T-cells attacking a damaged (in this cancerous) cell.
One of the immune system’s premier hunter-killer cells, cytotoxic (killer) T cells (CD8) find infected, cancerous, or damaged cells, and then bore holes into them and trigger their suicide program. We rely on them to mop up infections. If they don’t work well, infections can persist, and inflammation can rise as the highly inflammatory innate immune system tries its best to compensate.
Researchers have found abnormalities in these hunter-killer cells before, but they haven’t determined why they happen, and that “why” is crucial because it answers the bigger “what” question: which treatments to use.
The Painter study took a major step forward in our understanding of what these powerful cells do in long COVID. Interestingly, the answer dovetails with what we’re learning about them in ME/CFS.
THE GIST
T-cells attacking a cancer cell.
One of the immune system’s premier hunter-killer cells, cytotoxic (killer) T cells (CD8) find infected, cancerous, or damaged cells, and then bore holes into them and trigger their suicide program. If they’re not working well, infections can persist, and inflammation can skyrocket.
Researchers have found abnormalities in these hunter-killer cells before, but they haven’t determined why they happen, and that “why” is crucial because it answers the bigger “what” question: what treatments to use.
This large study took a major step forward in understanding what happens to these stalwarts of the immune system.
Cytotoxic T-cells find and destroy infected cells. As they roar into action, they produce hordes of clones trained to attack specific viruses.
Once the virus is vanquished, there’s no reason to keep these amped up, energy-intensive T-cells on their search-and-destroy missions. Some researchers have speculated that chronically activated T-cells drain energy from other parts of the body.
Within a couple of months, they usually disappear, leaving T-helper memory cells on standby. They’re sprinters, not marathon runners.
The study looked for specific cytotoxic, or killer, T-cells activated by SARS-CoV-2 (coronavirus) and/or the Epstein-Barr Virus (EBV). In some cases, they found that even 18 months later, a significant subset of long COVID patients still had activated cells.
These activated cells were present in about 40% of long COVID patients; i.e. they are not contributing to all long COVID patients.
That is highly unusual and suggests several possibilities: the coronavirus has not been fully eradicated but remains present and replicating, likely in a hidden reservoir in the body. EBV, which almost all of us harbor in our cells, has become activated. The coronavirus may also have been eradicated, but it left behind fragments that tweak the immune system. In EBV’s case, a smoldering infection could produce proteins that chronically tweak T-cells.
Whatever the reason, these chronically activated T-cells end up in the same place: exhausted and unable to mount the immune response we need to eradicate pathogens.
The next step is clear – determine exactly what – the whole virus, bits of virus, virus-produced proteins – is activating these cells. Note, though, that it’s also possible that the chronic activation has also triggered an autoimmune process. Once the trigger(s) are determined, the treatment pathway becomes clear.
A complementary approach in ME/CFS has come to the same conclusion. Instead of looking for specific pathogen-activated T-cells, though, the Selin-Kumar-Kohlburger group first found abnormal T-cells (damaged CD8 and CD4/CD8 double-positive cells) and determined that long-term activation is present.
They are now looking for the trigger that turned them on. Because they’re looking at all possible triggers (instead of coronavirus- or EBV-specific T-cells), their approach (which needs funding) is more comprehensive. They believe that if they can identify the triggers, they may find what’s driving ME/CFS.
Another approach from Ariza and Williams at Ohio State University shows that a smoldering herpesvirus infection that fails to replicate but still pumps out proteins that tweak the immune system is present in a substantial subset of ME/CFS patients. It could produce many ME/CFS symptoms.
In 2019, Manual Ruiz-Pablo’s group received a Solve ME Ramsay Award to investigate the idea that people with ME/CFS have an immune deficiency that prevents them from keeping EBV in check.
Ruiz-Pablo believes that all post-infectious diseases like ME/CFS and long COVID have one thing in common: they suffer from an inability to fully clear (or, in the case of EBV, inhibit) viral or bacterial attacks.
This “immune hole” hypothesis could explain why the 2008 Dubbo studies showed that very different pathogens are able to produce the same ME/CFS-like state.
Pablo-Ruiz believes the new long COVID study will ultimately open the door to precision-guided treatments that will help us solve long COVID and ME/CFS.
Finally, EBV research in the ME/CFS/long COVID arena is heating up! Leonard Jason and Nancy Klimas have just received grants/donations to assess EBV effects in these diseases.
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Going Deeper Works
The latest big long COVID study, “Persistently activated virus-specific CD8 T cells indicate antigen-driven immune aberrations in long COVID, from Mark Painter and colleagues, demonstrates one thing we’ve been learning in spades in the ME/CFS and long COVID fields: going deeper works.
The deeper you dig, the clearer these diseases become.
Ordinary tests simply don’t cut it in these diseases. The NIH’s long COVID Recover project learned that the hard way when they put a billion dollars or so into giving most unsophisticated tests to tens of thousands of long COVID patients and healthy controls and came up drawing blanks.
