CD26: A negative selection marker for human Treg cells
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Abstract A major obstacle hampering the therapeutic application of regulatory T (Treg) cells is the lack of suitable extracellular markers, which complicates their identification/isolation. Treg cells are normally isolated via CD25 (IL‐2Rα) targeting, but this protein is also expressed by activated CD4 + effector T (Teff) lymphocytes. Other extracellular (positive or negative) Treg selection markers (e.g., HLA‐DR, CD127) are also nonspecific. CD26 is an extracellular peptidase whose high expression has been traditionally used as an indicator of immune activation and effector functions in T cells. Now, we provide flow cytometry data showing high levels of CD26 within CD4 + CD25 − or CD4 + FoxP3 −/low effector T (Teff) lymphocytes, but negative or low levels (CD26 −/low ) in Treg cells selected according to the CD4 + CD25 high or the CD4 + FoxP3 high phenotype. Unlike the negative marker CD127 (IL‐7Rα), which is down modulated in CD4 + Teff lymphocytes after TCR triggering, most of these cells upregulate CD26 and take a CD4 + CD25 +/high CD26 + phenotype upon activation. In contrast, there is only a slight upregulation within Treg cells (CD4 + CD25 high CD26 −/low ). Thus, differences in CD26 levels between Treg and Teff subsets are stable, and assessment of this marker, in combination with others like CD25, FoxP3, or CD127, may be useful during the quantitative evaluation or the isolation of Treg cells in samples containing activated Teff lymphocytes (e.g., from patients with autoimmune/inflammatory diseases). © 2012 International Society for Advancement of CytometryKeywords:
Interleukin-7 receptor
Abnormalities in CD4+CD25+Foxp3+ regulatory T (T reg) cells have been implicated in susceptibility to allergic, autoimmune, and immunoinflammatory conditions. However, phenotypic and functional assessment of human T reg cells has been hampered by difficulty in distinguishing between CD25-expressing activated and regulatory T cells. Here, we show that expression of CD127, the α chain of the interleukin-7 receptor, allows an unambiguous flow cytometry–based distinction to be made between CD127lo T reg cells and CD127hi conventional T cells within the CD25+CD45RO+RA− effector/memory and CD45RA+RO− naive compartments in peripheral blood and lymph node. In healthy volunteers, peripheral blood CD25+CD127lo cells comprised 6.35 ± 0.26% of CD4+ T cells, of which 2.05 ± 0.14% expressed the naive subset marker CD45RA. Expression of FoxP3 protein and the CD127lo phenotype were highly correlated within the CD4+CD25+ population. Moreover, both effector/memory and naive CD25+CD127lo cells manifested suppressive activity in vitro, whereas CD25+CD127hi cells did not. Cell surface expression of CD127 therefore allows accurate estimation of T reg cell numbers and isolation of pure populations for in vitro studies and should contribute to our understanding of regulatory abnormalities in immunopathic diseases.
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Abstract Background Appearance of improper immune responses against the fetus and/or inadequate immunoregulatory mechanisms during pregnancy may lead to recurrent spontaneous abortion (RSA). T H 17 cells play a significant role in inducing inflammation, autoimmune disease, and acute transplant rejection, while regulatory T (Treg) cells moderate the function of immune system in order to retain homeostasis. Methods This case-control study was designed to evaluate T H 17 as well as Treg cells in 25 women with RSA and 25 age-matched healthy non-pregnant women. Flow cytometric assay was performed using monoclonal antibodies to detect CD4 + CD25 + Treg cells (CD25 dim and CD25 bright ). FoxP3 and RORγt expressions were compared using real-time PCR, and pro-inflammatory and anti-inflammatory cytokines were measured by ELISA kits. Independent-samples T test was employed for statistical analysis. Results The ratio of CD4 + CD25 bright T cells was remarkably lower in women with RSA ( P <0.05), and CD4 + CD25 dim T cells did not show any significant difference among the groups ( P >0.05). RORγt was up-regulated, and FoxP3 was down-regulated significantly in case group ( P <0.05). The significant increase of IL-6 and IL-17 as well as the decrease of TGF-β was indicated in RSA group ( P <0.05). Also, IL-10 did not vary among the groups ( P >0.05). Conclusion These remarks prove that the decrease in regulatory factors such as CD4 + CD25 bright T-cells, TGF-β and FoxP3 expression may disrupt immune tolerance and homeostasis during pregnancy. Also, the environment rich in RORγt, IL-6, and IL-17 suggests the detrimental role of T H 17 cells, which may lead to fetal rejection.
