【作者】 张子宁；

【导师】 尚红；

【作者基本信息】 中国医科大学 ， 皮肤病与性病学， 2007， 博士

【摘要】 目的在免疫系统中，CD4+CD25+调节性T细胞可通过细胞直接接触及分泌细胞因子等机制调节天然及获得性免疫系统的功能，控制针对自身抗原反应的T细胞过度生长，对于维持免疫耐受及稳态具有重要作用。HIV感染中持续抗原刺激、组织损伤及不成熟状态DC的产生等可诱导CD4+CD25+调节性T细胞产生，从而影响机体针对HIV的免疫应答。研究表明，HIV感染可导致CD4+CD25+调节性T细胞数量的变化，但由于研究人群及手段的不同，结论并不一致。调节性T细胞可抑制CD8+T细胞针对HIV蛋白的特异性免疫应答，其对于HIV特异性免疫抑制不依赖于IL-10、TGF-β等细胞因子的分泌，可能通过细胞直接接触实现的，其机制可能包括共刺激分子CTLA-4的作用。目前尚无中国HIV感染者CD4+CD25+Foxp3+调节性T细胞作用及机制研究，本研究首次对中国不同疾病进展阶段HIV感染者“CD4+CD25+Foxp3+调节性T细胞”数量、与细胞毒性淋巴细胞的相关性以及调节性T细胞内CTLA-4的作表达水平进行了研究，初步探讨了调节性T细胞与HIV感染疾病进展的关系。方法1．研究对象73例未经抗病毒治疗的HIV／AIDS患者来自中国辽宁、吉林和河南，其中男性35例，女性38例，年龄分布为30-68岁(40.4±11.0)。根据HIV感染时间和CD4+T细胞数量将上述患者分为HIV感染长期不进展组(感染时间＞10年，CD4+T细胞＞500个／μl)、典型进展HIV组(感染时间＞5年，CD4+T细胞介于200-500个／μl之间，无艾滋病指征性疾病)和典型进展AIDS组(感染时间＞5年，CD4+T细胞＜200个／μl或出现艾滋病指征性疾病)。73例HIV／AIDS患者中LTNP组24例、HIV组30例、AIDS组19例。16例健康对照为随机抽取的HIV抗体阴性、未暴露于HIV的健康人，其中男性7名，女性9名。2．CD4~+T细胞绝对值及病毒载量检测CD4绝对值依据1997年美国CDC关于HIV感染者CD4~+T细胞检测指导方法，应用BD公司TruCOUNT管、FACSCalibur流式细胞仪MULTISET软件检测。病毒载量采用RT-PCR方法，用美国Roche公司的COBAS AMPLICOR自动载量仪测定病毒载量。3．CD4~+CD25~+Foxp3~+调节性T细胞检测密度梯度离心法分离HIV-1感染者及健康人PBMC。Foxp3~+细胞的检测应用PE anti-human Foxp3染色试剂盒，方法如下：向流式管1x10~+PBMC中加入anti-CD3-PerCP、anti-CD25-APC、anti-CD4-PE-Cy7，4℃避光染色30分钟。用PBS洗涤后加入1ml破膜／固定剂，4℃避光染色40分钟。洗涤后加入2％(2μL)鼠血清，4℃避光15分钟，加入anti-Foxp3-PE及IgG2a-PE同型对照，4℃避光30分钟，洗涤后加入含1％多聚甲醛的PBS重悬，FACSAria流式细胞仪Diva软件进行分析。以FSC、SSC、CD3-PerCP、CD4-PE-Cy7定义CD4~+T细胞，计算这一细胞群内CD25~+Foxp3~+细胞的比率。4．淋巴细胞活化及凋亡水平检测取HIV-1感染者及健康人全血100μl，应用以下荧光标记的单抗进行染色：CD8-FITC／CD95-PE／CD3-PerCP／CD4-APC-Cy7、HLA-FITC／CD38-PE／CD8-APC／CD4-APC-Cy7，室温避光染色30min。加入溶血素3毫升，室温避光10分钟，1200r／min离心5min，弃上清。加入3ml PBS洗涤两次，加入1％多聚甲醛的PBS重悬，FACSAria流式细胞仪Diva软件进行分析。以FSC、SSC定义淋巴细胞，分析CD4、CDS~+T细胞表面HLA-DR、CD38及CD95表达水平。5．CD8~+T细胞绝对值检测将20μlC D4FITC／CD8PE／CD3PerCP试剂加入绝对计数管中，经反向加样法加入50μl抗凝血，室温避光15min，加入免洗溶血素450μl，室温避光15min，FACS MULTISET软件检测并进行自动分析，计算CD8+T淋巴细胞绝对值。6．NK细胞绝对值检测将20μl CD45FITC／CD16+56+PE／CD3PerCP试剂加入绝对计数管中，经反向加样法加入50μl抗凝血，室温避光15min，加入免洗溶血素450μl，室温避光15min，FACS MULTISET软件检测并进行自动分析，计算NK细胞绝对值。7．NK细胞、CD8+T淋巴细胞Perforin、Granzyme-B表达水平检测密度梯度离心法分离HIV-1感染者PBMC。向流式管1x10~6 PBMC中加入anti-CD8-PerCP、anti-CD56APC、anti-CD3-APC-Cy7，4℃避光染色30分钟。用PBS洗涤后加入1ml破膜／固定剂，4℃避光染色40分钟。洗涤后加入2％(2μL)鼠血清，4℃避光15分钟，加入anti-Granzyme-B-FITC、anti-perforin-PE及相应同型对照，4℃避光30分钟，洗涤后加入含1％多聚甲醛的PBS重悬，FACSAria流式细胞仪Diva软件进行分析。