Regulatory T cells outmatched in active disease
If immunology had its own comic book series, a recurring MS story line would pit a regulatory T cell (Treg) as a flawed superhero against its evil twin. This nemesis is an autoimmune effector T cell that infiltrates the brain, intent on destroying the myelin coating of neurons. In people with relapsing-remitting multiple sclerosis (RRMS), studies have suggested, it's a battle the Tregs often cannot win, presumably because of an unknown defect in them associated with MS. A new paper adds another dimension to the contest: Tregs may fall short in part because, in MS, their adversaries have acquired extra powers to evade their control.
The effector T cells of some people with RRMS are relatively resistant to Tregs, at least in test tubes, researchers reported 30 January in Science Translational Medicine (Schneider et al., 2013). Using blood samples from 24 people with the disease, researchers identified a key molecular pathway that underlies the resistance and that has been previously implicated in MS. The authors suggest that therapies targeting this pathway, including a drug already approved for rheumatoid arthritis, be tested in people with RRMS.
"It's a nice step forward in trying to understand how the immune system is dysregulated in MS patients," says Larry Steinman, a neurologist at Stanford University in California. Many immunologists "believe we have a set of T cells that regulate, or suppress, the activity of pathogenic T cells," he says. "It's an attractive hypothesis, that something in patients' T cells is making them less responsive to T regulatory cells."
"This study is beautifully simple and yet very sophisticated in its implications," says J. Theodore Phillips, an MS neurologist at Baylor Institute for Immunology Research in Dallas, Texas, who was not involved in the study. "It provides a nice symmetry to the picture of what is going on in MS. It's almost as though the autoimmune cells in patients with active MS have an extra layer of armor to protect them from actions by regulatory T cells that themselves are not quite up to the task."
The investigation was inspired by similar findings in other autoimmune diseases, says senior author Jane Hoyt Buckner, a rheumatologist at Benaroya Research Institute at Virginia Mason Medical Center in Seattle. Other groups first reported resistant T cells that escape control by regulatory T cells in people with psoriasis and lupus. In Buckner's laboratory, the phenomenon was extended to type 1 diabetes by the first author of the new paper, Anya Schneider, a postdoctoral researcher and neurologist (Schneider et al., 2008).
"I'm interested in understanding what is common and what is different in other autoimmune diseases," Buckner says. Autoimmune diseases tend to run in families, although family members may suffer from different specific diseases. Genome-wide association studies have implicated overlapping biological pathways for autoimmune diseases. Although the different diseases affect different tissues, such as the pancreas in type 1 diabetes, they have in common a failed immune tolerance. "The immune system is supposed to be tolerant to the body," Buckner says. "In MS, the immune system loses tolerance to the central nervous system and attacks it. T cell resistance is one mechanism of failed tolerance."
Normally, effector T cells are counted among the immunological champions, because they recognize and defend against foreign invaders, such as bacteria and viruses, as well as against a person's own proteins that might be mutated or overexpressed, such as in cancer. A few T cells that react to seemingly healthy tissue lurk throughout the body in lymph nodes and the spleen, usually harmlessly. But in autoimmune diseases, they somehow activate and turn their destructive power inward. Effector T cells come in two main types, CD4+ (helper T cells) and CD8+ (cytotoxic T cells) (see "Altered Immunity, Crippled Neurons"). Both types have been found in postmortem brain and spinal cord tissue of people with MS.
In the latest study, Buckner and her colleagues sought evidence of CD4+ T cell resistance in blood samples from RRMS patients who had not taken any immunomodulatory drugs within the past three months. The patients were newly diagnosed, untreated, or changing treatment.
Only a minority of CD4+ effector T cells target the myelin sheathing protecting axons in the brain and spinal cord, but the analysis “took on all comers,” she says, because myelin-specific T cells possibly involved in MS are so rare they are hard to study.
To test the effector T cell behavior independently of potentially defective Tregs from MS patients, the researchers isolated CD4+ effector T cells. Normally, effector T cells multiply to respond to infections and pathogens, while the Tregs suppress such proliferation to control collateral damage to the body, including autoimmunity. In their cell cultures, the researchers added Tregs from healthy people and stimulated proliferation of the effector T cells with beads treated with activating antibodies. Using fluorescent labels that dim proportionately with each cell division, the researchers counted the effector T cells with a flow cytometry machine. (The Tregs were unlabeled.)
Overall, effector T cells from MS patients were better able to escape control of the healthy Tregs and proliferate than were those from healthy people. The researchers first tested T cells from 14 people with RRMS (and compared cells from 11 healthy people) and repeated the analyses a year later in an independent cohort of 10 untreated patients (compared to 13 healthy people).
In both studies, effector T cells from some people with RRMS were suppressed normally. Additional lab tests of the cytokine environment of those blood samples and of the T cells themselves offered no explanation. So the scientists looked for clues in the clinical status of the patients. Co-author Mariko Kita, a neurologist at Virginia Mason, who had not seen the T cell data, evaluated the clinical records of the test subjects and rated the severity of their disease, based on symptomatic flare-ups or gadolinium-enhanced lesions on MRI images over the past two years.
Patients with the most severe disease had effector T cells that were most resistant to suppression by healthy Tregs. The T cells that were squelched normally by Tregs came from patients with mild disease. “We may have an immune marker that can differentiate disease activity between people diagnosed with RRMS," Buckner says.
