Loss Of Foxp3 Research Articles

IL-21 induces pyroptosis of Treg cells via Akt–mTOR–NLRP3–caspase 1 axis in eosinophilic chronic rhinosinusitis

Regulatory T (Treg) cells, which prevent inflammation-induced eosinophil infiltration, are deficient in nasal polyps (NPs) in patients with eosinophilic chronic rhinosinusitis (ECRS). It is concomitant with loss of Foxp3 after certain inflammatory stimuli. We sought to determine the inflammatory cytokines involved in inducing the loss of Treg cells in NPs. The abundance of cytokines in ECRS patients or mice were tested using ELISA, immunochemistry, immunofluorescence, quantitative reverse transcription PCR (qPCR), and/or flow cytometry. Expression of eosinophil cationic protein (ECP), CD4+ T cells, IL-4, and IL-17A and eosinophils in nasal mucosa of mouse model was investigated by immunochemistry, immunofluorescence, and hematoxylin and eosin staining. The percentage and death of induced Treg (iTreg) cells, source of IL-21 in NPs from ECRS and non-ECRS patients, and abundance of different systemic phenotypes of CD4+ T cells in a mouse model were studied by flow cytometry. Western blot analysis, scanning, and transmission electronic microscopy were used to detect pyroptosis of iTreg cells. IL-21 was highly expressed in nasal mucosa of ECRS patients and mice, causing pyroptosis and preventing development of iTreg cells invitro. The elevated IL-21 in NPs from ECRS patients was mainly produced by CD3+ T cells, including T follicular helper, T peripheral helper, TH2, and TH17 cells and CD3+CD4- T cells. T peripheral helper cells and CD3+CD4- T cells were the predominant source of IL-21 in NPs from non-ECRS patients. Blocking IL-21/IL-21R signaling significantly reduced the number of eosinophils and CD4+ T cells along with ECP, IL-4, and IL-17A expression in the nasal mucosa of ECRS mice. It also increased Treg cell percentage and systemically decreased TH2 and TH17 ratios. Akt-mTOR inhibition prevented IL-21-induced pyroptosis in human and mouse iTreg cells. Elevated IL-21 drives pyroptosis and prevents Treg cell development in ECRS patients. IL-21 induced pyroptosis via activating Akt-mTOR-NLRP3-caspase 1 signaling.

Loss of FOXP3+ CD25+ Tregs is independent of type I interferon signaling in lupus-prone mice

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by deposition of autoantibodies that leads to tissue damage and inflammation. Our lab has generated the ARE mice strain that has low, chronic levels of IFNg and lupus-like disease. Regulatory T cells (Tregs) play an important role in preventing autoimmunity by suppressing T cell activation, making them an important therapeutic target. In addition, type I interferon (IFN) has also been broadly implicated in SLE but its specific effect on Tregs remain unknown. To address the impact of a type I IFN signature on the dynamics of regulatory T cells (Tregs), we investigated marker expression on Tregs in type I interferon receptor (IFNAR)-deficient ARE mice. Single cell suspensions harvested from spleens and kidneys of ARE −/−, IFNAR −/−, and IFNAR −/−/ARE −/−female mice between 150 to 200 days old were stained with a panel of markers associated with Treg function for analysis by flow cytometry. In the spleen, the proportion of CD4+ FOXP3+ expressing CD25 (IL2Ra) decreased in ARE −/−and IFNAR −/−/ARE −/−, but not IFNAR −/−, compared to wild type (wt). Thus, the disease progression in ARE mice may be due to the loss of bona fide Treg population (FOXP3+ CD25+), as IL-2 contributes to Tregs’ expansion and function. Furthermore, the kidneys of ARE −/−and IFNAR −/−/ARE −/−experienced increased CD4 infiltration as well as a similar decrease in FOX3+ CD25+ Tregs, which might explain the increased inflammation of the kidneys. IFNg-mediated chronic inflammation appears to downregulate the population of FOXP3+ CD25+ Tregs, independent of type I IFN signaling. Future experiments aim to perform in vitroassays to compare the suppressive ability of Tregs with and without CD25.

