Surface expression of CD39 identifies an enriched Treg‐cell subset in the rheumatic joint,which does not suppress IL‐17A secretion

Treg cells are important for the maintenance of self‐tolerance and are implicated in autoimmunity. Despite enrichment of Treg cells in joints of rheumatoid arthritis (RA) patients, local inflammation persists. As expression of the ATP‐hydrolyzing enzymes CD39 and CD73 and the resulting anti‐inflammatory adenosine production have been implicated as an important mechanism of suppression, we characterized FOXP3⁺ Treg cells in blood and synovial fluid samples of RA patients in the context of CD39 and CD73 expression. Synovial FOXP3⁺ Treg cells displayed high expression levels of rate‐limiting CD39, whereas CD73 was diminished. FOXP3⁺CD39⁺ Treg cells were also abundant in synovial tissue. Furthermore, FOXP3⁺CD39⁺ Treg cells did not secrete the proinflammatory cytokines IFN‐γ and TNF after in vitro stimulation in contrast to FOXP3⁺CD39^? T cells. FOXP3⁺CD39⁺ Treg cells could be isolated by CD39 and CD25 coexpression, displayed a demethylated Treg‐specific demethylated region and coculture assays confirmed that CD25⁺CD39⁺ T cells have suppressive capacity, while their CD39^? counterparts do not. Overall, our data show that FOXP3⁺CD39⁺ Treg cells are enriched at the site of inflammation, do not produce proinflammatory cytokines, and are good suppressors of many effector T‐cell functions including production of IFN‐γ, TNF, and IL‐17F but do not limit IL‐17A secretion.     

Peripheral whole blood FOXP3 TSDR methylation: a potential marker in severity assessment of autoimmune diseases and chronic infections

Immune dysregulation is a cardinal feature of autoimmune diseases and chronic microbial infections. In particular, regulatory T cells are downregulated in autoimmune diseases while upregulated in chronic microbial infections. FOXP3 is the master regulator of Treg development. Treg-specific demethylated region (TSDR) is a highly conserved locus on the FOXP3 gene that is fully demethylated in natural Tregs but methylated in effector T cells. In our study, we used high resolution melt-polymerase chain reaction (HRM-PCR) to determine the FOXP3 TSDR methylation status in autoimmune diseases and chronic microbial infections. We found that FOXP3 TSDR to have the highest mean melting temperature (highly methylated) in active SLE patients compared to all the other groups (p?<?0.001). The psoriasis group also had a significantly high mean melting temperature (78.62?±?0.20) when compared with the inactive SLE group (78.49?±?0.29, p?<?0.05) and control group (78.44?±?0.25, p?<?0.01). There was no significant difference in melting temperature between inactive SLE and healthy controls. Disease activity in SLE was directly associated with methylation of the FOXP3 TSDR. On the other hand, patients with chronic microbial infections had significantly lower FOXP3 TSDR mean melting temperature (demethylated) when compared with healthy controls (78.28?±?0.21 vs 78.44?±?0.25, p?<?0.05). Our results suggest that the use of HRM-PCR to detect FOXP3 TSDR methylation status is a reliable and easy method to predict natural regulatory T cell levels in peripheral blood in different disease conditions. Determining FOXP3 TSDR methylation status can be a useful tool in diagnosis, and monitoring the severity of autoimmune diseases and chronic microbial infections.     

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation

The adoptive transfer of CD4⁺CD25⁺ natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high‐grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell‐specific surface markers. Depletion of CD127⁺ cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3⁺ cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4⁺CD25⁺CD127^low Treg, correlating with loss of FOXP3 expression and emergence of pro‐inflammatory cytokines. Further analysis identified CD45RA^?FOXP3⁺ memory‐type Treg as the main source of converting cells, whereas CD45RA⁺FOXP3⁺ Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell‐type‐specific characteristics after repetitive TCR stimulation.     

