Behind the Scenes of Immunity & Disease The Hidden Role of Immunity and the Frontiers of Therapeutic Applications

2022/04/06

In recent years, it has become clear that our immune system not only protects against infection but also plays a role in a variety of diseases, including cancer, allergies, stroke, and Alzheimer's disease. In cancer treatment, immunotherapy has attracted a great deal of attention because the patient's immune system can eradicate tumors. However, there are still limitations to cancer immunotherapy; T cells, the major immune cells that kill cancer cells, become exhausted by fighting cancer cells and eventually cease to function.

The underlying mechanism of T cell exhaustion has long been a mystery. However, a recent breakthrough could overcome one of these limitations. Through research on laboratory mice, Professor Akihiko Yoshimura and his colleagues in the Department of Microbiology and Immunology at Keio University School of Medicine have revealed for the first time the mechanism by which T cells become exhausted and cease to function inside cancerous tissue. The group discovered the gene NR4A, which is responsible for T cell exhaustion, and demonstrated that cancer treatment could be more effective by inhibiting NR4A.

“The cells of the immune system uniquely regulate the body’s immune responses, much like applying accelerators and brakes when driving a car. Immunological research is merely the investigation into understanding the mechanism of these accelerators and brakes.”

When a virus invades our body or when cancer cells emerge, cells of the immune system spring into action to prevent the intrusion of the virus or to attack and destroy the infected cells or cancer cells. However, if these immune responses are too strong, the immune system may attack the body itself, leading to autoimmune disorders or a condition known as a cytokine storm, which has been in the news recently due to its prevalence in severe COVID-19 infections.

“The immune system distinguishes between the ‘self’ (one's own body) and the ‘non-self’ (foreign substances present in the body) and works to eliminate these foreign substances. But what would happen if it attacked all foreign or ‘non-self’ substances? There’s no doubt that humans would not be able to survive. For example, food is a foreign substance, yet we need to eat it every day to survive. And when a woman becomes pregnant, her body nurtures the baby growing in the womb , even though half of the baby is more or less a stranger to the mother’s body. If our immune system attacked the fetus, the human species would not survive.

This regulation of the immune system and allowance of foreign substances without regarding them as intrusive is called immune tolerance.

“Immune tolerance is essentially applying the brakes of the immune system. These brakes are what allow us to eat and procreate. But an overactive immune system can lead to allergies, miscarriages, autoimmune diseases, cytokine storms, and other issues.”

Professor Yoshimura and his colleagues found that NR4A is the key to both immune tolerance and the mechanism by which T cells are exhausted and rendered ineffective in the fight against cancer cells. "NR4A is, so to speak, the main controller of the brakes of the immune system," says Professor Yoshimura.

So what exactly are these brakes that generate the immune tolerance that prevents allergies and autoimmune diseases?

“There are two types of immunity brakes: cellular and molecular. The cellular brakes are immune cells called regulatory T cells. Regulatory T cells are central to immune tolerance and suppress excessive immunity to prevent miscarriages, allergies such as hay fever, and autoimmune diseases. Molecular brakes, on the other hand, work mainly within cells and suppress the signaling of cytokines and antigen receptors that transmit information between cells.”

Molecular brakes include SOCS1, which was discovered by Professor Yoshimura and his colleagues in 1995, as well as PD-1 and CTLA-4, which are targets of immune checkpoint inhibitors that have emerged as cancer drugs.

“We were researching molecular brakes, but it turns out that regulatory T cells, which are cellular brakes, actually use the same molecular brakes when suppressing an immune response. CTLA-4 and SOCS1, for example, play a vital role in regulatory T cells.”

His findings led to research on regulatory T cells, and in 2013, Professor Yoshimura and his colleagues published their discovery of the gene NR4A and its role in regulating T cells in "Nature Immunology".

“We wanted to clarify what molecules generate regulatory T cells and how regulatory T cells function as regulators of the immune system at the molecular level. My approach to research style has always been focused on the molecular aspects. After moving my lab to Keio, we discovered that NR4A was the molecule we had been looking for for years —that is, the molecule that creates regulatory T cells.”

