IPEX syndrome from diagnosis to cure, learning along the way.

In the past 2 decades, a significant number of studies have been published describing the molecular and clinical aspects of immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome. These studies have refined our knowledge of this rare yet prototypic genetic autoimmune disease, advancing the diagnosis, broadening the clinical spectrum, and improving our understanding of the underlying immunologic mechanisms. Despite these advances, Forkhead box P3 mutations have devastating consequences, and treating patients with IPEX syndrome remains a challenge, even with safer strategies for hematopoietic stem cell transplantation and gene therapy becoming a promising reality. The aim of this review was to highlight novel features of the disease to further advance awareness and improve the diagnosis and treatment of patients with IPEX syndrome.

FOXP3 deficiency, from the mechanisms of the disease to curative strategies.

FOXP3 gene is a key transcription factor driving immune tolerance and its deficiency causes immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome (IPEX), a prototypic primary immune regulatory disorder (PIRD) with defective regulatory T (Treg) cells. Although life-threatening, the increased awareness and early diagnosis have contributed to improved control of the disease. IPEX currently comprises a broad spectrum of clinical autoimmune manifestations from severe early onset organ involvement to moderate, recurrent manifestations. This review focuses on the mechanistic advancements that, since the IPEX discovery in early 2000, have informed the role of the human FOXP3+ Treg cells in controlling peripheral tolerance and shaping the overall immune landscape of IPEX patients and carrier mothers, contributing to defining new treatments.

Analyses of thymocyte commitment to regulatory T cell lineage in thymus of healthy subjects and patients with 22q11.2 deletion syndrome.

The Chromosome 22q11.2 deletion syndrome (22q11.2DS) results in an inborn error of immunity due to defective thymic organogenesis. Immunological abnormalities in 22q11.2DS patients are thymic hypoplasia, reduced output of T lymphocytes by the thymus, immunodeficiency and increased incidence of autoimmunity. While the precise mechanism responsible for increased incidence of autoimmunity is not completely understood, a previous study suggested a defect in regulatory T cells (Treg) cell lineage commitment during T cell development in thymus. Here, we aimed to analyze this defect in more detail. Since Treg development in human is still ill-defined, we first analyzed where Treg lineage commitment occurs. We performed systematic epigenetic analyses of the Treg specific demethylation region (TSDR) of the FOXP3 gene in sorted thymocytes at different developmental stages. We defined CD3+CD4+CD8+ FOXP3+CD25+ as the T cell developmental stage in human where TSDR demethylation first occurs. Using this knowledge, we analyzed the intrathymic defect in Treg development in 22q11.2DS patients by combination of TSDR, CD3, CD4, CD8 locus epigenetics and multicolor flow cytometry. Our data showed no significant differences in Treg cell frequencies nor in their basic phenotype. Collectively, these data suggest that although 22q11.2DS patients present with reduced thymic size and T cell output, the frequencies and the phenotype of Treg cell at each developmental stage are surprisingly well preserved.

PAM-free cascaded strand displacement coupled with CRISPR-Cas12a for amplified electrochemical detection of SARS-CoV-2 RNA.

The early diagnosis of coronavirus disease 2019 (COVID-19) is dependent on the specific and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Herein, we develop a highly sensitive and specific electrochemical biosensor for SARS-CoV-2 target RNA detection based on the integration of protospacer adjacent motif (PAM)-free cascaded toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a (PfTSDR-CRISPR). In this study, each target is transformed into multiple DNA substrates with bubble structure in the seed region by the cascaded TSDR, which can directly hybridize with guide RNA (gRNA) without PAM requirement and then activate CRISPR-Cas12a's trans-cleavage activity. Subsequently, the hairpin DNA modified with methylene blue (MB-HP) is cleaved by activated CRISPR-Cas12a. Therefore, as MB leaves the electrode surface, a decreased current signal is obtained. With the involvement of PAM-free cascaded TSDRs and CRISPR-Cas12a amplification strategy, the PfTSDR-CRISPR-based electrochemical biosensor achieves the detection of target RNA as low as 40 aM. The biosensor has high sequence specificity, reliability and robustness. Thanks to the PAM-free cascaded TSDR, the biosensor can achieve universal detection of different target RNA without redesigning gRNA sequence of CRISPR-Cas12a. In addition, this biosensor successfully detects SARS-CoV-2 target RNA in complex samples, which highlights its potential for diagnosing COVID-19.

