Aging Helper T Cells Gain New Life as Regulatory Agents

There is a Chinese saying, “When things reach an extreme, they reverse to their opposite.” HMS researchers, guided in part by this adage, have discovered a remarkable turnabout: they found that a cell responsible for initiating the immune response can turn around and quell that response when the inflammatory fire gets too big or lasts too long.

The conversion—from helper T cell to regulatory T cell—entails a remarkable change in the cell’s appearance and activity. T helper cells are recognized by an array of cell surface markers, most notably the CD4 protein. Xin Xiao Zheng, Dong Zhang, and colleagues found that cells converted to a regulatory way of life stop expressing CD4.

Even more striking is the change in behavior. Helper T cells typically work by summoning other immune cells to destroy pathogens or cancer cells. Yet it appears that the regulatory cells develop a taste for killing. They start expressing high levels of cytotoxic proteins that they appear to direct at their non-converted helper T cell cousins. Zheng, HMS assistant professor of medicine at Beth Israel Deaconess Medical Center, Zhang, research fellow in medicine, and their colleagues found that in the presence of the converted cells, activated helper T cells exhibited high levels of cell death. Intriguingly, the converts, who are recruited from the oldest members of the helper T cell population, resist apoptosis.

The findings, which appear in the Dec. 29, 2006, Blood, have exciting implications. Immunologists have labored to find ways to control unwanted inflammation that occurs in autoimmune disease and transplant rejection. One approach may be to raise an army of regulatory converts. Zheng, Zhang, and colleagues have already taken a step in that direction. They converted helper T cells into regulatory cells, then transplanted the converts with skin grafts into mice. They performed similar experiments with pancreatic islet cells. In each case, the mice accepted the transplants, even though skin and islet cells are notoriously susceptible to immune attack.

Researchers have known for some time that inflammatory fires are lit and then put out by a variety of homeostatic mechanisms. Immune cells, such as helper T cells, appear to burn themselves out when an inflammatory response has done its work. CD4 cells have been observed to undergo apoptosis after four or five rounds of replication. The T cells may also be actively suppressed by external agents, the regulatory T cells. This immune contingent has attracted a great deal of attention, not least for its potential therapeutic value, but much about the cells remains mysterious. Several types have been identified, but little is known about how they arise in the immune system. Certainly, no one had observed them developing from aging helper T cells.

That is, essentially, what Zheng, Zhang, and colleagues have found—and they did so thanks, they say, to a combination of careful science, luck, and cultural perspective.

“Chinese philosophy was helpful—it provides a guide,” Zheng said. “You do not just expect a cell to be one thing or another, that a CD4-postive cell is always positive or a CD4-negative cell is always negative. You have the idea that things can be converted.”

The researchers did not set out to contradict the prevailing view. “It was an accident,” said Zhang. Their initial goal was quite practical. They wanted to spur proliferation of a well-known regulatory T cell, the so-called CD4+CD25+ T cells. “We thought there might be clinical applications for these cells,” he said.

D4+CD25+ T cells—the first regulatory T cells ever found—originate in the thymus as CD4+ T cells and are activated in the periphery. In vitro, the regulatory T cells proliferate when they encounter a particular class of antigen-presenting cell. Zheng and colleagues wanted to see if they could increase this proliferation by adding growth factors.

Zhang began by mixing highly purified CD4+ cells with the special antigen-presenting cells; to some samples, he added the growth factor IL2 or IL15. He had only run a couple of experiments when he noticed something strange. “Dong came to me and said, ‘We’ve got something nobody else has described—it looks like some of the proliferating CD4 cells are losing CD4 expression,’” Zheng said.

The researchers reran their experiments. If the CD4– cells were contaminants, they should have been evident almost immediately. In fact, they showed up only on day four, after a burst of proliferation. And the loss of the CD4 marker appeared only on cells that had already undergone the requisite four or five rounds of proliferation. That left an intriguing possibility: might the CD4– cells be a product of the proliferating helper T cells?

To get a better picture of the cells, the researchers examined the cell surface markers. There was no evidence of the CD8 marker, usually expressed on killer T cells. The reason this was significant is that in 2000, a Canadian researcher had identified a regulatory T cell that expressed neither the CD4 nor CD8 markers. But she had used CD8+ transgenic mice in her experiments, which meant the regulatory cells were derived from CD8+ cells. Zheng and colleagues began their experiments with purified CD4+ cells. To distinguish their new CD4–CD8– regulatory cells, Zhang decided to call them CD4+ T cell–converted double-negative (DN) cells.

Still, it was not clear how the double-negative cells arose or what they did. A gene expression panel indicated that the CD4 gene had, indeed, been silenced, but the mechanism remained a mystery. Also puzzling, an apoptosis-revealing stain showed that the cells were not dying, despite their age. Yet the death toll among non-converted helper T cells appeared to increase when cultured with the converts.

The researchers looked at what other genes were expressed by the double-negative cells and found an important clue. The cells had apparently turned down the production of cytokines, the signals normally employed by helper T cells, and turned up the expression of two cytotoxic enzymes, perforin and granzyme B. To see if the converts were using the enzymes to kill helper T cells, and in this manner control inflammation, the researchers repeated their experiments, this time using CD4+ cells taken from a perforin-knockout mouse. In culture, the perforin-deprived double-negative cells did not appear to have as great an effect on the helper T cells, which exhibited lower levels of apoptosis, presumably because they were not being damaged by perforin.

To test the converts’ mettle in a living animal, the researchers raised an army of them and introduced them, along with skin tissue from a donor mouse, into a recipient mouse. The grafts were protected, but only when the double-negative recruits had been trained to recognize antigens specific to that tissue. Skin transplants from a third mouse were rejected. The scientists then tried a similar experiment, this time using a single, relatively small dose of double-negative cells and pancreatic islet cells, which they introduced into diabetic mice. The small infusion led to prolonged and, in some cases, permanent engraftment of the islets.

Zheng hopes to move the discovery into humans. “We envision that healthy people might make a cell bank for themselves, for their future,” he said. “I am healthy now so if I convert some of my cells to double-negative now, there is no health impact. In the future, if I need immune suppression, I could take my own cells from the cell bank and have them put back into me.”