---
title: "Distal IRF4 Regulatory Element Enables Inflammation-Driven Destabilization of Human Regulatory T C"
id: "nature-immunology-0-deletion-of-a-distal-irf4-element-prevents-inflammation-induced-reprogramming"
canonical_url: "https://medichelpline.com/clinical-feed/nature-immunology-0-deletion-of-a-distal-irf4-element-prevents-inflammation-induced-reprogramming"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Nature Immunology"
source_url: "https://www.nature.com/articles/s41590-026-02655-8"
published_at: "2026-09-21T11:53:43.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Distal IRF4 Regulatory Element Enables Inflammation-Driven Destabilization of Human Regulatory T C
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/nature-immunology-0-deletion-of-a-distal-irf4-element-prevents-inflammation-induced-reprogramming
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Nature Immunology
- **Source URL:** [Original Journal Publication](https://www.nature.com/articles/s41590-026-02655-8)
- **Published At:** 2026-09-21T11:53:43.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Researchers developed an in vitro model in which primary human CD4+ CD25+ CD127lo/− **regulatory T (T reg)** cells were repeatedly stimulated via CD3/CD28 in the presence or absence of inflammatory cytokines to test lineage stability. - A triple cytokine combination (IL-6, IL-1β and IL-23) most consistently induced progressive loss of **FOXP3** and **HELIOS**, reduced suppressive function, increased proliferation and acquisition of proinflammatory cytokine production (IFN-γ, TNF, IL-17A). - Loss of FOXP3 and HELIOS was dose- and duration-dependent and persisted after transient exposure, indicating a durable cell-state transition rather than short-term repression. - FOXP3 TSDR (CNS2) methylation analysis showed HELIOS+ cells were largely demethylated (stable T reg), whereas FOXP3− HELIOS− cells exhibited partial remethylation consistent with a transition state rather than expansion of contaminating conventional T cells. - Destabilized T reg cells downregulated canonical T reg markers (CD25, CTLA-4, ICOS, TIGIT) and upregulated markers associated with dysfunction (CD226, CD40LG), with impaired in vitro suppression of responder T cell proliferation. - Single-cell multi-omic profiling (chromatin accessibility + transcriptomics) identified distinct clusters; destabilized clusters showed reduced FOXP3/HELIOS expression and increased expression of proinflammatory or cytotoxic genes (IFNG, TNF, IL17A, NKG7, GZMB, GZMA). - Chromatin changes matched transcriptional changes (loss of accessibility at FOXP3 CNS2; gain at IFNG and GZMB loci) and revealed open chromatin regions (OCRs) unique to destabilized T reg cells, indicating epigenetic reprogramming distinct from conventional T cells. - Motif enrichment in destabilized cells showed depletion of FOXP3 and bHLH E-box motifs and enrichment of **AP-1**, RUNX, ETS and **IRF4** motifs, suggesting cooperative AP-1–IRF4 activity contributes to destabilization. - A putative distal regulatory OCR at the **IRF4** locus became more accessible and its accessibility correlated with increased IRF4 expression during destabilization; excision of this distal element reduced susceptibility to cytokine-induced reprogramming. - Conversely, forced expression of IRF4 together with BATF promoted T reg cell destabilization, implicating an AP-1–IRF4 axis as a critical driver. - The study identifies a distal IRF4 regulatory element as a potential target to engineer more stable T reg cells for cellular therapies, while acknowledging that some experimental detail and extended mechanistic descriptions beyond the reported findings were not provided in the source.