Having identified NR4A as essential for the creation of regulatory T cells, Professor Yoshimura considers it to be the fundamental molecule among the many molecular brakes. The group’s research also showed that activation of NR4A in mice can make T cells become regulatory T cells, which have never been shown to differentiate by other methods.

“Allergic diseases such as hay fever and asthma, and autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease are caused by excessive immune responses, and regulatory T cells mediate these responses. NR4A creates these regulatory T cells, and it is NR4A that is responsible for controlling regulatory T cell-mediated immune tolerance in the first place.”

Professor Yoshimura and his colleagues also found that NR4A is responsible for the expression of PD-1, one of the molecular brakes.

“There is a system for suppressing an immune response to prevent it from reacting too strongly and attacking normal tissue. The molecular brakes in this system include molecules (proteins) known as ‘immune checkpoints.’ Typical examples of these immune checkpoints are PD-1 and CTLA-4. Many readers may recall that Dr. Tasuku Honjo of Japan was awarded the Nobel Prize several years ago for demonstrating that the antibodies that inhibit PD-1 and CTLA-4 were effective in cancer treatment. These immune checkpoint molecules are not expressed on young T cells, but they increase as T cells are stimulated and become exhausted by fighting cancer cells. The increased PD-1 and CTLA-4 expression on T cells works as brakes. Cancer cells exploit this system in order to step on molecular brakes that work against the immune system, allowing the cancer cells to evade attack from immune cells and proliferate.”

“This is the phenomenon by which T cells are overworked and exhausted, eventually rendering them ineffective. This behavior seems very similar to that of humans after hours of work, a phenomenon we call 'exhaustion.' The principle behind immune checkpoint inhibitors works by blocking the molecular brake receptors on the T cells with antibodies so that PD-1 and CTLA-4 cannot function on them. That allows the T cells to continue to work, enhancing the immune response within the tumor for a while."

While researching T cell exhaustion, Professor Yoshimura said that the mechanism was similar to that of immune tolerance.

“The term ‘exhaustion’ is used to describe how immune cells—specifically T cells—become tired, burn out, and cannot function properly. Against cancer or a coronavirus infection, the T cells keep up the fight, but this can prove to be too much work. When T cells become exhausted, they become worn out and can no longer keep fighting. NR4A is the gene that creates this state of exhaustion.”

Might it be better if exhaustion didn’t occur? That, too, would actually be a problem because if T cells continued functioning without tiring, it would lead to an excessive immune response.

“Thus, while immune tolerance and T cell exhaustion are two seemingly distinct phenomena, the underlying mechanisms are nearly identical. Regulatory T cells use molecular brakes to produce immune tolerance, while NR4A is what creates such regulatory T cells. In the case of T cell exhaustion, various molecular brakes cause T cells to become exhausted, and it is NR4A that induces these molecular brakes as well. In other words, NR4A is what is running these two systems behind the scenes. NR4A is central to both immune tolerance and exhaustion. The former suppresses the immune system allowing for an organism to live, and the latter maintains effective immune cells to stop them from working too hard.”

The research by Professor Yoshimura and his colleagues on the mechanism of T cell exhaustion was published for the first time in the scientific journal Nature in 2019.

“In this study, we found that when we destroyed NR4A in a mouse cancer model, the T cells remained effective and successfully destroyed the cancer. But this is a double-edged sword. When this happens, the T cells become too aggressive and attack the body as well, resulting in autoimmune diseases, which pose an equally serious threat to their lives.”

Professor Yoshimura and his colleagues are now researching whether there is a way to suppress the excessive immune response that attacks the body so that the immune system will only destroy cancer cells.

