High-frequency and activation of CD4+CD25+ T cells maintain persistent immunotolerance induced by congenital ALV-J infection.

Congenital avian leukosis virus subgroup J (ALV-J) infection can induce persistent immunotolerance in chicken, however, the underlying mechanism remains unclear. Here, we demonstrate that congenital ALV-J infection induces the production of high-frequency and activated CD4+CD25+ Tregs that maintain persistent immunotolerance. A model of congenital infection by ALV-J was established in fertilized eggs, and hatched chicks showed persistent immunotolerance characterized by persistent viremia, immune organ dysplasia, severe imbalance of the ratio of CD4+/CD8+ T cells in blood and immune organs, and significant decrease in CD3+ T cells and Bu-1+ B cells in the spleen. Concurrently, the mRNA levels of IL-2, IL-10, and IFN-γ showed significant fluctuations in immune organs. Moreover, the frequency of CD4+CD25+ Tregs in blood and immune organs significantly increased, and the frequency of CD4+CD25+ Tregs was positively correlated with changes in ALV-J load in immune organs. Interestingly, CD4+CD25+ Tregs increased in the marginal zone of splenic nodules in ALV-J-infected chickens and dispersed to the germinal center. In addition, the proliferation and activation of B cells in splenic nodules was inhibited, and the number of IgM+ and IgG+ cells in the marginal zone significantly decreased. We further found that the mRNA levels of TGF- ß and CTLA-4 in CD4+CD25+ Tregs of ALV-J-infected chickens significantly increased. Together, high-frequency and activated CD4+CD25+ Tregs inhibited B cells functions by expressing the inhibitory cytokine TGF-ß and inhibitory surface receptor CTLA-4, thereby maintaining persistent immunotolerance in congenital ALV-J-infected chickens.

[Peripheral T and B lymphocytes in patients with occupational medicamentosa-like dermatitis due to trichloroethylene].

Objective: To investigate the changes in liver function and peripheral regulatory lymphocytes before and after treatment in patients with occupational medicamentosa-like dermatitis due to trichloroethylene (OMDT) . Methods: In December 2019, 16 patients with OMDT (8 patients with erythema multiforme and 8 with erythema multiforme major) who were admitted from February 2017 to February 2019 were enrolled as subjects. Liver function parameters and percentages of peripheral regulatory lymphocytes were measured before and after treatment, and the changes in liver function and peripheral regulatory T and B lymphocytes and their correlation were analyzed. Results: Before treatment, compared with the healthy control group, the experimental group had significantly higher levels of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , total bilirubin (TBIL) , direct bilirubin (DBIL) and gamma-glutamyl transpeptidase (GGT) and significantly lower levels of total protein (TP) , albumin (ALB) and cholinesterase (CHE) (P<0.05) . Compared with the healthy control group, the experimental group had significantly lower percentages of lymphocytes, CD4(+) T cells, CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+) Bregs and CD4(+)/CD8(+) ratio, as well as a significantly higher percentage of CD8(+) T cells (P<0.05) . Before treatment, the levels of ALT, AST, GGT and DBIL were negatively correlated with the percentages of CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+) Bregs, CD4(+) T cells and CD4(+)/CD8(+) ratio (r=-0.386 to -0.809, P<0.05) and was positively correlated with the percentage of CD8(+) T cells (except DBIL) (r=0.503-0.568, P<0.05) . The levels of TP and ALB were positively correlated with the percentages of CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+)Bregs and CD4(+) T cells (r= 0.351-0.784, P<0.05) , ALB was negatively correlated with the percentage of CD8(+) T cells (r=-0.315, P<0.05) . CHE was positively correlated with the percentages of CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+)Bregs and CD4(+)/CD8(+) ratio (r=0.390-0.527, P<0.05) . Conclusion: Immune dysfunction is observed in patients with OMDT, which may be caused by the imbalance of regulatory lymphocytes. And liver injury may be associated with the increase of CD8(+) T cells and the reductions of percentages of CD4(+) T cells, CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+)Bregs and CD4(+)/CD8(+) ratio.