Regulatory T cell
Homeostasis
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To observe the changes in proportion of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in peripheral blood of different stages of CPOD and intervention of Qibai Pingfei Capsules.The rats were randomly divided into three groups,including normal group, COPD group and Qibai Pingfei Capsules(2. 88 g/kg)group. At the end of 7,14,21 and 28 days,eight rats were sacrificed in each group. CD4 + CD25 + Foxp3 + Treg cells in peripheral blood were measured by flow cytometry method.Compared with normal group, the percentages of CD4 + % in peripheral blood were not significantly different at the end of 7, 14, 21 and 28 days. However, CD4 + CD25 + % and CD4 + CD25 + Foxp3 +/CD4 + were significantly increased and CD4 + CD25 + Foxp3 + Treg% were significantly decreased at the end of 14,21 and 28 days. Compared with model group, CD4 + CD25 + %, CD4 + CD25 + Foxp3 +/CD4 + were significantly decreased, CD4 + CD25 + Foxp3 + Treg % were significantly increased at different stages of CODP.Immune disorders may exist in COPD, and Treg cells may be involved in the process of COPD. Meanwhile,the protective effect in COPD rats of Qibai Pingfei Capsules may be associated with improving the percentage of suppressive CD4 + CD25 + Foxp3 + Tregs.
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Abstract Investigation of the role of regulatory T cells (Treg) in model systems is facilitated by their depletion using anti-CD25 Abs, but there has been considerable debate about the effectiveness of this strategy. In this study, we have compared the depletion and repopulation of CD4+CD25+Foxp3+ Treg in uninfected and malaria-infected mice using 7D4 and/or PC61 anti-CD25 Abs. We find that numbers and percentages of CD25high cells, but not Foxp3+ cells, are transiently reduced after 7D4 treatment, whereas treatment with PC61 alone or in combination with 7D4 (7D4 plus PC61) reduces but does not eliminate Foxp3+ cells for up to 2 wk. Importantly, all protocols fail to eliminate significant populations of CD25−Foxp3+ or CD25lowFoxp3+ cells, which retain potent regulatory capacity. By adoptive transfer we show that repopulation of the spleen by CD25highFoxp3+ cells results from the re-expression of CD25 on peripheral populations of CD25−Foxp3+ but not from the conversion of peripheral Foxp3− cells. CD25highFoxp3+ repopulation occurs more rapidly in 7D4-treated mice than in 7D4 plus PC61-treated mice, reflecting ongoing clearance of emergent CD25+Foxp3+ cells by persistent PC61 Ab. However, in 7D4 plus PC61-treated mice undergoing acute malaria infection, repopulation of the spleen by CD25+Foxp3+ cells occurs extremely rapidly, with malaria infection driving proliferation and CD25 expression in peripheral CD4+CD25−Foxp3+ cells and/or conversion of CD4+CD25−Foxp3− cells. Finally, we reveal an essential role for IL-2 for the re-expression of CD25 by Foxp3+ cells after anti-CD25 treatment and observe that TGF-β is required, in the absence of CD25 and IL-2, to maintain splenic Foxp3+ cell numbers and a normal ratio of Treg:non-Treg cells.
