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Tantalizing, exhilarating, frustrating, hopeful – Solve M.E.’s recent “Immune Dysfunction & T-Cell Exhaustion via Single Cell Immune Profiling in ME/CFS & Long COVID” webinar was all of these things. Researchers have shied away from ME/CFS for decades, and National Institutes of Health (NIH) funding is still in the pits, but what I came away with from this webinar was a sense, once again, researchers are missing out by not tackling ME/CFS.

Don’t they know they’re missing the opportunity to blow the doors open on not just one but possibly many diseases? Apparently not. I have the feeling there will be some teeth gnashing at some point over paths not taken.

To be sure, T-cell exhaustion is one area the NIH didn’t miss. Kudos to them for giving Liisa Selin a major RO1 NIH grant to study it.

It’s always good to go back and see how something like that happened. Not surprisingly, it started with a small patient-funded grant (your dollars at work). Solve M.E.’s 2020 Ramsey award helped Liisa Selin and Anna Gil gather the data needed to score that $2.5 million big NIH grant in 2021. Then in 2023, the Patient-Led Research Collaborative, which knows a good thing when it sees it, kicked in more funding for Selin, and then in 2024, it added funding for Roshan Kumar, Anna Gil, and Selin.

Selin and Gil

Selin and Gil approached Rivka Solomon in 2019, stating they had found some unique immune abnormalities in ME/CFS.

Long-time ME/CFS and long-COVID advocate – and Patient Representative to the two labs working on this research – Rivka Solomon explained that Liisa Selin and Anna Gil told her at the 2019 Mass ME event that their two-woman immunology lab at the UMassChan Medical School, had, while researching EBV, uncovered some striking immune abnormalities in ME/CFS. By that time, Liisa Selin, an accomplished researcher, had been living with ME/CFS since 1975. In order to keep from being ostracized, she’d laid low about her ME/CFS for decades.

The Hypothesis

Their hypothesis—that a dysfunctional response to an immunological trigger (infection) results in a chronically hyperactive immune response—is not novel. It’s the last part—the T-cell exhaustion—that is so tantalizing because, as we’ll see, their T-cell work provides the potential for getting at the actual starting place—when these diseases began.

The work is in good hands. If anyone is a T-cell expert, it’s Liisa Selin: she’s co-authored almost 60 papers on T-cells over the past 35 years.

