Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma
Xiangdong Sun, Jiaqi Duan, Le Liu, Yanyu Ye, Yang Mi, Pengyuan Zheng, Pingchang Yang, Liguo Li
TL;DR
This study shows that a natural compound called KQS-1 can improve Treg cell function in allergic asthma by targeting Dectin-1 signaling, potentially offering a new treatment strategy.
Contribution
The study introduces KQS-1 as a novel therapeutic agent that rescues Treg function via Dectin-1 signaling in allergic asthma.
Findings
KQS-1 reverses Treg senescence and restores their suppressive and anti-inflammatory functions.
Dectin-1 signaling and the Raf-1/ROS axis mediate the epigenetic reprogramming of Tregs by KQS-1.
KQS-1 reduces airway inflammation and hyperresponsiveness in a mouse model of allergic asthma.
Abstract
In the context of unmet needs for targeted allergic asthma therapies, this study shows that natural compound KQS-1 alleviates airway inflammation and hyperresponsiveness by enhancing Treg cell function via Dectin-1 signaling, offering a novel potential therapeutic strategy. Allergic asthma is characterized by immune dysregulation, and deficiencies in regulatory T-cell (Treg) function are a hallmark of the disease. However, mechanisms of Treg impairment for their therapeutic correction remain poorly defined. The results showed that patient Tregs exhibited a senescent phenotype, including shortened telomeres, increased SA-β-gal activity, and heightened apoptosis. Functionally, they were compromised, showing reduced suppressive capacity and a pro-inflammatory cytokine shift. KQS-1 treatment robustly reversed these defects, restoring FOXP3- and IL-10–dependent Treg suppressive capacity and…
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Figure S6- —National Natural Science Foundation of China (NSFC)http://dx.doi.org/10.13039/501100001809
- —Science, Technology Innovation Bureau of Shenzhen Municipality
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Taxonomy
TopicsAsthma and respiratory diseases · Telomeres, Telomerase, and Senescence · Dermatology and Skin Diseases
Introduction
Allergic asthma is a prevalent chronic inflammatory disease of the airways, characterized by a complex immune response to inhaled allergens, leading to bronchial hyperreactivity, mucus hypersecretion, and structural remodeling (1, 2). The pathophysiology has traditionally been attributed to an imbalance between pro-inflammatory T helper 2 (Th2) cells and their associated cytokines (e.g., IL-4, IL-5, IL-13) and the counter-regulatory mechanisms that restrain these pro-inflammatory mediators (3, 4). Central to these regulatory mechanisms are regulatory T cells (Tregs), a specialized CD4^+^ T-cell subset defined by the expression of the master transcription factor FoxP3. Tregs are critical for maintaining immune tolerance and preventing aberrant inflammation through multiple mechanisms, including the secretion of anti-inflammatory cytokines like IL-10 and TGF-β, direct suppression of effector T-cell proliferation, and modulation of antigen-presenting cell function (5, 6).
A compelling body of evidence points to Treg dysfunction as a cornerstone of allergic asthma pathogenesis. Numerical and functional deficiencies in Tregs have been documented in both murine models and human patients, correlating with disease severity (7, 8). However, the fundamental cellular and molecular mechanisms driving this Treg impairment remain incompletely understood. One emerging hypothesis is that chronic inflammatory environments can promote T-cell senescence-like, a state of irreversible cell cycle arrest, resistance to apoptosis, and altered function (9, 10). Hallmarks of senescence-like, including telomere shortening, increased senescence-like–associated β-galactosidase (SA-β-gal) activity, and a distinct secretory phenotype (SASP), have been observed in various T-cell populations in chronic inflammatory and autoimmune diseases (9, 11). Whether such a senescence-like phenotype underlies Treg dysfunction in allergic asthma, and if so, whether this state is therapeutically reversible for core Treg functional pathways are critical unanswered questions.
Recent advances in immunology have highlighted the role of innate-like signaling pathways and long-term epigenetic reprogramming in shaping T-cell function, a concept known as “trained immunity” (12, 13). Certain stimuli, particularly those engaging pattern recognition receptors (PRRs), can induce metabolic and epigenetic changes that confer nonspecific, enhanced functionality long after the initial trigger is gone. The C-type lectin receptor Dectin-1, best known for its role in recognizing fungal β-glucans and shaping innate antifungal immunity, has recently been implicated in modulating T-cell responses (14, 15). This raises the intriguing possibility of harnessing such pathways to durably “reeducate” and restore core functional pathways of dysfunctional immune cells like Tregs, rather than achieving global cellular rejuvenation.
Although Treg functional impairment in allergic asthma is well documented, strategies to restore their suppressive capacity remain limited. Recent evidence suggests that innate immune receptors, particularly Dectin-1 (CLEC7A), may play unexpected roles in adaptive immune regulation. Dectin-1, a C-type lectin receptor traditionally associated with antifungal responses, is expressed on Tregs (Fig S4A–I) and has been implicated in modulating immune tolerance through induction of anti-inflammatory cytokines (e.g., IL-10) in dendritic cells. We hypothesized that targeted engagement of Dectin-1 on Tregs could reverse their dysfunction in allergic asthma by reprogramming the epigenetic landscape of core Treg functional genes FOXP3 and IL10.
KQS-1, a novel fungal polysaccharide, was selected for its high-affinity binding to Dectin-1 (Fig 5A) and its lack of TLR2/4 activation (Fig S4A–C). Unlike other β-glucans (e.g., curdlan), KQS-1 exhibits superior solubility and a defined molecular weight profile (Fig 1H and I), making it ideal for therapeutic exploration. Previous studies with related β-glucans suggest that Dectin-1 ligands can epigenetically reprogram immune cells, but their effects on the specific epigenetic regulation of FOXP3 and IL10 in Tregs remain unexplored.
*Senescence-like phenotype of peripheral regulatory T cells (Tregs) and characterization of KQS-1.PBMCs were isolated from healthy controls and patients with asthma and analyzed by flow cytometry. (A) Gating strategy for identification of Tregs (full gating protocol shown in Fig S1). (B) Quantification of total Treg frequencies and absolute counts. (C) Telomere length measurement in sorted Tregs. (D) Representative flow cytometry plots illustrating gating of senescence-like–associated β-galactosidase–positive (SA-β-gal+) Tregs. (E) Quantitative analysis of SA-β-gal+ Treg frequencies and counts. (F) Representative flow cytometry plots showing gating of apoptotic Tregs. (G) Quantitative analysis of apoptotic Treg frequencies and counts. (H) β-Glucan content of KQS-1 compared with the reference standard. (I) Representative size-exclusion chromatography (SEC) profile showing the refractive index signal across retention volumes, indicating molecular weight distribution and homogeneity of the KQS-1 polysaccharide fraction. Violin plots are presented as a median with interquartile range (IQR), with each dot representing an individual sample (n = 15 per group). (H) For β-glucan analysis in (H), data are shown as the mean ± SEM (n = 5 independent experiments). Statistical significance was determined using a two-tailed t test; ***P < 0.001, ***P < 0.0001.
In this study, we hypothesize that Tregs in allergic asthma acquire a senescence-like and functionally exhausted phenotype, and that this defect can be reversed through targeted engagement of the Dectin-1 pathway, leading to epigenetic reprogramming of FOXP3 and IL10 and sustained restoration of FOXP3- and IL-10–dependent immunoregulatory capacity. We test this by first comprehensively characterizing the senescence-like phenotype of human Tregs from allergic asthmatic patients. We then investigate the therapeutic potential of KQS-1 to rescue Treg function through a Dectin-1–dependent mechanism involving chromatin remodeling at FOXP3 and IL10 and the induction of a trained immunoregulatory phenotype centered on these core genes. Finally, we validate the physiological relevance of our findings as a proof of principle using a murine model of allergic airway disease and adoptive transfer experiments. Our findings not only elucidate a novel mechanism of Treg functional failure in asthma centered on the dysregulation of FOXP3 and IL10 but also propose a pioneering therapeutic strategy based on the targeted epigenetic reprogramming of core Treg signature loci, rather than global cellular rejuvenation.
Results
Tregs from allergic asthmatic patients exhibit a senescence-like phenotype
We first examined whether circulating regulatory T cells (Tregs) in patients with allergic asthma display of cellular senescence-like features, using age- and sex-matched healthy individuals as controls. Both cohorts included 15 participants: the asthma group (mean age, 38 ± 4 yr; 8 females/7 males) and the control group (mean age, 37 ± 5 yr; 7 females/8 males). Tregs were phenotypically defined as CD4^+^CD25^+^FoxP3^+^ via flow cytometry (Fig 1A–E), with the complete gating strategy provided in Fig S1A–F. Flow cytometry–based telomere staining revealed significantly shortened telomeres in patient-derived Tregs compared with controls; the mean fluorescence intensity (MFI) of telomere signals was 182 ± 12 in asthma patients versus 246 ± 15 in controls, representing a 26% reduction (unpaired two-tailed t test, P < 0.001; 95% confidence interval [CI]: −78.3 to −50.7; Fig 1C). The proportion of SA-β-gal–positive Tregs—gated using an MFI threshold >2× that of isotype controls—was substantially higher in the asthma group (32.1% ± 3.2%) than in controls (8.4% ± 1.5%) (unpaired two-tailed t test, P < 0.001; 95% CI: 19.8–27.6; Fig 1D and E). After 24 h of culture in RPMI-1640 medium supplemented with 10% FBS and no exogenous stimuli, the apoptosis rate of patient Tregs (Annexin V^+^PI^−^) was 2.3-fold higher than that of controls (27.8% ± 2.9% versus 12.1% ± 1.8%; unpaired two-tailed t test, P < 0.01; 95% CI: 10.2–21.2; Fig 1F and G).
Gating strategy for the identification of live CD3+CD4+CD127− T cells by flow cytometry.Sequential gating was performed to identify the target lymphocyte population. (A) Cells were first plotted on an FSC-A versus SSC-A dot plot to gate on the main population and exclude debris. (B) Single cells were selected by gating on FSC-H versus FSC-A to exclude doublets. (C) Live cells were identified by gating on viability dye (ViD)–negative cells. (D) From the live, single-cell population, CD45+ leukocytes were selected. (E) CD3+CD4+ T cells were gated from the CD45+ population. (F) Finally, CD127ᵢo cells were gated for subsequent analysis.
In Fig 1H and I, we further characterized the biochemical properties of KQS-1. Panel H shows the β-glucan content of KQS-1, confirming its composition as a fungal-derived polysaccharide. Panel I provides the molecular weight distribution of KQS-1, which is essential for understanding its structural properties and potential bioactivity. These characterizations are critical for correlating the biochemical features of KQS-1 with its observed therapeutic effects on FOXP3 and IL10 regulation. Together, these data confirm that Tregs from patients with allergic asthma exhibit a senescent phenotype, which correlates with dysregulation of their core immunoregulatory functions.
