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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.
ABSTRACT
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.
ABSTRACT
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.
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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.