Scientists at the Gladstone Institutes, in collaboration with researchers at the University of California, San Francisco (UCSF), and the Technical University of Munich (TUM), report that they say have mapped out the networks of genes that help differentiate regulatory T cells from other T cells. Their findings could lead to immune therapies that strengthen or weaken the function of regulatory T cells, according to Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology, and a senior author of the study.

“Piecing together the genetic networks that control the biology of regulatory T cells is a first step toward finding drug targets that change the function of these cells to treat cancer and autoimmune diseases,” he says referring to the study “Functional CRISPR dissection of gene networks controlling human regulatory T cell identity,” which appears in Nature Immunology.

“Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions,” write the investigators.

“We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell–mediated immunosuppression.”

“By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.”

Studies in mice have suggested that increasing the number of regulatory T cells—and therefore putting stronger “brakes” on the immune system—might help subdue symptoms of autoimmune diseases. On the other hand, blocking regulatory T cells, or lifting these molecular brakes, is suspected to help the immune system better fight cancer.

Therapies that boost populations of regulatory T cells—by removing the cells from patients’ bodies, expanding them, and infusing them back in—are already being tested in people with autoimmune disease, including type 1 diabetes, and organ transplant recipients. So far, however, such treatments generally haven’t involved actually altering the function of the immune cells.

“Most of our previous knowledge about regulatory T cells is from mouse models,” says Kathrin Schumann, PhD, a co-first and co-corresponding author of the paper and former UCSF postdoctoral fellow, now an assistant professor at the Technical University of Munich. “We wanted to genetically dissect human regulatory T cells to better understand how they’re wired and how we can manipulate them. Once we understand the functions of each gene, we can precisely edit cells to treat disease.”

In the new study, Marson, Schumann, and their collaborators used CRISPR-based gene-editing technology to alter regulatory T cells, selectively removing any of 40 different transcription factors. The 40 transcription factors were chosen because previously published data had already hinted that they might perform specific functions in the regulatory cells compared to other T cells.

The researchers then focused on the 10 transcription factors that had the strongest effect in this initial screen, and looked across tens of thousands of genes to see which ones were turned on or off in the altered cells. In all, they performed this analysis on 54,424 individual regulatory T cells.

By analyzing the subsets of genes activated or silenced by these 10 original transcription factors, the team put together vast networks of genetic programs involved in the biology of regulatory T cells. Among the most surprising results, the study revealed that the little-studied transcription factor HIVEP2 has a strong effect on regulatory T cell function. In follow-up studies in mice, the scientists found that removing the HIVEP2 gene reduced the ability of the regulatory T cells to quell inflammation.

“This was a significant hit,” said Sid Raju, a co-first author of the paper and former UCSF computational biologist who is now a graduate student at the Broad Institute of MIT and Harvard. “This gene had really never been implicated in regulatory T cell biology before.”

The team also says their study acts as a proof-of-principle for how powerful the combination of CRISPR gene editing and the analysis of individually edited cells can be in studying the genetics of human biology and human disease.

“Now, we can theoretically take any specialized cell from the body and start removing individual genes and study the consequences on the cells in much finer detail than ever before,” says Marson. “This really opens up human cells removed from the body as a tractable experimental system.”

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