Adoptive transfers of CD4+ CD25+ Tregs partially alleviate mouse premature ovarian insufficiency.

This study was designed to investigate the protective effect of CD4+ CD25+ regulatory T cells (Tregs) against zona pellucida glycoprotein 3 peptide (pZP3) immunization-induced premature ovarian insufficiency (POI) in mice. A mouse POI model was induced by two subcutaneous injections of pZP3 (50 nmol/L). Mice in the pZP3-Treg group were intraperitoneally injected with 5 × 105 CD4+ CD25+ Tregs after the POI model was established. Sex hormone levels, follicle numbers, apoptotic events, and the Akt/FOXO3a signaling pathway molecules in the ovaries were assessed. Compared with control group, the weight of ovaries in both pZP3 group and pZP3-Treg group was decreased and no difference was found between them. The number of follicles in the Treg transferred mice, like in pZP3 group, was significantly reduced compared to the control group, but showed a modest improvement when compared the pZP3 group alone. Significantly lower serum concentrations of follicle-stimulating hormone, luteinizing hormone, and anti-zona pellucida antibodies (AZPAbs) were found, while the concentrations of estradiol and anti-Mullerian hormone increased. In mechanism, Treg cell transfer to ZP3 treated mice restored the levels of Caspase3 to control levels, and partially restored Bax, however, had no effect on Bcl-2. Moreover, Treg cell transfer to ZP3 treated mice partially restored the levels of Akt and FOXO3a, and partially restored the ratios of p-Akt/Akt and p-FOXO3a/FOXO3a. In conclusion, Treg cells improved some aspects of ZP3-induced POI which may be mediate by suppressing ovarian cells apoptosis and involving the Akt/FOXO3a signaling pathway. Therefore, Treg cells may be protective against autoimmune POI.

Fibroblast activation proteins-α suppress tumor immunity by regulating T cells and tumor-associated macrophages.

Fibroblast activation protein-α (FAPα) is a type-II cell-surface-bound integral transmembrane serine protease and selectively overexpressed by tumor-associated stromal fibroblasts (TAFs), which are the main components in the tumor microenvironment, in >90% of malignant epithelial carcinomas. FAPα regulates the immunosuppression of tumor cells in the tumor microenvironment. Regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) are the major immunosuppressive cells in the tumor microenvironment. However, the effect of FAPα on Tregs and TAMs is unknown. The non-enzymatic function of FAPα on Treg and TAM was investigated. In this study, we confirm that FAPα can promote the generation of Tregs and TAMs, which suggests that FAPα plays a immunosuppressive role in the tumor microenvironment and provides evidence for FAP α as a potent immunotherapeutic target for cancer.

Changes in CD4+CD25+ Tregs in the pathogenesis of atherosclerosis in ApoE-/- mice.