Sin3A Controls FoxP3+ Treg Production, Stability and Function

Abstract Purpose: Foxp3 T-regulatory (Treg) cells are key to immune homeostasis but the function of paired amphipathic helix protein, Sin3a, is unknown. Methods: We studied the effects of Sin3a deletion in Treg cells. Results: Sin3a−/− FoxP3Cre mice died from severe autoimmunity by 2–3 weeks of birth. Mice had enlarged lymph nodes, severe inflammation in lungs, skin, livers and kidneys and autoantibodies to islet cells, striated muscles, keratin, endomysium and gastric parietal cells. Percentages of FoxP3+ Tregs in LNs and spleens were lower (p values ranging from >0.01 to >0.0001) than controls and FoxP3 mRNA expression (>20-fold) and FoxP3 protein per Treg (>3-fold) were also significantly reduced in Sin3a−/− mice. Reduction of FoxP3+ Tregs in the periphery of Sin3a−/− FoxP3cre mice was largely due to the loss of FoxP3, determined by an increase of peripheral ex-Tregs as compared to WT (p<0.001 in sLN and spleens) within Sin3a−/− FoxP3Cre tdTomatoROSA26 lineage tracing mice. Increased mRNA expression of pro-apoptotic genes, decreased mRNA expression of anti-apoptotic genes, and increased presence of Annexin V on the cell surface indicate that apoptosis also contributed to peripheral Treg reduction. Sin3a−/− Tregs produced increased amounts of granzyme B, IFN-γ and IL-2 (12–45% of cells) and had impaired suppressive function (p<0.01). Sin3a was also essential for peripheral iTreg conversion since CD4+CD25− cells from Sin3a−/− FoxP3Cre mice lacked ability to convert to FoxP3+ iTregs ex vivo. Conclusions: The nuclear co-regulator, Sin3a, helps maintain the unique homeostatic properties of Treg cells. Therapeutic modulation of Treg functions may be possible by pharmacologic targeting of components within the Sin3a complex.

Targeting glutamine metabolism mitigates Foxp3-deficiency mediated disease

Abstract Regulatory T (Treg) cells play a key role in enforcing peripheral immunological tolerance to self-antigens. Foxp3 has been widely described as a master regulator for Treg differentiation and maintenance of their suppressive functions. Upon Foxp3 loss of function mutations, Foxp3-deficient Tregs (ΔTreg) develop in the thymus, however, these cells present impaired suppressive capacity and an effector T cell (Teff)-like phenotype. Among the Teff-like characteristics is the shift in their metabolism towards glycolysis and enhanced mitochondrial respiration. Metabolomic analysis revealed an increase in glutamine consumption. Here, we used 6-diazo-5-oxo-norleucine (DON), a broad inhibitor of glutamine-utilizing enzymes to treat Foxp3 deficient mice (Foxp3 ΔeGFPiCre), and observed a significant decrease in Teff cell activation, and IFNγ production from both ΔTreg and Teff cells together with a significant increase in survival. This effect was enhanced when we combined DON treatment with a ΔTreg specific Rictor depletion (Foxp3 ΔeGFPiCreRictor Δ/Δ). Given that glutamine is a key amino acid for T cell activation, we evaluated if this was a pan-T cell effect by inducing Treg depletion in Foxp3 DTRmice with concomitant DON treatment. Surprisingly, in the absence of Tregs, DON was not able to prevent autoimmune disease. These results suggest that DON mitigates Foxp3-deficiency mediated disease in a ΔTreg dependent manner, by improving their suppressive capacities. We are currently performing metabolomic studies to better understand the role of glutamine usage in ΔTreg, more specifically if glutamine is directed to glutaminolysis, nucleotide or glutathione synthesis and how this contributes to Treg cell loss of function. NIH grant 1R01AI153174

TCR-induced FOXP3 expression by CD8+ T cells impairs their anti-tumor activity

Adoptive cell transfer therapy using CD8+ T lymphocytes showed promising results eradicating metastatic malignancies. However, several regulatory mechanisms limit its efficacy. We studied the role of the expression of the transcription factor FOXP3 on CD8+ T cell function and anti-tumor immunity. Here we show that suboptimal T cell receptor stimulation of CD8+ T cells upregulates FOXP3 in vitro. Similarly, CD8 T cells transferred into tumor-bearing mice upregulate FOXP3 in vivo. Cell-intrinsic loss of FOXP3 by CD8+ T cells resulted in improved functionality after TCR stimulation and better antitumor responses in vivo. Inhibition of the FOXP3/NFAT interaction likewise improved CD8+ T cell functionality. Transcriptomic analysis of cells after TCR stimulation revealed an enrichment of genes implicated in the response to IFN-γ, IFN-α, inflammatory response, IL-6/JAK/STAT, G2M checkpoint and IL-2/STAT signaling in FOXP3-deficient CD8+ T cells with respect to FOXP3-wt CD8+ T cells. Our results suggest that transient expression of FOXP3 by CD8+ T cells in the tumor microenvironment restrains their anti-tumor activity, with clear implications for improving T cell responses during immunotherapy.

