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Simon McGrath blogs: Mark Davis finds the strongest evidence yet for ME/CFS immune activation..

Jonathan Edwards

"Gibberish"
Messages
5,256
@Simon I was very pleased to hear Mark Davis say the techniques they are using can be applied to T and B cells.

In fact I listened twice to make sure I heard him correctly! Would love to hear more about B cells, i.e.,might B cell clonal expansion occur concurrently with the T cell expansion?

I don't think this technique can be applied to B cells in practice. T cells are circulating cells by nature. B cells less so. Most B cells live in follicles in lymph nodes where they are normally present as expanded clones. Clonal expansion has been demonstrated in abnormal B cell collections in joints in RA - you can do it in a solid tissue where the cells expand locally - but you do not have any baseline figures because locally there are always expanded clonalities.

I am also still unclear whether or not this is all about clonal expansion or similar TCRs.

I have not yet found a way to access the talk, is there a link somewhere?
 

Simon

Senior Member
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3,789
Location
Monmouth, UK
. But I am still confused about what this paper is about. Clonal expansion is something quite specific about lots of T cells having identical receptors. Lots of T cells having rather similar receptors is something quite different. And lots of T cells recognising the same peptide is again quite different. I am afraid I have not had time to look at the presentation itself but I am confused.
I'd love to kow what you think after you've seen the presentation: you can watch the meat of it starting here - it's only ten minutes or so. The method as outlined in the paper I highlighted (Identifying specificity groups in the T cell receptor repertoire : Nature) is based on recognising the same peptides (they focused on antigen-receptor binding points, not the common framework), so similar in that sense, and not identical. Hope that helps.
 

Jonathan Edwards

"Gibberish"
Messages
5,256
Thanks, watched it.

I am fairly sure the ME work is looking for clonal expansion - so identical cDNA sequences retrieved from individual cells. That makes things simple because there is none of the fuzziness you would get with similar sequences or similar peptide binding. Clonal expansion would make sense to me and would be very interesting.

I was disappointed not to see any controls for the ME patient data. Normal levels are given as if they have been taken from historical literature. That suggests that this is preliminary data. It is a bit of a worry that even at this stage ME patients are not being processed blind alongside controls. Maybe they are but why no control data?

I am also a bit surprised by the MS and cancer data. As far as I know the clonal expansions in cancer occur within the tumour tissue. Clonal expansion in local tissue is a normal universal feature of a defence response. It just means that some cells have stopped there and divided. Clonal expansion in blood would be something entirely different. For MS I am unclear where the data come from. Pubmed shows a few papers indicating CD8 T cell expansion in brain lesions, but as before, this simply implies that some cells have stopped by and divided. They might be recognising degraded host protein with post-translational changes like citrullination or free radical damage so might be non-specific heathy T cells.

There are a couple of papers suggesting CD8 T cell clonal expansion in blood but it does not look like a very big effect. The interest is mostly in that the clones seem to be the same ones as in brain. Again I would wonder if benign clearing up T cells might not be expanded during an episode of tissue damage.

I would like to see more directly comparable data done blind with the sae assay system.

I also worry a bit when an immunologist says that there is clearly lots of inflammation in ME. There isn't. That is the whole problem. 'Systemic inflammation' doesn't actually exist as a concept.Inflammation is by definition a local pathological change. You can have a systemic inflammatory disease but there the systemic is an adjective that qualifies the word 'disease', not the inflammation!

Quite possibly all this stuff together with the TGF beta will fall into place but I do wish people were a bit sharper about data presentation.
 

duncan

Senior Member
Messages
2,240
I also worry a bit when an immunologist says that there is clearly lots of inflammation in ME. There isn't. That is the whole problem. 'Systemic inflammation' doesn't actually exist as a concept.Inflammation is by definition a local pathological change. You can have a systemic inflammatory disease but there the systemic is an adjective that qualifies the word 'disease', not the inflammation!

A) What about fevers?
B) Maybe the inflammation is limited to multiple tissue reservoirs or areas like the brain or joints, so then technically it would not qualify as systemic, but the effect on the patient might be comparable?
C) What if the inflammation does not register via conventional metrics? Maybe this is where employing new inflammation diagnostics - like cytokines/chemokines or specific proteins - comes into play eventually?

Sorry if this makes little sense.
 
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Jonathan Edwards

"Gibberish"
Messages
5,256
A) What about fevers?
B) Maybe the inflammation is limited to multiple tissue reservoirs or areas like the brain or joints, so then technically it would not qualify as systemic, but the effect on the patient might be comparable?
C) What if the inflammation does not register via conventional metrics? Maybe this is where employing new inflammation diagnostics - like cytokines/chemokines or specific proteins - comes into play eventually?

Sorry if this makes little sense.