Probing deeper, the researchers explored the potential mechanism by which effector T cells evade Tregs. A major cytokine called interleukin 6 (IL-6) seemed to be a good candidate, based on results from the previous study on T cell resistance in psoriasis, as well as on human and mouse studies of MS (Steinman, 2013). "IL-6 is a pro-inflammatory cytokine," Buckner says. Even better, an approved therapeutic for rheumatoid arthritis, another autoimmune disease, targets IL-6.
The researchers found no increase in IL-6 in the serum of the MS patients, but when they added IL-6 to effector T cells in test tubes, the MS-derived T cells responded significantly more than did T cells of healthy controls, showing elevated pSTAT3 activity and phosphorylation. These traits—which indicate activation of the IL-6 pathway—correlated with impaired suppression of T cells in RRMS subjects. Inhibition of the STAT3 phosphorylation increased suppression of the MS T cells in the presence of healthy Tregs. Further digging revealed increased expression of the most common IL-6 receptors on the CD4+ T cells from people with MS, which the researchers propose helps explain the resistance.
"The story has two parts," Buckner says. "We've described general T cells in MS that are resistant to suppression by Tregs and showed that characteristic is seen in T cells of patients with more active or aggressive disease. Then we took another step and identified a molecular mechanism that leads to that defect, which is through the IL-6 pathway. This is one mechanism, and there may be others we need to explore and target for therapeutic purposes."
In the meantime, the researchers point out, the findings provide a biomarker to test drugs that target the IL-6 pathway, including tocilizumab (Actemra, Genentech), an approved rheumatoid arthritis drug that blocks the IL-6 receptor.
The findings make a strong case for testing inhibitors of the IL-6 receptor pathway in MS, Phillips says, especially the one already approved, tocilizumab. "The study finds a new mechanism that is fully complementary to everything else we have learned about MS, connects the mechanism to clinical activity, and identifies a key molecular component that, by pleasant coincidence, is an agency that is pharmaceutically available," he says.
Other researchers agree that the findings strengthen the case for testing an IL-6 receptor inhibitor, but are more cautious about other implications of the study. "We're calling them T effector cells, but we don't know if the resistant T cells [in the study] are causing MS," Steinman says. "Some of them are keeping you from having an attack by influenza or from an infection if you stab yourself with a rusty screwdriver." Further complicating matters, he points out, scientists have not yet elaborated the actual effector T cell mechanisms producing the tissue damage in MS. He also has reservations about the therapeutic potential of targeting CD4+ cells. About 20 years ago, he and his colleagues found only limited success in knocking out CD4+ cells in a Phase II clinical trial for MS.
As for how well an IL-6 receptor blocker might work in treating MS, Stephen Anderton, a T cell immunologist at the University of Edinburgh in Scotland, is skeptical, based on his and other studies of the experimental autoimmune encephalomyelitis (EAE) mouse model, which in some ways mimics MS and allows more detailed molecular studies of the brain. In humans, Anderton says, what happens in peripheral blood may not always translate past the blood-brain barrier, if recent mouse studies are any indication. In mice, blocking the IL-6 receptor can reduce EAE severity only if it is done very early, when the disease is induced, not later, because activated T cells lose their receptors (Leech et al., 2013). He and his colleagues found that all T cells (both effector and regulatory) in the brains of EAE mice have lost their IL-6 receptors, he reported at the January 2013 MS Keystone Symposia in Big Sky, Montana, making them unable to respond to IL-6 (O'Connor et al., 2012). However, the evidence for resistant T cells from Buckner's lab gives a mechanistic explanation of the ineffective Treg suppression of effector T cells in tissue cultures of the blood of MS patients, he says, even if the experiments on peripheral blood samples in the lab may not fully encapsulate the molecular scenario of MS.
In follow-up in vitro studies, Buckner and her colleagues are trying to understand the role of IL-6 signaling more broadly in the context of MS and other autoimmune diseases, including other effects on the immune system and the impact of genetic variations in the IL-6 pathway. Their plans include studying a larger cohort—including patients with progressive forms of MS—and investigating how the Treg resistance changes over time. Furthermore, they aim to study how approved MS drugs affect Treg resistance, whether resistance predicts disease course, and how to target those mechanisms therapeutically. In the meantime, she hopes someone will follow up with a clinical trial to learn whether blocking IL-6 could reduce the number and severity of attacks in people with RRMS.
To that list, Steinman adds two more suggestions. First, he suggests performing similar experiments on antigen-specific CD4+ T cells that might target tissue in the central nervous system, such as those that may attack myelin in MS. Recently, Steinman has become bullish on the role of autoreactive CD8+ cells (see “Bad Presentation”). So next, he says, it would be interesting to see if the newfound resistance of CD4+ T cells to Treg suppression extends to CD8+ T cells as well.
The study adds a new dimension to the tactical maneuvering and countermoves of CD4+ cells, which may be influencing disease severity in people with RRMS and may be vulnerable to therapeutic intervention. Yet, Steinman cautions, the final outcome of the clash of the T cells is likely to be more nuanced and complicated than their names imply.
Key open questions
- Will blocking the IL-6 receptor work to treat RRMS?
- Will the T cell resistance assay predict which patients will advance more quickly to progressive disease?
- How could the impaired resistance of T cells in the peripheral blood contribute to MS?
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