以FSC、SSC定义淋巴细胞，CD3-CD56+、CD3+CD8+T淋巴细胞内Perforin、Granzyme-B表达水平分别乘以NK、CD8+T淋巴细胞绝对值获得Perforin、Granzyme-B表达水平。8．CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达水平检测密度梯度离心法分离HIV-1感染者PBMC。向流式管1x10~6 PBMC中加入anti-CD3-PerCP、anti-CD25-APC、anti-CD4-PE-Cy7，4℃避光染色30分钟。用PBS洗涤后加入1ml破膜／固定剂，4℃避光染色40分钟。洗涤后加入2％(2μL)鼠血清，4℃避光15分钟，加入anti-Foxp3-FITC、anti-CTLA-4-PE及相应同型对照，4℃避光30分钟，洗涤后加入含1％多聚甲醛的PBS重悬，FACSAria流式细胞仪Diva软件进行分析。以FSC、SSC、CD3-PerCP、CD4-PE-Cy7、CD25-APC、Foxp3-FITC定义调节性T细胞，计算这一细胞群内CTLA-4的表达百分率。结果1．HIV感染者CD4~+CD25~+Foxp3~+调节性T细胞水平通过单细胞水平检测外周血PBMC中CD3~+CD4~+CD25~+Foxp3~+T细胞水平发现，LTNP组、健康对照组、HIV组、AIDS组CD4~+CD25~+Foxp3~+调节性T细胞数量依次升高，HIV组明显低于AIDS组(P＜0.05)，与健康对照组无显著性差异。LTNP组CD3~+CD4~+CD25~+Foxp3~+T细胞水平明显低于HIV组及AIDS组(P＜0.01)，值得注意的是，尽管LTNP组与健康对照组相比CD4+T细胞数量并无差异，但CD3~+CD4~+CD25~+Foxp3~+T细胞水平明显低于健康对照组(P＜0.05)。2．CD4~+CD25~+Foxp3~+调节性T细胞与CD4~+T细胞、病毒载量的相关性HIV感染者CD4~+CD25~+Foxp3~+调节性T细胞与CD4~+T细胞显著负相关(r=-0.571，P＜0.001)，与病毒载量明显正相关(r=0.453，P＜0.001)。健康人CD4~+CD25~+Foxp3~+调节性T细胞与CD4~+T细胞无显著相关性。3．CD4~+CD25~+Foxp3~+调节性T细胞与淋巴细胞活化及凋亡的相关性HIV-1感染者CD4~+CD25~+Foxp3~+调节性T细胞与CD4~+、CD8~+T细胞凋亡水平呈明显正相关(CD4~+T细胞凋亡：r=0.352，P=0.002；CD8~+T细胞凋亡：r=0.359，P=0.002)，与淋巴细胞活化水平无显著相关性。4．HIV感染者NK细胞、CD8+T淋巴细胞水平通过对HIV感染者细胞毒性淋巴细胞数量检测显示，随疾病进展，LTNP组、HIV组、AIDS组NK细胞水平依次下降，其中LTNP组明显高于HIV组及AIDS组(P＜0.05)，LTNP组高于健康对照组，但无显著差异。LTNP组、HIV组、AIDS组CD8+T细胞依次下降，LTNP组明显高于AIDS组(P＜0.05)，HIV组与AIDS组间无显著差异。5．HIV感染者NK细胞、CD8+T淋巴细胞内Perforin、Granzyme-B表达水平对HIV感染者细胞毒性淋巴细胞Perforin、Granzyme-B表达的研究结果显示，LTNP组、NC组、HIV组、AIDS组NK细胞Perforin、Granzyme-B表达依次下降，其中LTNP组及健康对照组明显高于HIV组、AIDS组(P＜0.05)，HIV组与AIDS组间无显著差异。CD8+T细胞内Granzyme-B表达NC组、LTNP组、HIV组、AIDS组依次下降，其中LTNP组、HIV组、AIDS组明显高于健康对照组，各组间的差异无显著意义。CD8+T细胞内Perforin表达HIV组显著高于NC组，其余各组间无显著差异。6．HIV感染者CD4+CD25+Foxp3+调节性T细胞与NK细胞、CD8+T淋巴细胞及Perforin、Granzyme-B表达水平的相关性HIV感染者CD4+CD25+Foxp3+调节性T细胞与NK细胞、CD8+T淋巴细胞数量呈明显负相关(NK细胞：r=-0.502，P＜0.01；CD8+T细胞：r=-0.437，P＜0.001)，与NK细胞内Perforin、Granzyme-B表达水平呈明显负相关(Perforin：r=-0.515，P＜0.01；Granzyme-B：r=-0.507，P＜0.01)，与CD8+T细胞内Granzyme-B表达水平呈明显负相关(Granzyme-B：r=-0.298，P＜0.01)，与CD8+T细胞内Perforin表达水平无明显相关性(Perforin：r=-0.240，P＞0.05)。7．