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To investigate the level of CD4+ CD25+ Foxp3+ regulatory T cells and observe relation between expression of Foxp3 and CD127 in peripheral blood of chronic HBV infection.CD4+ CD25+ Foxp3+ and CD4+ CD25+ CD127low Treg in peripheral blood from 34 patients of immune tolerance stage, 26 patients of immune clearance stage and 31 patients of non-active status were quantitatively analyzed by flow cytometry.Immune tolerance group presented a higher fraction of CD4+ CD25+ Foxp3+ and CD4+ CD25+ CD127low Treg than non-active group in chronic HBV infection (Z = -2.693, P = 0.007 and t = 3.251, P = 0.002), and HBV positive group also presented a higher fraction than non-active group (t = 2.266, P = 0.026 and t = 3.208, P = 0.002), But ALT normal group is similar to ALT abnormal group (P > 0.05). In this study, the relation between expression of CD127low and Foxp3+ from CD4+ CD25+ regulatory T cells was observed, and CD4+ CD25+ CD127low Treg presented a higher fraction than CD4+ CD25+ Foxp3+ Treg.Peripheral Treg in HBV active replication group is higher than HBV negative group of chronic HBV infection. Expression of CD127low is consistent with Foxp3+ in CD4+ CD25+ regulatory T cells, but the former is significantly higher than the latter.
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Regulatory T cells (Tregs, CD4 + CD25(high) Foxp3(+)) play a crucial role in allergy and other inflammatory diseases. However, the isolation of viable Tregs on the basis of intracellular expression of specific Forkhead Box Protein P3 (Foxp3) is difficult. In this study we checked if the expression of IL-7 receptor (CD127) on the Tregs could be a useful marker for isolation of viable Treg Foxp3(+) cells.Twenty-five patients sensitized to grass pollen with allergic rhinitis (AR) and ten healthy subjects were included. We compared Foxp3 expression in different CD4(+) T cell subsets by flow cytometry and we assessed the relationship between the expression of Foxp3 and CD127 within regulatory T cells.Within the CD(4)+ lymphocytes 3.68 ±2.0% showed expression of Foxp3, 51.82 ±8.03% of CD4(+)CD25(high) were Foxp3 positive (Foxp3(+)), whereas 82.12 ±5.4% of CD4(+)CD25(high)CD127(low) were Foxp3(+). High intracellular expression of Foxp3 correlated with low superficial CD127 expression (r = 0.42, p = 0.017). There were no significant differences regarding the analysed markers between AR patients and healthy controls.Regulatory T cells may be purified from the fresh peripheral blood as viable regulatory Foxp3 bright cells using CD4, high expression of CD25 and low expression of CD127 antigen.
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Background: We found previously that depletion of CD4+Foxp3+Tregs at early time (within 20 days) and later time (80 days) of transplantation abrogated pig-islet-xenograft tolerance in mice induced by short-term CTLA4-Fc/MR-1 treatment. We also identified memory-like CD127+/highCD4+GFP+/Foxp3+Tregs (CD127+/highTreg) in spleen of tolerant mice following CTLA4-Fc/MR-1 induction and demonstrated their potent suppressive capacity in an adaptive-transfer model. Aims: 1) Further characterise tissue CD127+/high Tregs. 2) Investigate transcriptional profile of CD4+Foxp3+Treg and non-Foxp3 CD4+ subsets in transplant tolerance. Methods: We used DEpletion of REGulatory T cells (DEREG) mice, which carry the enhanced GFP transgene under Foxp3 promoter as recipients of NICC transplantation tolerance model. Cell-subsets were selected with FACS/Cell Sorter based on positive or negative expressions of CD4, GFP, and CD127 or CD45, CD4 and GFP. mRNA expression of Il-10, Tgf-β, Blimp-1, Ebi3 (reflecting IL-35) of CD127+/high Tregs was assessed using TaqMan® Gene Expression Assay. Bulk RNA-Seq revealed the transcriptional profiles of CD127+/highTreg, CD127-/low Treg, CD4+GFP-Foxp3+ Treg, non-Foxp3 CD4+, and CD45+CD4- subsets from spleens (sp), graft draining-lymphocytes (DLN/dln), or grafts in mice with 100-day tolerant-graft induced by CTLA4-Fc/MR-1 blockade or naïve DEREG-mice. Results: RT-PCR showed Ebi3, Il-10, Blimp-1 significantly increased in splenic CD127+/high Tregs compared to naïve-CD4+GFP-Foxp3+Tregs or non-Foxp3 CD4+T cells. The proportion of CD127+/high Tregs was higher in tolerant grafts (25.6±3.1%) than tolerant spleens (14.8±0.4%). 