MicroRNA-31 regulates T-cell metabolism via HIF1α and promotes chronic GVHD pathogenesis in mice

AbstractChronic graft-versus-host disease (cGVHD) remains a major obstacle impeding successful allogeneic hematopoietic cell transplantation (HCT). MicroRNAs (miRs) play key roles in immune regulation during acute GVHD development. Preclinical studies to identify miRs that affect cGVHD pathogenesis are required to develop these as potential lifesaving interventions. Using oligonucleotide array, we identified miR-31, which was significantly elevated in allogeneic T cells after HCT in mice. Using genetic and pharmacologic approaches, we demonstrated a key role for miR-31 in mediating donor T-cell pathogenicity in cGVHD. Recipients of miR-31–deficient T cells displayed improved cutaneous and pulmonary cGVHD. Deficiency of miR-31 reduced T-cell expansion and T helper 17 (Th17) cell differentiation but increased generation and function of regulatory T cells (Tregs). MiR-31 facilitated neuropilin-1 downregulation, Foxp3 loss, and interferon-γ production in alloantigen-induced Tregs. Mechanistically, miR-31 was required for hypoxia-inducible factor 1α (HIF1α) upregulation in allogeneic T cells. Therefore, miR-31–deficient CD4 T cells displayed impaired activation, survival, Th17 cell differentiation, and glycolytic metabolism under hypoxia. Upregulation of factor-inhibiting HIF1, a direct target of miR-31, in miR-31–deficient T cells was essential for attenuating T-cell pathogenicity. However, miR-31–deficient CD8 T cells maintained intact glucose metabolism, cytolytic activity, and graft-versus-leukemia response. Importantly, systemic administration of a specific inhibitor of miR-31 effectively reduced donor T-cell expansion, improved Treg generation, and attenuated cGVHD. Taken together, miR-31 is a key driver for T-cell pathogenicity in cGVHD but not for antileukemia activity. MiR-31 is essential in driving cGVHD pathogenesis and represents a novel potential therapeutic target for controlling cGVHD.

GPR Expression in Intestinal Biopsies From SCT Patients Is Upregulated in GvHD and Is Suppressed by Broad-Spectrum Antibiotics.

Microbiota can exert immunomodulatory effects by short-chain fatty acids (SCFA) in experimental models of graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT). Therefore we aimed to analyze the expression of SCFAs sensing G-protein coupled receptor GPR109A and GPR43 by quantitative PCR in 338 gastrointestinal (GI) biopsies obtained from 199 adult patients undergoing allo-SCT and assessed the interaction of GPR with FOXP3 expression and regulatory T cell infiltrates. GPR expression was strongly upregulated in patients with stage II-IV GvHD (p=0.000 for GPR109A, p=0.01 for GPR43) and at the onset of GvHD (p 0.000 for GPR109A, p=0.006 for GPR43) and correlated strongly with FOXP3 and NLRP3 expression. The use of broad-spectrum antibiotics (Abx) drastically suppressed GPR expression as well as FOXP3 expression in patients’ gut biopsies (p=0.000 for GPRs, FOXP3 mRNA and FOXP3+ cellular infiltrates). Logistic regression analysis revealed treatment with Abx as an independent factor associated with GPR and FOXP3 loss. The upregulation of GPRs was evident only in the absence of Abx (p=0.001 for GPR109A, p=0.014 for GPR43) at GvHD onset. Thus, GPR expression seems to be upregulated in the presence of commensal bacteria and associates with infiltration of FOXP3+ T regs, suggesting a protective, regenerative immunomodulatory response. However, Abx, which has been shown to induce dysbiosis, interferes with this protective response.

Regulatory T cells function in established systemic inflammation and reverse fatal autoimmunity.