Epigenetic and immunological indicators of IPEX disease in subjects with FOXP3 gene mutation.

BACKGROUND: Forkhead box protein 3 (FOXP3) is the master transcription factor in CD4+CD25hiCD127lo regulatory T (Treg) cells. Mutations in FOXP3 result in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome. Clinical presentation of IPEX syndrome is broader than initially described, challenging the understanding of the disease, its evolution, and treatment choice. OBJECTIVE: We sought to study the type and extent of immunologic abnormalities that remain ill-defined in IPEX, across genetic and clinical heterogeneity. METHODS: We performed Treg-cell-specific epigenetic quantification and immunologic characterization of severe "typical" (n = 6) and "atypical" or asymptomatic (n = 9) patients with IPEX. RESULTS: Increased number of cells with Treg-cell-Specific Demethylated Region demethylation in FOXP3 is a consistent feature in patients with IPEX, with (1) highest values in those with typical IPEX, (2) increased values in subjects with pathogenic FOXP3 but still no symptoms, and (3) gradual increase over the course of disease progression. Large-scale profiling using Luminex identified plasma inflammatory signature of macrophage activation and TH2 polarization, with cytokines previously not associated with IPEX pathology, including CCL22, CCL17, CCL15, and IL-13, and the inflammatory markers TNF-α, IL-1A, IL-8, sFasL, and CXCL9. Similarly, both Treg-cell and Teff compartments, studied by Mass Cytometry by Time-Of-Flight, were skewed toward the TH2 compartment, especially in typical IPEX. CONCLUSIONS: Elevated TSDR-demethylated cells, combined with elevation of plasmatic and cellular markers of a polarized type 2 inflammatory immune response, extends our understanding of IPEX diagnosis and heterogeneity.

Epigenetic/genetic variations in CG-rich elements of immune-related genes contribute to food allergy development during childhood.

BACKGROUND: Genetic areas of FOXP3 TSDR, human leukocyte antigen-G (HLA-G) upstream of CpG island 96, CpG41 and CpG73 islands of the HLA-DRB1 and HLA-DQB1 genes respectively, previously documented to display immune-modulatory properties, were subjected to epigenetic/genetic analysis to assess their influence in IgE-mediated food allergy (FA) development in children. METHODS: Sixty-four orally challenged and IgE-tested food allergic subjects together with 44 controls were recruited. Targeted pyrosequencing analysis to detect DNA methylation status and genetic variations was utilized and experimental results obtained were analyzed by a statistical software platform and correlated to clinical data. Also, transcription factor (TF) binding sites in study areas were unmasked by the JASPAR prediction database. RESULTS: Parents' smoking was significantly correlated with aberrant methylation patterns, regardless of food allergic or control status. HLA-G promoter region showed a trend for hypomethylation in food allergic subjects, with one of the CG sites displaying significantly decreased methylation values. Rs1233333, residing within the HLA-G promoter region preserved a protective role toward DNA methylation. Variable methylation patterns were recorded for CpG41 of the HLA-DRB1 gene and hypermethylation of the region was significantly correlated with the presence of single nucleotide polymorphisms (SNPs). TFs' recognition sites, located in studied genetic areas and exerting pivotal regulatory biological roles, are potentially affected by divergent DNA methylation status. CONCLUSIONS: We propose that HLA-G expression is triggered by food-derived allergens, providing a TregFoxP3-/HLA-G+ subpopulation generation to promote direct immune tolerance. Furthermore, clear evidence is provided for the underlying co-operation of genetic polymorphisms with epigenetic events, mainly at the CpG41 island of the HLA-DRB1 gene, which needs an extended investigation and elucidation.

Protein phosphatase 6 (Pp6) is crucial for regulatory T cell function and stability in autoimmunity.

Regulatory T (Treg) cells constitute a dynamic population that is critical in autoimmunity. Treg cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities. However, recent studies demonstrated that certain inflammatory conditions induce Treg cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells. Therefore, the identification of novel targets crucial to both Treg cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity. In this study, we found that conditional Pp6 knockout (cKO) in Treg cells led to spontaneous autoinflammation, immune cell activation, and diminished levels of FoxP3 in CD4+ T cells in mice. Loss of Pp6 in Treg cells exacerbated two classical mouse models of Treg-related autoinflammation. Mechanistically, Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308, leading to impaired FoxP3 expression in Treg cells. In summary, our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling, thus maintaining Treg cell stability and preventing autoimmune diseases.