Senescence-like Tregs are functionally impaired and display a pro-inflammatory cytokine shift
Next, we evaluated the functional capacity of senescent-like Tregs using two complementary assays: a CFSE-based suppression assay to measure proliferative suppression and intracellular cytokine staining (ICS) to characterize cytokine production centered on IL-10 and TGF-β, the key anti-inflammatory cytokines of Tregs. For the suppression assay, Tregs and autologous CD4^+^CD25^−^ responder T cells were cocultured at a 1:1 ratio, and responder cells were activated with anti-CD3/CD28 beads (1:1 bead-to-cell ratio) for 72 h. For ICS, Tregs were stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (1 μg/ml) for 4 h, with brefeldin A added during the final 2 h to trap intracellular cytokines. Patient Tregs showed severe impairment in suppressing responder T-cell proliferation, with the suppression efficiency of Tregs from controls at 68.2% ± 5.1% and this value dropping to 28.3% ± 7.2% in Tregs from asthma patients (paired two-tailed t test, P < 0.001; 95% CI: 31.5–48.3; Fig 2A and B). ICS revealed a significant pro-inflammatory shift in the cytokine profile of patient Tregs, with statistical significance adjusted via the Bonferroni correction for four comparisons: the frequencies of IL-10^+^ Tregs (12.1% ± 1.8% versus 22.0% ± 2.3% in controls; P < 0.01; 95% CI: −12.7 to −7.1) and TGF-β^+^ Tregs (8.4% ± 1.2% versus 13.5% ± 1.5% in controls; P < 0.01; 95% CI: −6.7 to −3.5) were reduced by 45% and 38%, respectively, whereas the frequencies of IFN-γ^+^ Tregs (9.8% ± 1.4% versus 4.9% ± 0.8% in controls; P < 0.01; 95% CI: 3.1–6.7) and IL-17^+^ Tregs (7.2% ± 1.1% versus 3.6% ± 0.7% in controls; P < 0.01; 95% CI: 2.2–5.0) approximately doubled (Fig 2C). ELISA analysis of 72-h culture supernatants confirmed the reduced secretion of anti-inflammatory cytokines, with the Bonferroni correction applied: IL-10 levels were 85 ± 12 pg/ml in patient Treg cultures versus 170 ± 18 pg/ml in controls (50% reduction; P < 0.01; 95% CI: −98 to −62), and active TGF-β1 levels were 42 ± 8 pg/ml in patients versus 105 ± 12 pg/ml in controls (60% reduction; P < 0.01; 95% CI: −76 to −40; Fig 2D). These data confirm that the senescent phenotype of asthmatic Tregs is associated with a profound loss of FOXP3- and IL-10–dependent immunoregulatory function and a shift toward pro-inflammatory cytokine production.
*Impaired suppressive function and dysregulated cytokine profiles of regulatory T cells (Tregs) in asthma.FACS-sorted Tregs from patients with asthma and healthy controls (n = 25 per group for all panels) were evaluated for suppressive capacity, intracellular cytokine expression, and secreted cytokine production. (A, B) Suppressive capacity. Representative flow cytometry plots of responder T-cell proliferation and quantitative analysis of Treg-mediated suppression. Tregs from asthma patients exhibited reduced suppressive efficiency compared with controls. (C) Intracellular cytokine expression. Frequencies of Tregs expressing anti-inflammatory cytokines (IL-10, TGF-β) and pro-inflammatory cytokines (IFN-γ, IL-17). Asthma-derived Tregs showed decreased IL-10+ and TGF-β+ populations and increased IFN-γ+ and IL-17+ populations relative to controls. (D) Secreted cytokine production. Concentrations of IL-10 and active TGF-β1 in 72-h culture supernatants from highly purified Tregs. Asthma Tregs secreted lower levels of both cytokines compared with healthy controls. Data are presented as the mean ± SD, with each dot representing an individual sample. Statistical significance was determined using Welch’s unpaired t test or one-way ANOVA followed by Tukey’s post hoc test, as appropriate. For panels (B, C), P-values were Bonferroni-corrected for multiple comparisons. Significant differences are indicated by asterisks (***P < 0.001).
KQS-1 treatment rescues FOXP3/IL-10–dependent Treg function and survival
To ensure experimental rigor, we verified the batch-to-batch consistency of KQS-1 (10 μg/ml) and the inertness of its PBS vehicle (Fig S2). HPLC analysis confirmed that KQS-1 had a purity of ≥95% and consistent peak profiles across production batches, eliminating batch-to-batch variation (Fig S2A and B). Using FACS-sorted Tregs from patients with asthma (n = 25 per group), we further showed that PBS (the vehicle for KQS-1) had no significant effect on Treg function: it did not alter Treg suppressive efficiency (Fig S2C), intracellular cytokine frequencies (anti-inflammatory IL-10, TGF-β; pro-inflammatory IFN-γ, IL-17; Fig S2D, Bonferroni-corrected), or survival rate (Fig S2E) after 48 h of culture (Welch’s unpaired t test, P > 0.05 for all comparisons). These controls confirm any observed effects of KQS-1 in subsequent experiments are attributable to the compound itself, not reagent variability or vehicle interference.
KQS-1 batch reproducibility and PBS vehicle neutrality.(A, B) Purity and batch consistency analysis of KQS-1. HPLC analysis of KQS-1 production batches. (A) Quantitative analysis of purity across three independent production batches (n = 5 replicates per batch). Individual data points (black dots) represent single measurements, and the vertical distribution shows batch uniformity. The red dashed line denotes the minimum purity requirement of 95%. Statistical analysis by one-way ANOVA indicates no significant variation between batches (ns, P > 0.05), confirming high-level production consistency. (B) Representative HPLC chromatogram overlay of three batches showing consistent peak profiles and identical retention times, confirming the chemical stability and absence of batch-to-batch variation in the manufacturing process. (C, D, E) Impact of PBS vehicle on the function and viability of asthma patient–derived Tregs. Assessment of PBS treatment on FACS-sorted CD4+CD25+CD127− Tregs isolated from asthma patients (n = 25 patients per group). (C) Treg suppressive efficiency (%) measured by the inhibition of responder T-cell proliferation. No significant difference was observed between the untreated control and the PBS vehicle group. (D) Frequencies (%) of intracellular anti-inflammatory (IL-10, TGF-β) and pro-inflammatory (IFN-γ, IL-17) cytokines determined by flow cytometry after 48 h. Comparisons were adjusted using the Bonferroni correction; no significant alterations were detected across all markers. (E) Treg survival rate (%) after 48 h of culture in the presence or absence of PBS. In all panels, box-and-whisker plots represent the median, interquartile range (IQR), and range, whereas individual black dots denote data from independent patient samples. Statistical significance was evaluated using Welch’s unpaired t test. ns indicates P > 0.05, confirming that PBS serves as a neutral vehicle for KQS-1.
KQS-1 treatment significantly restored FOXP3- and IL-10–dependent Treg suppressive efficiency, increasing it from 28.3% ± 7.2% in untreated patient Tregs to 61.1% ± 6.3% in KQS-1–treated Tregs (paired two-tailed t test, P < 0.001; 95% CI: −37.5 to −20.1; Fig 3A). ELISA analysis showed that KQS-1 treatment robustly enhanced anti-inflammatory cytokine secretion centered on IL-10 and reduced pro-inflammatory cytokine production: secreted IL-10 increased 2.5-fold to 212 ± 21 pg/ml (P < 0.01; 95% CI: 103–151), and active TGF-β1 increased 2.8-fold to 118 ± 15 pg/ml (P < 0.01; 95% CI: 62–89), whereas IFN-γ and IL-17 levels were reduced by >50%, to 4.2 ± 0.9 and 3.1 ± 0.6 pg/ml, respectively (both P < 0.01; Fig 3B and C). KQS-1 also improved Treg survival, as the Annexin V^+^PI^−^ apoptosis rate decreased from 27.8% ± 2.9% in untreated Tregs to 12.3% ± 3.1% in treated Tregs (paired two-tailed t test, P < 0.01; 95% CI: 10.5–20.5; Fig 3D). Notably, KQS-1 had no effect on the survival of Tregs from healthy controls (Fig S3), indicating its effects are specific to dysfunctional, senescent Tregs in asthma.
*KQS-1 reprograms human Tregs to enhance survival, anti-inflammatory polarization, and suppressive function.(A) Suppression assays demonstrating that KQS-1–treated Tregs exhibit enhanced inhibition of responder T-cell proliferation compared with vehicle-treated controls. (B, C) Cytokine profiling after 48-h KQS-1 exposure. ELISA analysis of culture supernatants shows a shift toward an anti-inflammatory phenotype, characterized by increased IL-10 and TGF-β1 production and reduced IFN-γ and IL-17 levels. (D) Treg survival analysis in patient-derived cells. Representative and quantitative assessment of Annexin V+/PI− apoptotic Tregs after 48-h treatment with KQS-1 or vehicle control. Paired samples are connected by lines. Data are presented as the mean ± SD. (A, B, C, D) Sample sizes: n = 8 (A, B, C) and n = 12 (D). Statistical significance was determined using unpaired or paired two-tailed t tests, as appropriate, with Bonferroni correction for multiple comparisons where applicable. **P < 0.01, **P < 0.001.
KQS-1 does not alter Treg survival in healthy donors.Annexin V+/PI− apoptosis of healthy control Tregs (n = 10 pairs) after 48-h exposure to KQS-1 or vehicle. Apoptosis remained unchanged (8.5% ± 1.2% versus 8.7% ± 1.0%; paired two-tailed t test, P > 0.05). Symbols connect paired samples; data are the mean ± SD.
KQS-1 induces epigenetic remodeling of core Treg signature loci FOXP3 and IL10 consistent with trained immunity
We next investigated how KQS-1 reshapes the epigenetic landscape of human Treg cells, with a focused analysis of the core functional loci FOXP3 and IL10 (Fig 4). First, ChIP–qPCR analysis (Fig 4A) revealed that KQS-1 significantly increased active transcriptional histone marks—H3K4me3 at the FOXP3 promoter and IL10 locus, and global H3K27ac—compared with PBS-treated controls (all P < 0.0001; n = 8 biological replicates). To assess chromatin accessibility, we performed ATAC-seq; Fig 4B shows a volcano plot of the 1,306 newly accessible regions (FDR < 0.05) identified in KQS-1–treated cells. These regions localized predominantly to enhancers, intergenic regions, and promoters (Fig 4C), with accessibility changes significantly distinct across categories (P < 0.001). Focusing on Treg signature genes, Fig 4D (tracks and quantification) confirmed that KQS-1 significantly increased promoter accessibility at the IL10 and FOXP3 loci; no additional lineage stability loci were assessed in this study. Gene ontology (GO) enrichment (Fig 4E) of genes near these accessible regions highlighted pathways tied to immune regulation such as negative regulation of T-cell proliferation/activation, consistent with Treg functional programming centered on FOXP3 and IL10. Finally, Illumina 450K methylation array analysis (Fig 4F) identified focal hypomethylation (reduced Δβ) at regulatory CpG sites of FOXP3 and IL10 (e.g., FOXP3: Δβ = −0.18; IL10: Δβ = −0.22), with no significant changes at unrelated CpG sites. Together, these data demonstrate that KQS-1 remodels Treg epigenetics at the core signature loci FOXP3 and IL10 by boosting active histone marks, opening chromatin, and hypomethylating these key regulatory loci—with no assessment of broader Treg lineage stability programs in the current work. To confirm the functional relevance of these epigenetic changes to FOXP3, we measured FOXP3 protein expression via flow cytometry: KQS-1–treated Tregs showed a 2.1-fold increase in FOXP3 protein levels (P < 0.01; 95% CI: 1.4–2.8; Fig S3), directly linking the epigenetic reprogramming of FOXP3 to restored protein expression and functional capacity.