You have to dig deeper. That’s what Maureen Hanson and company and what Lissa Selin have been doing with their single-cell analyses in ME/CFS, and that’s what Painter and company did in their nice, big 300-person study. When they found nothing of significance in the entire circulating cytotoxic (killer) T-cell population, they didn’t throw up their hands…They dug deeper.
Instead of looking at the broad array of T-killer cells, Painter focused on looking for cytotoxic or killer T-cells activated by the SARS-CoV-2 (coronavirus) and/or the Epstein-Barr Virus (EBV). In some cases, they found that even 18 months later, a significant subset of long COVID patients still had activated cells.
That is highly unusual. Cytotoxic T-cells are meant to find and destroy infected cells, not hang around. As they roar into action, they produce hordes of clones trained to attack specific viruses. Once the virus is vanquished, there’s no reason to keep these amped up, energy-intensive T-cells on their search-and-destroy missions. Within a couple of months, they’ve usually disappeared, leaving T-helper memory cells behind to stay on the lookout. They’re sprinters, not marathon runners.
Researchers had found coronavirus-activated T-cells in long COVID before, but no one has been nearly as comprehensive as Painter was. His study was much larger, and used multiple tests (spectral flow cytometry, single-cell RNA sequencing, T-cell receptor sequencing, activation-marker analysis, and functional assays) that assessed activation.
Painter found long term activation of T-cells by the coronavirus and Epstein-Barr virus in about 40% of long COVID patients.
Significantly, this study also found that an inflammatory milieu (called “bystander activation”) did not turn the T-cells on. Because they assessed the T-cells repeatedly over time, they showed that the activation was constant.
If past studies suggested long-term T-cell activation was happening, Painter’s study proved it is.
Painter also found, not surprisingly, that these chronically activated cells were getting tired. They were no longer able to pump out normal levels of cytokines, they had trouble proliferating, and they were prone to dying. In short, they appeared to be exhausted.
The fact that the coronavirus and EBV activation tended to occur in the same patients suggested that some individuals harbor a susceptibility to multiple infections/reactivations.
Mysteries remain
What triggered the activation – a replicating virus, or pieces of a virus, or proteins produced by a virus is unclear.
These pathogen-activated T-cells are not present in all long COVID patients. This study found them in about 40% of long COVID patients, The findings were quite strong. The long COVID group was 3.6 times more likely to have these cells, with a highly significant p-value (p = 0.000016). Their presence in about 16% of the recovered COVID-19 patients, suggests, though, that T-cell activation plus another X factor may be needed to produce long COVID.
We don’t know what turned on these powerful immune cells. We know inflammation isn’t the cause, but it remains unclear whether replicating viruses, viral fragments, or viral proteins are responsible. Because each requires a different treatment approach, determining which one is responsible will be important.
This is surely the next step researchers will take, or probably already are taking. Hopefully, we’ll get bigger studies and answers sooner rather than later now that the pathogen activation question has been resolved.
Because Painter did not look for T-cells attacking the body, an autoimmune process also remains possible. In a process called molecular mimicry, an immune response to a viral antigen can attack the body at the same time. A recent long COVID paper found that people with long COVID a) carried a variety of autoantibodies able to attack central nervous system tissues, and b) transferring IgG antibodies from individuals with LC into mice produced fatigue, balance/coordination problems, pain sensitivity, and small fiber nerve damage (!).
Long-lived activated T-cells, in fact, increase the risk that the immune system will make a mistake and start attacking the body.
In any case, researchers now have a strong foundation to move forward to answer these questions.
Template for ME/CFS?
The study also potentially provides a nice template for ME/CFS. Most ME/CFS studies have used antibody levels to a virus to see if a virus is involved. While antibodies can show if an immune response has been mounted at some point they’re not as effective at telling if an active immune response is currently present.
Painter’s ability to isolate virus-specific T-cells and then assess activation markers (CD38, HLA-DR, CD39, and Ki-67) allowed him to determine that they’re currently active. Plus, he had those gene expression results which indicated the same.
Painter’s provided a template for resolving one of the great questions in ME/CFS – is a persistent virus at play?
ME/CFS researchers could presumably use these techniques to determine if EBV, for instance, is currently tweaking the T-cells. They may not have to, though, as a complementary and ultimately more promising approach is under way.
A Complementary ME/CFS Approach Is Underway
Because the Selin-Kumar-Kohlburger ME/CFS effort has a wider scope it may prove more fruitful – if it can get funded.
Lissa Selin, Rosh Kumar, and Ayano Kohlburger have been pursuing a complementary approach to the T-cells in ME/CFS. Their sample sizes are much smaller (this is ME/CFS after all (lol)), but their approach is broader and better matched to ME/CFS.
They started from a different place but reached the same conclusion: antigen-driven T-cells are activated in ME/CFS. Instead of looking for specific pathogen-activated T-cells, this group first found abnormal T-cells (damaged CD8 and CD4/CD8 double-positive cells), determined that they were pumping out specific clones, and is now looking for the trigger or antigen they believe turned them on.