The Gist

  • Tantalizing, exhilarating, frustrating, hopeful – Solve M.E.’s recent “Immune Dysfunction & T-Cell Exhaustion via Single Cell Immune Profiling in ME/CFS & Long COVID” webinar was all of these things.
  • It was exhilarating because Liisa Selin, Ana Gil, and Roshan Kumar believe they have a shot at figuring out what, at the immune level, started things off for ME/CFS and long COVID. It was frustrating because despite their progress, they lack the funding to get there right now.
  • The work got going with a 2020 Solve ME Ramsey Award grant which laid the foundation for a big 2021 NIH grant, and has been supported since then by two Patient Lead Research Foundation grants in 2023 and 2024.
  • Their hypothesis—that a dysfunctional response to an immunological trigger (infection) results in a chronically hyperactive immune response—is not novel. What’s so exciting is their T-cell exhaustion work, led by long-time T-cell researcher Liisa Selin (a long-time ME/CFS patient).
  • Talk about exhaustion. They’ve found that the T-cells in ME/CFS and long COVID patients are producing very little of the substances (perforin, granzymes) they use to kill virally infected cells, and very few of the cytokines (IFN-y, TNF-a) needed to orchestrate the immune response.
  • That means that one of the two main players in the adaptive immune response is essentially dead in the water. The cytotoxic T-cells appear to be so messed up that they’ve resorted to producing a strange, rarely seen kind of hybrid T-cell called a double-positive T-cell in great numbers in these diseases.
  • In an attempt to get the T-cells moving again, these cells produce high levels of a cytokine called IL-9, which also increases mast cell production and the risk of an autoreactive response.
  • Rosh Kumar, the head of research at HiFiBiO Therapeutics, joined the Selin/Gil team in 2024 in the Patient Lead Research Foundation Grant. (Kumar’s wife has had ME/CFS for 20 years.)
  • The grant allowed them to use single-cell analyses to dig deep into the gene expression, T receptor repertoires, and T-cell subsets in the cytotoxic T-cells. The goal is to uncover T-cell biomarkers and ultimately—and here’s where it gets really interesting—determine what antigen (pathogen, autoimmune factor) is keeping the T-cells and the immune system ME/CFS and long COVID up at night and driving it (and us) to exhaustion.
  • Four hundred thousand cytotoxic T-cells from 16 participants later, they’ve uncovered more intriguing issues.  A “quite unusual” pattern of both inflammatory and exhaustion markers is present plus two more types of unusual T-cells popped up.
  • Assessing the very, (very) complex cytotoxic T-cell receptor repertoires brought some welcome news. Because the T-cell receptors can be configured to identify virtually any antigen (danger signal) they provide a memory bank of all the immune insults a person has been subjected to. They’re very complex keys which, if researchers can decipher them, could unlock what started these diseases and is keeping them going.
  • That process has begun. They were able to identify several T-cell receptor clusters unique to ME/CFS and long-COVID patients that appeared associated with some sort of antigenic driver.
  • Getting to the “answer,” i.e., actually identifying the actual culprit(s); i.e. the pathogen and immune dysregulation present, is not going to be easy. For one, while it has long been clear that these diseases are very heterogeneous, it was still remarkable to see a fine-tuned analysis of a very specific part of the immune system (CD8 T-cells) in a small group of patients uncover substantial heterogeneity.
  • Even within the double-positive T-cells, they can identify distinct subsets, clonotypes (T-cells with a similar origin), and enriched sequence motifs (which can help them uncover the triggering factor).
  • Liisa Selin explained that this makes sense given the different pathogens we are exposed to and the different immune systems we have. Indeed, their ability to identify subsets at the molecular level is part of what makes their work so tantalizing.
  • Because T-cell exhaustion has been almost totally explored at the tumor level by uncovering T-cell exhaustion at the global or systemic level Selin, Gil, and Kumar’s work is, in effect, creating a new field of T-cell exhaustion.  one that Selin believes could help explain ME/CFS, long COVID, and other diseases.
  • Ultimately, these researchers want to uncover biomarkers corresponding to the different molecular subsets and produce tests that doctors can use in the clinic. That’s a big task, but Kumar reported that with the technology available today, it is eminently doable.
  • The only thing stopping them now is more funding.

 

 

Really Exhausted

Perforin production

Perforin production in unstimulated (left) and stimulated (right) T-cells. It’s easy to find the healthy controls (tall gray bars).

Do you feel exhausted? You are not alone. Your T-cells are probably just as exhausted.

Every once in a while, you see a graph that makes your jaws drop open, and the perforin graph was one. Perforin is the substance T and NK cells use to drill into an infected cell and kill it. Selin and Gill’s studies suggest that ME/CFS and long-COVID patients’ T cells produce almost no perforin…

They’re also producing 4-5 times lower amounts of IFN-y, a central orchestrator of the immune response. Low IFN-y levels alone could result in difficulty clearing pathogens, produce inflammation, impair T-cell metabolism, and promote T-cell exhaustion.

IFN-y and TNF-a

Greatly reduced IFN-y and TNF-a levels in the ME/CFS and long-COVID patients compared to healthy controls (light grey).

Strange T-cells Pop Up

Plus, Selin and Gill uncovered something brand new: a huge increase in a distinct type of T-cell called double-positive T-cell (CD4+ CD8+) in people with ME/CFS and/or long COVID. Instead of being differentiated into the two usual major T-cell subsets CD4 (helper/regulatory) and CD8 (killer) T-cells, these hybrid T-cells look like both CD4 and CD8 T-cells at the same time.