There is a confusion between inflammation, which is defined as a local process of increased blood flow, vascular permeability and cell migration and various systemic events that may precede or follow inflammation. Fever goes with inflammation some times and not others.

This is important because otherwise you get muddled thinking about what causes what. And in diseases where the normal sequence of events breaks down, like rheumatoid arthritis, or even tuberculosis, completely inappropriate conclusions may be drawn.
 

Jonathan Edwards

"Gibberish"
Messages
5,256
Could you expand on this if you haven't done so already?

I thought someone might ask that. I don't have a well articulated answer. However, my impression is that in post infective syndromes like Reiter's and some forms of Psoriasis T cells in specific compartments are too busy but without any clear evidence of them being directed at any particular antigen. For CD8s that should be an antigen present within cells and being presented on the surface. Clonality is normally associated with a response to specific antigen and in some sense it probably always is. However, I can envisage a situation where there is a general disturbance of clonal regulation in a compartment that means that instead of CD8 cells settling back to all being different after each insult, as they should do, they go on being very asymmetrical in clonal size. You would then get evidence for clonal expansion but it might not matter very much which clones were expanded. A significant possible for me would be that there are certain clones that play 'housekeeping' roles that are normally thinned out after each time they are needed but in some situations go on being dominant. An example might be as I indicated before, clones that recognise peptides with post-translational modification like citrullination of arginines. That seems to be the case for CD4 T cells in RA maybe so here it would probably be something else but lots of post-translational changes occur inside cells particularly if proteins are considered junk and suitable for degradation.

Not very crisp but something like that.
 

Jonathan Edwards

"Gibberish"
Messages
5,256
As an aside, I think if we are talking about TCRs just being similar in the sense of nearly the same that does not translate well into recognising the same peptide. Two peptides with one amino acid difference are likely to come from totally unrelated proteins. TCRs bdingin the two peptides may well only differ in one amino acid similarly. There are not doubt correlations you can find in vitro but these may be artefacts of the libraries used and the detection systems. T cells need to be able to tell one amino acid difference very precisely.
 

Marco

Grrrrrrr!
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2,386
Location
Near Cognac, France
However, I can envisage a situation where there is a general disturbance of clonal regulation in a compartment that means that instead of CD8 cells settling back to all being different after each insult, as they should do, they go on being very asymmetrical in clonal size. You would then get evidence for clonal expansion but it might not matter very much which clones were expanded.

Thanks Jonathan. I've re-read that three times and I'm non the wiser. As for the quoted passage, you say you can 'envisage'a situation etc - but can you point to any examples or is this 'novel'?

Secondly why do you feel this suggestion might be a good fit for ME/CFS; is it central to the pathology and how might it explain the constellation of symptoms? (no pressure!).
 

Jonathan Edwards

"Gibberish"
Messages
5,256
Thanks Jonathan. I've re-read that three times and I'm non the wiser. As for the quoted passage, you say you can 'envisage'a situation etc - but can you point to any examples or is this 'novel'?

Secondly why do you feel this suggestion might be a good fit for ME/CFS; is it central to the pathology and how might it explain the constellation of symptoms? (no pressure!).

Said it was poorly articulated. I will have to try an remember my motivations.
 

Manganus

Senior Member
Messages
166
Location
Canary islands
Said it was poorly articulated. I will have to try an remember my motivations.

Still, even poorly articulated ideas, educated guesses, hunches and intuitions are valuable and interesting, when they are based in experience.

My position is that it might be worth waiting for this work to be published. The presentation at the conference was clearly and wisely aimed at patients/clinicians rather than towards professors emeriti or the brain trust of Phoenix Rising.
 

Murph

:)
Messages
1,799
Could be that the charts are difficult to interpret because this is pre-publication data, the details of which they want to keep vague.

Now, in the closed sessions, that's where you get to see the charts. ;)

We saw Cort in photos of the closed sessions, right? He will of course be careful not to betray what he's heard in there and only use data from the open sessions. But when we see what he emphasises and what he focuses on when does his write ups*, we may be able to infer where the real treasure was buried.

*I"m assuming there will be write ups. From everything I've observed of that guy so far, I think it's a fair assumption.
 

Simon

Senior Member
Messages
3,789
Location
Monmouth, UK
Apologies in advance for a long post, I'm catching up.

I am fairly sure the ME work is looking for clonal expansion - so identical cDNA sequences retrieved from individual cells. That makes things simple because there is none of the fuzziness you would get with similar sequences or similar peptide binding. Clonal expansion would make sense to me and would be very interesting.
I think the pie charts are based on identical TCRs from single-cell work and so are clonal. The earlier "Charlie Brown" graph is based on similar (as in predicted to bind the same peptides) TCRs, though the high level in the top cluster seen for mecfs, but not controls, still indicate clonal expansion, as far as I can tell.