“There are three molecules in NR4A that have more or less the same function: NR4A1, NR4A2, and NR4A3. We destroy two of them and leave one. By doing so, it is possible to make adjustments such that the immune system will attack the cancer but not the body. Right now, we are trying to target cancer without causing autoimmune or allergic diseases. However, it’s not so easy. This is because NR4A is expressed throughout the body and serves various roles in addition to acting on T cells. NR4A, for example, is expressed in neurons and has been shown to be important in Parkinson's disease. Therefore, we are continuing to investigate ways to strengthen immunity against tumors by altering how NR4A works on T cells without affecting other areas.”

Since moving his lab to Keio, Professor Yoshimura has also begun a study that attempts to make exhausted T cells 'young' again.

"Essentially, it’s as if we are trying to turn an elderly person into their younger self. We want to find out if there is a way to reinvigorate truly burned-out T cells. If we can do that, we may be able to revive T cells that can no longer take up the fight against cancerous tissue. Immune checkpoint inhibitors that inhibit the molecular brake PD-1 can almost accomplish this task, but it has been found that they only restore T cells before the T cells have reached an exhausted and ineffective state."

So, does the inhibition of NR4A, which governs exhaustion, serve to rejuvenate T cells?

“We don’t fully understand that yet. Our goal is to rejuvenate the completely exhausted and inactive T cells. And in fact, we are having success rejuvenating T cells in the test tube.”

CAR T cell therapy is currently attracting attention as a treatment for blood cancers. In this therapy, T cells are harvested from the patient's blood, and a gene called CAR, which recognizes cancer, is introduced into the T cells in a test tube, after which they are returned to the patient’s body.

“CAR T cell therapy works well in children and young adults with high levels of healthy immune cells. This is because they have many healthy, young T cells that have just been born, and by reintroducing them to the body with improved cancer recognition, they are effective in fighting cancer. However, in the elderly, immune cells are also aging, and their CAR T cells are not as effective. Although the terms ‘aging’ and ‘exhaustion’ are different, the underlying mechanism is similar. If you try to use aged T cells, they quickly become exhausted after introducing the CAR gene.”

Professor Yoshimura and his colleagues are conducting experiments to restore aged T cells to their young state.

“Although not exactly the same as the original young cells, we are now able to confirm that exhausted cells can be rejuvenated in vitro. We have succeeded in rejuvenating T cells by adding several external factors such as cytokines and growth factors. Although we have yet to successfully rejuvenate T cells inside the human body, we believe that treatment will become even more effective if we can rejuvenate them as they proliferate outside the body in combination with CAR T cell therapy.”

Immune function, initially known for fighting infectious diseases, is now understood to be deeply involved in cancer, and Professor Yoshimura and his colleagues have further demonstrated that immunity is also involved in brain diseases, including stroke.

“It began when a medical student was looking at a stroke patient. The patient was not improving, so the student came to my laboratory to learn more about the mechanisms of stroke and apply that knowledge to devise a treatment.”

When Professor Yoshimura's laboratory—which makes extensive use of immune-related model mice—investigated the relationship between immunity and stroke, they discovered something unexpected.

“Mice that did not express certain cytokines were less likely to have a stroke , which was surprising. A stroke is simply damage, or injury, to the brain, in which a blood vessel becomes blocked, and the brain tissue dies. And yet, for some reason, certain immune system cytokines were making the stroke condition better or worse.”

Since then, Professor Yoshimura and his colleagues have continued their research and explained the inflammatory process after a stroke, focusing on macrophages of the immune system.

“This process occurs when tissue breaks down and dies anywhere in the body, not only brain tissue, but also in the lungs and kidneys, and wherever cancer grows. Stroke, caused by a clogged blood vessel, is probably one of the most common causes of this kind of damage. An immune response occurs even when physical trauma is sustained in a car accident. What happens is that when the body sustains a wound, the tissue dies, and immune cells gather in that location.”

“We have found that this has both beneficial and harmful effects. In the acute phase, immediately after stroke onset, inflammatory macrophages infiltrate the stroke site, recognize dead cell material, and release inflammatory cytokines to promote inflammation. This process promotes neuronal death. The inflammatory response then subsides in approximately one week, after which macrophages switch to having tissue-repairing properties to remove inflammatory substances and further promote regeneration of the nervous system.”

