Frequencies of β-cateninhigh RORγt+ Tregs increase in the PB and colonic mucosa of IBD patients during inflammation and malignant disease progression. We previously established that RORγt+ Tregs expand in sporadic CRC patients30. To understand the etiology of this expansion, we investigated IBD, which represents a unique platform to study the mechanisms underlying the emergence and distribution of RORγt+ Tregs during inflammation and progression to CRC. We analyzed colonic tissue and/or PB samples from 65 IBD patients, with or without dysplasia (IBD/Dys), and compared them to a group of 20 healthy donors (HD; Table S1).
We first determined the frequencies of circulating fractions (Fr.) of naïve Tregs (Fr.I=CD45RA+Foxp3int), activated Tregs (Fr.II=CD45RA-Foxp3high), and activated conventional T cells (Fr.III= CD45RA-Foxp3int) within CD4+ T cells as suggested by Sakaguchi and colleagues (Fig. 1a-e)32, 27. Previously, we reported that Fr.II of activated Tregs selectively increased in sporadic CRC compared to HDs30. Likewise, here we show a progressive enrichment of Fr.II Treg frequencies from HDs to IBD and IBD/Dys (Fig. 1c). We also observed a progressive increase of non-Treg Fr.III T cells, which were suggested to be prognostic for CRC clinical outcomes (Fig. 1c)27. We previously defined proinflammatory Tregs in patients by their expression of RORγt. We therefore assessed the proportions of RORγt-expressing cells within the Sakaguchi Treg fractions (Fig. 1b-d) as well as in the classically defined CD25+Foxp3+CD127- Treg population (Fig. S1a-c). In accordance with our previous work, RORγt+ cells were enriched within Fr.II of activated Tregs in IBD/Dys patients (Fig. 1d). Similarly, frequencies of RORγt+ within CD25hiFoxp3+CD127- Tregs also increased in IBD and IBD/Dys patients compared to HDs (Fig. S1b, c). Conclusively, PB RORγt+ Treg frequencies were elevated in IBD and IBD/Dys patients.
Previously, we causatively linked the skewing of CD4+ conventional T cells towards a Th17/pro-inflammatory phenotype to the activation of the Wnt/β-catenin pathway31. Hence, we assessed the expression of β-catenin in RORγt+ and RORγt- Fr.II Tregs and in CD25hiFoxp3+CD127- Treg cells. Indeed, RORγt expression in Tregs uniformly correlated with enhanced β-catenin protein levels (Fig. 1b, e and Fig. S1d-e). Specifically, β-catenin expression was significantly higher in RORγt+ compared to RORγt- Fr.II Tregs in patients and HDs (Fig. 1e). Further validating previous findings by our group and others21, 30, the RORγt+ but not the RORγt- CD25+Foxp3+CD127- PB Tregs produced proinflammatory cytokines upon stimulation with PMA/Ionomycin (Fig. 1f-i and Fig S1d-g). More precisely, RORγt+ PB Tregs intracellularly accumulated IL-17, IFNg, and TNFa (Fig. 1 f-h) and a fraction of them were even found to be double producers for TNFa and IL-17 (Fig. 1i). Importantly, a significantly higher percentage of RORgt+ PB Tregs produced IFNg in IBD and IBD/Dys patients compared to HDs (Fig. 1g-h). Furthermore, compared to RORgt-, co-expression of IL-17 and TNFa was significantly more frequent in RORgt+ PB Tregs of IBD and IBD/Dys patients but not of HDs (Fig. 1i). These findings show that chronic inflammation in IBD and IBD/Dys conincides with the increase in frequencies of the previously described RORgt+Treg population in the PB which produces multiple pro-inflammatory cytokines.
Next, we determined the frequencies of tissue-resident RORγt+ Tregs in IBD patients by purifying mononuclear cells (MNCs) from inflamed (INF) and less inflamed (‘margin’, M) colonic mucosa samples. We found substantially higher frequencies of total Foxp3+ T cells (~30%) within tissue-resident CD4+ T cells compared to the PB. Moreover, tissue-resident total Foxp3+ T cells were significantly increased in IBD/Dys compared to IBD patients (Fig. 1j) for both M and INF areas. We also found that frequencies of RORγt+ Tregs increased from the margin to the inflamed mucosa (Fig. 1k-l). Like their circulating counterparts, tissue-resident RORγt+ Tregs expressed significantly higher levels of β-catenin compared to RORγt- Tregs (Fig. 1m-n) and produced IL-17 upon stimulation (Fig. S1h, Fig. 1o). Thus, circulating RORγt+ Tregs in IBD/Dys patients share major similarities with tissue-resident RORγt+ Tregs and are likely tissue/tumor derived.