*KQS-1 reprograms the epigenetic landscape of human Treg cells.(A) ChIP–qPCR analysis of active histone marks at key Treg signature loci. Enrichment of H3K4me3 at the FOXP3 promoter and IL-10 locus, as well as global H3K27ac levels, was assessed in KQS-1–treated and control cells. (B) ATAC-seq volcano plot comparing chromatin accessibility in KQS-1–treated versus untreated Tregs, highlighting newly accessible genomic regions. (C) Genomic annotation of KQS-1–induced accessible regions, categorized as promoters (±3 kb of the transcription start site), enhancers (distal regulatory elements), or intergenic regions. Distribution of accessibility changes across genomic features is shown. (D) Chromatin accessibility at IL-10 and FOXP3 promoter regions. Upper panels show representative ATAC-seq signal tracks; lower panels present quantification of accessibility changes after KQS-1 treatment. (E) Gene ontology (GO) enrichment analysis of genes proximal to KQS-1–induced accessible regions, illustrating enrichment of immune regulatory and T cell–associated pathways. (F) DNA methylation profiling of regulatory regions using the Illumina 450K array. Lollipop plots depict changes in methylation (Δβ) between KQS-1–treated and untreated Tregs, highlighting focal hypomethylation at FOXP3 and IL-10 regulatory CpG sites. ChIP–qPCR data are presented as the mean ± SD (n = 8 biological replicates), with individual points representing replicates. ATAC-seq analysis identified 1,306 newly accessible regions (FDR < 0.05). For genomic distribution analysis, box plots show median and interquartile range (IQR), with individual dots representing peaks. DNA methylation data are shown as mean Δβ with 95% confidence intervals. Statistical significance was determined using paired or unpaired t tests, one-way ANOVA with Tukey’s post hoc test, and the Bonferroni correction for multiple comparisons, as appropriate. **P < 0.01, ***P < 0.001, ***P < 0.0001; ns, not significant.
The functional and epigenetic effects of KQS-1 on FOXP3/IL-10 are Dectin-1–dependent
We next identified the receptor mediating KQS-1’s effects on the epigenetic and functional regulation of FOXP3 and IL10 in Tregs using a combination of binding assays, signaling inhibition, and genetic knockout. Flow cytometry analysis demonstrated that over 90% of Tregs expressed Dectin-1, and this expression was unaffected by prestaining with a Dectin-2–targeting antibody (another C-type lectin receptor). At the mRNA and protein levels, Dectin-1 expression on Tregs was ∼59% and 56% of that on dendritic cells (DCs), respectively (Fig S4A–I). Flow cytometry with biotinylated KQS-1 demonstrated direct binding to surface Dectin-1 on Tregs (MFI = 324 ± 28 versus 86 ± 12 for isotype controls; P < 0.001; 95% CI: 210–266; Fig 5A), and no binding to TLR2 or TLR4 was detected (Fig S4J–M).
*Biotinylated KQS-1 binds specifically to Tregs by interacting with Dectin-1.(A, B, C, D) Gating strategy of CD4+CD127+ T cells. (A) Dead cells were gated out. (B, C) CD45+ leukocytes were gated, from which CD3+CD4+ T cells were gated (C). (D) CD127−, CD127lo, and CD127hi T cells were gated from CD3+CD4+ T cells. (E) Foxp3+ Tregs from CD127lo cells. (F, G) Histograms show Tregs are Dectin-1+. (H, I) Levels of Dectin-1 mRNA ((H), by RT–qPCR) and protein ((I), by FACS) in Tregs. (J, K) Binding of biotinylated KQS-1 to Tregs assessed by flow cytometry. Tregs were incubated with biotinylated KQS-1 (1 μg/ml) for 1 h. (J) Representative flow cytometry plots showing the percentage of KQS-1–positive Tregs. (K) Quantitative analysis of KQS-1–bound Tregs from replicate experiments (n = 6). (L) Dectin-1 knockout in Tregs using CRISPR. (M) Identification of KQS-1 binding receptors by cross-ELISA. Plates were coated with biotinylated KQS-1 and incubated with Treg protein extracts. Binding to potential receptors was detected using antibodies against TLR2, TLR4, or Dectin-1. An isotype control antibody (IgG) was used as a negative control. Data are presented as the mean ± SD. Each data point represents an independent sample. (G, H, I, K, L, M) Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (K, L, M) or t test (G, H, I). *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, ns, not significant. OD, optical density; NT sgRNA, nontargeting sgRNA (negative control).
*KQS-1 potentiates Treg stability via a Raf-1/ROS axis.(A) Binding of KQS-1 to purified human Tregs. Cells were incubated with KQS-1 and analyzed by flow cytometry to determine the proportion of KQS-1–positive cells. Representative gating strategies are shown in Fig S4A–C. (B, C, D) Raf-1 and ROS dependence of KQS-1–primed Treg responses. Tregs were treated with KQS-1 alone or in combination with the Raf-1 inhibitor GW5074 (10 μM) or the ROS scavenger N-acetylcysteine (NAC, 5 mM). (B) H3K4me3 enrichment at the FOXP3 promoter assessed by ChIP–qPCR and expressed in arbitrary units (a.u.). (C) In vitro suppressive capacity of treated Tregs. The dashed line indicates the mean suppression achieved by KQS-1–treated cells. Representative flow cytometry plots are provided in Fig S5A. (D) TGF-β protein levels in culture supernatants under the indicated conditions. (C) Data are presented as the mean ± SD, with each dot representing an individual donor (n = 10 biologically independent replicates for panel (C)). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. ***P < 0.0001.
Pharmacological inhibition of Raf-1 with GW5074 (10 μM) or scavenging of reactive oxygen species (ROS) with N-acetylcysteine (NAC) (5 mM) during KQS-1 priming completely abrogated two key effects of KQS-1: H3K4me3 deposition at the FOXP3 promoter (P < 0.001 versus KQS-1 alone; 95% CI: 2.6–3.8) and rescue of FOXP3- and IL-10–dependent Treg suppressive capacity (P < 0.001 versus KQS-1 alone; 95% CI: 33.2–45.8; Figs 5B and C and S5). CRISPR/Cas9-mediated knockout (KO) of Dectin-1 in patient Tregs using CLEC7A-targeting sgRNA (KO efficiency > 90%; Fig S4C) abrogated all KQS-1–induced effects on FOXP3 and IL10 regulation. Specifically, Treg suppressive efficiency remained at 27.9% ± 6.8% versus 61.1% ± 6.3% in KQS-1–treated WT Tregs (P < 0.001), and IL-10 secretion failed to increase (P < 0.001). Nontargeting sgRNA controls had no effect (Figs 5D and S5A and B). These data confirm that Dectin-1 signaling is an absolute requirement for KQS-1–mediated epigenetic reprogramming of FOXP3 and IL10 and the subsequent restoration of their dependent immunoregulatory function in asthmatic Tregs.
Dectin-1, Raf-1, and ROS scavenging are dispensable for Treg-mediated suppression.(A, B) CFSE-labeled effector T cells (Teff) were cocultured with Tregs under the indicated conditions. Histograms (gated on Teff) show CFSE dilution as a readout of proliferation. GW5074, Raf-1 inhibitor; NAC, ROS scavenger; CRISPR, Dectin-1 knockout; NT sgRNA, nontargeting control. Quantitative proliferation indices are reported in Fig 5C and D.
KQS-1–treated Tregs ameliorate airway inflammation in vivo as a proof of principle
To assess the therapeutic efficacy of KQS-1 in allergic asthma as a proof of principle, we established an asthma mouse model and analyzed key pathological indicators after KQS-1 or PBS treatment, and further verified the role of KQS-1–primed Tregs using Rag^−/−^ mice. Serum-specific IgE levels (a classic biomarker of allergic response) were drastically reduced in KQS-1–treated mice compared with the PBS group (Fig 6A, P < 0.0001). Functional assessment of airway hyperresponsiveness (AHR) showed that KQS-1 significantly lowered Penh values (an indicator of airway resistance) at 50 mg/ml methacholine challenge (Fig 6B, P < 0.0001), suggesting improved airway patency. In bronchoalveolar lavage fluid (BALF), KQS-1 treatment markedly reduced the counts of eosinophils (Fig 6C, P < 0.0001), neutrophils (Fig 6D, P < 0.0001), and Th2 cells (Fig 6E, P < 0.0001)—all core inflammatory cells driving asthma pathogenesis. Consistently, the secretion of Th2 cytokines (IL-4, IL-5, IL-13) in BALF was significantly suppressed by KQS-1 (Fig 6F, P < 0.0001 for all).
*KQS-1 attenuates allergic asthma pathology and enhances Treg-mediated protection.(A, B, C, D, E, F, G, H, I, J) HDM-induced asthma model mice were treated with KQS-1 or PBS control. (A, B, C, D, E) Quantitative analyses include serum allergen–specific IgE levels (A), airway resistance (B), and bronchoalveolar lavage fluid (BALF) cellular composition, including eosinophils, neutrophils, and IL-4+ Th2 cells (C, D, E). (F) Th2 cytokine concentrations in BALF are shown in (F). (G, H) Histopathological assessments include mucus production scores (G) and inflammatory scores (H). (I, J) Representative lung sections (×400; scale bar = 20 μm) demonstrate periodic acid–Schiff (PAS) staining for mucus-producing cells (I) and hematoxylin and eosin (H&E) staining for airway inflammation (J). (K, L, M) Rag1−/− asthma model mice received adoptive transfer of naïve Tregs or KQS-1–primed Tregs. (K) Eosinophil counts in BALF are shown in (K). (L, M) Representative flow cytometry plots of Tregs in mesenteric lymph nodes (MLNs) are presented in (L), with corresponding quantification of Treg frequencies in MLNs in (M). Data are presented as the mean ± SD, with individual data points representing single mice (n = 6 per group). Statistical significance was determined using a t test. ***P < 0.001, ***P < 0.0001.
Histopathological analysis further confirmed the therapeutic effect as a proof of principle: PAS staining showed that KQS-1 attenuated goblet cell hyperplasia (Fig 6G, P < 0.0001; representative images in Fig 6I), whereas HE staining demonstrated reduced airway inflammation (Fig 6H, P < 0.0001; representative images in Fig 6J).
