Regulatory T cells (Tregs) are the immune system’s peacekeepers. They suppress excessive immune responses to prevent autoimmune diseases and harmful inflammation. Because of this, Tregs are being developed as living medicines in adoptive cell therapy: isolating and engineering a patient’s own Tregs and infusing them back to dampen unwanted inflammatory and autoimmune reactions. For next-generation Treg therapies, it is essential to understand how Tregs respond to inflammatory signals (such as those released during infections) and how these signals interact with the T cell receptor (TCR), which tells the cells when and where to act.
This project focused on the effects of type I interferons, powerful alarm signals produced during inflammation.
Aim 1: What does type I IFN do to Tregs?
Earlier studies suggested that inflammatory signals like IFN-I “destabilize” Tregs, causing them to lose their identity (marked by the protein FoxP3) and their ability to suppress inflammation. However, when we used very pure human Tregs (isolated using the marker GPA33), we found the opposite: IFN-I (even together with the inflammatory cytokines IL-1 and IL-6), did not cause loss of FoxP3 or suppressive capacity and the Tregs did not start producing inflammatory cytokines like IL-2 or IFNγ.
The difference with earlier studies seems to come down to purity. Impure Treg populations (contaminated with ordinary T cells) showed overgrowth of unstable cells that lost control and produced inflammatory signals. Pure, genuine Tregs proved remarkably stable.
Type I IFN did have two clear effects:
• It strongly inhibited Treg proliferation (slowing their multiplication). This matches mouse studies showing that blocking IFN-I signaling in Tregs leads to excessive Treg numbers and poor control of infections or cancer.
• It increased expression of cell surface proteins that help Tregs migrate to inflamed tissues.
Conclusion: Type I IFN has mixed effects. It temporarily brakes Treg expansion early in an infection (allowing a strong protective immune response) but equips Tregs to travel later to sites of inflammation for tissue repair and damage control. If the negative (anti-proliferation) and positive (migration) effects can be separated at the molecular level, it could help create more potent therapeutic Tregs for patients.
Aim 2: How is type I IFN signaling wired inside Tregs?
Using phospho-proteomics and RNA sequencing, we mapped how IFN-I changes protein phosphorylation and gene expression in Tregs compared with conventional T cells.
IFN-I activated expected JAK-STAT pathways but also triggered signals linked to cell-cycle arrest, DNA damage responses, and tumor suppressors (p53, PML), while suppressing genes that drive cell division (Myc targets). This likely explains the observed block in proliferation.
An interesting candidate was the chromatin modifier EP400. A previously unknown phosphorylation site on EP400 was induced by TCR stimulation plus costimulation but prevented by IFN-I. EP400 helps control cell-cycle genes such as Myc and E2F1. Technical challenges prevented full functional testing of EP400 (antibody generation failed and shRNA knockdown was unsuccessful), but CRISPR tools for editing primary human Tregs were successfully developed.
Aim 3: Does the IKZF1-STAT4-FoxP3 axis protect Tregs from destabilization?
Tregs normally express very little STAT4. Forcing high STAT4 makes them vulnerable to IFN-I-induced destabilization. We originally hypothesized that the transcription factor Ikaros (IKZF1) keeps STAT4 levels low in Tregs, thereby protecting their stability. Treatment with lenalidomide (which degrades Ikaros) caused only modest effects and no Treg destabilization. CRISPR knockout experiments are planned to clarify Ikaros function.
Additional results
• TCR signaling differences: Tregs show generally weaker TCR signaling than conventional T cells, especially in the Ras/MAPK pathway. This presumably helps prevent IL-2 production and ensures Tregs only fully activate suppression in response to high-affinity antigens.
• TNF signaling: Engagement of TNFR-II enhanced aspects of TCR signaling strength, possibly explaining its ability to costimulate Treg prolifration. As with the other inflammatory cytokines tested, engagement of TNFR-II did not destabilize genuinely pure Tregs.
• We developed advanced CRISPR methods (including safe TCR knock-in) that will allow engineering of more effective therapeutic Tregs.
Overall Takeaway
Pure human Tregs are more stable than previously thought when exposed to inflammatory cytokines. Type I IFN does not break them but temporarily slows their expansion while preparing them for migration to inflamed sites. The project revealed key molecular differences between Tregs and other T cells in their responses to inflammation and TCR signals. These insights, together with newly developed gene-editing tools, bring us closer to designing smarter, more potent Treg therapies for autoimmune diseases and transplant rejecton.