The goal of this study was to observe the pathological characteristics of atherosclerotic plaques in the aortic walls of ApoE-/- and C57BL/6J mice and the changes of CD4+CD25+ regulatory T cells (Tregs) in atherosclerotic mice. Twenty ApoE-/- mice were split into high-fat diet (AH) and normal diet (AN) groups and 10 C57BL/6J male mice were designated as the control group (BN). The serum concentrations of IL-10 and TGF-ß1 were detected by enzyme-linked immunosorbent assay; paraffin sections of the aorta were stained with hematoxylin & eosin, and morphometric parameters were measured using the Image Pro Plus 6.0 system. Verhoeff stain was used to observe the distribution of elastic fibers, and immunohistochemical staining was performed to verify the phenotype of the forkhead box protein 3 (Foxp3+) CD25+ cells in the atherosclerotic tissue. The proportion of CD4+CD25+ Tregs in the spleen was calculated by flow cytometry. The thickness of the intima, the intima/media ratio, the plaque area, and the plaque/lumen ratio of mice in AN group were significantly larger than those of mice in BN group. The thickness of the intima, the plaque area, and the plaque/lumen ratio of the mice in AH group were significantly increased compared with those of the AN group mice. The serum concentrations of IL-10 and TGF-ß1 and the percentage of splenic CD4+CD25+ Tregs in AN group mice were significantly decreased compared with the control group. The serum concentrations of IL-10 and TGF-ß1 and the percentage of splenic CD4+CD25+ Tregs in the mice in AH group were significantly decreased compared with those in AN group. The proportions of Foxp3+ and CD25+ cells within the total lymphocyte population were significantly decreased in AH group mice compared with those in AN group mice. Atherosclerosis in an experimental mouse model was correlated with Treg depletion in the lymphoid tissues and plaques, indicating the important antiatherosclerotic role of CD4+CD25+ Tregs. Impact statement In this article, we conclude that Tregs decreased with atherosclerosis (AS) as determined in ApoE knockout mice fed a high fat diet. It is an important matter for understanding the AS pathology.

Spectrum drift characteristics of CD4+ CD25+ Tregs TCR CDR3 in hepatitis B patients before and after treatment / 中国免疫学杂志

Objective:To determine the spectrum drift characteristics of CI4+CD25+Tregs TCR β chain CDR3 in patients with different phases of acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients before and after the entecavir treatment.Methods:Anticoagulation venous blood was collected from 4 normal control subjects,3 AHB patients with acute phase and convalescent phase,and 4 CHB patients before and after the entecavir treatment;and peripheral blood mononuclear cells were isolated;CD4+ CD25+ Tregs were separated by using the magnetic beads,and total RNAs were extracted from CD4+ CD25+ Tregs and used for reverse transcription.The TRBV CDR3 was amplified by polymerase chain reaction (PCR) with forward primers specific for 24 TRBV families and one fluorescence-labeled common reverse primer specific for the BC region.The PCR products were sent out for Genescan,and results were analyzed for the TRBV family CDR3 spectrum characteristics by using the Peak Scanner Software vl.0.Data were analyzed with the comparative t-test to perform the statistical analysis.Results:The CDR3 spectral types of the TRBV family showed drift characteristics in 3 cases of AHB patients with acute and convalescent phases;single/oligo peak spectral type family was observed in most of patients with acute phase;multiple peak spectral type was seen in patients with convalescent phase;and the common spectrum shift of TRBV4,10,14,16,19 families seen in patients with acute phase was changed to multiple peak spectral type.The clonal expansion of TRBV family in the CD4+CD25+Tregs in PBMC from AHB patients with convalescent phase was significantly lower than AHB patients with acute phase (t =9.456,P =0.011).The clonal expansion of Tregs TRBV13.2,15,16,18,20 family seen in C HB patients before treatment may interfere the virus removal through down-regulating the body's immune response;and with the decline of viral load in serum after the antiviral treatment,the clonal expansion of Tregs TRBV1,5.2,6,12,14,24 family may help body induce immune tolerance and result in the HBV persistence.The clonal expansion of TRBV family in the CI4+CD25+Tregs in PBMC from of CHB patients after antiviral treatment was increased (t =-0.666,P =0.553).Conclusion:TRBV4,10,14,16,19 family of spectrum shift seen in AHB patients with acute phase was changed to multiple peak spectral type in patients with convalescent phase,suggesting this transition may be associated with HBsAg and HBeAg turning to negative.The clonal expansion of Tregs TRBV13.2,15,16,18,20 family seen in CHB patients before treatment may interfere the virus removal through down-regulating the body's immune response;and with the decline of viral load in serum after the antiviral treatment,the clonal expansion of Tregs TRBV1,5.2,6,12,14,24 family may help body induce immune tolerance and result in the HBV persistence.

Lentinan ameliorates burn sepsis by attenuating CD4+ CD25+ Tregs.

