SAN FRANCISCO—The immune system faces a delicate balancing act: it must be aggressive enough to fight infections and cancer, yet restrained enough to avoid attacking the body's own tissues.
More than two decades ago, researchers identified a gene called FOXP3 as playing a critical role in maintaining this balance and preventing autoimmune disease—work that garnered this year's Nobel Prize in Physiology or Medicine.
Now, scientists at Gladstone Institutes and UC San Francisco (UCSF) have mapped the intricate network of genetic switches that immune cells use to fine-tune levels of FOXP3. Their findings, published in Immunity, have important implications for developing immune therapies and address a long-standing mystery about why this gene behaves differently in humans than in mice.
"FOXP3 is absolutely essential for regulating our immune systems," says Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology, who led the study. "How it's controlled is a fundamental question of immunology, and the detailed answer could offer clues to develop future therapies for autoimmune diseases or cancer."
A Search for Dimmer Switches
The gene FOXP3 is active in all regulatory T cells, which keep immune reactions in check. Without this gene, regulatory T cells cannot function properly and the immune system spirals out of control, attacking the body's own tissues. People with mutations in FOXP3 develop rare and severe autoimmune diseases.
In mice, FOXP3 is only switched on in regulatory T cells. But in humans, conventional T cells—the inflammatory cells that fight infections—can also briefly activate FOXP3. This difference has puzzled immunologists for years.
In the new work, Marson's lab used CRISPR-based gene silencing technology to systematically test 15,000 sites in the DNA surrounding the gene FOXP3. They were looking for genetic regulatory elements—nearby stretches of DNA that act like dimmer switches, controlling when and how much a gene is turned on or off.
By disrupting thousands of locations in both human and mouse regulatory and conventional T cells and then measuring effects on FOXP3 levels, the team identified which nearby DNA sequences control FOXP3.
"We essentially created a functional map of the entire FOXP3 control system," says Jenny Umhoefer, PhD, a former postdoctoral fellow in Marson's lab and first author of the new paper.
Immune Control Panels
The experiments revealed that different human cell types have different control systems for the gene FOXP3. In regulatory T cells, where FOXP3 must remain constantly active, multiple enhancers—DNA sequences that boost the levels of a gene—work together to ensure the gene stays on. Because they work redundantly, disrupting just one of those enhancers had only a small effect on FOXP3 levels.
In conventional T cells, only two enhancers were mapped. But the researchers also discovered an unexpected repressor that acts as a brake on the FOXP3 gene.
"What we're seeing is a sophisticated regulatory circuit," Umhoefer says. "The cell has gas pedals and brakes, and it coordinates them to achieve precise control."
To understand not just where these genetic switches are located, but also what controls them, the team conducted a second massive CRISPR screen. This time, they systematically disrupted nearly 1,350 genes throughout the genome to identify specific proteins that control FOXP3 levels.
Then, working with Gladstone Affiliate Investigator Ansuman Satpathy, MD, PhD, the team used a technique called ChIP-seq to physically map where the proteins are located on the DNA in relation to the FOXP3 gene.
"This was a big step forward in developing ways to link the local regulatory elements with the proteins that actually bind to them," says Satpathy, who is also an associate professor in the Department of Pathology at the Stanford School of Medicine. "No one had put together these tools in such a broad, systematic way before."
A Species Mystery
Marson's lab had initially hypothesized that in humans, conventional T cells may have an enhancer to turn on FOXP3 that is missing in mice, explaining why the mouse cells never flip the gene on. Surprisingly, they found that conventional T cells in mice have all the same enhancer elements as humans.
The difference, the scientists realized, may lie in the repressor they discovered. In mouse conventional T cells, this repressor keeps FOXP3 constantly off. When the researchers used CRISPR to delete the repressor from mice DNA, the conventional T cells began to express the FOXP3 gene like human cells.
"This was a striking result," Marson says. "By removing a single repressive element, we could break the species difference and enable conventional T cells in mice to express FOXP3. This offers new hints as to how regulation of key genes might evolve across species."
The findings point to the importance of studying gene regulation in human cells, and underscore the need to look broadly for repressors—not just the more common enhancer elements.
Precision Cell Engineering
The new study provides a foundation for ongoing efforts to discover and develop new treatments for a range of diseases. Armed with a full map of the different elements involved in controlling the levels of the FOXP3 gene, researchers can begin to develop new ways of tweaking these levels for immunotherapies.
Treatments for autoimmune diseases, for instance, may benefit from increased levels of FOXP3, while treatments for cancer may work better with lower FOXP3 activity.
"There are enormous efforts right now to drug regulatory T cells, either to promote their activity or reduce it," Marson says. "As we understand new aspects of the circuitry that distinguishes regulatory T cells from conventional cells, we can think about strategies to rationally manipulate it."