## Clinical Analysis & Structured Key Points
Deletion of a distal IRF4 element prevents inflammation-induced reprogramming of human regulatory T cell fate | Nature Immunology Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Deletion of a distal IRF4 element prevents inflammation-induced reprogramming of human regulatory T cell fate Download PDF Download PDF Abstract Regulatory T (T reg ) cells in mice can lose lineage identity and acquire proinflammatory functions, but whether human T reg cells are similarly susceptible to cytokine-driven destabilization remains unclear. Here we established an in vitro model of human T reg cell destabilization defined by silencing of the lineage-specifying transcription factor FOXP3, loss of suppressive function and acquisition of proinflammatory activity. Single-cell chromatin accessibility and transcriptomic profiling revealed a genome-wide increase in accessibility at AP-1-binding sites, including a putative regulatory element distal to IRF4 . Increased accessibility at this element correlated with increased IRF4 expression during T reg cell destabilization, and its excision conferred resistance to inflammatory cytokine-induced reprogramming. Conversely, forced expression of IRF4 together with BATF promoted T reg cell destabilization. These data identify a distal IRF4 regulatory element as a critical node enabling heightened AP-1-IRF4 cooperative activity to drive T reg cell destabilization, with implications for the design of more stable and effective T reg cell-based therapies. Explore related subjects Discover the latest articles and news in related subjects. Autoimmunity Immunosuppression Peripheral tolerance Main Regulatory T (T reg ) cells are a specialized subset of CD4 + T cells characterized by constitutive FOXP3 expression and an ability to suppress the activities of other immune cells. T reg cells play a dominant role in peripheral immune tolerance through a myriad of mechanisms that dampen antigen presentation, compete for T cell growth factors, convert metabolites, promote tissue repair and induce neighboring cells to develop tolerogenic activities 1 . The consequences of T reg cell dysfunction are exemplified by FOXP3 mutations that prevent T reg cell differentiation, leading to systemic immune dysregulation in mice and humans 2 . Boosting or infusing T reg cells has shown remarkable efficacy in many preclinical models of transplantation and autoimmune diseases, which has inspired clinical translation of T reg cell-enhancing strategies to promote immune tolerance 3 . However, lineage-tracing studies have shown that mouse T reg cells can undergo epigenetic reprogramming within chronically inflamed environments 4 , 5 . The resulting ex-Foxp3 cells show reduced suppressive activity and acquisition of proinflammatory and cytotoxic functions. Some evidence suggests that human T reg cells can also lose lineage identity in chronic inflammation 6 , 7 , 8 , 9 , but the molecular mechanisms underlying T reg cell destabilization are not well understood. T reg cell identity depends on a unique epigenetic landscape marked by hundreds of differentially methylated regions that guide T reg cell-specific transcriptional programs 10 . For example, the T reg cell-exclusive DNA demethylation at the FOXP3 conserved noncoding sequence (CNS)2 allows an embedded enhancer to drive constitutive FOXP3 expression. This aptly termed T reg -specific demethylated region (TSDR) is widely considered the most reliable indicator of T reg cell lineage commitment 11 . Although the stepwise imprinting of T reg cell epigenetic signatures during thymic T reg cell development has been mapped, maintenance of the T reg cell epigenome in the periphery remains poorly understood. In particular, several transcription factors (TFs) have been reported to reinforce or undermine T reg cell identity 12 , and the likely interplay between dysregulated gene regulatory networks and epigenetic rewiring in T reg cell fate decisions requires more thorough investigation. Here we present an in vitro model of inflammatory cytokine-driven human T reg cell instability, which enabled a molecular dissection of human T reg cell lineage maintenance and the development of cell engineering strategies to preserve T reg cell identity and function. Results In vitro model of human T reg cell instability To investigate whether human T reg cells are susceptible to losing lineage identity, CD4 + CD25 + CD127 lo/− fluorescence-activated cell sorting (FACS)-isolated primary human T reg cells (Extended Data Fig. 1a ) were subjected to repeated cycles of in vitro CD3/CD28 stimulation in the presence or absence of proinflammatory cytokines (Fig. 1a ). As a preliminary gauge of T reg cell identity, we focused on FOXP3 and HELIOS, a key pair of TFs with well-established, complementary roles in regulating T reg cell fitness and suppressive function 13 , 14 (Extended Data Fig. 1b ). As human conventional CD4 + T (T conv ) cells transiently express FOXP3 following T cell receptor (TCR) activation, expression profiles were evaluated 7 days into each stimulation cycle, after FOXP3 levels had mostly waned in non-T reg cells 15 . Fig. 1: Primary human T reg cells undergo progressive destabilization during repeated in vitro CD3/CD28 stimulation under sustained inflammatory cytokine exposure. Full size image a , Primary human CD4 + CD25 + CD127 lo/− T reg cells were stimulated with anti-CD3/CD28 every 9 days in the presence or absence of IL-6, IL-1β and IL-23. The expression of FOXP3 and HELIOS was assessed at day 7 of each stimulation cycle via flow cytometry. b , Representative FOXP3 and HELIOS flow cytometry profiles at days 7, 16 and 25. Labels indicate the percentage of cells in the gated subpopulations for HELIOS + (top), FOXP3 + HELIOS − (bottom right) and FOXP3 − HELIOS − (bottom left). Profiles of CD4 + T conv cells are shown for comparison. c , T reg cells were cultured as in a and challenged with the triple combination of IL-6, IL-1β and IL-23 across a 50-fold concentration range. Average distribution of cells within each gated subpopulation from b at days 7, 16 and 25 is summarized (mean ± s.d., n = 8 unrelated normal donors except the highest dose had five donors). d , Percentage of unmethylated CpG sites at the FOXP3 CNS2 (TSDR) from each of the FACS-purified HELIOS + , FOXP3 + HELIOS − (F + H − ) or FOXP3 − HELIOS − (F − H − ) subpopulations, as determined by pyrosequencing of bisulfite-converted genomic DNA. Results shown are mean ± s.d.; the numbers of biological replicates are listed. Statistical significance was calculated via Kruskal–Wallis test with Dunn’s test. Only adjusted P values <0.05 are shown. e , After 25 days of in vitro culture, T reg cells were co-cultured with PBMCs at the indicated T reg :PBMC ratios. In vitro suppression of responder CD4 + (left) and CD8 + (right) T cell proliferation relative to proliferation of the respective responder population in PBMC-only samples. Results shown are mean ± s.d. ( n = 3 technical replicates). Statistical significance was calculated via two-way ANOVA and Sidak’s test. Only adjusted P values <0.05 are shown. f , Cumulative fold-expansion of T reg and T conv cells over 25 days of in vitro culture in the presence or absence of IL-6, IL-1β and IL-23. Results are shown as mean (lines) ± s.d. (shaded regions) ( n = 8 unrelated normal donors). g , At day 25 after culturing in IL-2 only or with the addition of IL-6, IL-1β and IL-23 (25, 20 and 20 ng ml −1 , respectively), cells were stimulated for 5 h with PMA and ionomycin and the expression of proinflammatory cytokines was assessed via intracellular staining and flow cytometry. Percentages of cells expressing IFN-γ, TNF, IL-17A and IL-4 among HELIOS + cells, HELIOS − T reg cells and T conv cells are summarized. Boxes represent the interquartile ranges, lines inside the boxes represent medians and the whiskers represent ranges from minimum to maximum ( n = 16 unrelated normal donors). Statistical significance was calculated via repeated-measure two-way ANOVA with the Geisser–Greenhouse correction and Sidak’s test for comparisons within the same cell subsets. Only adjusted P values <0.05 for T reg cell subsets are shown. Source data Previous studies on the impact of inflammatory cytokines on human T reg cell lineage stability have implicated a negative role of IL-12 14 , 16 , whereas the effects of IL-6 have been more controversial 17 , 18 , 19 , 20 . We thus started with unbiased comparisons of Th1-, Th2- and Th17-driving cytokines, singly and in combinations (Extended Data Fig. 1c ). We found that the Th17-promoting combination of IL-6, IL-1β and IL-23 was the most consistent at inducing FOXP3 and HELIOS silencing (Extended Data Fig. 1c ). The extent of T reg cell destabilization by this triple cytokine combination was dependent on both the concentrations and duration of cytokine exposure. Under prolonged exposure, primary human T reg cells progressively downregulated both FOXP3 and HELIOS (Fig. 1b ). The fraction of FOXP3 − HELIOS − cells scaled with the concentration of IL-6, IL-1β and IL-23 across a 50-fold range (Fig. 1c ), within the range reported in inflamed tissues in humans (Supplementary Table 1 ). Moreover, transient exposure to the inflammatory cytokines for 7 days during the first cycle of stimulation was sufficient to drive the loss of FOXP3 and HELIOS in subsequent cycles, albeit in a smaller proportion of cells than under conditions of persistent cytokine exposure (Extended Data Fig. 1d,e ). This suggested that the appearance of FOXP3 − HELIOS − cells was not due to direct repression of the expression of these genes by the cytokine cocktail but rather a more permanent change in cell identity. As T conv cells rapidly out-proliferate T reg cells in vitro, an increase in FOXP3 − HELIOS − cells could also