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss-of-function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of “redeemed” Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease, and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

Transient Depletion of Foxp3+ Regulatory T Cells Selectively Promotes Aggressive β Cell Autoimmunity in Genetically Susceptible DEREG Mice.

Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3+ regulatory T (Treg) cell function due to Foxp3 gene mutations, but whether the loss of Foxp3+ Treg cell activity is indeed sufficient to promote β cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, β cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive Foxp3 deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3+ Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3+ Treg cell ablation focused on Foxp3DTR NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4+ TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3+ Treg cell ablation on β cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3+ Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3+ Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8+ T cells in terminal β cell destruction. Despite the severity of destructive β cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced β cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3+ Treg cell activity for the control of genetically pre-installed autoimmune diabetes.

The STAT Signaling Biosignature at the Single Cell Level Reveals Novel Insights in the Association of FOXP3+ T Regulatory Cells with Recurrent Spontaneous Abortions (RSA)

BackgroundTregs play an important role in the establishment and maintenance of immune tolerance during pregnancy and they are expanded early after embryo implantation. Decreased frequency and function of Tregs in women suffering from recurrent spontaneous abortions (RSA) has been reported in literature. Signal transducer and activator of transcription (STAT) proteins play a prominent role in the epigenetic control of Treg differentiation. Deficiency of STAT5 leads to loss of Foxp3+ T regulatory cells (Tregs) and inability to induce Tregs in vitro whereas constitutive activation of STAT5β enforces Foxp3 positive Treg development. Aberrant immune signaling via STATs is involved in disease pathobiology in numerous autoimmune conditions, therefore explored the STAT signaling biosignature of Tregs by single-cell phospho-specific flow cytometry in women with RSA before and after treatment with lymphocyte immunotherapy (LIT).Materials and methodsPeripheral blood mononuclear cells from 31 women with either RSA (n=23) or with infertility and ≥2 unsuccessful IVF (n=8) and 10 age-matched females without RSA who had at least one successful pregnancy, were obtained before and after LIT. Basal levels of phospho-STAT1, STAT3 and STAT5 and potentiated, i.e. target/stimuli, nodes (IFNα/pSTAT1, IFNα/pSTAT5, IL-6/pSTAT1, IL-6/pSTAT3 and IL-2/pSTAT5) were measured by phospho-specific flow cytometry. Treg signaling profiles were grouped by using unsupervised hierarchical clustering and correlated with clinical parameters by using χ2 or Fisher Exact tests. Associations of single signaling nodes with the aforementioned parameters were performed by Mann-Whitney and Wilcoxon signed-rank tests as appropriate.ResultsNo differences were observed between women with RSA and age matched women without RSA concerning the pretreatment and post treatment levels of Tregs. By contrast, clustering of pretreatment signaling nodes identified three signaling clusters (SC, figure 1a). SC#3 was enriched in women without RSA (p<0.001) and characterized by high potentiated responses of STATs, whereas women with RSA segregated in SC#1 and SC#2 which were characterized by blunted STAT responses in cytokine stimulation. Of note, all women with successful pregnancy after LIT (n=7) were clustered in SC#1 (p=0.028) which was characterized by high basal levels of pSTAT1 and pSTAT5 but weak potentiated responses of all nodes. Also, Tregs in women with a successful pregnancy after LIT displayed a significantly decreased response of pSTAT5 to IFNα (p= 0.05) and increased basal pSTAT5 levels (p=0.047), compared to women who failed LIT. Τhe post-treatment STAT signaling biosignature remained abnormal in women with RSA (p<0.001, figure 1b). However, compared to women with at least one miscarriage (n=23), women with infertility and unsuccessful IVFs displayed decreased post treatment basal levels of pSTAT1 (p=0.004) and pSTAT3 (p=0.01) and increased responses of pSTAT1 (p=0.047) and pSTAT3 (p=0.047) to IL6. Also, clustering the fold change of pre versus post-treatment STAT signaling profiles in Tregs revealed two signaling clusters. The #SC2 was marginally enriched in women with successful pregnancy after LIT (p=0.1) and was characterized by upregulation of all potentiated nodes after treatment (figure 1c).Collectively, we show for the first time that Tregs in women with RSA bear a pathological STAT biosignature which correlates with LIT outcome. Moreover, LIT can potentially induce a beneficial modulation of STAT signaling by upregulating Treg responsiveness to cytokines. Our work provides novel biological data on the mechanisms of immune tolerance in pregnancy and may point to therapies that can overcome pregnancy loss by modifying cell and cytokine responses. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract IA16: Role of PI3K signaling in T cell-mediated immune responses