Selin found that these cells increased 8-9 fold in ME/CFS and long-COVID patients. We want dramatic findings like that. We don’t want subtle findings, we don’t want findings that researchers have to do a double pirouette to fit into their hypothesis—we want major differences that stand out like a sore thumb—and that’s what Selin found.

We also want findings that align with symptoms, and that’s what we got as well: the double-positive T-cells were highly correlated with symptoms, but in a strange way. Instead of hurting things, they appeared to result from an attempt to rebalance the immune system. A positive correlation between double-positive T-cell levels and perforin production (more double-positive T-cells equaled increased perforin production) suggested these odd cells were both: a) intimately tied to the reduced T cell cytotoxicity; and b) might be functioning in a compensatory manner.

On the other hand, a correlation analysis suggested that the compensatory response was not, as so often seems to happen with compensatory responses, going so well. The few double-positive T-cells in the healthy controls existed within a tightly integrated system, but the loose correlations found in the ME/CFS and long-COVID patients suggested that a “highly dysregulated” cytotoxic T-cell immune response was present. That suggested that the immune system was kind of flailing around, trying to come up with an answer.

Correlation analysis

Tightly integrated immune response by healthy controls (left); loosely integrated immune response (ME/CFS/long COVID).

It’s perhaps no surprise that men and women tended to be different – but similar as well. Women produced more of the double-positive T-cells, which produced IL-9. The IL-9 connection seemed particularly apt as it drives T-cell proliferation, prevents apoptosis, and enhances the killing ability of CD8 T cells. Thus, it may be a beneficial compensation acting to help retain cytotoxic function in CD8 T cells.

The Model

CD8 Exhaustion Model

The grand model.

In all, if I got it right, the hypothesis goes something like this – an infection triggers a chronically activated exhausted immune response which either fails to clear the infection, reactivates persistent viruses (herpesviruses) or produces an autoimmune/autoreactive response. Our now-wired and tired immune systems try to compensate by producing scads of double-positive T-cells (as well as other unusual types of T-cells).

wired and tired T-cells

High activation (right box) paired with low effector molecule production (granzymes/perforin—left boxes) in the ME/CFS/long-COVID patients (orange/blue) spells wired and tired cytotoxic T-cells.

Double-positive T-cells produce IL-9, which enhances T-cell proliferation, increases mast cell production, and increases the risk of an autoreactive response. Chronic immune activation alters our metabolism, resulting in increased oxidative stress and lactic acid. Brain fog and autonomic nervous system instability result.

In this scenario, T-cell exhaustion comes first, ultimately resulting in B-cell dysregulation. (Note that Nath believes B-cell dysregulation comes first.) The authors did not explain how T-cell dysregulation could throw the B cells off, but several scenarios exist. Reduced cytokine production could prevent the B-cells from getting on track when pathogens present themselves. Exhausted T cells also produce a factor called PD-1, which inhibits B-cell proliferation and antibody production.

The dramatic reduction in cytotoxic killing ability, the equally dramatic increase in the levels of the strange T-cells, and the high IL-9 levels that may be driving T-proliferation (and exhaustion) were all pretty exciting stuff. The deeper Selin and Gil dug, the more they seemed to find.

It was the next step, though, that was really eye-opening.

Getting at the Source?

Roshan Kumar

Roshan Kumar’s wife has had ME/CFS for 20 years.

Roshan Kumar, PhD, has a special connection to ME/CFS—his wife has had it for 20 years. Kumar is the head of research at HiFiBiO Therapeutics, which develops immunotherapies. He joined the show when he, Selin, and Gill received a PLRC grant in 2024, which funded them to assess the gene expression and receptor repertoires of T cells in ME/CFS and long-COVID patients using single-cell analyses.