Antigenic Challenge Influences Epigenetic Changes in Antigen-Specific T Regulatory Cells.

Background: Human regulatory T cells (Tregs) are the fundamental component of the immune system imposing immune tolerance via control of effector T cells (Teffs). Ongoing attempts to improve Tregs function have led to the creation of a protocol that produces antigen-specific Tregs, when polyclonal Tregs are stimulated with monocytes loaded with antigens specific for type 1 diabetes. Nevertheless, the efficiency of the suppression exerted by the produced Tregs depended on the antigen with the best results when insulin ß chain peptide 9-23 was used. Here, we examined epigenetic modifications, which could influence these functional differences. Methods: The analysis was pefromed in the sorted specific (SPEC, proliferating) and unspecific (UNSPEC, non-proliferating) subsets of Tregs and Teffs generated by the stimulation with monocytes loaded with either whole insulin (INS) or insulin ß chain peptide 9-23 (B:9-23) or polyclonal cells stimulated with anti-CD3/anti-CD28 beads (POLY). A relative expression of crucial Tregs genes was determined by qRT-PCR. The Treg-specific demethylated region (TSDR) in FoxP3 gene methylation levels were assessed by Quantitative Methylation Specific PCR (qMSP). ELISA was used to measure genomic DNA methylation and histone H3 post-translational modifications (PTMs). Results: Tregs SPECB:9-23 was the only subset expressing all assessed genes necessary for regulatory function with the highest level of expression among all analyzed conditions. The methylation of global DNA as well as TSDR were significantly lower in Tregs SPECB:9-23 than in Tregs SPECINS. When compared to Teffs, Tregs were characterized by a relatively lower level of PTMs but it varied in respective Tregs/Teffs pairs. Importantly, whenever the difference in PTM within Tregs/Teffs pair was significant, it was always low in one subset from the pair and high in the other. It was always low in Tregs SPECINS and high in Teffs SPECINS, while it was high in Tregs UNSPECINS and low in Teffs UNSPECINS. There were no differences in Tregs/Teffs SPECB:9-23 pair and the level of modifications was low in Tregs UNSPECB:9-23 and high in Teffs UNSPECB:9-23. The regions of PTMs in which differences were significant overlapped only partially between particular Tregs/Teffs pairs. Conclusions: Whole insulin and insulin ß chain peptide 9-23 affected epigenetic changes in CD4+ T cells differently, when presented by monocytes. The peptide preferably favored specific Tregs, while whole insulin activated both Tregs and Teffs.

Lower Functional and Proportional Characteristics of Cord Blood Treg of Male Newborns Compared with Female Newborns.

Understanding the early events involved in the induction of immune tolerance to harmless environmental antigens and microbiota compounds could reveal potential targets for allergic disease therapy or prevention. Regulatory T cells (Treg), particularly induced Treg (iTreg), are crucial for the induction and maintenance of tolerance against environmental antigens including allergens. A decrease in the number and/or function of Treg or iTreg could represent an early predictor of allergy development. We analyzed proportional and functional properties of Treg in the cord blood of children of allergic mothers (neonates at high risk of allergy development) and healthy mothers (neonates with relatively low risk of allergy development). We observed a higher number of induced Treg in the cord blood of females compared to males, suggesting an impaired capacity of male immunity to set up tolerance to allergens, which could contribute to the higher incidence of allergy observed in male infants. The decreased proportion of iTreg in cord blood compared with maternal peripheral blood documents the general immaturity of the neonatal immune system. We observed a positive correlation in the demethylation of the Treg-specific demethylated region (TSDR) and the proportion of Treg in cord blood. Our data suggest that immaturity of the neonatal immune system is more severe in males, predisposing them to increased risk of allergy development.

Attenuation of canonical NF-κB signaling maintains function and stability of human Treg.

Nuclear factor 'κ-light-chain-enhancer' of activated B cells (NF-κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR-derived signals, thereby maintaining their suppressive features. However, the role of NF-κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF-κB proteins p50, p65, and c-Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF-κB signaling in FOXP3-transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF-κB signaling. Additionally, repression of residual NF-κB signaling in Treg further enhances interleukin-10 (IL-10) production. Blockade of NF-κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF-κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg-specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF-κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy.

DL-propargylglycine administration inhibits TET2 and FOXP3 expression and alleviates symptoms of neonatal Cows' milk allergy in mouse model.