Painters’ extensive testing of coronavirus- and EBV-activated cytotoxic T-cells has allowed him to definitively determine they are in play. The Selin-Kumar-Kohlburger group hasn’t been able to do that in ME/CFS, but their approach gives them an edge in another way. Painter looked for coronavirus/EBV specific T-cells, but the ME/CFS group is looking at every possible antigen. They have the potential to a) pick up more pathogens, and b) determine if an autoimmune process is present.
They’re confident that, given the resources (they’re seeking funding), they can identify what is tweaking the abnormal T-cells in ME/CFS. If they found that EBV antigens (antigens trigger an immune response) were activating T-cells in a subset of ME/CFS patients, we would have a nice overlap with long COVID.
Smoldering Herpesviruses?
The ME/CFS field has another trick up its sleeve. For over a decade, Marshall Williams and Maria Ariza at Ohio State University have received a rather remarkable string of large NIH grants to explore the role a smoldering herpesvirus may play in ME/CFS and other diseases. Their string of ME/CFS wins is unprecedented at the NIH.
They’re illustrating that a hybrid approach may be at play. EBV and other herpesviruses are trying in ME/CFS to replicate but are only getting halfway. A strong immune system should keep them bottled up, but in a significant subset of ME/CFS patients, it’s failing to do so. EBV starts to replicate but fails to complete it. As this happens, EBV-infected cells produce proteins such as dUTPase that tweak the immune system.
Mouse and other studies show that herpesvirus dUTPases can affect the blood-brain barrier, pain, neurotransmitter activity, sickness behavior, and anxiety, and can also affect T-cell regulation.
Their smoldering herpesvirus findings could help explain another knotty question that has dogged the ME/CFS field: “If EBV is driving or contributing to the symptoms in ME/CFS, how is it doing so without replicating?”
In their most recent paper, they associated antibodies to EBV and HHV-6 dUTPase with increased pain and fatigue in ME/CFS.
Ruiz-Pablo’s Answer to ME/CFS and Long COVID
“I believe we are getting closer to developing a stronger scientific basis for antiviral treatment in selected patients with these diseases” Ruiz-Pablo
In 2021, “Epstein-Barr Virus and the Origin of Myalgic Encephalomyelitis or Chronic Fatigue Syndrome” Manual Ruiz-Pablo and his collaborators proposed that EBV activation is driving ME/CFS in a subset of patients. They believed that an immune hole in genetically susceptible individuals made it difficult for their immune systems to detect EBV in its latent state. That ultimately led to chronic immune activation and, eventually, immune exhaustion. They received a 2019 Solve ME Ramsay Award to investigate this hypothesis.
Ruiz-Pablo proposes that post-infectious diseases share an inability to totally clear a pathogen. This can result in autoimmunity and chronic immune dysfunction.
Ruiz-Pablo heralded the Painter paper because it validated the idea that a persistent virus – either whole or pieces of it – are present in long COVID. He was particularly taken by the fact that the long COVID patients tended to have prolonged T-cell activation to both the coronavirus and the Epstein-Barr virus.
He proposed that the initial coronavirus infection dysregulated the immune system and allowed EBV to flourish by impairing the ability of the immune system to detect EBV-infected cells.
He then attempted to address perhaps the greatest question of all. Referencing the 2006 Dubbo study findings, he asked how it is that dramatically different pathogens can produce the same ME/CFS state?
He came up with a surprisingly straightforward answer: “antigen” persistence. (Again – an antigen is a part of a virus, bacteria, toxin, or autoimmune target that tweaks the immune system.)
Post-infectious diseases begin with an infection that isn’t fully resolved. Something, whether it’s the replicating pathogen itself, pieces of it, or proteins it produces in its latent state, gets left behind. That foreign object keeps the immune system persistently activated.
Over time, that activation leads to immune exhaustion, which can allow pathogens like EBV to reactivate and may ultimately result in autoimmunity.
The key, though, is a chronically activated immune system which becomes more dysregulated and exhausted over time.
Targeted Treatments – The Antiviral Plus Proposal
For Ruiz-Pablo, the most intriguing aspect of Painter’s study is the opportunity he believes it presents for targeted treatments. We’re not there yet: we don’t know whether persistent virus activation, viral fragments, or an autoimmune process is driving T-cell activation, but once we do, the treatment pathways become clear.
Until we know that, treating the immune abnormalities may not help much if a pathogen is still present.
Ruiz-Pablo does not believe, though, that antiviral strategies are necessarily the be-all and end-all. If immune abnormalities have developed over time, they should be addressed at the same time.
EBV Research Heating Up
Epstein-Barr virus research is heating up. 🙂
Leonard Jason just reported that his team was awarded an NIH grant, “SARS-CoV-2 and EBV-orchestrated activation of the kynurenine pathway,” that will explore how the coronavirus and EBV activate the kynurenine pathway to produce long COVID and/or ME/CFS in a significant subset of patients.
Nancy Klimas has also received funding to explore EBV’s effects in these diseases.
I think Nath and his team in the NIH study suggested antigen persistence could be the driving causative factor.
Yet…. Painter’s study found this abnormality in 40% of patients and 19% of controls. To me, that suggests some sort of associative factor rather than a factor actually driving the illness.
In terms of EBV – let’s see if Polybio’s trial of Truvada yields any positive results.