AIM: The aim of our study was to investigate the effect of lentinan on regulatory T cells (Tregs) in sepsis, especially on the generation of interleukin (IL)-10 via regulation of Erk-FoxO1 signaling. METHODS: BalB/c mice were randomized into five groups: sham group, the group with burns plus Pseudomonas aeruginosa infection, and the groups with burns plus P. aeruginosa infection administered 40, 100, and 250mg/kg of lentinan. The mice were sacrificed on postburn days 0, 1, 2, 3, and 4, respectively, with eight animals per group at each time point. The peripheral blood CD4+ CD25+ Tregs and CD4+ T cells were isolated using magnetic microbeads. The phenotypes were analyzed by flow cytometry. The cytokine levels were determined with enzyme-linked immunosorbent assay (ELISA). Signal transduction was studied by Western blot, quantitative polymerase chain reaction (qPCR), and luciferase assay. RESULTS: The IL-10-producing capacity of CD4+ CD25+ Tregs was significantly enhanced in the group with burns plus P. aeruginosa infection, compared with the sham group. Administration of lentinan significantly decreased IL-10 production and FoxP3 expression of CD4+ CD25+ Tregs. The proliferative activities of CD4+ T cells, however, were restored. Lentinan decreased lipopolysaccharide (LPS)-induced IL-10 production in the Tregs isolated from burned mice. In addition, lentinan attenuated LPS-stimulated Erk-FoxO1 activation. CONCLUSIONS: Lentinan may improve the outcome of postburn sepsis by suppressing LPS-triggered Erk-FoxO1 activation. Consequently, the hyperfunction of CD4+ CD25+ Tregs is inhibited, leading to a shift in the inflammatory status from Th2 to Th1 in postburn sepsis.

[Effect and mechanism of CD4+CD25+ regulatory T cells on protective efficacy of protein vaccine against Schistosoma japonicum in mice].

OBJECTIVE: To explore the effect and mechanism of CD4+CD25+ Tregs (Tregs) on the protective efficacy of gluthatione-S-transferase (GST) against Schistosoma japonicum in mice. METHODS: Female BALB/c mice were divided randomly into five groups：a normal control group, an infected control group, an anti-CD25mAb group, a GST immunization group and a combination group with GST immunization and anti-CD25 mAb. The GST group and combination group were injected percutaneously with GST 50 µg each mouse, the other two groups were injected with equal volume PBS. The immunization was performed for 3 times for two-week interval, and 2 weeks after the last immunization, each mouse was challenged with 40 S. japonicum cercaria. Two weeks post-infection, the combination group and anti-CD25 mAb group were injected intraperitoneally with 300 µg antiCD25 mAb each mouse. The mice were succumbed 2 weeks, 3 weeks, 4 weeks and 5 weeks post-infection respectively. The percentages of CD4+CD25+ Tregs in splenocytes of mice were measured with flow cytometer. The levels of IFN-γ, IL-2, IL-4, IL-5 and TGF-ß in cell cultural supernatants were determined by sandwich-ELISA after stimulation with Con A. The liver sections were stained with hematoxylin and eosin. RESULTS: The worm burden in the combination group (15.80±2.74) was significantly lower than those of the infected control group (27.78±3.15), anti-CD25 mAb group (21.50±4.21), and GST group (20.84± 6.46). Compared to those of the infected control group, the percentages of CD4+CD25+ Tregs were significantly higher in the GST group, while the percentages of CD4+CD25+ Tregs were significantly lower post-anti-CD25 mAb-administration. Regardless of GST administration, the levels of IFN-γ, IL-2, IL-4 and IL-5 after anti-CD25 mAb were significantly higher than those of the infected control groups. There were no significant differences of egg granuloma and the level of TGF-ß between each group. CONCLUSIONS: CD4+CD25+ Tregs could be partially blocked by anti-CD25 mAb while Th1 and Th2 type immunization response could be enhanced, which plays a role in improving the protective efficacy of GST against of S. japonicum.

