Regulatory T Cells Tactful in Controlling Killer Cousins

On Video, Regulators Prevent Cytotoxic T Cells from Releasing Poison Vesicles

The thymus is a harsh mistress of an organ. Millions of nascent T cells enter the twin-lobed structure and yet the vast majority will die there. Only those that have learned the thymus’s crucial lesson—attack invaders while doing no harm to the body’s own tissue—have a chance of leaving as full-fledged T cells. Occasionally self-reactive T cells do escape and travel to the far reaches of the body. Thirty years ago, a pair of researchers proposed that these renegade cells are intercepted and subdued by a population of suppressor cells. Researchers raced to find the mysterious cells, but came up empty-handed.

Photo by Graham Ramsay

“Our study highlights one way that regulatory T cells blunt the immune response,” said Thorsten Mempel (front left). “We are not saying this is the exclusive way. But under certain circumstances, it is a very important means of regulation.” He is shown with co-authors Mikael Pittet (back left) and Ulrich von Andrian (front right). Roberto Bonasio (back right), a graduate student in von Andrian’s lab, is working on the role of dendritic cells in the immune response.

“After years, and shelves full of publications on suppressors, some people conducted a critical review of the literature and said what many thought and did not dare to say, which is basically, there is no good evidence,” said Ulrich von Andrian, the Edward Mallinckrodt Jr. professor of immunopathology at HMS. The whole concept of a suppressor cell fell into disrepute until the mid-1990s when a researcher identified a CD25-bearing T cell that stopped self-reactive T cells from attacking the body. A decade of intense scrutiny later, regulatory T cells remain enigmatic. It is still not clear how exactly they keep self-reactive T cells from killing. In vitro experiments suggested they stop the cells from proliferating, but recent studies on living animals contradict this idea. Cytotoxic T cells kill by embracing their prey and shooting them with poison. Some researchers speculated that regulatory T cells might be stopping the cells from making their toxin.

The arrest appears to be more subtle. Thorsten Mempel, Mikael Pittet, von Andrian, and their colleagues observed—for the first time—individual cytotoxic T cells attacking their prey. They found that in the presence of regulatory T cells, the poison is made and loaded into dartlike granules, but the granules are not released. They simply remain in the cellular quiver. These findings appear in the July 29 Immunity.

Under Wraps
One reason regulatory T cells have remained so mysterious is that they are hard to isolate and observe. Cytotoxic T cells are also elusive. “No one has been able to see cytotoxic killing in a living animal at the single cell level,” said von Andrian. Over the past few years, Mempel, HMS research fellow in pathology, and colleagues in the von Andrian lab have developed a method for fluorescently staining, visualizing, and filming immune cells in the lymph nodes of living organisms (see Focus Jan. 23, 2004). Using a palette of red, green, and blue fluorescent protein, Mempel and Pittet compared the activity of cytotoxic T cells in the presence and absence of regulatory T cells.

In the absence of regulation, as videos show, green-stained cytotoxic T cells approach and latch on to blue-red prey, in this case, antigen-bearing B cells. Once paired, they roll around, often furiously, as though engaged in a wild dance. At some point, the blue-red changes to blue-white. “That is the kiss of death—the B cell was just killed,” said Mempel. (The videos are available at http://cmir.mgh.harvard.edu/cip/movies.)

In the presence of regulatory T cells, the cytotoxic T cells and B cells engage in the same frenzied polka, but the color of the B cells does not change. “What’s missing is the lytic event, the kiss of death,” said Pittet, HMS assistant professor of radiology. Once the regulatory T cells are removed, the killing starts again. “So it is not something that is imprinted on the cells and sticks with them,” said Pittet, who is at the Center for Molecular Imaging Research at MGH, headed by HMS professor of radiology Ralph Weissleder.

This reversibility means that regulatory T cells could be used to tip the balance between tolerance and immunity. By activating them, one might prevent autoimmune disease. Blocking their activity could enhance the immune response to pathogens or tumors. It is not surprising that drug companies have been pushing to harness their power. Though such efforts are promising, results of at least one recent attempt suggests that regulatory T cells may not be so easily domesticated. A drug designed to quell arthritis and other autoimmune disease by activating regulatory T cells produced the opposite effect in clinical trials—low levels of regulatory T cells and devastatingly high levels of autoimmune activity, which caused serious illness in several subjects.

Out on a Limb
The current project grew out of an interest in using the immune system to fight cancer. Pittet, with Khashayarsha Khazaie, HMS assistant professor of radiology, and Harald von Boehmer, HMS professor of pathology, both at the Dana–Farber Cancer Institute, found that tumors implanted in the foot pads of mice were rejected in the absence of regulatory T cells, but not in their presence. Based on previous experiments, they thought the regulatory T cells might be keeping cytotoxic T cells from expanding their numbers. In fact, the regulated cytotoxic T cells were proliferating normally, but they were not killing the tumor cells. Along with Mempel, Pittet set out to determine why.

Image courtesy of Mikael Pittet The kiss of death. A cytotoxic T cell (top, green) leaves its victim after delivering poison. The targeted B cell has burst and, in its death throes, is leaking out cytoplasm (the cell is still red because it has not yet lost all of its contents). At bottom, another cytotoxic T cell grabs hold of a still living B cell (purple) and delivers its poison. Videos can be seen at http://cmir.mgh.harvard.edu/cip/movies.

The researchers introduced tumor cells, specially engineered to bear the hemagglutinin antigen (HA), into the mouse foot pads. Next, they gave the mice a transfusion of green fluorescent cytotoxic T cells, equipped to recognize the HA antigen. Having met their antigenic match, the T cell population expanded and differentiated over the course of the following week. Ideally, the researchers would have watched the immune cells attacking the actual tumor cells, but that kind of visualization is not yet possible. Mempel and Pittet decided to focus their intravital approach on the tumor-draining lymph node behind the knee. They surgically exposed the node and trained their multiphoton microscope on it at the same time that they introduced a population of HA-bearing B cells, essentially stand-ins for the HA-bearing tumor cells. Then they watched to see whether the surrogate target cells, stained blue-red, were killed.

Observing that the B cells’ color did not change in the presence of regulatory T cells, the researchers wanted to know why. “Our first speculation was that the cells are less equipped with the cellular tools to kill a cell,” said Mempel. To destroy their prey, cytotoxic T cells must make toxin and load it into vesicles, or granules, that then travel to the cell surface and are released. By staining a marker found on the granules, the researchers were able to watch the process of degranulation occur in the presence and absence of regulatory T cells. In their absence, the granules could be seen forming and eventually approaching and fusing with the cell membrane. In the presence of regulatory T cells, they stayed buried below the surface. “That is where we found a big difference—that was the aha moment,” Mempel said. “It was one of those experiments we did very late at night.”

The pair may have more midnight-oil experiments ahead of them. They do not know yet how regulatory T cells stop the poison packets from surfacing. Nor is it clear what is turning on the regulatory cells in the first place. It is known that they require TGF-beta to survive. “Tumor cells make copious amounts of TGF-beta,” said Pittet, which raises the unsettling possibility that cancer cells may be using regulatory T cells to hide from the immune system. “Tumor cells seem to be quite good at turning on regulatory T cells,” he said. What this means is that drugs designed to activate regulatory T cells for the purpose of dampening autoimmune disease could put patients at higher risk for cancer.

“It will be crucial to understand more of the biology of regulatory cells in order to better predict what kind of therapeutic regimen will be successful in a clinical trial,” said Mempel. “You know something is powerful, you try it in patients, you see it is not working, you don’t know why. You have to better understand the biology. Our study is hopefully a small contribution to that.”

—Misia Landau

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