025-1-15_10-45-16.png

This was posted as a May 2021 article on the Sato et al work.
https://www.ncnp.go.jp/topics/docs/NCNP0520press.pdf
In particular, the BCR family with IGHV3-30 (and closely related 3-30-3) was significantly increased in patients who developed ME/CFS after infection-like episodes. CDR3 (complementarity determining
regions 3), is the most important site for binding to an antigen. They found that a specific length of CDR3 of IGHV3-30 (3-30-3) was particularly increased in patients with especially abundant IGHV3-30,
suggesting strong selective process by specific antigens.
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Previous reports indicate that IGHV3-30 (and 3-30-3) are B cell receptors that are induced by influenza virus, malaria, and COVID-19 infections. Since various pathogens are thought to react with B
cells having this B cell receptor, the antigen is not necessarily derived from the pathogen, but may be derived from autologous or symbiotic microorganisms such as gut commensal bacteria.
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I'm thinking the IgG/IgM ratio is probably not that important. These are very cheap tests so I would think this would be reported previously if it's a real effect, but I don't see anything from a quick search of the literature. Might just be something like how recently people in this specific study had an infection.

The IGHV3-30 seems interesting since it's now been high in three studies.

Edit: In regards to IgG and IgM: from the Lipkin paper:
Guenther et al (2015) [56] found a decrease in IgG3 and 4, but an increase in IgG2 and IgM. We do not see any overall changes to Ig isotypes. Although ME/CFS patients with sr-IBS had decreased IgG1 (aOR = 0.361, p-value = 0.037) and increased IGHA2 (aOR = 2.852, p-value = 0.046) levels, these findings were not significant after adjustment for multiple testing.
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Thanks @ME/CFS Skeptic, for getting the citations up.

I think it is interesting that the one thing that replicates is the VH3-30 weighting. Audrey and Jo C and I chatted about these data last Wednesday before the seminar at UCL. I think it is important that Audrey has not been tempted to trot out a story about an antigen drive. She didn't find evidence and I think the phenomenon is very likely not related to a specific antigen drive. The potential interest I see as much greater if not.

Audrey is also stickler for things like statistical power and not afraid to produce negative data. These are still only hints but I am very encouraged that we are beginning to see serious science panning out.
 
Wrote a short section about this in our 2021 review of ME/CFS studies:
We start off with an interesting study from Japan that looked at B cells, the white blood cells that produce antibodies. Dr. Wakiro Sato and colleagues from the National Center of Neurology and Psychiatry were interested in the receptors on B cells. In several immune-mediated diseases, the repertoire of these B cell receptors is skewed. The researchers wanted to test if this was also true in ME/CFS.

Using next-generation sequencing, Sato and colleagues found clues that this might indeed be the case. Although their initial sample size was quite small, they were able to confirm their findings in a new cohort. The abnormalities were more pronounced in patients with an infectious onset of ME/CFS.

Interestingly, the Japanese findings confirmed those of a study from last year by the research team of Prof. Ian Lipkin at Columbia University despite using different methods. Lipkin’s team reported a “significant association between ME/CFS and the immunoglobulin heavy variable (IGHV) region 3-23/30” using mass spectrometry. Sato and colleagues found that the same IGHV region was significantly increased in ME/CFS patients in both cohorts using DNA sequencing. During an online conference, Lipkin said that the Japanese researchers had independently confirmed their findings.

Another top scientist who’s enthusiastic about the B cell findings is NIH researcher Dr. Avindra Nath. During a fascinating discussion organized by the Japanese ME/CFS Association, Nath showed his admiration of Sato’s research, stating: “Your findings are very remarkable and I think you are on the right track. B-cell abnormalities make a lot of sense.”
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Jonathan Edwards said:
So we cannot actually say anything about what antigen any of these antibodies using VH3-30 bind to, just that they were made in a particular factory (if you like).
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Any ideas at what this may point to? If it is not specific antigen driven expansion, what else might be causing this? And why would B-cell abnormalities make sense, as Nath said, if it is likely not related to a specific antigen drive?
 
We compared the B cell receptor (BCR) repertoires of 25 patients with mild-moderate ME/CFS, 36 patients with severe ME/CFS
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One of two Immunoglobulin Heavy Variable (IGHV) genes, IGHV3-30, reported to be increased in ME/CFS patients in a previous study, was replicated in patients with mild/moderate disease in our cohort
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Does this imply the finding was not replicated in severe people?
 
ME/CFS Skeptic said:
Any ideas at what this may point to? If it is not specific antigen driven expansion, what else might be causing this?
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I think it may point to some sort of 'gear-changing' in immune activity. It might fit loosely with ideas like a 'hibernation response' or a 'batten down the hatches' response that normally follows an acute response to infection and doesn't get switched off. Or maybe getting stuck in first gear with an early phase response.

In Crohn's, Reiter's and Psoriasis there doesn't seem to be much evidence of a specific antigen driven response but more a sort of general 'hyper-vigilance response that might be similar to the effect of adjuvants in lots of animal models. Almost like a crush at a football crowd when people trying to leave, beyond a certain point tripping over each other for no good reason other than all moving at once.

An awful lot of the symptomatology of infections is due to non-specific signals that proclaim various phases of defence response. Those signals are likely to do lots of different things ('pleiotropic') including shifting gears in immune cell expansions.

It's all very vague but we are looking for a framework of explanation a bit different from the stories of the past. This is an area I think might be fertile.
 
Just posting the main results here:

The Japanese used next-generation sequencing (NGS) and got the following results:
upload_2025-1-15_21-54-29.png

They also found that it was related to an infectious-onset and shorter illness duration (perhaps that explains why Ryback only seem to find it in the mild/moderate but not severe group, perhaps the latter were ill for a longer time?)
upload_2025-1-15_21-54-33.png

The Lipkin group had 39 ME/CFS patients and 41 healthy controls and used liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). They use the term IGHV3-23/30 because their assay could not distinguish between IGHV3-23 and IGHV3-30. They do not plot the data but report the results of a logistic regression:
increasing the protein levels from 51,000 to 100,000 was associated with an increased risk of ME/CFS with an aOR of 4.44 (95% CI: 1.29–15.29, p-value = 0.018), while decreasing the protein level from 51,000 to 25,000 yielded an aOR of 5.65 (95% CI: 1.18–27.04, pvalue = 0.030)
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Later they also note:
Whilst 12 patients had extremely high levels (>100,000) of IGHV3-23/30, only 3 patients had extremely low levels (<25,000) of this protein
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Paper said:
There were no detectable repertoire signatures associated with infection or autoimmunity in repertoires from ME/CFS patients
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So is there like a library of antibody signatures for different pathogens and self-antigens, and the antibodies here didn't match up with anything in the library? Is this the main reason you think it's not likely to be antigen-driven, @Jonathan Edwards?
 
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