To explore possible RORγt+ Treg tumor infiltration we analyzed datasets from The Cancer Genome Atlas (TCGA, Research Network: https://www.cancer.gov/tcga) and determined how expression changes of Wnt/β-catenin, Treg, and Th17 pathway genes were connected. To do this, we calculated the average z-scores over all genes in the ‘human WNT signature’ (KEGG_human_WNT)33, 34 and signatures containing genes that are transcriptionally up-regulated in Th17 cells (TH17_UP) and Tregs (Treg_UP) that were kindly provided to us by Dr. Benoist and colleagues10. The TH17_UP positively correlated with the KEGG_human_WNT signature (p<0.001, Spearman-score r=0.5604, red dots Fig. 1p) and mirrored the equally strong positive correlation between the Treg_UP and the KEGG_human_WNT (p<0.001, Spearman-score r=0.6161, blue dots Fig. 1p) signatures. Also, the TH17_UP and Treg_UP signatures showed a strong positive correlation (p<0.001, Spearman-score r=0.7836, Fig. S1i). This suggests that the Treg infiltrate in CRC tumors with an activated Wnt signature may possess Th17-like traits.
We further assessed whether the enhanced expression of Treg/Th17 signature genes could be correlated with adverse survival in the TCGA cohort, as the expression of Th17 associated genes was previously linked to detrimental outcome in CRC35. The effect on survival was tested with a machine learning approach. Coefficient values were derived from the machine learning approach for each gene in the TH17_UP, Treg_UP, and the combined TH17_UP/Treg_UP signatures via Cox proportional-hazards regression (example for the TH17_UP signature is shown in Fig. S1k,l) (Table S2). The genes whose enhanced expression predicted decreased survival included, amongst others, LEF-1 and MAF. We then divided patients, based on the median score of the TH17_UP, Treg_UP and combined TH17_UP/Treg_UP signature (TH17_UP signature shown as example in Fig. S1i). We interogated the survival outcomes of groups with above versus below the median score of the weighted signatures. These analyses indicated that above-median signature scores for the TH17_UP (Fig. 1q, p<0.0001, log-rank test, n=188 – below and above median) Treg_UP (Fig. 1r, p<0.0001) and most importantly for the combined TH17_UP/Treg_UP genes (Fig. 1s, p<0.0001) correlated with reduced overall survival. The significant detriment in survival for the combined weighted signature (TH17_UP/Treg_UP) further supports the suggestion from the unweighted Spearman correlations that a Treg tumor infiltrate with Th17-traits might results in adverse survival.
Ex vivo stabilization of β-catenin in human Tregs is sufficient to induce the pro-inflammatory phenotype. Given our finding that RORγt expression in Tregs correlates with increased β-catenin levels in IBD(/Dys) patients, we investigated whether β-catenin stabilization was sufficient to induce RORγt and pro-inflammatory cytokine expression in HD Tregs. Therefore, we treated HD PBMCs ex vivo with the GSK-3β inhibitor Chiron (CHIR99021) to stabilize β-catenin protein. Levels of β-catenin (Fig. 2a) and RORγt (Fig. 2c) were assessed in CD4+CD25+Foxp3+ Tregs after 4 (d4) and 7 (d7) days of culture with Chiron. Intracellular β-catenin (Fig. 2b) as well as RORγt (Fig. 2d) levels were significantly elevated in Tregs after Chiron treatment at both time points. While β-catenin expression plateaued on d4, RORγt expression in Chiron-treated Tregs increased significantly between d4 and d7 (Fig. 2d).
