| ObjectiveCD4+CD25+ regulatory T cells (Treg cells) were cellular subsets with constitutive immunosuppressive activity to preventing the emergence of autoimmune disease, dampening the intensity of immune responses to pathogens and mediating peripheral transplantation tolerance.Repeated antigen exposure, tissue damage and abnormal presence of semimature dendritic cells have been suggested to be sufficient to generate T reg cells.T regs have been demonstrated to affetct the function of HIV specific CD4+ and CD8+ T cell responses thus influence the progression of HIV. Some reports suggest that the number of T regs was altered by HIV infection but the results were inconsistent due to the different method to identify T regs and inclusion criteria of patients. The results of studies on the function of Tregs showed that they can suppress the proliferation and cytoxity of CD8+ T cells towards the HIV infected cells but the influence of T regs to NK cells, which is also an important cytotoxic cell in natural immune system were absent. Suppressive activity of Tregs in HIV infection was not dependent of the secretion of cytokines. It seems cell-cell contact mediated uppression, which may need the expression of CTLA-4 on Tregs, is important in the suppressive activity of T regs in HIV infection. In HIV infected Chinese, there has no study on the numbers, effect and mechanism of Tregs in disease progression In the present paper, we studied the nmber and function of Foxp3+CD25+CD4+ Treg cells and their relation to disease progression of HIV-1 infected patients in China.. Material and MethodsStudy subjects and specimensA total of 73 treatment naive HIV-infected patients from Liaoning, Jilin and Henan provinces of China were enrolled (35 males, 38 females; median age 40 years, range 30-68 years). HIV-infected subjects were classified into 3 stages. The first stage was long term nonprogressors (LTNP) and included subjects with persistent CD4+ T-cell counts more than 500 cell/μl and no clinical sign of disease for at least 10 years. The second stage was chronical HIV-infected subjects and included patients who had CD4+ T-cell counts between 200-500 cell/μl, and no AIDS-defining condition. The third stage was AIDS and consisted of patients with an AIDS-defining condition according to the World Health Organization classification, including CD4+ T-cells less than 200 cells/μl, presence or previous opportunistic infections or HIV-related neoplasms. There were 24 LTNPs, 30 HIV and 19 AIDS in the HIV infected patients. People from the First Affiliated Hospital Physical Examination Center were used as healthy control. They were randomly enrolled and then matched for age and gender with the subjects' population. 16 seronegative subjects that never exposed to HIV-1 were selected as normal controls and there were 7 males and 9 females. Blood was drawn by venipuncture from each subject in EDTA tubes (Becton Dickinson) for FACS analysis and the detection of viral load. Controls were 14 healthy HIV-1 seronegative blood donors.Determination of T-cell and viral loadT cell absolute counts were defined by using the TruCOUNT tubes and MultiSET software with a FACSCalibur cytometer (Becton Dickinson). HIV-1 RNA level was determined from plasma using Roche Amplicor 1.5 assay (Roche). All undetectable values (below 400 copies) were assigned a value of 399. Phenotyping of CD4+CD25 regulatory T cellsPBMCs were obtained from HIV-1 infected individuals and healthy controls by Ficoll-Hypaque density gradient centrifugation. Kits from eBioscience (72-5776) were used to identify FoxP3+ cells. Briefly, 100μL of prepared cells (1x106) were first stained with anti-CD3-PerCP, anti-CD25-APC, anti-CD4-PE-Cy7(BD Biosciences) for 30 min at 4℃. The cells were washed in cold PBS and 1 ml of freshly prepared eBioscience Fix/Perm Buffer was added to each sample and incubated at 4℃for 40 minutes in the dark. After the cells were washed again, 2% (2μL) normal rat serum was added and at 4℃for 15 minutes. Then anti-human Foxp3-PE (PCH101) antibody or IgG2a-PE was added and incubate at 4℃for 30 minutes in the dark. The cells were washed twice with PBS and fixed in 1% polyformaldehyde. Cells were analyzed on a FACSAria (Becton Dickinson) with FACSDiva software. T lymphocytes were identified by gating on CD3+T cells and side scatter, then CD4+T cells were gated and the percentage of CD25 and Foxp3 expression were determined. Expression of activation and apoptosis markersWhole blood samples were incubated with the following monoclonal antibody combinations: anti-CD8-FITC/anti-CD95-PE/anti-CD3-PerCP/anti-CD4-APC-Cy7, anti-HLA-FITC/anti-CD38-PE/anti-CD8-APC/anti-CD4-APC-Cy7 for 30 minutes at room temperature. After lysis of red blood cells by FACS lysis buffer (Becton Dickinson), cells