Scientists find evidence of Epstein-Barr virus activity in spinal fluid of multiple sclerosis patients

Emerging research has provided fresh evidence regarding the role of viral infection in the development of multiple sclerosis. By analyzing immune cells extracted from the spinal fluid of patients, scientists identified a specific population of “killer” T cells that appear to target the Epstein-Barr virus. The findings suggest that an immune response directed at this common pathogen may drive the neurological damage associated with the disease. The study was published in the journal Nature Immunology.

Multiple sclerosis is a chronic condition in which the immune system mistakenly attacks myelin, the protective sheath covering nerve fibers in the central nervous system. This damage disrupts communication between the brain and the rest of the body. For decades, scientific inquiry focused heavily on CD4+ T cells. These are immune cells that help coordinate the body’s defense response.

However, pathologists have observed that a different type of immune cell is actually more abundant in the brain lesions of patients. These are CD8+ T cells, also known as cytotoxic or “killer” T cells. Their primary function is to destroy cells that have been damaged or infected by viruses. Despite their prevalence at the site of injury, the specific targets they hunt in the central nervous system have remained largely unknown.

There is a strong epidemiological link between the Epstein-Barr virus and multiple sclerosis. Almost every person diagnosed with the condition tests positive for previous exposure to this virus. Yet, because the virus infects the vast majority of the global population, the mere presence of the virus does not explain why some individuals develop the disease while others do not.

Joseph J. Sabatino Jr., a researcher at the University of California, San Francisco, and his colleagues sought to resolve this ambiguity. They aimed to determine what specific proteins the CD8+ T cells in the central nervous system were recognizing. The team hypothesized that identifying the targets of these cells could reveal the mechanism driving the disease.

Free daily newsletter

The researchers collected samples of cerebrospinal fluid and blood from human participants. The study group included 13 individuals with multiple sclerosis or clinically isolated syndrome, a precursor to the disease. For comparison, they also collected samples from five control participants who were healthy or had other neurological conditions.

Obtaining cerebrospinal fluid is an invasive procedure. This makes such samples relatively rare and difficult to acquire, particularly from patients in the early stages of the disease. The team used a technology called single-cell RNA sequencing to analyze these samples. This method allows scientists to examine the genetic activity of thousands of individual cells simultaneously.

The investigators paid particular attention to the T cell receptors found on the surface of the immune cells. These receptors function like unique identification cards or keys. Each one is shaped to bind with a specific protein fragment, or antigen. When a T cell encounters its specific target, it clones itself repeatedly to create an army capable of eliminating the threat.

In the spinal fluid of patients with multiple sclerosis, the researchers found groups of CD8+ T cells that were genetically identical. This indicated they had undergone clonal expansion. These expanded groups were found in much higher concentrations in the spinal fluid than in the blood of the same patients. This suggests that these cells were not just passing through but were actively recruited to the central nervous system to fight a specific target.

To identify that target, the research team employed several antigen discovery strategies. One method involved a technique known as yeast display. The researchers created a library of hundreds of millions of yeast cells, each displaying a different protein fragment on its surface. They exposed the T cell receptors from the patients to this library to see which proteins they would bind.

This screening process initially identified synthetic protein fragments that acted as “mimics” for the true target. While these mimics bound to the receptors, they did not necessarily provoke a functional immune response. To find the naturally occurring target, the researchers compared the genetic sequences of the receptors against databases of known viral antigens.

This comparison yielded a match for the Epstein-Barr virus. Specifically, the receptors from the expanded CD8+ T cells matched those known to target proteins produced by the virus. To validate this finding, the team used CRISPR gene-editing technology. They engineered fresh T cells from healthy donors to express the exact receptors found in the multiple sclerosis patients.

When these engineered cells were exposed to Epstein-Barr virus peptides, they became activated and released inflammatory cytokines. This confirmed that the receptors identified in the spinal fluid were indeed specific for the virus. The team found that these virus-specific cells were highly activated and possessed the molecular machinery necessary to migrate into tissues and kill cells.