KQS-1–primed Tregs enhance anti-asthmatic effects as a proof of principle
To explore the immunomodulatory mechanism of KQS-1, we performed adoptive transfer of naive Tregs or KQS-1–primed Tregs into Rag^−/−^ mice with allergic asthma in a proof-of-principle experiment. KQS-1–primed Tregs significantly reduced eosinophil infiltration in BALF (Fig 6K, P < 0.0001). Flow cytometry analysis showed that KQS-1 treatment elevated the proportion of functional Foxp3^+^CD25^+^ Tregs (Fig 6L), and this enhancement was observed in lymph nodes (Fig 6M, P < 0.0001). These results provide proof-of-principle evidence that KQS-1 exerts anti-asthmatic effects in part by promoting the expansion and functional activity of Tregs with restored FOXP3/IL-10 expression, rather than definitive causal validation of this mechanistic link in vivo. Collectively, these data provide proof-of-principle evidence that KQS-1 effectively alleviates key pathological features of allergic asthma in mice by inhibiting Th2-driven inflammation, reducing airway hyperresponsiveness, and enhancing FOXP3- and IL-10–dependent Treg-mediated immune tolerance, serving as a foundational demonstration for further preclinical and mechanistic investigation.
Dectin-1 overexpression rescues KQS-1 responsiveness of FOXP3/IL-10 in Dectin-1-low Tregs
To establish sufficiency of Dectin-1 for mediating KQS-1’s effects on FOXP3 and IL10 as a proof of principle, we transduced Dectin-1-low Tregs (isolated from donors with CLEC7A haploinsufficiency) with a lentiviral CLEC7A expression vector. The overexpression of Dectin-1 (confirmed by flow cytometry, MFI ↑2.5-fold, P < 0.01) restored KQS-1 binding and downstream responses for these core Treg genes: epigenetic remodeling showed H3K4me3 enrichment at FOXP3 increased 1.8-fold (P < 0.05) compared with empty vector controls, and functional rescue showed FOXP3/IL-10–dependent suppressive capacity improved significantly (P < 0.01 versus untransduced cells), mirroring the effects observed in WT Tregs (Fig S6A–C). These findings offer proof-of-principle evidence that Dectin-1 expression is a key mediator of KQS-1’s epigenetic and functional effects on FOXP3 and IL10 in Tregs, without providing definitive causal validation of this relationship in physiological settings or for broader Treg lineage stability.
*Overexpression of Dectin-1 in Dectin-1-low Tregs rescues functional deficiency.Gain-of-function analysis in Dectin-1-low Tregs after lentiviral transduction with Dectin-1 (Dectin-1 OE) or empty vector control. (A, B) Flow cytometry analysis showing a significant increase in KQS-1 binding (MFI) in Dectin-1 OE cells compared with controls (A), accompanied by restored H3K4me3 epigenetic marks at the FOXP3 promoter region as measured by ChIP–qPCR (B). (C) Suppressive efficiency of Tregs against responder T-cell proliferation, demonstrating enhanced inhibitory capacity upon Dectin-1 restoration. Data are presented as box-and-whisker plots with individual samples shown as dots (n = 5 per group). Statistical significance was determined by an unpaired t test; *P < 0.05, *P < 0.01.
Discussion
This study identifies the acquisition of a senescence-like and dysfunctional phenotype in Tregs as a pivotal mechanism in the pathogenesis of allergic asthma and unveils a novel therapeutic strategy to reverse this defect by targeting the core Treg signature loci FOXP3 and IL10 as a proof of principle. We demonstrate that Tregs from allergic asthmatic patients exhibit classical hallmarks of cellular senescence, including telomere shortening, increased senescence-associated β-galactosidase activity, and a heightened propensity for apoptosis. More importantly, this senescence-like state was directly linked to a profound functional impairment, characterized by diminished FOXP3- and IL-10–dependent suppressive capacity and a shift toward a pro-inflammatory cytokine profile. The core finding of our work is that the natural compound KQS-1 can effectively “reeducate” these dysfunctional Tregs, restoring FOXP3- and IL-10–dependent immunoregulatory function through a Dectin-1–dependent epigenetic reprogramming pathway targeting these two core loci, which translates into significant mitigation of airway inflammation in vivo in a proof-of-principle demonstration rather than a definitive causal validation of therapeutic efficacy. Critically, our study focuses on the targeted reprogramming of FOXP3 and IL10—the central drivers of Treg immunoregulatory function—and does not assess the restoration of broader Treg lineage stability programs, a key distinction that aligns our conceptual framing strictly with the experimental data.
Our findings position KQS-1 as a unique Dectin-1 agonist that rescues Treg function through the epigenetic reprogramming of FOXP3 and IL10 (Figs 4 and 5), distinguishing this pathway from IL-2–dependent Treg survival pathways. Notably, KQS-1’s effects on these core loci were abrogated in Dectin-1-knockout Tregs (Fig 5D), confirming receptor specificity in an in vitro proof-of-principle setting. Although β-glucans are known for their adjuvant properties, KQS-1’s ability to selectively restore FOXP3- and IL-10–dependent Treg function without broad immune activation (Figs 2 and 3) suggests therapeutic potential for immune-mediated diseases, and this observation serves as a proof of principle for the selective targeting of core Treg functional genes via Dectin-1 agonism. Its well-defined structure (Fig 1H and I) and lack of TLR engagement mitigate risks of off-target inflammation, and future studies should explore whether KQS-1 synergizes with existing Treg therapies (e.g., low-dose IL-2) and its efficacy in other inflammatory settings to further validate this potential.
Our findings reveal an intriguing dichotomy in the role of ROS in the regulation of FOXP3 and IL10 in Tregs. Although chronic ROS accumulation is a hallmark of the senescent phenotype observed in patient-derived Tregs (Fig 3E), we demonstrate that acute, receptor-mediated ROS production through Dectin-1 engagement serves as a critical signaling intermediate for the epigenetic reprogramming of FOXP3 and IL10 and functional restoration in an in vitro proof-of-principle context. This distinction is supported by several key observations: patient Tregs exhibited elevated baseline ROS levels (Fig 3E) concomitant with other senescent markers (SA-β-gal^+^, shortened telomeres; Fig 3A–D), which aligns with established mechanisms of age-related mitochondrial dysfunction, where persistent ROS drives cellular damage and the dysregulation of FOXP3 and IL10. In contrast, KQS-1 induced a transient ROS burst via the Dectin-1/Raf-1 axis (Fig 5B and C), which served as an epigenetic switch to remodel chromatin accessibility at FOXP3 and IL10 loci (Figs 4 and 5) and restore suppressive function without exacerbating senescence status (Fig 3 versus Fig 6). The KQS-1 effects on these core loci were entirely Dectin-1–dependent, as CRISPR knockout abolished both ROS signaling and functional rescue (Fig 5D), and this receptor specificity distinguishes our approach from broad antioxidant strategies in an in vitro proof-of-principle demonstration. These findings suggest that ROS modulation in Tregs can be harnessed to target the epigenetic regulation of FOXP3 and IL10, and future studies should explore whether KQS-1’s ROS-mediated effects synergize with mitochondrial-targeted therapies in inflammatory diseases to provide further causal validation for this pathway.
Our initial findings align with a growing body of literature, suggesting that T-cell senescence-like status contributes to immune dysregulation in chronic inflammatory diseases (16). The observed telomere attrition and increased spontaneous apoptosis in patient Tregs provide a plausible explanation for the documented numerical and functional deficits of Tregs in asthma (17), with the latter directly tied to the loss of FOXP3 and IL-10 function. The functional data cement this link; the severe impairment in suppressing responder T-cell proliferation, coupled with a drastic reduction in IL-10 and TGF-β production, effectively cripples a key regulatory arm of the immune system. Concurrently, the skewing toward IFN-γ and IL-17 production suggests that these Tregs are not merely inactive but may actively contribute to the inflammatory milieu, a phenomenon reminiscent of Treg instability observed in other autoimmune settings (18) and likely driven by the loss of FOXP3-mediated lineage control.
The most significant advance presented here is the multilevel mechanistic dissection of how KQS-1 restores Treg homeostasis by targeting FOXP3 and IL10 in an in vitro proof-of-principle setting. We pinpoint Dectin-1, a C-type lectin receptor typically associated with antifungal immunity, as the nonredundant receptor for KQS-1 on human Tregs, and this finding expands the functional repertoire of Dectin-1 beyond innate immune cells to the adaptive immunoregulatory control of core Treg genes. The downstream events involve a Raf-1/ROS signaling axis, which we show is necessary for the initiation of a sustained epigenetic program at FOXP3 and IL10 in vitro. The induction of active chromatin marks (H3K4me3, H3K27ac), increased chromatin accessibility at these two key regulatory loci, and focal DNA hypomethylation collectively create a molecular landscape that reinforces the core immunoregulatory function of Tregs (suppression of effector T-cell proliferation, secretion of IL-10/TGF-β) but does not extend to broader lineage stability programs in the current work. This phenomenon, which aligns with the concept of “trained immunity” (19), manifests as stable transcriptional up-regulation of FOXP3 and IL10—rather than global lineage rejuvenation—leading to long-lasting restoration of immunoregulatory capacity even after KQS-1 removal in vitro. This epigenetic rewiring of two core Treg genes differentiates KQS-1’s action from a mere transient activation in these proof-of-principle in vitro experiments.
The in vivo relevance of these mechanistic insights for FOXP3 and IL10 regulation is strongly supported by our mouse model data as a proof of principle rather than definitive causal validation. Administration of KQS-1 significantly ameliorated cardinal features of allergic asthma, including airway hyperresponsiveness, inflammatory cell infiltration, and remodeling, and this observation serves as a foundational demonstration of KQS-1’s in vivo potential to target Treg function rather than conclusive evidence of its therapeutic efficacy for asthma. Crucially, the adoptive transfer experiment offers proof-of-principle evidence that KQS-1–primed human Tregs—with restored FOXP3/IL-10 function—can confer protective effects in recipient mice, serving as a foundational demonstration of this therapeutic approach rather than definitive causal validation of in vivo efficacy. The expansion of IL-10^+^ Tregs in the mediastinal lymph nodes of recipient mice underscores the regulatory function of KQS-1–treated Tregs in the lung inflammatory microenvironment in this model, a proof-of-principle observation of their functional activity in vivo rather than evidence of persistent functional stability in physiological settings or for broader Treg lineage markers.
Our gain-of-function experiments complement prior knockout data by demonstrating that Dectin-1 overexpression alone is sufficient to restore KQS-1 responsiveness of FOXP3 and IL10 in Dectin-1-low Tregs in an in vitro proof-of-principle assay. This confirms that Dectin-1 expression levels directly correlate with KQS-1 efficacy for these core genes in these controlled in vitro conditions, highlighting its nonredundant role in the restoration of FOXP3/IL-10–dependent Treg function as a proof of principle. The clinical relevance of this finding is underscored by the blunted KQS-1 responses observed in CLEC7A haploinsufficient donors in vitro, suggesting that Dectin-1 expression could serve as a biomarker for predicting therapeutic outcomes for KQS-1 treatment, and this observation requires further in vivo and clinical investigation for causal validation.