Furthermore, Professor Yoshimura and his colleagues found that in the chronic phase beginning more than two weeks after the stroke onset, regulatory T cells accumulate in large numbers in the brain and play an essential role in improving neurological symptoms. The detailed mechanism of this process was published in Nature in 2019. It has become clear that when a stroke or other condition results in physical tissue damage, the immune system plays an important role in tissue damage and repair, including in the brain.

His research on cytokines has revealed previously unknown features of the immune system. Although Professor Yoshimura is now known worldwide as an expert in immunology, it was not until after university and a career as a researcher that he began to study immunology in earnest.

“When I was a student, immunology was still a chaotic and complicated subject. I was a molecular biology and biochemistry researcher who specialized in signal transduction. I had no idea that I would end up shifting to immunology."

As a young researcher, Professor Yoshimura had a reputation among colleagues for having "masterful (or “god-like,” in Japanese) hands," which is an industry phrase to describe one who has mastered the art and science of experimentation. In his early thirties, Professor Yoshimura traveled overseas to improve his research abilities. In months, he completed experiments that would typically take a year or more, more than once succeeding in uncovering the genes he sought. These super-human feats earned him a reputation among researchers, so we asked him about the secret to his success.

“When I was young and starting my molecular biology experiments, I was forced to teach myself experimental techniques because the field was different from what I had studied in graduate school. I figured out the principles that usually lead to successful experiments through repeated trial and error. These four principles are (1) positive controls, (2) negative controls, (3) experimental value, and (4) hard work .”

“Positive and negative controls are controls to be compared with the target of the research. You should prepare and test the results of the positive and negative controls together in the experiment. The third principle of experimental value is understanding the purpose of your experiment. Quite often, students cannot answer this question when asked. If you understand why you are doing what you are doing and how it fits in your overall research, then I think you can conduct a proper experiment.”

“When I was working in the US as a postdoctoral fellow, a friend told me a story about his father, biochemist Dr. Kaji, who became a professor in the US after graduating from the University of Tokyo. He said that his father had told him the three principles needed to become a decent researcher were ‘positive controls, negative controls, and the experimental value.’ When I heard this, I realized that this principle is true. As Professor Kaji’s words suggest, researchers who fail in their experiments usually cut corners somewhere. I am constantly telling students who fail their experiments that they must include positive and negative controls.’ I understand that it can be a hassle, but it is important to do the work without cutting corners.”

“The fourth principle, hard work , means that ordinary people like myself have to work that much harder and longer to reach the same level as those who are highly gifted. I was aware that I wasn’t exceptionally bright and that my only merit was that I was good at experiments, so when I was young, I worked hard in the laboratory from morning till night and only went home to sleep.”

So Professor Yoshimura reveals his secret to success and how he became an immunology researcher unlocking the mysteries of the immune system.

“Stroke and cancer may seem unrelated, but in my mind, they are connected by immunity. We now know that immunity is deeply connected to almost every field of medicine. Moving forward, I hope to conduct further research in areas where immunity may have traditionally been overlooked in hopes of shedding more light on the hidden world of immunity.”

Akihiko Yoshimura

Professor Yoshimura graduated from the Faculty of Science at Kyoto University in 1981. He obtained a master’s degree from the Graduate School of Science at Kyoto University in 1983 and received his Ph.D. in 1985. After working at the Oita Medical University Department of Chemistry, he became an assistant professor at the Cancer Research Institute at Kagoshima University’s School of Medicine in 1987. After training as a postdoctoral fellow at the Massachusetts Institute of Technology, he became an associate professor at the Kagoshima University School of Medicine Cancer Research Institute in 1989 before serving as a professor at the Kurume University Institute of Life Science in 1995 and a professor at the Kyushu University Medical Institute of Bioregulation in 2001. He assumed his current position in 2008. In 2021, he was awarded the Uehara Prize and Japan’s Medal of Honor with Purple Ribbon.