The researchers also investigated whether the virus itself was present in the central nervous system. They analyzed the cerebrospinal fluid for viral DNA. They detected genetic material from the Epstein-Barr virus in the fluid of both patients and controls. However, the presence of DNA alone only indicates that the virus is there, not necessarily that it is active.

To assess viral activity, the team looked for viral RNA transcripts. These are produced when the virus is reading its own genes to make proteins. They found higher levels of a specific transcript called BamHI-W in the fluid of patients with multiple sclerosis compared to the control group. This transcript is associated with the virus’s lytic phase, a period when it is actively replicating.

The detection of lytic transcripts suggests that the virus is not dormant in these patients. Instead, it appears to be reactivating within the central nervous system or the immune cells trafficking there. This reactivation could be the trigger that causes the immune system to expand its army of CD8+ T cells.

Some theories of autoimmune disease propose a mechanism called molecular mimicry. This occurs when a viral protein looks so similar to a human protein that the immune system attacks both. The researchers tested the Epstein-Barr virus-specific receptors against human proteins that resembled the viral targets. They found no evidence of cross-reactivity. The T cells attacked the virus but ignored the human proteins.

This finding implies that the immune system in multiple sclerosis may not be confused. It may be accurately targeting a viral invader. The collateral damage to the nervous system could be a side effect of this ongoing battle between the immune system and the reactivated virus.

The gene expression profile of these cells supported this idea. The virus-specific T cells expressed high levels of genes associated with migrating to tissues and persisting there. They appeared to be an “effector” population, primed for immediate defense rather than long-term memory storage.

“Looking at these understudied CD8+ T cells connects a lot of different dots and gives us a new window on how EBV is likely contributing to this disease,” said senior author Joe Sabatino in a press release. The study provides a clearer picture of the cellular machinery at work in the disease.

There are limitations to the study that warrant consideration. The sample size was small, involving only 18 participants in total. This is a common challenge in studies requiring invasive spinal fluid collection. While the researchers identified Epstein-Barr virus targets for some of the expanded T cell clones, the targets for the majority of the expanded cells remain unidentified.

It is also not yet clear if the viral reactivation causes the disease or if the disease state allows the virus to reactivate. The immune system is complex, and inflammation in the brain could theoretically create an environment that favors viral replication. Further research will be necessary to establish the direction of causality.

Future studies will likely focus on larger cohorts of patients. Researchers will need to determine if these virus-specific cells are present at all stages of the disease or only during early development. Additionally, understanding where the virus resides within the central nervous system remains a priority. The virus typically infects B cells, another type of immune cell, and their presence in the brain is a hallmark of multiple sclerosis.

The implications for treatment are notable. Current therapies for multiple sclerosis largely function by suppressing the immune system broadly or by trapping immune cells in the lymph nodes so they cannot enter the brain. If the disease is driven by a viral infection, therapies targeting the virus itself could offer a new approach. Antiviral drugs or vaccines designed to suppress the Epstein-Barr virus might help reduce the immune activation that leads to neurological damage.

The study, “Antigen specificity of clonally enriched CD8+ T cells in multiple sclerosis,” was authored by Fumie Hayashi, Kristen Mittl, Ravi Dandekar, Josiah Gerdts, Ebtesam Hassan, Ryan D. Schubert, Lindsay Oshiro, Rita Loudermilk, Ariele Greenfield, Danillo G. Augusto, Gregory Havton, Shriya Anumarlu, Arhan Surapaneni, Akshaya Ramesh, Edwina Tran, Kanishka Koshal, Kerry Kizer, Joanna Dreux, Alaina K. Cagalingan, Florian Schustek, Lena Flood, Tamson Moore, Lisa L. Kirkemo, Isabelle J. Fisher, Tiffany Cooper, Meagan Harms, Refujia Gomez, University of California, San Francisco MS-EPIC Team, Claire D. Clelland, Leah Sibener, Bruce A. C. Cree, Stephen L. Hauser, Jill A. Hollenbach, Marvin Gee, Michael R. Wilson, Scott S. Zamvil & Joseph J. Sabatino Jr.