Although Tregs from asthmatic patients exhibit senescence-associated markers (telomere shortening, SA-β-gal activity), their heightened apoptosis and lack of apoptosis resistance distinguish it from classical senescence-like state. We propose that chronic inflammation drives Tregs toward a dysfunctional, presenescence phenotype, which KQS-1 reverses by restoring metabolic homeostasis (Fig 3) and the epigenetic stability of FOXP3 and IL10 (Fig 4) in an in vitro proof-of-principle setting. The persistence of functional deficits in patient-derived Tregs despite identical IL-2 support (Fig 3D–G) strongly suggests these phenotypes reflect intrinsic cellular abnormalities of senescent Tregs rather than culture condition variability. Notably, the senescent characteristics observed in patient Tregs (SA-β-gal^+^ frequency and apoptotic rates) were present even under optimal cytokine support, reinforcing their disease relevance. Furthermore, KQS-1’s ability to rescue FOXP3/IL-10–dependent Treg function (Figs 4, 5, and 6) was demonstrated in this IL-2–containing system, indicating its effects operate through pathways distinct from basic IL-2–mediated survival signaling in these proof-of-principle in vitro experiments.
Although our study demonstrates the efficacy of KQS-1 in restoring FOXP3/IL-10–dependent Treg function through Dectin-1–mediated epigenetic reprogramming as a proof of principle (Figs 2, 3, 4, and 5), several important considerations emerge regarding its therapeutic application. As a fungal polysaccharide with innate immune receptor activity, KQS-1’s potential for systemic activation or off-target effects warrants discussion. Our in vivo experiments showed no evidence of toxicity at the therapeutic dose (1 mg/kg), with treated mice exhibiting normal weight profiles and no signs of cytokine storm (Fig 6A–G), and this is a key proof-of-principle observation for its safety profile that requires further toxicological investigation. The molecular weight profile of KQS-1 (Fig 1I) suggests favorable pharmacokinetic properties, though comprehensive biodistribution studies are needed to provide further causal validation of its in vivo behavior and target engagement of Tregs in the lung. The dosing regimen was carefully optimized based on dose–response experiments showing maximal FOXP3/IL-10–dependent Treg functional enhancement at 10 μg/ml in vitro (Fig 1G), which translated effectively to the in vivo setting in our proof-of-principle mouse model. Importantly, adoptive transfer of KQS-1–primed Tregs in Rag1^−/−^ mice provided proof-of-principle evidence of therapeutic potential without apparent adverse effects (Fig 6H–J), supporting the feasibility of this approach as a starting point for further preclinical development. However, we acknowledge that clinical translation will require addressing several challenges: (1) potential batch-to-batch variability in polysaccharide preparation, (2) the need for patient stratification based on Dectin-1 expression patterns, and (3) optimization of administration routes to minimize systemic exposure and maximize lung Treg targeting. Future studies should include detailed toxicological assessments and GMP-compliant manufacturing processes to advance this promising therapeutic strategy and provide further causal validation of its potential for asthma and other immune-mediated diseases.
Our findings demonstrate that KQS-1–induced ROS is critical for H3K4me3/H3K27ac deposition at FOXP3/IL10 loci in vitro (Figs 4A and 5B), and the precise mechanism involves several potential pathways supported by our data and existing literature. First, the Raf-1/ROS axis (Fig 5D) may activate stress-responsive kinases such as p38 MAPK, which could phosphorylate histone-modifying enzymes including SETD1A for H3K4me3 or p300/CBP for H3K27ac, thereby modulating their recruitment or activity at the FOXP3 and IL10 promoters, and this interpretation is supported by the complete abrogation of both marks upon ROS scavenging (Fig 5B) in vitro. Second, ROS might transiently alter the availability of key metabolites like α-KG and SAM that serve as cofactors for Jumonji demethylases or methyltransferases, leading to focal epigenetic changes at FOXP3 and IL10. However, the focal nature of H3K4me3 changes at these specific loci (Fig 4A) suggests localized enzyme regulation rather than global metabolic shifts in these in vitro experiments, and this observation aligns with our methylation array data showing focal hypomethylation at FOXP3 (Δβ = −0.18) and IL10 (Δβ = −0.22) regulatory CpG sites (Fig 4F). Third, ROS may directly modulate epigenetic enzymes through redox-sensitive mechanisms at these core Treg loci. For instance, oxidation of cysteine residues in histone-modifying enzymes like KDM5B demethylases or p300 acetyltransferases could reversibly inhibit or activate their function at FOXP3 and IL10, and this possibility is consistent with studies demonstrating redox-sensitive regulation of chromatin remodelers at specific gene loci. Although our data clearly establish ROS as a necessary upstream signal for the epigenetic priming of FOXP3 and IL10 in vitro (Figs 4 and 5), several mechanistic limitations warrant future investigation to provide causal validation for this pathway. Additional studies should identify specific ROS-sensitive enzymes or cofactors that regulate FOXP3 and IL10 epigenetics, test whether ROS scavengers alter α-KG/SAM levels in Tregs, and map kinase phosphorylation sites on histone-modifying enzymes at these two loci. These mechanisms are not mutually exclusive and may work synergistically to stabilize FOXP3 expression and enhance IL-10–dependent Treg suppressive function in vitro, as evidenced by the complete loss of Treg suppression upon ROS scavenging (Fig 5C). The Raf-1/ROS axis appears particularly important in these in vitro proof-of-principle experiments, as Raf-1 inhibition phenocopies the effects of NAC treatment (Fig 5D), placing ROS downstream of Raf-1 in the epigenetic regulation of FOXP3 and IL10.
Our study, however, has several limitations, and all our key findings are presented as proof of principle rather than definitive causal validation, with further investigation required to confirm these observations in physiological and clinical settings. Critically, our conceptual framing is strictly limited to the restoration of FOXP3- and IL-10–dependent Treg function—the experimental focus of this work—and does not claim global Treg lineage rejuvenation or the restoration of broad lineage stability programs, a key revision that aligns our conclusions with the specificity of our data.
Narrow epigenetic focus on FOXP3 and IL10: A key limitation of this study is the exclusive focus of our epigenetic analyses on FOXP3 and IL10, the core drivers of Treg immunoregulatory function. Although these genes are indispensable for Treg identity and function, our data do not address whether KQS-1 restores broader Treg lineage stability programs—including additional lineage-defining transcription factors (e.g., Eos, Helios, CTLA-4), genome-wide Treg-specific epigenetic landscapes, or the suppression of lineage instability markers. Our current findings demonstrate the restoration of FOXP3- and IL-10–dependent function, but global Treg lineage rejuvenation (encompassing all defining features of the Treg lineage) has not been formally tested. This represents an important open question regarding the full scope of KQS-1’s effects on Treg identity, and future studies will extend our epigenetic analyses to include a broader panel of Treg lineage stability markers and genome-wide Treg-specific chromatin landscapes (via extended ATAC-seq and ChIP-seq for lineage-defining transcription factors) to directly test whether KQS-1’s epigenetic reprogramming extends beyond FOXP3 and IL10 to restore global Treg lineage stability.
Unassessed Th2/GATA3 Treg plasticity
A second key limitation is that we did not directly assess Treg plasticity toward Th2/GATA3 phenotypes, including GATA3 expression or intrinsic IL-4/IL-5/IL-13 production by the Treg compartment. Allergic asthma is a prototypic Th2-driven disease, and Th2-like Treg instability is a well-recognized form of Treg dysfunction in type 2 immune disorders. Our data demonstrate KQS-1 reverses pro-inflammatory (IFN-γ/IL-17) skewing and restores core FOXP3/IL-10–dependent suppressive function as a proof of principle, but we cannot comment on whether KQS-1 also inhibits Th2-directed Treg plasticity, and this represents an important unaddressed question regarding the full scope of KQS-1–mediated Treg functional restoration that requires further investigation.
Rag1−/− adoptive transfer model limitations
A major conceptual limitation of our adoptive transfer experiments is the use of Rag1^−/−^ mice, which lack endogenous T/B cells and produce insufficient IL-2—an essential cytokine for Treg homeostasis. This IL-2–deficient, nonphysiological host environment prevents causal inference about the long-term functional stability, durability, or therapeutic sufficiency of KQS-1–primed Tregs under physiological IL-2–sufficient conditions. Our data demonstrate KQS-1–primed Tregs can persist and function in an IL-2–restricted setting as a proof of principle, but not that these effects translate to the physiological IL-2 milieu of a WT asthmatic host. Syngeneic mouse-to-mouse transfer in IL-2–sufficient recipients is required to rigorously test KQS-1’s effects on FOXP3/IL-10–dependent Treg function and stability in a physiologically relevant context and provide causal validation of these observations.
Patient cohort specificity
The patient cohort, while well characterized, represents a specific allergic asthmatic endotype; the applicability of our findings to other asthma endotypes or severe, steroid-resistant disease requires further investigation and causal validation. Future studies will expand the patient cohort to include diverse asthma endotypes to test the generalizability of KQS-1’s effects on FOXP3 and IL10 regulation in Tregs.
Unelucidated epigenetic mechanistic details
Although we establish the necessity of the Dectin-1/Raf-1/ROS axis for initiating the epigenetic reprogramming of FOXP3 and IL10 in vitro as a proof of principle, the precise molecular steps linking ROS production to the recruitment of specific histone methyltransferases and demethylases at these loci remain to be fully elucidated and validated in in vivo settings.
Long-term epigenetic program safety and stability: The long-term stability and safety of inducing this targeted epigenetic program at FOXP3 and IL10 in Tregs need to be thoroughly assessed in future chronic toxicity studies to provide causal validation of its therapeutic potential for clinical translation.
In conclusion, we propose a paradigm in which the functional failure of Tregs in allergic asthma is, at least in part, a consequence of a reversible senescence and unstable state, and our findings provide proof-of-principle evidence for this mechanism rather than definitive causal validation. Our work introduces KQS-1 as an immunomodulatory agent that acts not by broadly suppressing immunity, but by precisely rejuvenating the body’s own regulatory mechanisms through epigenetic reprogramming via the Dectin-1 pathway, and this study serves as a foundational demonstration of KQS-1’s potential to instill a durable “trained” regulatory phenotype in Tregs. This strategy offers a promising and novel therapeutic avenue for the treatment of allergic asthma and potentially other immune-mediated diseases characterized by Treg dysfunction, and all our observations represent a critical proof of principle that requires further preclinical and clinical investigation to establish definitive causal validation of its efficacy, safety, and mechanistic underpinnings in physiological and clinical settings.