In addition to β-catenin regulation, GSK-3β phosphorylates STAT proteins in T cells36, 37, which promote the expression of pro-inflammatory cytokines IL-17 (STAT3)38, 39 and IFN-γ (STAT1)40, 41. Hence, GSK-3β inhibition cannot be used to assess downstream cytokine production in these ex vivo cultures. Alternatively, in human primary T cell cultures19 and in murine thymocytes42 it was shown that TCR engagement stabilizes β-catenin. To determine the physiological effect of β-catenin activation, we cultured HD PBMCs for 4d in the presence of its natural activator, Wnt3a, in combination with CD3/CD28-beads, CD3/CD28-beads alone, or vehicle control (Fig. 2e). CD3/CD28-beads were removed from cultures on d4 and Fr.II Treg cells (Fig. 2f) were analyzed for β-catenin and RORγt expression (Fig. 2g,h) as well as cytokine production (Fig. 2i,j) on d6. Intracellular levels of β-catenin and RORgt directly correlated (Fig 2g) and increased significantly in Wnt3a/CD3/CD28 and CD3/CD28 compared to vehicle control treated Fr.II Tregs (Fig. 2h). Similarly, a significantly higher frequency of Wnt3a/CD3/CD28 and CD3/CD28 treated Fr.II Tregs expressed the proinflammatory cytokines IL-17, IFNγ, and TNFa (Fig. 2j) after PMA/Ionomycin stimulation. As observed in IBD(/Dys) patients, expression of IL-17 and TNFα (Fig. 2i,k) coincided and a smaller proportion of Fr.II Tregs even co-expressed IL-17 and IFNγ. Interestingly, gut homing receptor CCR9 became strongly upregulated (Fig. 2l) in Fr.II Tregs upon Wnt/ꞵ-catenin-activating stimulation. Conclusively, TCR stimulation in combination with activating Wnt-signals induced a pro-inflammatory phenotype in Fr.II Tregs that is indentical with that observed in IBD(/Dys) patients (Fig. 1). Hence, these observations imply that β-catenin stabilization in primary human PBMCs is sufficient to drive the proinflammatory phenotype of Tregs observed in IBD and CRC patients.
Activated RORγt+ sub-populations of Tregs expressing gut homing receptors peripherally expand during disease progression in a murine IBD/CRC model. To recapitulate our findings in patients and further analyze IBD/CRC-associated RORγt+ Tregs, we used our previously established murine APCΔ468 (APCΔ) polyposis model43. In this model, translation of the adenomatous polyposis coli (APC) protein prematurely terminates due to ablation of exons 11 and 12, resulting in a truncated, nonfunctional 468 aa protein. The pathology of APCΔ resembles APCMin/+ mice. They develop intestinal polyposis that spreads to the colon over time, and succumb to disease between 6-8 month of age44, 45. In previous work we used this model to demonstrate that Treg-specific ablation of Rorc reduced polyp burden in APCΔ mice30. Treatment of APCMin/+ mice with dextran sodium sulfate (DSS), which causes colitis in mice46, leads to the development of invasive CRC47, 48. We established invasive colonic lesions (Fig. 3b), by treating 3-4-month-old APCΔ mice with 3 rounds of 2% DSS in the drinking water for 7 days followed by 2 weeks recovery (Fig. 3a). This regimen led to colon shortening and a highly significant increase in colonic adenomas compared to untreated APCΔ mice (Fig. 3c).
Next, we assessed the frequencies of total colonic Tregs (Fig. 3d) and RORγt+ Tregs (Fig. 3e). Each of the three pathologic conditions colitis (WT+DSS), polyposis (APCΔ-DSS), and IBD/CRC (APCΔ+DSS) displayed increased colonic CD25+Foxp3+ Treg (Fig. 3d) and RORγt+ Treg (Fig. 3e) frequencies compared to naïve mice (WT-DSS). As observed in IBD patients, β-catenin expression was significantly higher in RORγt+ Tregs than in RORγt- Tregs for all treatment groups, (Fig. 3f). However, total Tregs (Fig. 3d) and RORγt+ Tregs (Fig. 3e) trended towards even higher frequencies in IBD/CRC compared to colitis or polyposis alone. We speculated that specific sub-populations of RORγt+ Tregs increased under IBD/CRC conditions, but the effect was masked by the overall increase of Tregs. Therefore, we designed two flow cytometric panels comprising CD4, Foxp3, CD25, RORγt, and β-catenin to distinguish distinct RORγt+ and