Abstract A number of receptors are capable of activating the PI3K pathway in T cells, most prominently the T-cell receptor for antigen (TCR), the costimulatory receptor CD28, and the interleukin 2 receptor (IL-2R). PI3K signaling is critical for the initiation of effector T-cell responses, and activated multiple central downstream cascades including mTOR. Consistent with this, drugs that target mTOR, such as rapamycin, are effective inhibitors of T cell-mediated immune responses. Despite the important role of PI3K in T-cell activation, mTOR inhibition has been reported to “spare” regulatory T-cell (Treg) responses. This was of particular interest as Tregs, in contrast to all other T cells, constitutively express the high-affinity IL-2R, and thus should be poised for continual PI3K activation. Tregs, defined as cells that express the lineage-defining transcription factor Foxp3, are required for normal immune homeostasis. Foxp3-deficient mice (scurfy) and humans (patients with IPEX) are characterized by widespread and severe multiorgan autoimmunity and inflammation. As well, deletion of Tregs in adult mice leads to rapid and fulminant autoimmunity and death. In a series of studies conducted by members of my group over the past several years, as well as a variety of other laboratories, we have gained insight into the role of PI3K signals in Tregs, and a complex picture has emerged. We have observed that signals that elicit PI3K pathway activation in stimulated T cells, such as the cytokine IL-2, fail to do so in Tregs, despite the fact that Tregs express the IL-2R. Moreover, we have found that this is a selective “defect” in IL-2R signaling as the other major pathway linked to this receptor, STAT5, is intact. Specific loss of PI3K signaling in Tregs can be linked to expression of the lipid phosphatase PTEN. All T cells express PTEN in their resting state. However, while “conventional” T cells (i.e., non-Tregs) downregulate PTEN expression upon activation, Tregs uniquely do not. To test the importance of maintained PTEN expression in Tregs, we created mice with constitutive and with inducible deletion of PTEN specifically in Foxp3+ Tregs. These mice developed a dramatic phenotype of lupus-like autoimmunity, despite the presence of large numbers of Tregs, indeed more than in control mice. Moreover, we observed that loss of PTEN was followed by lineage instability, i.e., loss of Foxp3, and expression of proinflammatory cytokines such as IL-17 and IFNg. PI3K signals activate pathways important for T-cell metabolism, including c-myc, HIF-1a, and mTOR, and promote aerobic glycolysis. In contrast to most activated T cells, Tregs have been reported to be more reliant on fatty acid oxidation for their energy demands, and indeed loss of PTEN in Tregs dramatically augments aerobic glycolysis in these cells. However, most of the studies of Treg metabolism have focused on cells that were differentiated to become Tregs in vitro. In this system, naïve T cells are activated in the presence of the anti-inflammatory cytokine TGF-b, which induces them to express Foxp3. Such cells, “iTregs,” are easy to generate in large numbers and believed to be induced in vivo when antigen is encountered in noninflammatory settings in visceral organs and tissues. However, most T cells in lymphoid organs are thought to have originated in the thymus, are called tTregs, and are important for the prevention of immune responses to self-antigens. We have recently characterized the metabolism of these two Treg subsets. While, as reported, iTregs make minimal use of glycolysis, tTregs are in fact as glycolytic as effector T cells. We have further found that exposure of tTregs to TGF-b represses PI3K-mediated mTOR signaling, inhibits glucose transporter expression, and reprograms their metabolism to favor oxidative phosphorylation. Conversely, replicating the effects of inflammation via elevation of PI3K signaling has minimal effects on tTregs but dramatically enhances the glycolysis of normally oxidative iTregs, resulting in reduction of Foxp3 expression. Collectively, these findings suggest unique signaling and metabolic demands of Tregs and Treg subsets that may be leveraged in the design of therapeutics, both in vivo agents and adoptive cellular therapies. Citation Format: Laurence Turka. Role of PI3K signaling in T cell-mediated immune responses [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA16.

Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B 100 –Reactive CD4 + T-Regulatory Cells

Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4+ T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (TH1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4+ T cells with an atheroprotective, regulatory T cell (Treg) phenotype in healthy individuals. Yet, the function of apoB-reactive Tregs and their relationship with pathogenic TH1 cells remain unknown. To interrogate the function of autoreactive CD4+ T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B978-993 (apoB+) at the single-cell level. We found that apoB+ T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a Treg-like transcriptome, although only 21% of all apoB+ T cells expressed the Treg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB+ T cells formed several clusters with mixed TH signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of TH1, T helper cell type 2 (TH2), and T helper cell type 17 (TH17), and of follicular-helper T cells. ApoB+ T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic TH1/TH17-like cells with proinflammatory properties and only a residual Treg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed TH1/TH17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB+ Tregs in lineage tracing of hyperlipidemic Apoe-/- mice. In adoptive transfer experiments, converting apoB+ Tregs failed to protect from atherosclerosis. Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive Tregs as a novel cellular target in atherosclerosis.

Helios: still behind the clouds

Regulatory T (Treg) cells are a subset of CD4+ T cells that are critical for the maintenance of self-tolerance. The forkhead box transcription factor Foxp3 is a master regulator for the Treg phenotype and function and its expression is essential in Treg cells, as the loss of Foxp3 results in lethal autoimmunity. Two major subsets of Treg cells have been described in vivo; thymus-derived Treg (tTreg) cells that develop in the thymus and peripherally induced Treg (pTreg) cells that are derived from conventional CD4+ Foxp3- T cells and are converted in peripheral tissues to cells that express Foxp3 and acquire suppressive ability. The transcription factor Helios, a member of the Ikaros transcription factor family, is expressed in 60-70% of Treg cells in both mouse and man, and is believed to be a marker of tTreg cells. In this review, we discuss the role and function of Helios in Treg cells, the controversy surrounding the use of Helios as a marker of tTreg cells, and how Helios controls specific aspects of the Treg cell program.

Loss of FOXP3 and TSC1 Accelerates Prostate Cancer Progression through Synergistic Transcriptional and Posttranslational Regulation of c-MYC.

Although c-MYC and mTOR are frequently activated proteins in prostate cancer, any interaction between the two is largely untested. Here, we characterize the functional cross-talk between FOXP3-c-MYC and TSC1-mTOR signaling during tumor progression. Deletion of Tsc1 in mouse embryonic fibroblasts (MEF) decreased phosphorylation of c-MYC at threonine 58 (pT58) and increased phosphorylation at serine 62 (pS62), an observation validated in prostate cancer cells. Conversely, inhibition of mTOR increased pT58 but decreased pS62. Loss of both FOXP3 and TSC1 in prostate cancer cells synergistically enhanced c-MYC expression via regulation of c-Myc transcription and protein phosphorylation. This crosstalk between FOXP3 and TSC1 appeared to be mediated by both the mTOR-4EBP1-c-MYC and FOXP3-c-MYC pathways. In mice, Tsc1 and Foxp3 double deletions in the prostate led to prostate carcinomas at an early age; this did not occur in these mice with an added c-Myc deletion. In addition, we observed synergistic antitumor effects of cotreating mice with inhibitors of mTOR and c-MYC in prostate cancer cells and in Foxp3 and Tsc1 double-mutant mice. In human prostate cancer, loss of nuclear FOXP3 is often accompanied by low expression of TSC1. Because loss of FOXP3 transcriptionally induces c-Myc expression and loss of TSC1 activates mTOR signaling, these data suggest cross-talk between FOXP3-c-MYC and TSC1-mTOR signaling that converges on c-MYC to regulate tumor progression. Coadministration of c-MYC and mTOR inhibitors may overcome the resistance to mTOR inhibition commonly observed in prostate cancer cells. SIGNIFICANCE: These results establish the principle of a synergistic action of TSC1 and FOXP3 during prostate cancer progression and provide new therapeutic targets for patients who have prostate cancer with two signaling defects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1413/F1.large.jpg.

Cutting Edge: ICOS-Deficient Regulatory T Cells Display Normal Induction of Il10 but Readily Downregulate Expression of Foxp3.