were washed twice, fixed with 1% paraformaldehyde, and analyzed with the FACSAria. Lymphocytes were identified by gating on forward scatter and side scatter, then CD4+ or CD8+ T cells were gated and the percentage of HLA-DR, CD38 expression of CD4+,CD8+ T cells were determined. To measure the CD95 expression,, CD3+T cells were gated and the percentage of CD95 expression on CD4+,CD8+ T cells were calculated.Absolute count of CD8+T, NK cellsPipette 20μl of TriTEST CD4FITC/CD8PE/CD3PerCP or CD3FITC/ CD56CD16PE/CD45PerCP reagent and add 50μl anticoagulated whole blood into the bottom of the TruCOUNT Tube using reverse pipetting and incubate for 15 minutes in dark at room temperature(20-25℃). Add 450μl 1X FACS Lysing Solution to the tube, incubate for 15minutes in the dark at room temperature and analyze the samples on the flow cytometer using FACS MULTISET software to acquire T lymphocyte counts, NK cell counts and corresponding ratios.Expression of Perform,Granzyme-B in CD8+T and NK cells PBMCs were obtained from HIV-1 infected individuals and healthy controls by Ficoll-Hypaque density gradient centrifugation. 100μL of prepared cells (1×106) were first stained with anti-CD8-PerCP, anti-CD56-APC, anti-CD3-APC-Cy7(BD Biosciences) for 30 min at 4℃. The cells were washed in cold PBS and 1 ml of freshly prepared eBioscience Fix/Perm Buffer was added to each sample and incubated at 4℃for 40 minutes in the dark. After the cells were washed again, 2% (2μL) normal rat serum was added and at 4℃for 15 minutes. Anti-Granzyme-B-FITC>anti-perforin-PE and isotypes was added and incubate at 4℃for 30 minutes in the dark. The cells were washed twice with PBS and fixed in 1% polyformaldehyde. Cells were analyzed onG a FACSAria (Becton Dickinson) with FACSDiva software. T lymphocytes were identified by gating on CD3+T cells and side scatter, then CD3-CD56+,CD8+T cells were gated and the percentage of Perforin,Granzyme-B expression were determined. Expression of CTLA-4 in CD4+CD25+Foxp3+ regulatory T cellsPBMCs were obtained from HIV-1 infected individuals and healthy controls by Ficoll-Hypaque density gradient centrifugation. 100μL of prepared cells (1×106) were first stained with anti-CD3-PerCP,anti-CD25-APC,anti-CD4-PE-Cy7 (BD Biosciences) for 30 min at 4℃. The cells were washed in cold PBS and 1 ml of freshly prepared eBioscience Fix/Perm Buffer was added to each sample and incubated at 4℃for 40 minutes in the dark. After the cells were washed again, 2% (2μL) normal rat serum was added and at 4℃for 15 minutes. anti-Foxp3-FITC,Anti-CTLA-4-PE and isotypes was added and incubate at 4℃for 30 minutes in the dark. The cells were washed twice with PBS and fixed in 1% polyformaldehyde. Cells were analyzed onG a FACSAria (Becton Dickinson) with FACSDiva software. Regualtory T cells were gated by FSC, SSC, CD3-PerCP, CD4-PE-Cy7, CD25-APC and Foxp3-FITC and CTLA-4 expression within this cell population were determined.ResultsFrequency of CD4+CD25+Foxp3+Treg cells in peripheral blood of patients with HIV infection By using the MAb of Foxp3, we found the percentage of Foxp3+ cells in the CD3+CD4+CD25+ T cells continuously increased in LTNP, NC, HIV and AIDS group. The frequency of CD4+CD25+Foxp3+ regulatory T cells in HIV group siginificantly lower than that in AIDS group(P<0.05). No significant differences in the frequency of CD4+CD25+Foxp3+Treg cells were observed between normal controls and HIV , AIDS group. The frequency of CD4+CD25+Foxp3+ regulatory T cells in LTNP group siginificantly lower than that in HIV and AIDS group(P<0.05). Although the absolute count of CD4+T cells has no significantly difference between the LTNP group and normal controls, we observed a significantly lower frequency of CD4+CD25+Foxp3+Treg in LTNP groups than in normal controls. Relationship between frequency of CD4+CD25+Foxp3+Treg cells and disease progressionWe then examined the relationship between CD4+CD25+Foxp3+Treg cell frequency and CD4+T cells and viral load. The frequency ofCD4+CD25+Foxp3+Treg cells frequency was closely related to viral load (r=0.453, P<0.001) and inversely related to CD4+T cells (r=-0.571, P<0.001) in HIV infected patients. The relationship of the frequency of CD4+CD25+Foxp3+Treg cells and CD4+T cells was not found in normal controls.Relationship between frequency of CD4+CD25+Foxp3+Treg cells and activation and apoptosis of T lymphocytesTo test whether the frequency of CD4+CD25+Foxp3+Treg cells correlated to the activation and apoptosis of T lymphocytes, we also analyzed the HLA-DR, CD38 and CD95 expression on CD4+, CD8+T cells. The frequency of CD4+CD25+Foxp3+Treg cells in HIV infected patients closely correlated to the apoptosis of T cells (CD4+CD95+T cells: r=0.352, P=0.002, CD8+CD95+T cells: r=0.359, P=0.002). Therelationship between the