RORγt- Treg subsets. The first panel assessed tissue and inflammation homing markers; gut-homing receptor CCR9, inflammation-homing receptor CCR6, and tissue-residency marker CD103. The second panel focused on proliferation and activation markers Ki67, CD44, CD69, and CD62L (Fig. 3g). We gated on CD4+CD25+Foxp3+ Tregs from spleen (SPL) and colon MNCs, concatenated the populations from all experimental groups, and performed tSNE analysis. RORγt+ Treg gates were then superimposed onto the respective tSNE landscape revealing unique populations (Fig. S2a,b). These landscapes show that the complexity of RORγt+ Treg populations in the colon is greater than in the spleen (tissue and inflammation homing panel: colon=8, SPL=2 populations, activation panel: colon=8, SPL=3 populations, Fig 3g). To trace the origin of these cells, we focused on populations that expressed the same markers between the spleen and colon (red arrows, Fig. 3g). Analysis of expression profiles (Fig. S2a,b) revealed that RORγt+ Treg populations shared between the colon and spleen expressed CCR9/CD103, and Ki67/CD44 (Fig. 3g). Cumulative analysis confirmed that CCR9+CD103+ (Fig. 3h) and CD44+Ki67+ (Fig. 3i) RORγt+ Treg frequencies in the IBD/CRC group (APCΔ+DSS) increased compared to the untreated WT, colitis (WT+DSS), and polyposis (APCΔ-DSS) groups for colonic and splenic samples. Moreover, polyposis (APCΔ-DSS) or colitis (WT+DSS) alone also showed elevated frequencies of the shared Treg populations in the colon, compared to the untreated group. This suggested that during IBD/CRC carcinogenesis a sub-population of highly activated and proliferative RORγt+ Tregs with gut-homing properties expanded in the colon and the periphery (SPL). It further indicated diversity within the RORγt+ Treg subset, particularly in the colon.
Treg-specific β-catenin stabilization in mice results in a pro-inflammatory Treg phenotype in mice. Ex vivo Wnt/β-catenin pathway activation in primary human HD Tregs recapitulated the phenotype of RORγt+ Tregs observed in patients (Fig. 2). Accordingly, we examined in mice whether Treg-specific activation of the Wnt/β-catenin pathway was sufficient to induce RORγt+ Tregs. We introduced the ctnnb1fl(ex3) allele49 into the Foxp3YFP-Cre mice established by Rudensky and colleagues50. Cre-excision of exon 3 encoding the β-catenin degradation domain leads to intracellular accumulation of β-catenin protein.
As the Foxp3 gene resides on the X-chromosome, male mice are hemizygous. Male Foxp3YFP-Cre Ctnnb1fl(ex3) (CAT) mice showed severe X-linked, scurfy-like51, 52 immune-pathology, (Fig. S3b) they were smaller than Foxp3YFP-Cre WT (Cre) littermates and died ~3-4 weeks after birth (Fig. S3a). They further presented enlarged secondary lymphoid organs, particularly peripheral lymph nodes (pLN), and splenomegaly (Fig. S3c). Histological assessment via H&E staining of paraffin-embedded tissue sections revealed a reduced thymic cortex and severe immune infiltrates in lungs and liver (Fig. S3d, blue arrows). These observations mirrored those of a recent study using a different Foxp3Cre53 with the same floxed β-catenin allele21.
The observed pathology in CAT mice implied a lymphoproliferative disorder. Hence, we next assessed the activation status of conventional T cells in the peripheral lymphoid organs and thymi of 21-day-old mice. Compared to Cre-only, CAT mice had increased frequencies of total CD3+ cells in the spleen and pLNs; with a decrease of CD4+ T helper (TH) cells in the pLNs and mLNs and an increase of CD8+ cytotoxic T cells (CTLs) in the spleen (Fig. S3e), indicating imbalanced T cell proliferation. This was further supported by the increase of Ki67+TH cells and CTLs in CAT compared to WT mice (Fig S4a-b). Moreover, CD25-Foxp3-CD4+ TH cells and CTLs were strongly activated in CAT mice. The frequencies of TH cells and CTLs expressing the activation markers CD44, CD69, and CD25 were significantly increased in all peripheral lymphoid organs whereas the fractions of T cells expressing the naïve T cell marker CD62L was reduced. (Fig. S4a-b).