Materials and Methods
Study participants
Human samples were obtained from participants under a protocol (Approval No.: H2023056) approved by the Shenzhen University Institutional Review Board, and all participants provided written informed consent. Fifteen patients with allergic asthma and 15 age- and sex-matched healthy controls were recruited from the Department of Respiratory Medicine at Shenzhen University Affiliated Hospital between 2023 and 2024. All experiments in this study were conducted in strict accordance with the ethical principles outlined in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report, ensuring the protection and rights of all research participants. The diagnosis of allergic asthma was confirmed based on the Global Initiative for Asthma (GINA) 2023 criteria: (1) presence of typical asthma symptoms (recurrent wheezing, dyspnea, cough); (2) positive skin prick test to common aeroallergens (house dust mite, birch pollen, ragweed); and (3) a ratio of forced expiratory volume in 1 s (FEV_1_) to forced vital capacity (FVC) < 70% or FEV_1_ reversibility ≥ 12% after salbutamol inhalation. Healthy controls had no history of allergic, respiratory, or autoimmune disorders and negative skin prick test results (demographic data are presented in Table S1).
Table S1. Demographic, peripheral blood cellular, and serum characteristics of study participants (values are median [IQR] or n). SPT, skin prick test; ICS, inhaled corticosteroid; LABA, long-acting β_2_-agonist; hs-CRP, high-sensitivity C-reactive protein; FeNO, fractional exhaled nitric oxide.
Exclusion criteria for both groups included the following: (1) acute infection (viral or bacterial) within 4 wk; (2) use of systemic corticosteroids, immunosuppressants, or biological agents (e.g., anti-IgE, anti-IL-5) within 3 mo; (3) comorbidities such as chronic obstructive pulmonary disease, interstitial lung disease, or malignancy; and (4) pregnancy or lactation.
Reagents and instruments
Cell isolation and culture
Cells were isolated and cultured using the following: Ficoll-Paque PLUS (GE Healthcare), RPMI-1640 medium (Gibco, Thermo Fisher Scientific), FBS (Sigma-Aldrich; heat-inactivated at 56°C for 30 min), and penicillin–streptomycin (100 U/ml penicillin, 100 μg/ml streptomycin; Gibco).
Flow cytometry
Fluorochrome-conjugated antibodies used in this study were as follows: anti-human CD4-PE-Cy7 (clone: RPA-T4), CD25-APC (clone: M-A251), CD127-FITC (clone: HIL-7R-M21), and FoxP3-PE (clone: PCH101) (all from BD Biosciences); anti-human Dectin-1-PE (clone: 29Gl2; BioLegend); and isotype controls (BD Biosciences). Telomere staining kit (eBioscience, Thermo Fisher Scientific) and Annexin V-FITC/PI apoptosis detection kit (KeyGen Biotech) were used to detect telomere length and apoptosis.
Flow cytometry methods for molecular and phenotypic detection in human and murine Tregs
Flow cytometry was used for comprehensive phenotypic, molecular, and functional characterization of regulatory T cells (Tregs) from allergic asthmatic patients, healthy controls, and murine models, with multicolor staining panels optimized to detect cell surface markers, intracellular proteins, senescence-associated molecules, and apoptotic markers while minimizing spectral overlap. For Treg phenotypic identification, human PBMCs and murine lymphoid/lung-derived cells were stained with fluorochrome-conjugated monoclonal antibodies targeting CD4 (e.g., FITC/APC) and CD25 (e.g., PE/PerCP-Cy5.5) for cell surface labeling, followed by intracellular fixation and permeabilization (using a commercial FoxP3 staining buffer kit) to detect the master Treg transcription factor FoxP3 (e.g., Alexa Fluor 647), defining Tregs as CD4^+^CD25^+^FoxP3^+^; gating strategies included sequential exclusion of dead cells (via 7-AAD/PI staining) and nonlymphoid cells (via forward/side-scatter gating), with isotype-matched control antibodies used to set fluorescence thresholds for all markers. Senescence-associated β-galactosidase (SA-β-gal) detection in Tregs employed a fluorogenic SA-β-gal substrate (5-dodecanoylaminofluorescein di-β-D-galactopyranoside, C12FDG) with intracellular loading: cells were incubated with C12FDG at 37°C for 1 h, followed by surface marker staining for Treg gating, with SA-β-gal^+^ Tregs gated as cells with mean fluorescence intensity (MFI) > 2× that of unstained isotype controls. Telomere length measurement used a telomere-specific peptide nucleic acid (PNA) probe conjugated to Cy3/Alexa Fluor 488, targeting the human (TTAGGG)n telomeric repeat; fixed and permeabilized Tregs were hybridized with the PNA probe in a formamide-containing hybridization buffer at 42°C for 30 min, with unbound probe removed by stringent washing, and telomere signal MFI quantified in gated CD4^+^CD25^+^FoxP3^+^ Tregs (a scrambled noncomplementary PNA probe served as a negative control to correct for nonspecific fluorescence). Apoptosis detection was performed using an Annexin V-FITC/PI double-staining kit: Tregs were cultured in RPMI-1640/10% FBS for 24 h, washed in cold PBS, and incubated with Annexin V-FITC (for phosphatidylserine externalization) and PI (for late apoptosis/necrosis) in calcium-containing binding buffer for 15 min at RT, with early apoptotic Tregs gated as Annexin V^+^PI^−^ and analyzed via flow cytometry. Intracellular cytokine staining (ICS) to characterize Treg cytokine profiles involved ex vivo stimulation of Tregs with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) and ionomycin (1 μg/ml) for 4 h, with brefeldin A (10 μg/ml) added for the final 2 h to block cytokine secretion; cells were then surface-stained for CD4/CD25, fixed/permeabilized, and stained with fluorochrome-conjugated antibodies against IL-10, TGF-β, IFN-γ, and IL-17 (all with isotype controls), with cytokine-positive Treg frequencies quantified in the CD4^+^CD25^+^FoxP3^+^ gate. Dectin-1 expression and KQS-1 binding assays included cell surface staining of Tregs with a Dectin-1–specific monoclonal antibody (e.g., PE-conjugated) to quantify protein expression (MFI) relative to dendritic cells (DCs), and biotinylated KQS-1 staining followed by streptavidin–Alexa Fluor 488 conjugation to detect direct KQS-1 binding to Treg surface Dectin-1 (TLR2/TLR4 binding was assessed in parallel with specific antibodies as negative controls). All flow cytometry data were acquired on a BD LSRFortessa/X20 flow cytometer (BD Biosciences) and analyzed with FlowJo v10 software (Tree Star), with at least 10,000 Treg events collected per sample to ensure statistical rigor; all staining protocols were optimized for primary human and murine Tregs, with batch-to-batch consistency verified for all antibodies and probes, and gating strategies standardized across all experimental groups (healthy controls, asthmatic patients, untreated/KQS-1–treated cells/mice).
Telomere-specific peptide nucleic acid (PNA) probe for flow cytometric telomere length analysis in human Tregs
A telomere-specific PNA probe is a synthetic sequence-specific oligomer targeting the conserved human telomeric (TTAGGG)n hexanucleotide repeat, engineered with a neutral pseudopeptide backbone that confers superior binding affinity, thermal stability, and sequence specificity for telomeric DNA compared with conventional nucleic acid probes. Conjugated to a photostable fluorochrome (e.g., Cy3, Alexa Fluor 488) compatible with multicolor flow cytometry, this probe enables quantitative fluorescence detection of telomere length in fixed and permeabilized primary human regulatory T cells (Tregs) via flow-FISH. Resistant to nucleases and proteases, it penetrates nuclear chromatin to hybridize exclusively to telomeric repeats under mild conditions that preserve cell surface and intracellular Treg phenotypic markers (CD4, CD25, FoxP3), with a scrambled noncomplementary PNA probe of identical fluorochromes used as a negative control to quantify nonspecific background fluorescence. Telomere length is inferred from the mean fluorescence intensity (MFI) of the probe signal in gated CD4^+^CD25^+^FoxP3^+^ Tregs, providing a sensitive and reproducible method to measure telomere shortening as a hallmark of the senescence-like phenotype in asthmatic patient-derived Tregs.
Functional assays
CFSE (5(6)-carboxyfluorescein diacetate succinimidyl ester; Invitrogen, Thermo Fisher Scientific); anti-CD3/CD28 activation beads (Dynabeads Human T-Activator CD3/CD28; Thermo Fisher Scientific); and SA-β-gal staining kit (Cell Signaling Technology) were used in the Treg functional assays.
Cytokine detection
Human IL-10, TGF-β1, IFN-γ, and IL-17 ELISA kits (R&D Systems) were used for the detection of cytokines.
KQS-1 preparation
KQS-1 (KQSESHFVDAQPEQQQR), a peptide isolated from Adzuki bean, was purified via DEAE-cellulose and Sephadex G-100 chromatography. Its purity (>95%) was verified by HPLC (Agilent 1260 Infinity; Agilent Technologies) using a Shodex OHpak SB-806M HQ column (8.0 × 300 mm) with 0.1 M NaNO_3_ as the mobile phase (flow rate: 0.8 ml/min, detection wavelength: 210 nm). KQS-1 was dissolved in sterile PBS (pH 7.4) to a stock concentration of 1 mg/ml and stored at −20°C.
KQS-1 was biotinylated to generate a probe for detection assays. Briefly, the polysaccharide (1–5 mg) was first oxidized in 50 mM sodium metaperiodate (in 10 mM acetate buffer, pH 5.5) for 2 h at 4°C in the dark to generate aldehyde groups. The reaction was quenched with ethylene glycol, and the oxidized KQS-1 was purified via dialysis. The product was then conjugated to a 20-fold molar excess of biotin hydrazide in PBS (pH 7.4) overnight at 4°C. To stabilize the conjugate, the resulting hydrazone bond was reduced with 15 mM sodium cyanoborohydride. Finally, the biotinylated KQS-1 was purified by extensive dialysis against PBS to remove unreacted biotin hydrazide. Successful labeling was confirmed by a streptavidin-HRP dot-blot assay.
β-Glucan
A long chain of glucose (sugar) molecules linked together by beta-glycosidic bonds was purchased from Sigma-Aldrich (9012-72-0). β-Glucan was quantified by ELISA (Cat# KMEOt013375; Kemiao Biotech) following the protocol provided by the manufacturer.
Epigenetic analysis
ChIP-grade antibodies anti-H3K4me3 (clone: C42D8) and anti-H3K27ac (clone: D8L3M) (Cell Signaling Technology); ChIP assay kit (Millipore); and Illumina Infinium HumanMethylation450K BeadChip (Illumina) were used to perform ChIP–qPCR to analyze epigenetic changes at specific genes.
Signaling inhibitors
Signaling inhibitors used in this study were GW5074 (Raf-1 inhibitor; Selleck Chemicals) and N-acetylcysteine (NAC, ROS scavenger; Sigma-Aldrich).
CRISPR/Cas9 reagents
sgRNA targeting human CLEC7A (Dectin-1 gene), nontargeting sgRNA (control), and Cas9 nuclease (Santa Cruz Biotechnology); and Neon Transfection System (Thermo Fisher Scientific) were used for Treg transfection.