The ICOS pathway has been implicated in the development and functions of regulatory T (Treg) cells, including those producing IL-10. Treg cell-derived IL-10 is indispensable for the establishment and maintenance of intestinal immune homeostasis. We examined the possible involvement of the ICOS pathway in the accumulation of murine colonic Foxp3- and/or IL-10-expressing cells. We show that ICOS deficiency does not impair induction of IL-10 by intestinal CD4 T cells but, instead, triggers substantial reductions in gut-resident and peripherally derived Foxp3+ Treg cells. ICOS deficiency is associated with reduced demethylation of Foxp3 CNS2 and enhanced loss of Foxp3. This instability significantly limits the ability of ICOS-deficient Treg cells to reverse ongoing inflammation. Collectively, our results identify a novel role for ICOS costimulation in imprinting the functional stability of Foxp3 that is required for the retention of full Treg cell function in the periphery.

Reduced expression of phosphatase PTPN2 promotes pathogenic conversion of Tregs in autoimmunity.

Genetic variants at the PTPN2 locus, which encodes the tyrosine phosphatase PTPN2, cause reduced gene expression and are linked to rheumatoid arthritis (RA) and other autoimmune diseases. PTPN2 inhibits signaling through the T cell and cytokine receptors, and loss of PTPN2 promotes T cell expansion and CD4- and CD8-driven autoimmunity. However, it remains unknown whether loss of PTPN2 in FoxP3+ regulatory T cells (Tregs) plays a role in autoimmunity. Here we aimed to model human autoimmune-predisposing PTPN2 variants, the presence of which results in a partial loss of PTPN2 expression, in mouse models of RA. We identified that reduced expression of Ptpn2 enhanced the severity of autoimmune arthritis in the T cell-dependent SKG mouse model and demonstrated that this phenotype was mediated through a Treg-intrinsic mechanism. Mechanistically, we found that through dephosphorylation of STAT3, PTPN2 inhibits IL-6-driven pathogenic loss of FoxP3 after Tregs have acquired RORγt expression, at a stage when chromatin accessibility for STAT3-targeted IL-17-associated transcription factors is maximized. We conclude that PTPN2 promotes FoxP3 stability in mouse RORγt+ Tregs and that loss of function of PTPN2 in Tregs contributes to the association between PTPN2 and autoimmunity.

Pharmacological inhibition of USP7 promotes antitumor immunity and contributes to colon cancer therapy.

BackgroundEffectiveness of clinical therapy such as chemotherapy for solid tumors is limited by acquired drug resistance and side effects. Available antitumor immunity methods showed promising prospect of cancer therapy. However, more drug targets for boosting antitumor immunity still need to be explored and selective and effective compounds are yet to be developed.PurposeTo study the effect and possible mechanism of compound P5091, a selective USP7 inhibitor, on CT26 xenografts growth in mice.Materials and methodsCT26 xenografts model was employed to examine the anti-tumor effect of P5091. RT-PCR and ELISA analysis were used to detect the level of IFN-γ, TNF-α and IL-10 in tumor tissue and serum, respectively. IFN-γ expression in CD4+ and CD8+ T cells was analyzed by intracellular stain. The level of FOXP3 in Treg cells was confirmed by intracellular stain and western blotting.ResultsCompound P5091, a selective USP7 inhibitor, was found to inhibit CT26 xenografts growth in mice, which is comparable to the effect of Anti-PD-1 antibody. RT-PCR analysis showed that P5091 treatment decreased IL-10 mRNA level in tumor tissue while elevated mRNA level of IFN-γ and TNF-α. Moreover, ELISA analysis manifested decreased of IL-10 and elevation of IFN-γ and TNF-α in serum from tumor bearing mice. Intracellular stain showed increased IFN-g expression both in CD4+ and CD8+ T cells after P5091 treatment. Furthermore, P5091 treatment caused FOXP3 loss in Treg cells decreased the proportion of Treg cells in tumor bearing mice.ConclusionOur study here showed that P5091 may be a candidate for cancer immunotherapy.

FOXP3 and miR-155 cooperate to control the invasive potential of human breast cancer cells by down regulating ZEB2 independently of ZEB1.