frequency of CD4+CD25+Foxp3+Treg cells and immune activation is not seen in both HIV infected patients and normal controls. Absolute count of CD8+T cells and NK cells of HIV infected patientsWe found the absolute count of NK cells continuously decreased in LTNP, HFV and AIDS group. The number of NK cells in LTNP group significantly higher than that in HIV and AIDS group(P<0.05). No significant differences in the number of NK cells were observed between normal controls and LTNP group. The number of CD8+T cells continuously decreased in LTNP, HIV and AIDS group and the level of CD8+T cells in LTNP group siginificantly higher than that in AIDS group(P<0.05). No significant differences in the number of CD8+T cells were observed between HIV and AIDS group.Frequency of Perform and Granzyme-B expression NK or CD8+T cells in peripheral blood of patients with HIV infectionBy the detection of Perform and Granzyme-B expression in the cytotoxic lymphocytes, we found the expression of Perform and Granzyme-B within the NK cells continuously decreased in LTNP, NC, HIV and AIDS group. The level of the expression of Perform and Granzyme-B within the NK cells siginificantly higher than that in HIV and AIDS group(P<0.05). No significant differences were observed between HIV and AIDS group. The expression of Granzyme-B within the CD8+T cells continuously decreased in NC, LTNP, HIV and AIDS group and is significantly higher in HIV infected patients than in normal controls. Relationship between frequency of CD4+CD25+Foxp3+Treg cells and Perform and Granzyme-B expression within cytotoxic lymphoctyesThe frequency of CD4+CD25+Foxp3+Treg cells in HIV infected patients closely correlated to the apoptosis of T cells (CD4+CD95+T cells: r=0.352, P=0.002, CD8+CD95+T cells: r=0.359, P=0.002). The relationship between the frequency of CD4+CD25+Foxp3+Treg cells and immune activation is not seen in both HIV infected patients and normal controls.Expression of CTLA-4 in CD4+CD25+Foxp3+ regulatory T cells in peripheral blood of patients with HIV infectionBy the detection of CTLA-4 expression in the CD4+CD25+Foxp3+ regulatory T cells, we found the expression of CTLA-4 continuously increased in NC, LTNP, HIV and AIDS group. The level of the expression of CTLA-4 within regulatory T cells is siginificantly lower in LTNP group than that in HIV and AIDS group (P<0.05). No significant differences were observed between normal control and LTNP group. The level of the expression of CTLA-4 within regulatory T cells is siginificantly lower in normal controls than that in HIV and AIDS group (P<0.05) while there is no significant differences between HIV and AIDS group.Relationship between CTLA-4 expression in CD4+CD25+Foxp3+Treg cellsand and disease progressionThe frequency of CTLA-4 expression in CD4+CD25+Foxp3+Treg cells inversely related to CD4+T cells (r=-0.419, P<0.05) but not correlated with viral load in HIV infected patients.Relationship between CTLA-4 expression in CD4+CD25+Foxp3+Treg cells and Perform and Granzyme-B expression within T lymphoctyesWe did not observe the correlation between the frequency of CTLA - 4+CD4+ CD25+Foxp3+Treg cells and Perform and Granzyme-B expression within cytotoxic lymphoctyes in total HIV infected patients. When study the correlation in each group, we found the expression of CTLA-4 in CD4+CD25+Foxp3+Treg cells inversely correlated with CD4+Granzyme-B+, CD8+Granzyme-B+, CD4+Perforin+ cells in LTNP group(P<0.05) while this significant was not found in HIV and AIDS patients.Conclusion1.We found the frequency of CD3+CD4+CD25+ Foxp3+ T cells continuously increased in LTNP, NC, HIV and AIDS group and correlated with disease progression of HIV infected Chinese.2.The frequency of CD4+CD25+Foxp3+ regulatory T cells in LTNP group siginificantly lower than that in HIV, AIDS group and normal controls, which indicates the lower level of regulatory T cells may be one of the protective factors to delay the disease progression of HIV infection.3.The frequency of CD4+CD25+Foxp3+ regulatory T cells correlated with the change of the number and Perforin, Grazyme-B expression of cytotoxic lymphocytes, which indicates the high level of regulatory T cells may influence the function of cytotoxic lymphocyes.4.The expression of CTLA-4 within regulatory T cells continuously increased in NC, LTNP, HIV and AIDS group and correlated with disease progression of HIV infection. 5.The level of the expression of CTLA-4 within regulatory T cells is siginificantly lower in LTNP group than that in HIV and AIDS group and was inversely correlated with the level of Granzyme-B+, Perforin+ T cells. The results indicates the lower level of CTLA-4 within regulatory T cells may delay the disease progression of HIV infection. |
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