Using new technologies to study thousands of genes simultaneously within immune cells, researchers at the Gladstone Institutes, UC San Francisco (UCSF) and the Stanford School of Medicine have created the most detailed map yet of how complex networks of genes work together. New insights into how these genes relate to one another sheds light on basic factors in immune cell function and immune disease.
“These results help us to develop a systematic network map that can serve as a how-to manual on how human immune cells work and how we can engineer them for our benefit,” says Alex Marson, MD, PhD, director of the Gladstone Institute -UCSF Professor of Genomic Immunology and co-senior author of the new study, published in Nature genetics.
The study, conducted in collaboration with Jonathan Pritchard, PhD, professor of genetics and biology at Stanford School of Medicine, is also critical to better understanding how variations in a person’s genes are connected to risk for autoimmune diseases.
CRISPR immunological insights
Researchers know that when the immune system’s T cells — white blood cells that can fight infections and cancer — are activated, the levels of thousands of proteins within the cells change. They also know that many of the proteins are interconnected in such a way that changes in the level of one protein can cause changes in the level of another.
Scientists represent these connections between proteins and genes as networks that look like a subway map. Mapping these networks is important because they can help explain why mutations in two different immune genes can lead to the same disease or how a drug can impact many immune proteins at once.
In the past, scientists have mapped a part of these networks by removing the gene from each protein one at a time and studying the impact on other genes and proteins, as well as the overall function of immune cells. But this kind of “downstream” approach reveals only half the picture.
“We really wanted to see what’s controlling key immune genes,” says Jacob Freimer, PhD, a postdoctoral fellow in the Marson and Pritchard labs and first author of the new paper. “This kind of upstream approach has not been done before in primary human cells.”
This upstream approach would be like mapping subway routes by first identifying the main hubs and then figuring out the routes to those key stations, rather than painstakingly reconstructing the entire network from different satellite stations.
Freimer and his collaborators turned to the CRISPR-Cas9 gene editing system, which allowed them to disrupt thousands of genes at once. They focused on genes that produce a type of protein known as transcription factors. Transcription factors are the switches that turn other genes on or off and can control many genes at the same time. The scientists then studied the impact of disrupting these transcription factors on three immune genes known to play an important role in T cell function: IL2RA, IL-2 and CTLA4. These three genes were hubs that anchored upstream mapping efforts.
This allows us to analyze over a thousand transcription factors and see which ones impact these immune genes.”
Jacob Freimer, PhD, first author
an interconnected network
The researchers suspected they would find connections between the genes that regulate IL2RA, IL-2 and CTLA, but were surprised by the extent of connectivity they discovered. Among 117 regulators found to control the levels of at least one of the three genes, 39 controlled two of the three, and 10 regulators simultaneously altered the levels of all three genes.
To help further populate the immune gene map, the team took a more traditional downstream approach, removing 24 of the identified T-cell regulators to show the full list of genes they regulate; in addition to IL2RA, IL-2 and CTLA4.
The researchers showed that many of the regulators controlled each other. The transcription factor IRF4, for example, altered the activity of 9 other regulators and was regulated by another 15 regulators; all 24 controlled levels of IL2RA. In other cases, the regulators themselves were regulated by the IL2RA, in so-called “feedback loops”.
As in a dense subway network, each hub was connected to many others, and the connections worked both ways.
“There were cases where a transcription factor regulated IL2RA, but IIL2RA itself also controlled that same transcription factor,” says Freimer. “It appears that these types of feedback loops and regulatory networks are much more interconnected than we previously thought.”
Back to Patients
Among the complete list of genes controlled by the regulators studied, the research team found a large number of genes already linked to immune diseases, including multiple sclerosis, lupus and rheumatoid arthritis.
The new map helped reveal how the genetic changes associated with these diseases can appear in different genes, but – because of the regulatory connections between the genes – end up having the same net effect on cells. It also points to key groups of genes that could be targeted by drugs to treat immune disorders. The study suggests that there is a core network of important genes, and when this network is disrupted, it can increase a person’s risk of disease.
“When we understand the ways in which these networks and pathways are connected, it starts to help us understand the key collections of genes that need to function properly to prevent immune system disease,” says Marson.
Freimer, JW, et al. (2022) Systematic discovery and disruption of regulatory genes in human T cells reveals the architecture of immune networks. Nature Genetics. doi.org/10.1038/s41588-022-01106-y.