The highly activated effector T cell compartment in CAT mice suggested that β-catenin stabilization altered Treg function. Treg frequencies strongly decrease within mLNs and spleens of CAT mice, however thymic Treg generation was unaltered (Fig. 4a, Fig. S4c). In peripheral Tregs, Foxp3 expression mildly increased (Fig. 4b), whereas expression of the Treg marker Neuropilin (Fig. S4d) did not change. Similar to the human ex vivo cultures, β-catenin stabilization (Fig. 4c) was sufficient to uniformely upregulate RORγt (Fig. 4d) levels in CAT (β-cateninhigh) compared to Cre Tregs. Moreover, β-cateninhigh Tregs were highly activated, as evidenced by increased frequencies of CD44+ (Fig. 4e) and CD69+ (Fig. 4f) Tregs and decreased CD62L+ Tregs (Fig. 4g) in CAT mice. Likewise, Tregs in CAT mice were more proliferative, demonstrated by Ki67 staining (Fig. 4h). Lastly, we tested the ability of β-cateninhigh Tregs to suppress proliferation of polyclonally activated, CFSE-labeled CD4+CD25- T effector cells (Teff) in vitro. Compared to control Cre Tregs, β-cateninhigh Tregs had significantly reduced suppressive function for all Treg:Teff ratios tested (Fig. 4i).
Conclusively, β-catenin stabilization in murine Tregs was sufficient to up-regulate RORγt and induce an activated, pro-inflammatory phenotype that mirrored the systemically expanding RORγt+ Treg sub-population observed in the APCΔ/DSS model (Fig. 3i). The lymphoproliferative disease in CAT mice could thus be attributed to a combination of the activated phenotype and the reduced suppressive function of β-cateninhigh Tregs.
β-cateninhigh Tregs are competitively disadvantaged in a chimeric setting and spontaneously express proinflammatory cytokines. Foxp3YFP-Cre heterozygous females represent natural chimeras of β-cateninhigh/YFP+ versus WT/YFP- Tregs due to random X-chromosome inactivation. These mice provide the opportunity to test the pathogenic potential of β-cateninhigh/RORγthigh Tregs in a competitive chimeric setting. Interestingly, compared to hemizygous males, heterozygous Foxp3Cre(+/-) females that carry the Ctnnb1fl(ex3) allele are healthy.
When comparing Cre (Foxp3YFP-Cre(+/-) WT) and CAT (Foxp3YFP-Cre(+/-) Ctnnb1fl(ex3)) female mice, total Treg numbers in the spleen and thymus were identical between genotypes (Fig. 5a). The frequency of YFP+ Tregs, however, was drastically reduced in CAT compared to Cre females (Fig. 5b). Cumulative analysis of YFP+ and YFP- Treg fractions showed that YFP+ Tregs only accounted for 1-3% of total Tregs in all peripheral lymphoid organs of female CAT mice (Fig. 5c). The thymic output of YFP+ Tregs was also reduced to 20 % in CAT females. In Cre females the ratio of YFP+/YFP- Tregs was also slightly reduced to 40%/60% (Fig. 5b,c). This could be attributed to the YFP-Cre transgene rendering Foxp3 expression in YFP+ Tregs mildly hypomorphic compared to Tregs with an unaltered Foxp3 locus54. The persisting YFP+/β-cateninhigh Tregs had elevated RORγt expression (Fig. 5d) and an activated phenotype, as evidenced by increased CD44 expression (Fig. 5e) compared to CAT YFP- Tregs but also compared to Cre YFP+/YFP- Treg populations. In accordance to our findings in patients’ RORgt+ Tregs, CAT YFP+/β-cateninhigh compared to YFP- and to Cre YFP+/YFP- Treg populations spontaneously expressed proinflammatory cytokines IL-17, IFNg, and TNFa. Further mirroring our finding in patients (Fig. 1), a significant proportion of these YFP+/β-cateninhigh Tregs co-expressed IL-17 and IFNg or IL-17 and TNFa (Fig. 5f-i). In accordance to findings in human samples, the gut-homing receptor CCR9 was upregulated (Fig. S5a) as shown for wnt3a/anti-CD3/28-treated human Tregs (Fig. 2l). Conclusively, in a non-inflammed setting, β-cateninhigh/RORγthigh Tregs have a competitive disadvantage in chimeric mice. Although they do not spontaneously cause disease, they are poly-functional in producing proinflammatory cytokines.