indicate imperfect T reg cell purity during the initial isolation. However, the very low percentages of FOXP3 − HELIOS − cells in parallel cultures without inflammatory cytokines signified high initial cell purity (Fig. 1b,c ) and supported an inflammatory cytokine-dependent effect. To determine if the loss of FOXP3 and HELIOS expression reflected an epigenetically delineated change in T reg cell fate, the HELIOS + , FOXP3 + HELIOS − and FOXP3 − HELIOS − populations were FACS purified and evaluated for DNA demethylation at the TSDR (Fig. 1d ). This evolutionarily conserved FOXP3 enhancer is uniquely demethylated in T reg cells; it is fully methylated in T conv cells 21 , 22 . The TSDR was predominantly uniformly demethylated in HELIOS + cells, consistent with a highly stable, committed T reg cell population. Meanwhile, FOXP3 − HELIOS − cells exhibited substantial but incomplete TSDR remethylation. To determine whether the partial methylation reflected a distinct transitional cellular state or a mixed population of demethylated T reg cells and methylated non-T reg cells, we examined individual CpG sites within TSDR and observed varying degrees of remethylation at different sites (Extended Data Fig. 2 ). This suggested that the FOXP3 − HELIOS − cells were undergoing cell state transition and provided additional evidence that this population did not expand from TSDR-methylated T conv cells. Together, these data showed that patterns of HELIOS + or FOXP3 − HELIOS − expression could serve as an accurate proxy for committed or destabilized T reg cell identity, respectively. We next sought to determine whether the function of T reg cells was affected by cytokine exposure. T reg cells treated with IL-6, IL-1β and IL-23 progressively downregulated canonical T reg cell markers CD25, CTLA-4, ICOS and TIGIT and upregulated CD226 and CD40LG (Extended Data Fig. 3a,b ), markers that have been implicated in T reg cell dysfunction 23 , 24 . The FOXP3 − HELIOS − population exhibited a phenotypic profile distinct from that of T conv cells (Extended Data Fig. 3c ), further supporting a change in cell state rather than the outgrowth of T conv cell impurities from the initial isolation. Cytokine-exposed T reg cells displayed reduced in vitro suppressive activity (Fig. 1e ), increased cell expansion (Fig. 1f ) and acquisition of IFN-γ, TNF and IL-17A production (Fig. 1g and Extended Data Fig. 4a,b ), highlighting the potential of these T reg cells to acquire proinflammatory functions. Collectively, these data demonstrate that human T reg cells can lose lineage identity and become dysfunctional during repeated in vitro TCR stimulation in the presence of inflammatory cytokines. Molecular hallmarks of T reg cell destabilization Equipped with a robust model of human T reg cell destabilization, we next set out to elucidate molecular hallmarks and key regulators of T reg cell instability. Multi-omic single-cell transcriptional and chromatin accessibility profiles were generated from the nuclei of primary human T reg cells actively undergoing destabilization following 16 and 25 days of IL-6, IL-1β and IL-23 exposure. Donor-matched T reg cells from IL-2-only cultures and T conv cells were also profiled to serve as references. Ten clusters were identified on a weighted nearest-neighbor (WNN) Uniform Manifold Approximation and Projection (UMAP) that accounted for both sequenced modalities (Fig. 2a ). While there was little change in the cluster distribution between day 16 and day 25 for samples maintained in IL-2 alone, the IL-6, IL-1β and IL-23 exposure drove a subset of cells toward a progressively T conv cell-like state (Fig. 2b ). Inspection of cluster-defining transcriptional features revealed that the resting T reg (Tr rest), effector T reg (Tr eff) and TNFR2-hi T reg (Tr TNFR2hi) cell clusters shared high expression of stable T reg cell markers, including FOXP3, IKZF2 (encoding HELIOS ), CD27, CD25, CTLA4, ICOS and TIGIT (Fig. 2c ). Conversely, the cycling T reg (Tr cyc), cytotoxic T reg (Tr cytox) and inflammatory T reg (Tr infl) cell clusters all exhibited transcriptional profiles consistent with a loss of T reg cell lineage identity, with downregulation of FOXP3 and HELIOS and upregulation of proinflammatory cytokine and/or cytotoxicity genes, such as IFNG , TNF and IL17A or NKG7 , GZMB and GZMA , respectively (Fig. 2c ). We will refer to cells in these collective clusters as destabilized or putative (ex)T reg cells hereafter. Fig. 2: Single-cell multi-omic profiles of gene expression and chromatin accessibility reveal molecular hallmarks of T reg cell lineage decommitment. Full size image a , Single-cell multi-omic profiles were generated from T reg and T conv cells isolated from three unrelated normal human donors. The cells were stimulated and cultured in the presence or absence of IL-6, IL-1β and IL-23 as shown in Fig. 1a . Cells were collected on days 16 and 25 of culture