BACKGROUND: Cows' milk allergy (CMA) is a hypersensitivity immune reaction brought on by specific immunologic mechanisms to cow's milk proteins. As one of the most common food allergies in infants, the incidence of CMA during the first year of life is estimated to be nearly 7.5%. Due to the limitation in the knowledge of the pathological mechanism underlying CMA, however, the clinical interventions and therapies remain very unsatisfactory. AIM OF THE STUDY: The transcriptional factor FOXP3 possesses crucial roles in CMA, and increased FOXP3 mRNA expression has a predictive function in faster acquisition of tolerance in infants with CMA. But the exact mechanism remains not fully elucidated. METHODS: For PAG treatment, PAG (dissolved in saline 30 mg/mL, 0, 5, 10, 20 mg/kg BW) was administered daily intraperitoneally (ip) for one week at the time that 6 weeks after the CMP sensitisation. RESULTS: In the present study, we revealed that the expression of FOXP3 is significantly up-regulated in PBMCs from CMA patients and CMA mice on mRNA and protein level. Furthermore, a dramatic reduction in the FOXP3 TSDR methylation and a significant increase in the expression of TET2 are observed in CMA patients and CMA mice. More importantly, we found that propargylglycine (PAG) significantly alleviates symptoms of CMA in mice by suppressing the expression of FOXP3 through restoring TET2 expression. CONCLUSIONS: Our work revealed a novel function of PAG on CMA, which may provide a deeper insight into the pathomechanism of CMA and a novel therapy target for CMA clinical interventions.

Decipher manifestations and Treg /Th17 imbalance in multi-staging rheumatoid arthritis and correlation with TSDR/RORC methylation.

T regulatory (Treg)/T-helper (Th) 17 imbalance has been shown to integrate with epigenetics to result in the development of autoimmune diseases. We aim to investigate the influence of disease staging on Treg/Th17 cells and whether the aberrant DNA methylation is implicated in the development of rheumatoid arthritis (RA). By recruiting 65 patients with multi-staging RA and 20 healthy controls (HC), we found that patients with active RA exhibited relative lymphopenia and higher WBC, neutrophils, and PLT. Circulating Treg/Th17 in patients with early active RA was significantly decreased. The expression of IL-6 and IL-17A was significantly increased in early active RA, whereas that of IL-10 and TGF-ß was on the contrary. Furthermore, the frequency of Treg cells and Treg/Th17 were negatively correlated with DAS28, and the frequency of Th17 cells was on the contrary. Levels of DNA methylation related enzymes had significant difference between early active RA and HC. Relative hypermethylation was observed at the gene level for Treg-specific demethylated region (TSDR) and hypomethylation for retinoic acid-related orphan receptor (ROR)-C in early active RA. Thus, manifestations of RA and Treg/Th17 imbalance vary with disease staging, and the aberrant DNA methylation pattern may contribute to RA disease pathogenesis. Our results highlight the importance of disease staging in clinical research.

Development of Quantitative Methylation-Specific Droplet Digital PCR (ddMSP) for Assessment of Natural Tregs.

Regulatory T cells (Tregs) suppress immune responses in vivo in an antigen-specific manner. Of clinical relevance, Tregs can be isolated and expanded in vitro while maintaining immunoregulatory function. Tregs are classified as CD4+CD25highCD127low FOXP3+ cells. Demethylation of the Treg-specific demethylation region (TSDR) of FOXP3 is found in natural Tregs (nTregs). We report a method for the characterization of the differential methylation pattern of the FOXP3 TSDR in patient-derived and expanded nTregs. Human TSDR sequences from nTregs (unmethylated sequence) and pancreatic (methylated sequence) cells were amplified and cloned into plasmids. A droplet digital TaqMan probe-based qPCR (ddPCR) assay using methylation-specific primers and probes was employed to quantify unmethylated and methylated sequences. The methylation-specific droplet digital PCR (ddMSP) assay was specific and selective for unmethylated DNA in mixtures with methylated DNA in the range of 5000 copies/µL to less than 1 copy/µL (R 2 = 0.99) even in the presence of non-selective gDNAs. CD4+CD25highCD127lowFOXP3+ human nTregs, in the presence of Dynabeads or activators, were expanded for 21 days. There was a decrease in the unmethylated ratio of Tregs after expansion with essentially the same ratio at days 10, 14, and 17. However, the activator expanded group showed a significant decrease in unmethylated targets at day 21. The suppression activity of activator-expanded nTregs at day 21 was decreased compared to cells expanded with Dynabeads. These data suggest that the ddMSP can quantitatively monitor nTreg expansion in vitro. These data also indicate that the assay is sensitive and specific at differentiating nTregs from other cells and may be useful for rapid screening of nTregs in clinical protocols.