Effect and mechanism of CD4+CD25+regulatory T cells on protective effica-cy of protein vaccine against Schistosoma japonicum in mice / 中国血吸虫病防治杂志

Objective To explore the effect and mechanism of CD4+CD25+Tregs(Tregs)on the protective efficacy of glutha?tione?S?transferase(GST)against Schistosoma japonicum in mice. Methods Female BALB/c mice were divided randomly into five groups:a normal control group,an infected control group,an anti?CD25mAb group,a GST immunization group and a com?bination group with GST immunization and anti?CD25 mAb. The GST group and combination group were injected percutaneously with GST 50μg each mouse,the other two groups were injected with equal volume PBS. The immunization was performed for 3 times for two?week interval,and 2 weeks after the last immunization,each mouse was challenged with 40 S. japonicum cercaria. Two weeks post?infection,the combination group and anti?CD25 mAb group were injected intraperitoneally with 300μg anti?CD25 mAb each mouse. The mice were succumbed 2 weeks,3 weeks,4 weeks and 5 weeks post?infection respectively. The per?centages of CD4+CD25+Tregs in splenocytes of mice were measured with flow cytometer. The levels of IFN?γ,IL?2,IL?4,IL?5 and TGF?βin cell cultural supernatants were determined by sandwich?ELISA after stimulation with Con A. The liver sections were stained with hematoxylin and eosin. Results The worm burden in the combination group(15.80 ± 2.74)was significantly lower than those of the infected control group(27.78 ± 3.15),anti?CD25 mAb group(21.50 ± 4.21),and GST group(20.84 ± 6.46). Compared to those of the infected control group,the percentages of CD4+CD25+Tregs were significantly higher in the GST group,while the percentages of CD4+CD25+Tregs were significantly lower post?anti?CD25 mAb?administration. Regardless of GST administration,the levels of IFN?γ,IL?2,IL?4 and IL?5 after anti?CD25 mAb were significantly higher than those of the in?fected control groups. There were no significant differences of egg granuloma and the level of TGF?βbetween each group. Con?clusion CD4+CD25+Tregs could be partially blocked by anti?CD25 mAb while Th1 and Th2 type immunization response could be enhanced,which plays a role in improving the protective efficacy of GST against of S. japonicum.

Effect of cytotoxic T-lymphocyte-associated protein 4 on CD4(+)CD25(+) regulatory T cells in murine schistosomiasis japonica.

In a previous study we demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) contributed to the escape of Schistosoma japonicum (S. japonicum) from the host's immune responses. In this paper, we studied the effect of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on CD4(+)CD25(+) Tregs in murine Schistosomiasis japonica and its corresponding role in the immune evasion of S. japonicum in mice. The results showed substantial reductions of worm burden and egg production in worm groups treated with anti-CD25 or anti-CTLA-4 monoclonal antibodies (mAb) compared to an infected but untreated control. The reduction effect was even enhanced in an experimental group co-treated with both mAbs. Compared to the control group, the percentage of CD4(+)CD25(+) Tregs was very much lower in the anti-CD25 mAb group as determined by FACS analyses and higher in the anti-CTLA-4 mAb group. ELISA analyses showed that both the anti-CTLA-4 mAb and the co-treated groups had higher levels of cytokines compared to the control group as well as larger egg granuloma sizes as determined by microscopical analyses of liver sections of infected mice. These results suggest that treatment with an anti-CTLA-4 mAb allows the host to clear S. japonicum, but at the cost of elevated pathological damage. The latter indicated a role of CTLA-4 in granuloma formation. Moreover, CD4(+)CD25(+) Tregs and CTLA-4 may exert synergistic effects during immune evasion processes by enhancing Th1-type immune response.