The DNA-binding partner of β-catenin, TCF-1, and Foxp3 co-bind accessible chromatin at gene loci that are crucial for Treg function. Previous work indicated that β-catenin20/TCF-113 occupy DNA together with Foxp3. Thus, we anticipated that precise mapping of TCF-1 and Foxp3 co-binding in Tregs, could provide a molecular explanation for the change in the phenotype of Tregs that overexpress β-catenin. To address this, we analyzed TCF-1 DNA binding in Tregs through chromatin immunoprecipitation and deep sequencing (ChIPseq) in Foxp3YFP-Cre WT Tregs. Furthermore, we assessed regions of accessible chromatin in Tregs via transposase-accessible chromatin approach and deep sequencing (ATAC-seq). We also utilized available data55 for Foxp3-binding (Foxp3-ChIPseq), histone marks including; monomethylated histone 3 at Lys4 (H3K4me1), tri-methylated H3 Lys4 (H3K4me3), acetylated H3 at Lys27 (K3K27Ac), trimethylated H3 at Lys27 (H3K27me3), and Methyl-CpG binding domain-based capture and sequencing (MBD-seq).
We assigned TCF-1 (Fig. S6a-c) and Foxp3 (Fig. S7a-c) ChIPseq-peaks to specific gene regulatory regions by performing K-means clustering guided by the surrounding chromatin modifications. 14.6% of TCF-1 (Fig. S6a) and 24.0% of Foxp3 (Fig. S7a) binding sites, were assigned to active enhancers (AE, enriched for H3K4me1, H3K27Ac), 41.6% and 39.3% of transcription factor binding sites to poised enhancers (PE, enriched for H3K4me1, lacking H3K27Ac), and 44.0% and 36.7% to promoters (Pr, enriched for H3K4me3, H3K27ac, and accessible chromatin (ATAC-seq signal)). Both TCF-1 and Foxp3 preferentially bound open and poised chromatin as their binding sites rarely overlapped with the repressive histone mark H3K27me3 (Fig. S6b-c, Fig. S7b-c). We next performed motif analysis for TCF-1- and Foxp3-bound sites. TCF-1-bound enhancer sites were enriched for the TCF-1 consensus motif, while both promoter and enhancer Foxp3-bound sites were enriched for the Foxp3 motif (Fig S6d, Fig. S7d). The high enrichment for motifs of other factors like Ets, RUNX and YY family members agreed with previous evidence that TCF-114 and Foxp312 act in multi-molecular complexes. Pathway enrichment analysis (http://www.metascape.org) revealed that TCF-1 (Fig. S6e) and Foxp3 (Fig. S7e) binding to promoter and enhancer sites marked genes involved in T cell activation and other T cell processes, as well as in general DNA and RNA metabolism processes.
We further analyzed directly overlapping TCF-1 and Foxp3 co-bound sites (Fig. 6), which included 504 AE, 389 PE, and 1101 Pr (Fig. 6a). Examples for TCF-1 and Foxp3 co-bound gene loci at AE (Tcf7), PE (Ccr7), and Pr (Stat1) sites are visualized as IGB tracks (Fig. 6f). The TCF-1 consensus motif, but not the Foxp3 binding motif, was highly enriched in the overlapping AE and Pr sites (Fig. 6d). Pathway enrichment analysis revealed that co-binding of TCF-1 and Foxp3, particularly at AE sites, marked genes involved in Th17 differentiation, T cell activation, and cytokine production pathways (Fig. 6e). Thus, in Tregs TCF-1 and Foxp3 together occupied genes whose upregulation could explain the phenotype of RORγt+ Tregs observed in IBD/CRC patients and mouse models.
β-catenin stabilization drives the proinflammatory phenotype of RORγt+ Tregs through epigenetic and transcriptional regulation of critical Foxp3 and TCF-1 co-bound genes. The classical Wnt signaling model posits that TCF-1 acts as a transcriptional repressor that becomes an activator once β-catenin binds and mediates epigenetic changes. Hence, one could speculate that, in a Wnt-off state, TCF-1 is part of Foxp3 repressor complexes12, which act together to limit the expression of genes that should not be permanently expressed in bona fide Tregs. Thus, we postulated that β-catenin stabilization specifically affects the transcription and epigenetic states of these TCF-1/Foxp3 co-bound genes.