for single-cell multi-omic analysis. Unsupervised clustering on a WNN UMAP resulted in the identification of ten cell clusters shown in distinct colors. b , Cluster distributions for day 16 and day 25 samples from T reg cells expanded in IL-2 only or IL-2 plus IL-6, IL-1β and IL-23. c , Transcriptional signatures of cell clusters identified in a are shown, with additional annotations to group phenotypically suppressive (stable) T reg , destabilized (ex)T reg and T conv cells. Proinfl., proinflammatory; RM, resident memory; EM, effector memory; TE, effector T cell. d , Pseudobulk chromatin accessibility tracks and corresponding gene expression violin plots are shown for each cluster at the FOXP3 (CNS2 highlighted), IFNG and GZMB loci. ATAC-seq-defined OCRs (peaks) and correlations between peaks and transcript expression (links) are shown for each locus. e , Heat maps of chromatin accessibility for stable T reg , (ex)T reg and T conv cells at OCRs identified as differentially accessible between the indicated cell states. f , Venn diagram of shared and unique OCRs in stable T reg , (ex)T reg and T conv cells. Source data Differences in gene expression often correlated with altered chromatin accessibility at the encoding locus. For example, downregulation of FOXP3 transcription in the destabilized T reg cell clusters matched a loss of chromatin accessibility at the FOXP3 CNS2 (Fig. 2d ). Conversely, increased chromatin accessibility at IFNG and GZMB loci was linked to transcriptional upregulation in the destabilized T reg cell clusters (Fig. 2d ). These specific examples of epigenetic reprogramming mirrored a broader pattern of distinct open chromatin regions (OCRs) in destabilized T reg cells, with a pronounced reduction in chromatin accessibility at stable T reg cell OCRs as well as acquisition of OCRs normally inaccessible in stable T reg cells (Fig. 2e ). Many of these changes overlapped with differences in chromatin accessibility that distinguish T conv cells from T reg cells, but a subset of OCRs appeared unique to destabilized T reg cells (Fig. 2f ), suggesting that destabilized T reg cells are still epigenetically distinct from T conv cells. Together with the absence of cells in the (ex)T reg and T conv cell clusters that originated from IL-2-only T reg cell samples, these findings further highlight that the emergence of FOXP3 − HELIOS − cells was highly unlikely to be due to outgrowth of a contaminating T conv cell population. Chromatin alteration at the IRF4 locus in (ex)T reg cells To identify drivers of these distinct T reg cell fates, we separated cells from the IL-6, IL-1β and IL-23-exposed samples into stable and (ex)T reg cell states (Fig. 3a ) and analyzed their OCRs for differential TF motif enrichment. Destabilized T reg cells showed a relative depletion of FOXP3 and E-box elements targeted by bHLH TFs and an enrichment of AP-1, RUNX, ETS and IRF4 motifs (Fig. 3b ). Intriguingly, members of the AP-1 and ETS TF families can heterodimerize with IRF4 to drive distinct transcriptional programs 25 . The enrichment of AP-1, ETS and IRF4 motifs in (ex)T reg cells suggested that these TFs might cooperate to drive cell-state transition. Fig. 3: T reg cells undergo a reciprocal switch in chromatin accessibility at the IRF4 locus. Full size image a , TF motif enrichment analysis was performed on stable T reg and (ex)T reg cell clusters from IL-6, IL-1β and IL-23-treated samples isolated from three unrelated normal human donors to identify putative drivers of T reg cell destabilization. b , Volcano plot of differentially enriched TF motifs in stable T reg cells and destabilized T reg cells, with the E-box, AP-1 and ETS TF families highlighted. Benjamini–Hochberg correction was used for adjusted P value calculation. c , Pseudobulk chromatin accessibility tracks and corresponding gene expression violin plots for the IRF4 locus. The peak linkages (blue lines, bottom) indicate regions that coordinately influence gene expression. The OCR prox and OCR dist are highlighted in the shaded regions. d , Bulk ATAC-seq was performed on T reg cells treated with IL-2 only or IL-2 and one or more cytokines as indicated after three rounds of TCR stimulation; the PCA plot incorporating all ATAC peaks is displayed. e , CPM at the ATAC peaks for OCR dist and OCR prox ( n = 4 unrelated normal donors, except the IL-23 condition had three donors). Results shown are mean ± s.d., and significance was calculated via one-way ANOVA with Dunnett’s test for the IL-2-only condition. Only adjusted P values <0.05 are shown. f , Pearson correlation and simple linear regression model of OCR dist and OCR prox accessibility among all samples. Source data Inspection of the IRF4 locus revealed a gradual loss of chromatin accessibility at a proximal OCR (OCR prox ) located within intron 6 (Fig. 3c ). In T reg cells, IRF4 expression
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