The goal is to uncover T-cell biomarkers, and ultimately, what antigen (pathogen, autoimmune factor) might be keeping the T-cells in ME/CFS and long COVID up at night and driving them to exhaustion. They also want to learn more about those mysterious double-positive T-cells that showed up.

Four hundred thousand CD8 and double-positive T-cells from six ME/CFS patients, six long-COVID patients, and four healthy controls later, they uncovered a new suite of issues.

For one, a “quite unusual” pattern of both inflammatory and exhaustion markers was present. Plus two more types of unusual T-cells popped up in the ME/CFS/long-COVID patients. The strange cytotoxic T cells that resembled CD4 memory cells and naïve-like T-cells with distinct expression profiles provided more signs of dysregulation within the cytotoxic T-cell subset.

The deep dive

The deep dive: exploring the gene expression of T-cell subsets and T-cell receptors in 400,000 T-cells from each patient.

Next, they looked for commonalities in sequences of the receptors on the exhausted T-cells. Because T-cell receptors are looking for anything that can tweak the body, they’re amazingly complex, and trillions of possible combinations of T-cell receptors exist. These receptors sitting on the outside of the T-cells provide a memory bank of what the cells have been exposed to. They’re very complex keys that, if researchers can decipher them, could unlock what started these diseases and keep them going.

CD8 cell types

More strange types of cytotoxic T-cells show up.

Focusing on the gene sequences that deal specifically with antigen recognition, they were able to identify several clusters of similar T-cell receptors unique to the ME/CFS and long-COVID patients. Those clusters suggested that the receptors in the exhausted cytotoxic T-cells in the ME/CFS and long-COVID patients were responding to a common antigen; i.e. the team had made a “first step” in identifying what is turning on these T-cells – and perhaps what turned on these diseases.

Getting to the “answer,” i.e., actually identifying the actual culprit(s), is not going to be easy – but it is doable. For one, while it has long been clear that these diseases are very heterogeneous, it was still remarkable to see a fine-tuned analysis of a very specific part of the immune system (CD8 T-cells) in a small group of patients uncover substantial heterogeneity.

Even within the double-positive T-cells, they can identify distinct subsets, clonotypes (T-cells with a similar origin), and enriched sequence motifs (which can help them uncover the triggering factor).

Selin noted that the heterogeneity makes sense. We are all, after all, exposed to different pathogens, and all have different immune systems. Indeed, their ability to identify subsets at the molecular level is part of what makes their work so tantalizing. To get at these diseases, we need molecular signatures that can tease out the subsets and point to treatments. Once that happens, Kumar said, many immune modulators are available.

On that note, Kumar, whose company develops checkpoint inhibitors, feels we need to understand more about the immune system before using them. You wouldn’t want, for instance, to stimulate autoreactive T-cells and cause more inflammation.

Keys

Disentangling what’s happening with the T-cell receptors could be the key to understanding the immune aspects of ME/CFS, long COVID, and perhaps the whole diseases.

No standard blood tests are currently available that patients can get to assess T-cell exhaustion. Even in the lab, researchers usually only assess T-cell exhaustion within tumors, which is how checkpoint inhibitors came about. Until ME/CFS and long COVID, no one had been looking at “global” or systemic T-cell exhaustion. Selin, Gil, and Kumar’s work is, in effect, creating a new field of T-cell exhaustion – one that Selin believes could help explain ME/CFS, long COVID, and other diseases.

Ultimately, these researchers want to uncover biomarkers corresponding to the different molecular subsets and produce tests that doctors can use in the clinic.

Many studies are helping us understand the molecular problems in these illnesses, but what makes this work enticing is its potential to dig into the T-cells—”the sentinels of the immune system,” as Kumar called them—and possibly get at the prime mover of these illnesses.

Kumar says that it’s entirely possible to do that with the technology available now. The PLRC grant did what patient organizations do best—it allowed them to start down this path. The only thing stopping them now is more funding.

 

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