HIV感染者CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达水平通过单细胞水平检测外周血PBMC中CD4~+CD25~+Foxp3~+T细胞内CTLA-4表达水平发现，健康对照组、LTNP组、HIV组、AIDS组CTLA-4表达水平依次升高，其中LTNP表达水平(28.67±4.61)明显低于HIV组(36.71±9.99)及AIDS组(39.37±11.82，P＜0.05)，与健康对照组无显著差异(27.48±13.75)。健康对照组表达水平明显低于HIV组及AIDS组(P＜0.05)，HIV组及AIDS组间无显著差异，8．CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达水平与CD4~+T细胞、病毒载量的相关性HIV感染者CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达水平与CD4+T细胞显著负相关(r=-0.419，P＜0.05)，与病毒载量无显著相关性。9．CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达与淋巴细胞Perforin、Granzyme-B表达水平的相关性LTNP组CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达水平与CD4+、CD8+T淋巴细胞Granzyme-B表达、CD4+T淋巴细胞Perforin表达呈明显负相关(P＜0.05)，HIV组、AIDS组CD4~+D25~+Foxp3~+调节性T细胞内CTLA-4表达水平与CD4+、CD8+T淋巴细胞Perforin、Granzyme-B表达无显著相关性。结论1．LTNP组、健康对照组、HIV组、AIDS组CD4~+CD25~+Foxp3~+调节性T细胞数量依次升高，与HIV感染疾病进展明显相关。2．LTNP组CD4~+CD25~+Foxp3~+调节性T细胞表达水平低于HIV组及AIDS组，而且明显低于健康对照组，提示低水平的调节性T细胞数量对感染后疾病不进展具有保护作用。3．中国HIV感染者调节性T细胞与细胞毒性淋巴细胞数量功能的变化密切相关，提示高水平的调节性T细胞可导致细胞毒性淋巴细胞数量功能的下降，从而影响疾病进程。4．健康对照组、LTNP组、HIV组、AIDS组CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达水平依次升高，且与CD4~+T细胞数量显著负相关，提示CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达水平与疾病进展密切相关。5．HIV感染LTNP组CD4~+CD25~+Foxp3~+调节性T细胞内CTLA-4表达水平明显低于HIV组及AIDS组，与CD4+、CDS+T淋巴细胞Granzyme-B表达、CD4+T淋巴细胞Perforin表达明显负相关，提示调节性T细胞内低水平CTLA-4表达对感染后疾病不进展具有保护作用。更多还原

【Abstract】 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⁺T_reg 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⁺T_reg cells and activation and apoptosis of T lymphocytesTo test whether the frequency of CD4⁺CD25⁺Foxp3⁺T_reg 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⁺T_reg 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⁺T_reg 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⁺T_reg cells and Perform and Granzyme-B expression within cytotoxic lymphoctyesThe frequency of CD4⁺CD25⁺Foxp3⁺T_reg 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⁺T_reg 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⁺T_reg 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⁺T_reg cells and Perform and Granzyme-B expression within T lymphoctyesWe did not observe the correlation between the frequency of CTLA - 4+CD4⁺ CD25⁺Foxp3⁺T_reg 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⁺T_reg 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.更多还原