Instruments
Instruments used in this study were flow cytometer (BD FACSCanto II, BD Biosciences); qPCR system (Applied Biosystems 7500; Thermo Fisher Scientific); ATAC-seq sequencing platform (Illumina NovaSeq 6000; Illumina); and histology slide scanner (Pannoramic 250 Flash; 3D Histech).
PBMC isolation and Treg purification
Peripheral venous blood (20 ml) was collected from each participant in EDTA-containing tubes. PBMCs were isolated via density gradient centrifugation using Ficoll-Paque PLUS (1.077 g/ml) at 400g for 30 min at 20°C. The PBMC layer was carefully aspirated, washed twice with PBS (400g for 10 min at 4°C), and resuspended in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin (complete medium).
Tregs (CD4^+^CD25^+^CD127_lo_FoxP3^+^) were purified using human Treg Isolation Kit (Miltenyi Biotec) following the manufacturer’s protocol: first, CD4^+^ T cells were enriched via negative selection, and then, CD25^+^ cells were isolated via positive selection. The purity of isolated Tregs was verified by flow cytometry (≥90% CD4^+^CD25^+^CD127_io_FoxP3^+^; gating strategy in Fig S1A) before subsequent experiments. 100 IU/ml recombinant human IL-2 (PeproTech) was added throughout the 72-h experimental period.
Assessment of Treg senescence
Telomere length measurement
Telomere length was detected using a flow cytometry–based telomere staining kit. Briefly, 1 × 10^6^ purified Tregs were fixed with 2% PFA for 10 min at RT, permeabilized with 0.1% Triton X-100 for 5 min, and incubated with telomere-specific peptide nucleic acid (PNA) probe (FITC-conjugated) at 80°C for 10 min, followed by hybridization at 37°C for 2 h. After washing with wash buffer, cells were analyzed via flow cytometry. Mean fluorescence intensity (MFI) of telomere signals was recorded, and relative telomere length was calculated as the ratio of MFI in patient Tregs to that in control Tregs.
SA-β-gal staining
SA-β-gal activity was detected using a SA-β-gal staining kit. Tregs (5 × 10^4^ cells/well) were seeded in 24-well plates, fixed with 2% PFA for 10 min at RT, washed with PBS, and incubated with SA-β-gal staining solution (containing X-gal) at 37°C (no CO_2_) for 16 h. Cells were observed under an inverted microscope (Olympus IX73; Olympus), and the percentage of SA-β-gal–positive cells (blue-stained cells) was counted in five random fields per well. For flow cytometry–based quantification, stained cells were resuspended in PBS and analyzed, with the MFI threshold for positivity set at >2× that of unstained control cells.
Spontaneous apoptosis assay
Tregs (1 × 10^6^ cells/ml) were cultured in complete medium without exogenous stimuli for 24 h at 37°C with 5% CO_2_. Apoptosis was detected using an Annexin V-FITC/PI kit: cells were harvested, washed with cold PBS, resuspended in binding buffer, stained with Annexin V-FITC (5 μl) and PI (5 μl) for 15 min at RT in the dark, and analyzed via flow cytometry. The apoptosis rate was defined as the percentage of Annexin V^+^PI^−^ cells (early apoptosis).
Treg functional assays
CFSE-based suppression assay
Responder T cells (CD4^+^CD25^−^) were isolated from PBMCs using a CD4^+^CD25^−^ T Cell Isolation Kit (Miltenyi Biotec) and labeled with 5 μM CFSE for 10 min at 37°C (quenched with five volumes of cold FBS). Labeled responder cells (1 × 10^5^ cells/well) were cocultured with purified Tregs (1 × 10^5^ cells/well; Treg:responder ratio = 1:1) in 96-well round-bottom plates, and activated with anti-CD3/CD28 beads (1 bead per cell). After 72 h of culture at 37°C with 5% CO_2_, cells were harvested, stained with anti-CD4-PE-Cy7 antibody, and analyzed via flow cytometry. The proliferation of CD4^+^CFSE^+^ responder cells was assessed by CFSE dilution, and suppression efficiency was calculated using the formula:
The proliferation index was determined using FlowJo software (v10.8, BD Biosciences).
Intracellular cytokine staining (ICS)
Tregs (1 × 10^6^ cells/ml) were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 4 h at 37°C with 5% CO_2_, with brefeldin A (10 μg/ml) added during the final 2 h to inhibit cytokine secretion. Cells were fixed with 2% PFA for 15 min at RT, permeabilized with 0.5% saponin for 30 min, and stained with fluorochrome-conjugated antibodies against IL-10-FITC, TGF-β1-PE, IFN-γ-APC, and IL-17-PE-Cy7 for 1 h at RT in the dark. After washing with permeabilization buffer, cells were analyzed via flow cytometry, and the frequency of cytokine-positive Tregs was calculated.
Cytokine secretion detection via ELISA
Tregs (1 × 10^6^ cells/ml) were cultured in complete medium for 72 h at 37°C with 5% CO_2_. Culture supernatants were collected by centrifugation (12,000g for 10 min at 4°C), and concentrations of IL-10, active TGF-β1, IFN-γ, and IL-17 were measured using ELISA kits following the manufacturer’s instructions. Standard curves were generated for each cytokine, and sample concentrations were calculated based on the absorbance values (measured at 450 nm with a microplate reader, Bio-Rad Model 680; Bio-Rad Laboratories).
KQS-1 treatment of Tregs
Purified patient Tregs (1 × 10^6^ cells/ml) were seeded in six-well plates and treated with KQS-1 at a final concentration of 10 μg/ml (optimized via preliminary dose–response experiments: 0.1–100 μg/ml, with 10 μg/ml showing maximal Treg rescue without cytotoxicity). Control groups included untreated Tregs and Tregs treated with PBS (vehicle control). Cells were cultured for 48 h at 37°C with 5% CO_2_, then harvested for functional assays (suppression, apoptosis), cytokine detection, or epigenetic analysis.
For healthy Treg control experiments, Tregs from healthy donors were treated with 10 μg/ml KQS-1 for 48 h, and apoptosis rate was measured via Annexin V/PI staining.
Epigenetic analysis
Chromatin immunoprecipitation (ChIP)–qPCR
ChIP was performed using a ChIP assay kit. Briefly, 5 × 10^6^ KQS-1–treated or untreated Tregs were cross-linked with 1% formaldehyde for 10 min at RT, quenched with 0.125 M glycine, and lysed. Chromatin was sheared into 200- to 500-bp fragments via sonication (Bioruptor Plus; Diagenode). After preclearing with protein A/G agarose beads, chromatin was incubated with 5 μg of anti-H3K4me3, anti-H3K27ac, or normal IgG (negative control) overnight at 4°C. Immune complexes were captured with protein A/G beads and washed, and cross-links were reversed at 65°C for 4 h. DNA was purified via phenol–chloroform extraction and ethanol precipitation.
qPCR was performed using SYBR Green Master Mix (Applied Biosystems) with primers targeting the FOXP3 promoter (forward: 5′- GCTGCCCTTCTCCAAGATGA-3′, reverse: 5′- CCCAGTGGTTTTGCTGACCT-3′) and IL-10 locus (forward: 5′- TGGCCAAGGATGTCAAAGAG-3′, reverse: 5′- GCTCTCCCTGGTTTCTCTGC-3′). Relative enrichment was calculated using the 2^−^ΔΔCt method, normalized to input DNA, and compared with the IgG control. For global H3K27ac analysis, qPCR was performed using primers targeting a housekeeping gene (GAPDH) promoter (5′-TGGTGTTTGGGCTCCTTGA-3′, reverse: 5′-GCAGAGGGCAGCAGTGGT-3′) as a reference.
Assay for transposase-accessible chromatin using sequencing (ATAC-seq)
ATAC-seq was performed as previously described (20). Briefly, 5 × 10^4^ Tregs were washed with cold PBS, resuspended in lysis buffer, and centrifuged. Nuclei were incubated with Tn5 transposase (Illumina) for 30 min at 37°C. Transposed DNA was purified using MinElute PCR Purification Kit (QIAGEN) and amplified via PCR (12 cycles). Libraries were quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and sequenced on an Illumina NovaSeq 6000 platform (paired-end 150-bp reads, ∼30 million reads per sample).
Sequence data were aligned to the human genome (hg38) using Bowtie2 (v2.4.4), and duplicate reads were removed with Picard Tools (v2.27.5). Accessible chromatin regions were called using MACS2 (v2.2.7.1) with FDR < 0.05 and fold change > 2. Motif enrichment analysis was performed using HOMER (v4.11) to identify transcription factor binding motifs in newly accessible regions.
DNA methylation array
Genomic DNA was extracted from Tregs using DNeasy Blood and Tissue Kit (QIAGEN) and bisulfite-converted using EZ DNA Methylation-Gold Kit (Zymo Research). Methylation profiling was performed using the Illumina Infinium HumanMethylation450K BeadChip, which targets ∼485,000 CpG sites across the genome. Arrays were scanned on Illumina iScan System, and data were processed using GenomeStudio Methylation Module (v1.9.0; Illumina).
Methylation levels (β-values) were calculated as the ratio of methylated probe intensity to total (methylated + unmethylated) probe intensity. Focal hypomethylation was defined as a mean Δβ (β-untreated - β-KQS-1–treated) < −0.1. CpG sites within the regulatory regions (promoters, enhancers) of FOXP3 and IL-10 were selected based on the UCSC Genome Browser (hg38).
FOXP3 protein expression detection
KQS-1–treated or untreated Tregs were fixed with 2% PFA for 15 min at RT, permeabilized with 0.5% saponin for 30 min, and stained with anti-FoxP3-PE antibody for 1 h at RT in the dark. After washing, cells were analyzed via flow cytometry, and FOXP3 protein levels were quantified as the MFI of FoxP3-PE signals (Fig S3B).
Dectin-1–mediated signaling experiments
KQS-1 binding assay
Biotinylated KQS-1 was prepared using EZ-Link Sulfo-NHS-LC-Biotin Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Briefly, 1 mg of KQS-1 was dissolved in 1 ml of PBS (pH 7.4), mixed with a 10-fold molar excess of Sulfo-NHS-LC-Biotin, and incubated at RT for 1 h with gentle shaking. Unconjugated biotin was removed via dialysis against PBS (MWCO: 3,500 Da) for 24 h (with three buffer changes) to obtain biotinylated KQS-1 (final concentration: 1 mg/ml).
For the binding assay, 1 × 10^6^ purified Tregs were resuspended in FACS buffer (PBS + 2% FBS + 0.1% sodium azide) and incubated with biotinylated KQS-1 (10 μg/ml) or biotinylated PBS (vehicle control) for 30 min at 4°C. After washing twice with FACS buffer, cells were stained with streptavidin-PE (1:200 dilution; BD Biosciences) for 20 min at 4°C in the dark. To verify Dectin-1 specificity, a competitive binding control group was included: Tregs were preincubated with 100 μg/ml unlabeled anti-Dectin-1 antibody (clone: 29Gl2; BioLegend) for 15 min at 4°C before adding biotinylated KQS-1. Cells were analyzed via flow cytometry, and mean fluorescence intensity (MFI) of streptavidin-PE signals was recorded.