15 pairwise-comparisons identified 1740 differentially expressed genes (DEGs) (FDR<0.05) that clearly distinguished between CD45+CD4-, Foxp3-CD4+T, and Treg subsets; with no striking differences seen for CD45+CD4- cells (spleen) and mild differences in Foxp3-CD4+T cells (spleen) between naive and tolerant-groups; and diverse differences within Treg subsets. Next, 9 paired cross-comparisons between different Treg subsets identified 427 DEGs and showed large difference between graft-Treg and Treg subsets of spleen or DLN; moderate differences between spTreg and dlnTreg subsets; and minor differences within the three Treg subsets of spleen or DLN. Further, compared to naïve-Treg or CD127-/low Treg subsets, graft-Tregs shared many upregulated-DEGs across dlnCD127+/high Treg, and/or spCD127+/high Treg including Il7r, Kctd12, Cxcr6, Ctla2a, Anxa1, H2-Ab1 (an MHC-II gene), Klrk1, Klrg1, Ccl5, Id2, Ccr2, Adam8, Il18r1, Il1rl that have been reported in multiple tissue/tumour Treg subsets with memory features and high suppressive functions in both mice and/or humans. Conclusion: Tissue-Tregs (CD127+/high Tregs) developed in graft, spleen and DLN of transplant-tolerant mice share a transcriptional trajectory with other tissue/tumour Tregs.
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Objective To investigate the expression of CD4+CD25+FOXP3+regulatory T cells(Tregs), IL-6 and TNF-α in patients with juvenile idiopathic arthritis (JIA) and its clinical significance. Methods The percentages of CD4+CD25+FOXP3+ Tregs in 58 children with JIA and 40 healthy controls were detected by flow cytometry. Serum levels of IL-6 and TNF-α in each patient was detected by chemiluminescence. The correlations between the expression of CD4+CD25+FOXP3+ Tregs and IL-6, TNF-α were analyzed by pearson correlation analysis. We measured the expression level of CD4+CD25+FOXP3+regulatory T cells, IL-6 and TNF-α of 18 cases six weeks after the treatment of tocilizumab (TCZ) in order to figure out the dynamic changes using methods above. Results The percentages of CD4+CD25+FOXP3+Tregs in juvenile idiopathic arthritis were significantly lower than those in 40 healthy volunteers, while levels of IL-6 and TNF-α were significantly higher. However, no obvious difference in the levels of CD4+CD25+FOXP3+Tregs, IL-6 or TNF-α was observed between patients with systemic and poly-articular JIA. Pearson correlation analysis showed that the percentages of CD4+CD25+FOXP3+Tregs negatively correlated with the levels of IL-6 and TNF-α, while levels of IL-6 positively correlated with the levels of TNF-α. Compared with pre-treatment of TCZ, levels of CD4+CD25+FOXP3+Tregs in post-treatment markedly increased, which however were still lower than control group while the levels of IL-6 significantly decreased, yet remained higher than control group. There was no statistical difference between post and pre-treatment in the levels of TNF-α. Conclusion The percentages of CD4+CD25+FOXP3+ Tregs in peripheral blood of JIA children decreases, and it has a negative correlation with IL-6 and TNF-α. Furthermore, the levels of CD4+CD25+FOXP3+ Tregs and IL-6 are partially restored after treatment with TCZ, which may be helpful to assess the activity of systemic JIA and the efficacy of therapy.
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