Control of oncogenes, including ZEB1 and ZEB2, is a major checkpoint for preventing cancer, and loss of this control contributes to many cancers, including breast cancer. Thus tumour suppressors, such as FOXP3, which is mutated or lost in many cancer tissues, play an important role in maintaining normal tissue homeostasis. Here we show for the first time that ZEB2 is selectively down regulated by FOXP3 and also by the FOXP3 induced microRNA, miR-155. Interestingly, neither FOXP3 nor miR-155 directly altered the expression of ZEB1. In breast cancer cells repression of ZEB2, independently of ZEB1, resulted in reduced expression of a mesenchymal marker, Vimentin and reduced invasion. However, there was no de-repression of E-cadherin and migration was enhanced. Small interfering RNAs targeting ZEB2 suggest that this was a direct effect of ZEB2 and not FOXP3/miR-155. In normal human mammary epithelial cells, depletion of endogenous FOXP3 resulted in de-repression of ZEB2, accompanied by upregulated expression of vimentin, increased E-cadherin expression and cell morphological changes. We suggest that FOXP3 may help maintain normal breast epithelial characteristics through regulation of ZEB2, and loss of FOXP3 in breast cancer cells results in deregulation of ZEB2.

Cambogin suppresses dextran sulphate sodium-induced colitis by enhancing Treg cell stability and function.

Background and PurposeInflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract, and an impaired immune response plays a critical role in IBD. The current drugs and therapies for IBD treatment are of limited use, therefore, there is a need to find novel drugs or therapies for this disease. We investigated the effect of cambogin in a mouse model of dextran sulphate sodium (DSS)‐induced colitis and whether cambogin attenuates inflammation via a Treg‐cell‐mediated effect on the immune response.Experimental ApproachChronic colitis was established in mice using 2% DSS, and cambogin (10 mg·kg−1, p.o.) was administered for 10 days. Body weight, colon length and colon histology were assessed. Cytokine production was measured using elisa and quantitative real‐time PCR. To evaluate the mechanism of cambogin, human CD4+CD25hiCD127lo Treg cells were isolated from peripheral blood mononuclear cells. Major signalling profiles involved in Treg cell stability were measured.Key ResultsCambogin attenuated diarrhoea, colon shortening and colon histological injury and IL‐6, IFN‐γ and TNF‐α production in DSS‐treated mice. Cambogin also up‐regulated Treg cell numbers in both the spleen and mesenteric lymph nodes. Furthermore, cambogin (10 μM) prevented Foxp3 loss in human primary Treg cells in vitro, and promoted USP7‐mediated Foxp3 deubiquitination and increased Foxp3 protein expression in LPS‐treated cells.Conclusions and ImplicationsThe effect of cambogin on DSS‐induced colitis is expedited by a Treg‐cell‐mediated modification of the immune response, suggesting that cambogin could be applied as a novel agent for treating colitis and other Treg cell‐related diseases.

Loss of regulatory characteristics in CD4+ CD25+/hi T cells induced by impaired transforming growth factor beta secretion in pneumoconiosis.

Pneumoconiosis is caused by the accumulation of airborne dust in the lung, which stimulates a progressive inflammatory response that ultimately results in lung fibrosis and respiratory failure. It is possible that regulatory cells in the immune system could function to suppress inflammation and possibly slow or reverse disease progression. However, results in this study suggest that in pneumoconiosis patients, the regulatory T cells (Tregs) and B cells are functionally impaired. First, we found that pneumoconiosis patients presented an upregulation of CD4+ CD25+ T cells compared to controls, whereas the CD4+ CD25+ and CD4+ CD25hi T cells were enriched with Th1- and Th17-like cells but not Foxp3-expressing Treg cells and evidenced by significantly higher T-bet, interferon (IFN)-γ, and interleukin (IL)-17 expression but lower Foxp3 and transforming growth factor (TGF)-β expression. Regarding the CD4+ CD25hi T-cell subset, the frequency of this cell type in pneumoconiosis patients was significantly reduced compared to controls, together with a reduction in Foxp3 and TGF-β and an enrichment in T-bet, RORγt, IFN-γ, and IL-17. This skewing toward Th1 and Th17 types of inflammation could be driven by monocytes and B cells, since after depleting CD14+ monocytes and CD19+ B cells, the levels of IFN-γ and IL-17 were significantly decreased. Whole peripheral blood mononuclear cells and isolated monocytes and B cells in pneumoconiosis patients also presented reduced capacity of TGF-β secretion. Furthermore, monocytes and B cells from pneumoconiosis patients presented reduced capacity in inducing Foxp3 upregulation, a function that could be rescued by exogenous TGF-β. Together, these data indicated a potential pathway for the progression of pneumoconiosis through a loss of Foxp3+ Treg cells associated with impaired TGF-β secretion.