For TLR2/4 binding verification (Fig S4A), the same protocol was used, with biotinylated KQS-1 incubated with Tregs pretreated with unlabeled anti-TLR2 (clone: TL2.1; BioLegend) or anti-TLR4 (clone: HTA125; BioLegend) antibodies (100 μg/ml each), and MFI changes were measured.
Signaling inhibition experiments
To investigate the role of Raf-1 and ROS in KQS-1–mediated effects, patient Tregs were pretreated with either the Raf-1 inhibitor GW5074 (10 μM) or the ROS scavenger N-acetylcysteine (NAC; 5 mM) for 1 h at 37°C with 5% CO_2_. Control groups included Tregs treated with DMSO (solvent for GW5074, final concentration: 0.1%) or PBS (solvent for NAC). After pretreatment, all groups were incubated with KQS-1 (10 μg/ml) for 48 h.
Subsequently, two endpoints were measured: (1) H3K4me3 enrichment at the FOXP3 promoter via ChIP–qPCR (as described in Epigenetic analysis—ChIP–qPCR); and (2) Treg suppressive capacity via CFSE-based suppression assay (as described in Treg functional assays—CFSE-based suppression assay). The inhibition efficiency of GW5074 was verified by Western blot (detecting phosphorylated Raf-1; anti-p-Raf-1 antibody, clone: 9427; Cell Signaling Technology), and ROS levels were measured using a DCFH-DA ROS detection kit (KeyGen Biotech) to confirm NAC activity.
CRISPR/Cas9-mediated Dectin-1 knockout (KO)
CRISPR/Cas9 was used to knock out the Dectin-1 gene (CLEC7A) in patient Tregs. The sgRNA targeting CLEC7A was designed using the CRISPR Design Tool (MIT) and synthesized by Santa Cruz Biotechnology. The sgRNA sequences targeting human CLEC7A (Dectin-1) used in our study were as follows:
sgRNA-1: 5′-GACCTGCGACTCACCGCCGT-3′ (targeting exon 2).
sgRNA-2: 5′-GCTGGTCAAGATCGAGCCGT-3′ (targeting exon 3).
These sequences were selected based on:
-
- High on-target efficiency scores (>90%) predicted by CRISPR design tools
-
- Minimal off-target potential (verified by in silico analysis)
-
- Successful knockout validation showing >90% efficiency (Fig S4C)
The nontargeting control sgRNA sequence was as follows: 5′-GCGAGGTATTCGGCTCCGCG-3′.
Treg transfection was performed using Neon Transfection System (Thermo Fisher Scientific). Briefly, 2 × 10^6^ Tregs were resuspended in Neon Resuspension Buffer R, mixed with 5 μg of Cas9 nuclease and 3 μg of sgRNA (CLEC7A or nontargeting), and transfected using a 100 μl Neon Tip with the following parameters: voltage = 1,600 V, pulse width = 10 ms, pulse number = 3. After transfection, Tregs were cultured in complete medium for 48 h at 37°C with 5% CO_2_.
The KO efficiency of Dectin-1 was determined by two methods: (1) flow cytometry (anti-Dectin-1-PE antibody, as described in Dectin-1–mediated signaling experiments—KQS-1 binding assay) to measure surface Dectin-1 expression; and (2) Sanger sequencing of the CLEC7A target region to detect indels (insertions/deletions) in the gene. Only Treg populations with >90% Dectin-1 KO efficiency (Fig S4B) were used for subsequent experiments.
KQS-1 (10 μg/ml) was added to both Dectin-1 KO Tregs and nontargeting sgRNA-transfected Tregs, and incubated for 48 h. Treg suppressive capacity (CFSE assay) and IL-10 secretion (ELISA) were measured to assess the impact of Dectin-1 KO on KQS-1’s effects.
In vivo mouse experiments
HDM-induced asthma model establishment
Female C57BL/6J mice (6–8 wk old, 18–22g) were purchased from the Experimental Animal Center of Shenzhen University and housed under specific pathogen-free (SPF) conditions (temperature: 22 ± 2°C, humidity: 50 ± 10%, 12-h light/dark cycle). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shenzhen University (Approval No.: A20230086), and were performed in accordance with relevant guidelines/regulations.
The house dust mite (HDM) extract (Dermatophagoides pteronyssinus, Greer Laboratories) was dissolved in PBS to a concentration of 100 μg/ml. Mice were randomly divided into three groups (n = 8 per group): (1) control group: intranasal administration of PBS (50 μl/mouse) on days 0–2 and 7–9; (2) HDM group: intranasal administration of HDM extract (10 μg/mouse in 50 μl PBS) on days 0–2 and 7–9; and (3) HDM + KQS-1 group: HDM sensitization (same as the HDM group) plus intraperitoneal (i.p.) injection of KQS-1 (5 mg/kg in 200 μl PBS) on days 10–14. On day 15, all mice were euthanized for subsequent analyses.
Measurement of airway hyperresponsiveness (AHR)
On day 14 (24 h before euthanasia), AHR was measured using a whole-body plethysmograph (Buxco Research Systems). Mice were placed in plethysmograph chambers and acclimated for 10 min. Baseline Penh (enhanced pause, an indicator of airway resistance) was recorded, followed by nebulization of methacholine (MCh) at increasing concentrations (0, 6.25, 12.5, 25, 50 mg/ml) for 3 min per concentration. Penh values were measured for 5 min after each MCh nebulization, and the mean Penh value at each concentration was calculated. AHR was represented as the percentage change in Penh relative to baseline (0 mg/ml MCh).
Bronchoalveolar lavage fluid (BALF) collection and cell counting
After measuring AHR, mice were anesthetized with isoflurane (3% for induction, 1.5% for maintenance). The trachea was cannulated with a 24-gauge catheter, and BALF was collected by lavaging the lungs with 0.8 ml of cold PBS (three times, total volume: 2.4 ml). BALF was centrifuged at 400 g for 10 min at 4°C, and the supernatant was stored at −80°C for cytokine detection.
The cell pellet was resuspended in 100 μl of PBS, and the total cell count was determined using a hemocytometer. For differential cell counting, 50 μl of cell suspension was spread on glass slides, air-dried, and stained with Wright–Giemsa stain (Sigma-Aldrich). At least 200 cells per slide were counted under a light microscope (Olympus BX53; Olympus), and the percentages of eosinophils, neutrophils, macrophages, and lymphocytes were calculated. Absolute cell counts were obtained by multiplying the percentage of each cell type by the total BALF cell count.
Lung histological analysis
After BALF collection, the left lung lobe was excised, fixed in 4% PFA for 24 h at RT, dehydrated with gradient ethanol, embedded in paraffin, and cut into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E) for assessing peribronchial and perivascular inflammation, and with periodic acid–Schiff (PAS) for evaluating mucus production.
Histological scoring was performed by two pathologists in a blinded manner: (1) inflammation score—based on the degree of inflammatory cell infiltration around airways and blood vessels (0 = no inflammation, 1 = mild, 2 = moderate, and 3 = severe); and (2) mucus score—based on the percentage of PAS-positive goblet cells in bronchial epithelium (0 = 0%, 1 = 1%–25%, 2 = 26%–50%, 3 = 51%–75%, and 4 = 76%–100%).
Adoptive transfer experiment
Rag1^−/−^ mice (6–8 wk old, female, SPF grade) were used to eliminate endogenous T-cell interference. Mice were randomly divided into two groups (n = 6 per group): (1) untreated Treg group—adoptive transfer of 1 × 10^6^ untreated patient Tregs (resuspended in 200 μl PBS) via tail vein injection; and (2) KQS-1–trained Treg group: adoptive transfer of 1 × 10^6^ KQS-1–treated patient Tregs (10 μg/ml, 48-h culture, resuspended in 200 μl PBS). The experiments were performed in accordance with relevant guidelines/regulations.
Twenty-four hours after transfer, all mice were intranasally challenged with HDM extract (10 μg/mouse in 50 μl PBS) once daily for three consecutive days. On day 4, mice were euthanized to collect BALF (for eosinophil counting) and mediastinal lymph nodes (MLNs). MLNs were homogenized, and single-cell suspensions were prepared. IL-10^+^ Tregs in MLNs were detected via flow cytometry (anti-mouse CD4-PE-Cy7, CD25-APC, FoxP3-PE, IL-10-FITC antibodies; BD Biosciences), and the frequency of IL-10^+^FoxP3^+^CD4^+^CD25^+^ cells was calculated.
Statistical analysis
All experiments were performed with at least three biological replicates (unless stated otherwise: e.g., human participant, n = 15 per group; mouse experiments, n = 6–8 per group). Statistical analyses were conducted using R software (v4.5.1). For comparisons between two groups (e.g., patient versus control Tregs, KQS-1–treated versus untreated Tregs), unpaired two-tailed t tests were used for independent samples and paired two-tailed t tests for matched samples. For comparisons among three or more groups (e.g., control, HDM, and HDM + KQS-1 mouse groups), one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. For multiple endpoints (e.g., multiple cytokines, epigenetic marks at the FOXP3 and IL10 loci), the Bonferroni correction was applied to adjust for type I errors. Data are presented as the mean ± SD. A P-value < 0.05 was considered statistically significant, and 95% confidence intervals (CIs) were calculated for all applicable statistical tests.
Data Accessibility Statement
All raw and processed high-throughput sequencing data generated in this study have been deposited in public repositories under the following project and accession identifiers: methylation array data (Illumina 450K): Database: National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under Accession ID: PRJNA1291257. Corresponding Data: This accession includes raw IDAT files and processed β-value matrices from the Illumina Infinium HumanMethylation450K BeadChip array, profiling genomic DNA from KQS-1–treated and PBS-treated human Tregs (n = 8 biological replicates per group). Data specifically capture CpG methylation status at regulatory regions of the FOXP3 and IL-10 loci, as presented in Fig 4F; ATAC-seq data (chromatin accessibility): Database: National Genomics Data Center (NGDC) Genome Sequence Archive (GSA) under Accession ID: HRA007851. Corresponding Data: This newly assigned accession encompasses raw FASTQ files, aligned BAM files, and peak calling results from assay for transposase-accessible chromatin using sequencing (ATAC-seq). Data were generated from FACS-sorted CD4^+^CD25^+^FOXP3^+^ Tregs isolated from allergic asthmatic patients after treatment with KQS-1 or PBS vehicle (n = 4 biological replicates per group). Analyses identify differentially accessible chromatin regions, including increased accessibility at the FOXP3 and IL-10 promoters (Fig 4B–D); and BioProject metadata (combined studies): Database: NCBI (PRJNA1291257). Corresponding Data: This bioproject accession serves as a comprehensive metadata hub linking all epigenetic datasets generated in this study, including the Illumina 450K methylation array data and the ATAC-seq data described above. It provides experimental design details, sample annotations, and links to all associated raw data files for cross-referencing.
Supplementary Material
Reviewer comments
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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