Ex vivo expansion of regulatory T cells from long-term Belatacept-treated kidney transplant patients restores their phenotype and suppressive function but not their FOXP3 TSDR demethylation status.

We recently reported that Tregs from long-term Belatacept-treated kidney transplant patients displayed an altered phenotype and impaired suppressive function compared to Tregs from healthy controls. However, it remains unknown whether ex vivo expansion of Tregs from patients who underwent long-term immunosuppression may be feasible to be used in their treatment. In this work, Tregs from Belatacept-treated patients were polyclonally expanded in vitro in the presence of rapamycin and IL-2. After four weeks of expansion, Tregs from patients expressed high levels of FOXP3, CD25, CTLA-4, Helios and CCR7, and showed strong suppressive activity, even in the presence of pro-inflammatory cytokines. However, FOXP3 TSDR demethylation remained lower in expanded Tregs from Belatacept-treated patients compared to healthy control Tregs. These data suggest that ex vivo expansion of Tregs from patients undergoing long-term immunosuppression may require the use of epigenetic modifying agents to stabilize FOXP3 expression to be considered as treatment in kidney transplant patients.

Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells.

Regulatory T (Treg) cells mainly develop within the thymus and arise from CD25+Foxp3- (CD25+ TregP) or CD25-Foxp3+ (Foxp3+ TregP) Treg cell precursors resulting in Treg cells harboring distinct transcriptomic profiles and complementary T cell receptor repertoires. The stable and long-term expression of Foxp3 in Treg cells and their stable suppressive phenotype are controlled by the demethylation of Treg cell-specific epigenetic signature genes including an evolutionarily conserved CpG-rich element within the Foxp3 locus, the Treg-specific demethylated region (TSDR). Here we analyzed the dynamics of the imprinting of the Treg cell-specific epigenetic signature genes in thymic Treg cells. We could demonstrate that CD25+Foxp3+ Treg cells show a progressive demethylation of most signature genes during maturation within the thymus. Interestingly, a partial demethylation of several Treg cell-specific epigenetic signature genes was already observed in Foxp3+ TregP but not in CD25+ TregP. Furthermore, Foxp3+ TregP were very transient in nature and arose at a more mature developmental stage when compared to CD25+ TregP. When the two Treg cell precursors were cultured in presence of IL-2, a factor known to be critical for thymic Treg cell development, we observed a major impact of IL-2 on the demethylation of the TSDR with a more pronounced effect on Foxp3+ TregP. Together, these results suggest that the establishment of the Treg cell-specific hypomethylation pattern is a continuous process throughout thymic Treg cell development and that the two known Treg cell precursors display distinct dynamics for the imprinting of the Treg cell-specific epigenetic signature genes.

Human LAP+GARP+FOXP3+ regulatory T cells attenuate xenogeneic graft versus host disease.

Adoptive transfer of regulatory T cells (FOXP3+ Tregs) has been developed as a potential curative immune therapy to prevent and treat autoimmune and graft-versus-host diseases (GVHD). A major limitation that has hindered the use of Treg immunotherapy in humans is the difficulty of consistently isolating and obtaining highly purified Tregs after ex vivo expansion. Methods: We isolated bona fide Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). The TCR Vß diversity and intracellular cytokine production of Tregs were determined by flow cytometer. The TSDR methylation was determined by epigenetic human FOXP3 qPCR Assay. Their in vitro and in vivo potency was confirmed with suppression assay and humanized xenogeneic GVHD (xGVHD) murine model, respectively. Results: LAP+ repurification results in >90% LAP+FOXP3+ Tregs, leaving behind FOXP3- and FOXP3+ nonTregs within the LAP- population. After 4-week expansion, the LAP+ Tregs were >1 billion cells, highly suppressive and anergic in vitro, >90% demethylated in the TSDR and able to maintain TCR Vß diversity. In the xGVHD model, exogenous CD25-PBMC administered alone results in a median survival of 32 days. The co-transfer of LAP+ Tregs increased median survival to 47 days, while the LAP parent (CD25+) and LAP- nonTregs had median survival of 39 and 31 days, respectively. Conclusions: These preclinical data together provide evidence that LAP+ Tregs are highly purified with fully suppressive function for cell therapy. This population results in a more effective and safer product for immunotherapy to treat GVHD and provides the necessary preclinical data for transition into a clinical trial with LAP+ Tregs to prevent or treat GVHD and other autoimmune diseases.