As an aside, I think if we are talking about TCRs just being similar in the sense of nearly the same that does not translate well into recognising the same peptide. Two peptides with one amino acid difference are likely to come from totally unrelated proteins. TCRs bdingin the two peptides may well only differ in one amino acid similarly. There are not doubt correlations you can find in vitro but these may be artefacts of the libraries used and the detection systems. T cells need to be able to tell one amino acid difference very precisely.
Of course, and that's what makes the new approach so impressive. I promise I won't mention it again, but here's the abstract. The validation on TB argues against it being an artefact.

Identifying specificity groups in the T cell receptor repertoire : Nature)
Abstract T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual1, 2, 3, 4. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.
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More about it in a presentation here.

I'm sure Davis used this technique because Dr Joseph Breen at the NIH said it was - he'd seen Davis's presentation at an earlier (FOCIS?) conference, and presumably had seen the supplementary grant applicatiion too (see below). I checked with Breen that it was this specific paper.

Looks to me like the graph based on similar TCR sequences is the only place where this approach would apply.

I was disappointed not to see any controls for the ME patient data. Normal levels are given as if they have been taken from historical literature. That suggests that this is preliminary data. It is a bit of a worry that even at this stage ME patients are not being processed blind alongside controls. Maybe they are but why no control data?
This work was funded by the NIH as a supplementary grant, specifically aimed at researchers already doing other work who could tag a mecfs group onto an existing study. That might explain things: it literally was an afterthought.

As far as I know the clonal expansions in cancer occur within the tumour tissue. Clonal expansion in local tissue is a normal universal feature of a defence response. It just means that some cells have stopped there and divided. Clonal expansion in blood would be something entirely different.
That's actually what he showed, clonal expansion in tumour tissue vs not in adjacent tissue. Though looking again, I see that data is for CD4 cells, not CD8.

For MS I am unclear where the data come from. Pubmed shows a few papers indicating CD8 T cell expansion in brain lesions, but as before, this simply implies that some cells have stopped by and divided. They might be recognising degraded host protein with post-translational changes like citrullination or free radical damage so might be non-specific healthy T cells.

There are a couple of papers suggesting CD8 T cell clonal expansion in blood but it does not look like a very big effect. The interest is mostly in that the clones seem to be the same ones as in the brain. Again I would wonder if benign clearing up T cells might not be expanded during an episode of tissue damage.
Davis showed slides of mouse EAE with similar clonal expansion levels in blood and CNS for both CD4 and CD8. I think he may have said there, or somewhere else, they were the same clones in blood and brain, but I'm not totally sure. He suggested the mouse model data meant the same was likely to be true for humans (how do you check for humans: CSF, or are brain biopsies needed?).

Clonality is normally associated with a response to specific antigen and in some sense it probably always is. However, I can envisage a situation where there is a general disturbance of clonal regulation in a compartment that means that instead of CD8 cells settling back to all being different after each insult, as they should do, they go on being very asymmetrical in clonal size. You would then get evidence for clonal expansion but it might not matter very much which clones were expanded.
Somewhere else, possibly in the discussion, Davis does suggest it could be a non-specific T cell effect.

I think it is computationally impossible because we are talking about recognising something like decamer peptides with 20 amino acid options at each point ( peptide alternatives = 100,000,000,000,000,000,000).
Ah, 10^13, that's a good point. I'm sure that somewhere Davis refers to the same number; he saays the library is up to 10^9 and suggests that's just about enough, though it's still only 0.01% of the theoretical possibility.
 
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Jonathan Edwards

"Gibberish"
Messages
5,256
Apologies in advance for a long post, I'm catching up.

....
Davis showed slides of mouse EAE with similar clonal expansion levels in blood and CNS for both CD4 and CD8. I think he may have said there, or somewhere else, they were the same clones in blood and brain, but I'm not totally sure. He suggested the mouse model data meant the same was likely to be true for humans (how do you check for humans: CSF, or are brain biopsies needed?).

Thanks for the comments Simon - with which I agree. The one thing I would not put weight on is mouse EAE, which I do not think we have reason to think is immunological similar to human MS at all. Talking to colleagues today nobody seems to know about CD8 expansion in MS blood. There seems to be some sharing of TCRs with blood and brain CD8 cells in one study but I am not sure what to make of it. Everything I know about MS indicates it is a B/CD4 cell based problem.

So what we really want is for someone to sort cells from the UK ME Biobank and clone the TCRs to see what is there.
 

aimossy

Senior Member
Messages
1,106
I've been excited about the findings Mark Davis presented and hope we hear more from him soon. The work looks possibly very promising. I was impressed with his presentation and answers to questions. I also didn't detect any overselling of his findings or hyperbole. If anything he seemed cautious to me. Another immunologist in the top of their field looking at ME/CFS is brilliant.

It's heartening to me that Jonathan mentions the UK biobank!