FOXP3和GITR/GITRL mRNA在类风湿关节炎和结直肠癌患者外周血中的表达研究

探讨FOXP3和糖皮质激素诱导的肿瘤坏死因子受体(glucocorticoid-induced tumor necrosis factor receptor,GITR)及其配体(GITRL)在类风湿关节炎(rheumatoid arthritis,RA)和结直肠癌患者中的表达变化及GITR与FOXP3表达的相关性。运用实时荧光定量RT-PCR方法,检测55例健康体检者、55例RA患者、50例结直肠癌患者的外周血单个核细胞(peripheral blood monocytes,PBMC)中FOXP3、GITR和GITRL的基因表达水平。结果:在RA患者组FOXP3、GITR和GITRL均显著低于健康对照组(P<0.01),经过转换后的mRNA表达水平分别为对照组的18.5%、7.5%和1.7%,GITR和FOXP3的表达水平呈显著正相关(r=0.86,P<0.01);而在结直肠癌患者组,FOXP3的表达水平显著高于对照组(P<0.01),表达水平为对照组的173.7%,GITR和GITRL的表达水平显著低于对照组(P<0.01),mRNA表达水平为对照组的45.7%和50.1%,GITR和FOXP3的表达有一定的相关性(P=0.046)。RA和结直肠癌患者均存在调节性T细胞(regulatory Tcells,Treg)异常。在RA中,GITR表达趋势与FOXP3一致,均反映Treg数量或功能降低。但在结直肠癌患者中,两者表达不尽一致。     

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.     

Dendritic Cells from Rheumatoid Arthritis Patient Peripheral Blood Induce Th17 Cell Differentiation via miR‐363/Integrin αv/TGF‐β Axis

Dendritic cells (DCs) are critical regulators of immune responses. This study was to observe the effect of DCs from peripheral blood on the differentiation of Th17 in patients with rheumatoid arthritis (RA). Peripheral blood samples were collected from 30 patients with RA and 20 healthy controls, respectively. Flow cytometry results showed that in contrast to Treg cells, the proportion of Th17 cells in T cells and the Th17/Treg ratio were both increased in patients with RA. The RT‐PCR results showed that Foxp3、ROR γt and miR‐363 expression in PBMC of patients with RA were reduced, but the ITGAV expression was increased, which was negatively related to miR‐363 expression. IL‐17, TGF‐β and IL‐6 levels detected by ELISA were increased in peripheral blood serum of patients with RA. Moreover, we noted that the CD11C⁺αν⁺/CD11C⁺ DCs ratio was obvious increased in patients with RA and has positive correlation to the Th17/Treg ratio. In cocultured system, Th17 cell differentiation was significantly inhibited in the presence of ITGF‐β suggesting that Th17 cell differentiation was controlled by active TGF‐β (aTGF‐β). After DCs transfecting with miR‐363 mimics and cocultured with T cells, Th17 cell number, IL‐17 level and ROR‐γt expression were significantly reduced in the presence of latent TGF‐β (ITGF‐β). In addition, the integrin αv protein expression was both reduced in the presence of aTGF‐β or ITGF‐β. These data demonstrated that DCs induced Th17 cell differentiation through miR‐363/Integrin αv/TGF‐β pathway in patients with RA.     

Short‐term cytokine stimulation reveals regulatory T cells with down‐regulated Foxp3 expression in human peripheral blood

The identification of regulatory T cells (Treg cells) in human peripheral blood is an important tool in diagnosis, research, and therapeutic intervention. As compared to lymphoid tissues, the frequencies of circulating Treg cells identified as CD4⁺CD25⁺Foxp3⁺ are, however, low. We here show that many of these cells remain undetected due to transient down regulation of Foxp3, which rapidly decays in the absence of cytokine‐mediated STAT5 signals. Short‐term incubation of PBMCs or isolated CD4⁺ T cells, but not of lymph node cells, with IL‐2, ‐7, or ‐15 more than doubles the frequency of Foxp3⁺CD25⁺ among CD4⁺ T cells detectable by flow cytometry. This increase is not due to cell division but to upregulation of both proteins. At the same time, the uncovered Treg cells up‐regulate CD25 and down‐regulate CD127, making them accessible to viable cell sorting. “Latent” Treg cells have a demethylated FOXP3 TSDR sequence, are enriched in naïve, non‐cycling cells, and are functional. The confirmation of our findings in RA and SLE patients shows the feasibility of uncovering latent Treg cells for immune monitoring in clinical settings. Finally, our results suggest that unmasking of latent Treg cells contributes to the increase in circulating CD4⁺CD25⁺Foxp3⁺ cells reported in IL‐2 treated patients.     

Identification of FOXP3-negative regulatory T-like (CD4(+)CD25(+)CD127(low)) cells in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune disorder caused by mutations in the FOXP3 gene, which plays a key role in the generation of CD4⁺CD25⁺regulatory T (Treg) cells. We selected CD127 as the surface marker of Treg cells to illustrate the development and function of Treg cells in IPEX syndrome. CD4⁺CD25⁺FOXP3⁺ T cells, the putative Treg cells, were almost completely absent in all patients. Importantly, a substantial number of CD4⁺CD25⁺CD127^low T cells were observed in 3 IPEX patients with hypomorphic mutations in the FOXP3 gene. We demonstrated that CD4⁺CD25⁺CD127^low T cells isolated from these 3 patients exhibited an appreciable suppressive activity on effector T cell proliferation, although less than that displayed by Treg cells from healthy controls. These results suggest that genetically altered FOXP3 can drive the generation of functionally immature Treg cells, but that intact FOXP3 is necessary for the complete function of Treg cells.     

Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

CD40L demethylation in CD4(+) T cells from women with rheumatoid arthritis

We have previously demonstrated that DNA demethylation of CD40L on the X chromosome is responsible for female susceptibility to systemic lupus erythematosus (SLE). It is unknown whether aberrant methylation of the CD40L gene also contributes to the higher incidence of rheumatoid arthritis (RA) in females. In this study, we used real-time RT-PCR and flow cytometry to compare CD40L expression levels, and bisulfite sequencing to assess the methylation status of the CD40L promoter region. The results show that CD40L is upregrulated in CD4(+) T cells of female patients with RA. In addition, the CD40L promoter region in CD4(+) T cells from female RA patients was found to be demethylated, which corresponded with increased CD40L mRNA expression. These findings suggest that DNA demethylation contributes to CD40L expression in RA CD4(+) T cells and may in part explain the female preponderance of this disease.     

Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4+ Foxp3+ regulatory T cells

Recently, we demonstrated elevated numbers of CD4⁺ Foxp3⁺ regulatory T (Treg) cells in Plasmodium yoelii‐infected mice contributing to the regulation of anti‐malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus‐derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3⁺ Treg cells expressing neuropilin‐1 (Nrp‐1) decreased at early time‐points during P. yoelii infection, whereas percentages of Helios⁺ Foxp3⁺ Treg cells remained unchanged. Both Foxp3⁺ Nrp‐1⁺ and Foxp3⁺ Nrp‐1^? Treg cells from P. yoelii‐infected mice exhibited a similar T‐cell receptor Vβ chain usage and methylation pattern in the Treg‐specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3^? T cells adoptively transferred to P. yoelii‐infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3⁺ Treg cells.     

Inducible reprogramming of human T cells into Treg cells by a conditionally active form of FOXP3

FOXP3 is required for the development of Treg and its expression is often used as a surrogate marker of functional suppression. However, it is now known that activated human T effector cells can also express FOXP3 without acquiring regulatory activity. To more closely examine the requirements for FOXP3 to reprogram human T cells into Treg, we developed a conditionally active form of FOXP3 and show here that full acquisition of Treg phenotype and function is strictly dependent on the amount of active FOXP3 a T cell expresses. In addition, the phenotypic and functional alterations induced by FOXP3 are only fully manifested following prolonged induction of protein activity. Induction of FOXP3 activity does not upregulate EBI3 or p35 mRNA, providing evidence that secretion of IL‐35 does not substantially contribute to the suppressive mechanism of human Treg. These data represent the first formal evidence that FOXP3 acts as a quantitative regulator rather than a simple molecular switch for Treg.     

Overrepresentation of highly functional T regulatory cells in patients with nonfunctioning pituitary adenoma

Nonfunctioning pituitary adenoma is a common intracranial tumor. Though benign in the majority of cases, complications can be excruciating to the affected individual, and recurrences after tumor removal may happen with more aggressive clinical features. T regulatory (Treg) cells are generally considered a tumor-promoting immune cell type in malignant cancers with currently unclear roles in pituitary adenoma patients. Therefore, we investigated the frequency and functional characteristics of Treg cells in nonfunctioning pituitary adenoma patients before and after tumor removal. Compared to healthy controls, untreated patients with nonfunctioning pituitary adenomas presented an overrepresentation of highly functional circulating FOXP3⁺ Treg cells. Specifically, the FOXP3⁺ Treg cells in patients were slightly upregulated in frequency and displayed markedly elevated capacity to co-produce TGF-β and IL-10. TIM-3 is a negative regulator of proinflammatory immune responses and is expressed by highly activated Treg cells. In both healthy controls and pituitary adenoma patients, TIM-3⁺ Treg cells presented significantly higher levels of TGF-β and IL-10 co-producing cells than TIM-3⁻ Treg cells but compared to healthy controls, patients with nonfunctioning pituitary adenomas showed significantly higher levels of TIM-3⁺ FOXP3⁺ Treg cells. Interestingly, surgical removal of the tumor significantly reduced the extent of Treg upregulation in patients. Also, resected pituitary adenomas contained highly functional FOXP3⁺ Treg cells, with high levels of TIM-3 expression and high frequency of TGF-β and IL-10 co-producers in the TIM-3⁺ fraction. Overall, these results demonstrate that patients with nonfunctioning pituitary adenomas are characterized by an overrepresentation of highly functional Treg cells.