To explore this possibility, we performed ATAC-seq analysis of YFP+CD25+ β-cateninhigh Tregs FACS sorted from CAT (Foxp3YFP-Cre Ctnnb1fl(ex3)) and WT (Foxp3YFP-Cre) mice (Fig. 7a). β-cateninhigh Tregs (23,084 peaks) had fewer accessible sites than Cre Tregs (29,566 peaks). However, the sites that lost accessibility from Cre to β-cateninhigh (Cre unique sites, 9831 peaks), already had low accessibility in Cre Tregs and yet were not fully closed in β-cateninhigh Tregs. The majority of sites remained consistently open for both genotypes (common, 19735 peaks) and 3349 sites gained de novo accessibility in β-cateninhigh Tregs. We specifically analyzed the newly accessible sites for transcription factor binding motifs (Fig. 7b). One of the most strongly enriched motifs was that of TCF-1 (Tcf7), indicating that β-catenin together with TCF-1 may directly alter the accessibility of corresponding genes. Additionally, motifs of transcription factors that are involved in Th17 differentiation (Maf, RARg) and T cell activation (JunB) were highly enriched. The 3349 newly accessible sites corresponded to 2582 genes (Fig. 7a) of which 370 were co-bound by TCF-1 and Foxp3 (Fig. 7c). Pathway enrichment analysis of these 370 genes revealed that T cell activation, cytokine production, and Th17 differentiation pathways were most significantly enriched (Fig. 7d).
We next performed RNA-seq expression profiling to investigate how the changes in chromatin accessibility impacted transcription in β-cateninhigh Tregs. Differential expression testing yielded 3190 up- and 2795 down-regulated (q-value≤0.05) genes in β-cateninhigh compared to Cre Tregs. The upregulated list comprised genes that are essential for Treg function, including CTLA-4 and Il2ra (CD25, Fig. 7e). Pathway enrichment analysis for significantly up-regulated genes that were co-bound by TCF-1 and Foxp3 (Fig. 7f-g) revealed an enrichment for the very same pathways identified by the newly accessible chromatin sites, namely, T cell activation and Th17 differentiation (Fig. S8).
We next assessed the dynamic chromatin accessibility changes following β-catenin stabilization of genes co-bound by TCF-1/Foxp3 in different regulatory regions. Therefore, we compared the log2-fold accessibility changes in β-cateninhigh Tregs over Cre Tregs for promoter, PE, and AE co-bound to that of all differentially accessible genes (Fig. 8a). Although, genes co-bound by TCF-1/Foxp3 always gained accessibility, the AE-bound genes showed the strongest increase in accessibility in β-cateninhigh Tregs (Fig. 8a). As AE-bound genes are most affected by β-catenin stabilization, we specifically searched for those that were up-regulated, gained accessibility, and were TCF-1/Foxp3 co-bound at AE sites in Tregs (Fig. 8b). This yielded in 49 genes, that were identified to belong to T cell activation and Th17 differentiation pathways (Fig. 8b). We further validated these findings via an inverse, non-supervised approach. We retrieved gene lists for the Th17 differentiation (102 genes) pathway (GSEA/MSigDB database), and used the same Treg_UP signature (195 genes) used for TCGA screening. The leukocyte migration (242 genes) pathway (GSEA/MSigDB database) was also assessed due to the observed increase in CCR9 expression in RORγt+ Tregs in humans (Fig. 2l) and mice (Fig. 3, Fig. S5d) suggesting their migration from the colon to the periphery. Comparing accessibility of genes in these pathways showed strong de novo accessible sites that arise after β-catenin activation in Tregs (red lines) compared to common (black lines) and WT sites (green lines, Fig. 8c). Moreover, gene set enrichment analysis (GSEA56) showed a significant positive upregulation for Th17 differentiation and leukocyte migration pathways (Fig. 8d, Fig. S8). The Treg_UP signature, however, was not consistently changed between WT and β-cateninhigh Tregs (Fig. 8d). This indicated that the core Treg program was not drastically altered, but non-Treg/effector T cell functions were acquired upon β-catenin activation. For instance, activation of β-catenin led to the acquisition of a newly accessible site in the IFNγ locus (Fig. 5e, black arrow), which may account for the ability of β-cateninhigh Tregs to express IFNγ. Similarly, the IL-17 locus gained accessibility (Fig. 8e), which may account for spontaneous IL-17 production in β-cateninhigh Tregs (Fig 5h,i).
Overall, our findings indicated that β-catenin activation caused epigenetic and transcriptional changes of crucial Foxp3/TCF-1 co-regulated genes. This resulted in the induction of transcriptional programs responsible for Th17 differentiation/T cell activation that were super-imposed onto the Treg program and potentially conferred the observed pro-inflammatory phenotype to β-cateninhigh Tregs.