Effects of IL-7 and IL-2 on CD4+CD25-T cells proliferation in vitro / 中国免疫学杂志

Objective:To explore the promoting effects of IL-7 and IL-2 on CD4+CD25-T cells proliferation in vitro and construct a stable culture system in vitro for CD 4+CD25+regulatory T cells from human umbilical cord blood.To compare the inhibiting effects between induced proliferated CD 4+CD25+Tregs and naturally isolated CD 4+CD25+Tregs on PBMCs functional activity.Methods:CD4+CD25-T cells and CD4+CD25+T cells were isolated from human umbilical cord blood mononuclear cells by magnetic activated cell sorting ( MACS) system and then expanded in vitro.Four different concentration levels of IL-7 combined with proper concentration of IL-2 were added as inducer and the efficiency and optimal concentration of IL-7 on inducing,CD4+CD25-T cells were analyzed via 4 different methods.Flow cytometry method was used to detect the changes of CD 4+CD25-T cells.The inhibitory effect of expanded CD 4+CD25+T cells on peripheral blood mononuclear cells (PBMCs) was tested by MTS.The expressions of Foxp3,IL-10 and TGF-βgenes in CD4+CD25+T cells were test by RT-PCR.Results:The CD4+CD25+T cells from each groups were expanded significantly after three weeks of culture.The results indicated that use of IL-7 combined with IL-2 resulted in the highest cell expansion comparing to the other groups.It was shown by the inhibitory test that the expanded CD 4+CD25+regulatory T cells could inhibit the proliferation of PBMCs ,but IL-7 induced CD4+CD25+regulatory T cells exerted weaker suppressor activity than natural regulatory T cells .Only IL-7 (4 ng/ml) and IL-2 (2 000 U/ml) induced CD4+CD25+regulatory T cells showed the strongest killing activity.Conclusion:We successfully expand CD4+CD25+regulatory T cells in vitro.The protocol is established in which the use of mAbCD 3/CD28 combined with IL-7 and IL-2 resulted in the highest cell expansion ,and intensely expressed cell phenotype of CD 4 and CD25.

Circulating CD4~+CD25~+ and CD8~+CD28~- T regulate cells in multiple myeloma / 中国免疫学杂志

Objective: The study was designed to evaluate the changes and significance of circulating CD4~+CD25~+ and CD8~+CD28~- regulatory T cells (Tregs) in patients with multiple myeloma (MM).Methods:CD4~+CD25~+ and CD8~+CD28~-Tregs in peripheral blood of 38 patients with MM and of 20 healthy doners were measured by flow cytometry.Serum albumin and β_2-MG in patients with MM were measured using bromocresol green method,transmission turbidimetry respectively.Results:Compared to those of the controls,the proportions of CD4~+CD25~(+/high),CD4~+CD25~(high) CD127~(low) and CD8~+CD28~-Treg cells in newly diagnosed MM patients were elevated.Furthermore,the proportions of CD4~+CD25~(high) and CD4~+CD25~(high)CD127~(low) Tregs in each clinical stage were elevated when compared to those of the controls.The number of the Tregs were increasing with clinical stages and were significantly higher in stage Ⅲ MM than in stageⅠ MM;In stageⅡand Ⅲ MM,there were also elevated proportions of CD8~+CD28~- Tregs,increasing with clinical stages.However,there were no differences when compared between stage Ⅰ MM and the controls;Both the proportions of CD4~+CD25~(+/high) and CD4~+CD25~(high)CD127~(low) Tregs in active MM were not different from stable MM,although all of them were higher than those of controls.The proportion of CD8~+CD28~- Tregs was higher in active MM than in stable MM and controls,but there were no differences when compared between active and stable MM.The proportions of both CD4~+CD25~(high) Tregs and CD4~+CD25~(high)CD127~(low)Tregs had negative correlation with the levels of serum albumin.Conclusion:MM patients have elevated levels of circulating CD4~+CD25~+ and CD8~+CD28~-Tregs,which may be an important mechanism of MM immune evasion,and may be associated with clinical stages,disease progression and prognosis of MM to some extent.