Vision-Based Traffic Sign Detection and Recognition Systems: Current Trends and Challenges.

The automatic traffic sign detection and recognition (TSDR) system is very important research in the development of advanced driver assistance systems (ADAS). Investigations on vision-based TSDR have received substantial interest in the research community, which is mainly motivated by three factors, which are detection, tracking and classification. During the last decade, a substantial number of techniques have been reported for TSDR. This paper provides a comprehensive survey on traffic sign detection, tracking and classification. The details of algorithms, methods and their specifications on detection, tracking and classification are investigated and summarized in the tables along with the corresponding key references. A comparative study on each section has been provided to evaluate the TSDR data, performance metrics and their availability. Current issues and challenges of the existing technologies are illustrated with brief suggestions and a discussion on the progress of driver assistance system research in the future. This review will hopefully lead to increasing efforts towards the development of future vision-based TSDR system.

Analysis of Helios gene expression and Foxp3 TSDR methylation in the newly diagnosed Rheumatoid Arthritis patients.

BACKGROUND: The control of auto-reactive cells is defective in rheumatoid arthritis (RA). Regulatory T (Treg) cells which play a key role in the modulation of immune responses have an impaired function in RA. Foxp3 is a master regulator of Treg cells which its expression is under the tight control of epigenetic mechanisms. In the current study, we analyzed the epigenetic modulation of the Foxp3 Treg-specific demethylated region (TSDR) and Helios gene expression to determine Treg cells alteration in RA patients. METHODS: We have recruited 20 newly diagnosed patients with RA and 41 healthy controls in our study. The measurement of Foxp3 and Helios gene expression was performed by the real-time PCR technique and the methylation level of TSDR was analyzed by bisulfite treatment and quantitative methylation-specific PCR (Q-MSP). RESULTS: We found that RA patients had significantly lower level of Foxp3 gene expression and TSDR demethylation compared to healthy subjects (P < 0.001 and P = 0.006, respectively). Inversely, the Helios gene expression was elevated significantly in RA patients group (P = 0.048). We also observed a significant correlation between Foxp3 and Helios gene expression (P = 0.016) as well as a significant correlation between FoxP3 expression and demethylation rate of TSDR (P = 0.010). CONCLUSION: Our results suggested that both epigenetic modifications and Helios gene expression may have important roles in the pathogenesis of RA through their effects on Foxp3 gene expression.

Cells with Treg-specific FOXP3 demethylation but low CD25 are prevalent in autoimmunity.

Identification of alterations in the cellular composition of the human immune system is key to understanding the autoimmune process. Recently, a subset of FOXP3+ cells with low CD25 expression was found to be increased in peripheral blood from systemic lupus erythematosus (SLE) patients, although its functional significance remains controversial. Here we find in comparisons with healthy donors that the frequency of FOXP3+ cells within CD127lowCD25low CD4+ T cells (here defined as CD25lowFOXP3+ T cells) is increased in patients affected by autoimmune disease of varying severity, from combined immunodeficiency with active autoimmunity, SLE to type 1 diabetes. We show that CD25lowFOXP3+ T cells share phenotypic features resembling conventional CD127lowCD25highFOXP3+ Tregs, including demethylation of the Treg-specific epigenetic control region in FOXP3, HELIOS expression, and lack of IL-2 production. As compared to conventional Tregs, more CD25lowFOXP3+HELIOS+ T cells are in cell cycle (33.0% vs 20.7% Ki-67+; P = 1.3 × 10-9) and express the late-stage inhibitory receptor PD-1 (67.2% vs 35.5%; P = 4.0 × 10-18), while having reduced expression of the early-stage inhibitory receptor CTLA-4, as well as other Treg markers, such as FOXP3 and CD15s. The number of CD25lowFOXP3+ T cells is correlated (P = 3.1 × 10-7) with the proportion of CD25highFOXP3+ T cells in cell cycle (Ki-67+). These findings suggest that CD25lowFOXP3+ T cells represent a subset of Tregs that are derived from CD25highFOXP3+ T cells, and are a peripheral marker of recent Treg expansion in response to an autoimmune reaction in tissues.