HIV-1感染疾病长期不进展者CD8+T淋巴细胞非细胞毒性免疫应答作用的研究
摘要
目的
CD8+T淋巴细胞在机体抗HIV-1感染的免疫反应中具有细胞毒性和非细胞毒性的免疫应答(CD8+T cell non-cytotoxic anti-virus responses，CNAR)。细胞毒性T淋巴细胞(Cytotoxic T lymphocyte，CTL)是细胞免疫反应中杀灭HIV的主要效应细胞，但是单纯靠CTL介导的细胞免疫作用不足以保护机体免受HIV感染。CNAR功能通过分泌抗病毒因子抑制HIV感染细胞及HIV复制，在抗HIV感染中具有重要作用。近年来，国外关于CNAR功能的研究主要以无症状感染者为研究对象，对疾病长期不进展者(Long term non-progressors，LTNP)和艾滋病期的患者研究较少。LTNP是HIV-1感染超过10年，未经任何抗病毒治疗，CD4+T淋巴细胞数大于或等于500x10~6个／l，未出现艾滋病指征性症状的患者，疾病呈现长期不进展状态。CNAR功能可能在维持LTNP较好的免疫功能中发挥了重要作用，但其机制尚未明确。IL-15是一种多功能的细胞因子，能够促进体内NK细胞和记忆性T淋巴细胞分化和增殖，在机体抗病毒感染中发挥重要作用。国外研究显示IL-15能够增强无症状感染者CNAR功能，但CNAR功能在LTNP中的变化以及是否与IL-15相关国内外均未见报道。本研究以中国HIV感染LTNP为对象，研究其外周血中CD8+T淋巴细胞CNAR功能的变化及IL-15对CNAR功能的影响，并与HIV组、AIDS组及健康对照组进行比较，探讨其与疾病进展的关系。
方法
1、研究对象：
由中国医科大学艾滋病研究所、辽宁省疾病预防与控制中心以及吉林省疾病预防与控制中心、河南省疾病预防与控制中心艾滋病确认实验室经Western blot试验确认为HIV阳性标本52例，所有标本均在未接受抗病毒治疗前采集。根据HIV感染者CD4+T细胞数量和感染时间进行分组。LTNP组：CD4+T淋巴细胞≥500个／μl，HIV感染10年以上，未经抗病毒治疗，无艾滋病指征性症状；HIV组：CD4+T淋巴细胞介于200-500个／μl之间，无艾滋病指征性症状；AIDS组：CD4+T淋巴细胞＜200个／μl或出现艾滋病指征性症状。其中LTNP组17例，HIV组28例，AIDS组7例，健康对照组8例。每例采集EDTA抗凝全血9ml。
2、T淋巴细胞绝对计数：
将20μlCD4／CD8／CD3 TriTEST试剂加入TruCOUNT管中，加入50μlEDTA抗凝血，室温避光15min，加入免洗溶血450μl，室温避光15min，用FACS MULTISET软件检测并进行自动分析，计算CD4+、CD8+、CD3+T淋巴细胞绝对值及相应比值等。
3、病毒株TCID_(50)检测：
HIV病毒株为中国CDC参比实验室惠赠的实验室毒株SF33。提取健康人外周血单个核细胞(Peripheral blood monouclear cells，PBMC)，PHA刺激3天，用RPMI-1640(含100U／ml IL-2)重悬，调整浓度为4×10~6个／ml，放入37℃、5％CO_2孵箱中待用。在96孔板中部选12孔，每孔加入180μl RPMI-1640，PBMC 50μl。每孔加入200μl病毒，在板上做4倍系列稀释。3天后换液，第7天测p24，计算TCID_(50)。
4、CD4+T／CD8+T淋巴细胞的分选：
用密度梯度离心法提取8例健康对照和52例HIV感染者的PBMC。采用德国Miltenyi公司的MACS免疫磁珠分选装置，严格按照说明书进行操作。每1×10~7个细胞加20μl抗CD4或抗CD8免疫磁珠，10℃结合25min。300g离心μlin，500μ1缓冲液重悬细胞，经LS柱子进行阳性分选。取少量分选后细胞加入6μlCD4-FITC、6μl CD8-PE及6μl CD3-PerCP，用流式细胞仪Cellquest软件检测分选细胞纯度。
5、T淋巴细胞的体外培养：
用37℃水浴复苏病毒。准备15ml离心管，每管加入5×10~5个CD4+T细胞和600TCID_(50)的病毒，并用1ml吸管轻轻吹打20次，混匀后37℃、5％CO_2孵箱共培养3h。300g10min离心两次，洗去游离的病毒，向每管加入0.5ml RPMI-1640 (IL-2100U／ml)，重悬使细胞浓度为1×10~6个／ml。将预刺激72h的CD8+T细胞重悬，调整浓度到1×10~6个／ml。将CD8+T细胞和CD4+T细胞以2：1、1：1、0.5：1的比例加入到48孔板中，固定CD4+T细胞的数量为0.4×10~6个／孔，每个浓度设2个复孔，同时设定2个阴性对照。调整每孔的液体总量为1.2ml。37℃、5％CO_2孵育，每3天换液收集上清600μl，同时补充600μl RPMI-1640，培养至15天。在实验孔中加入IL-15，浓度为300ng／ml，对照孔中只加入细胞，培养15天。
6、p24检测：
采用Biomerieux公司的p24检测试剂盒，严格按照操作说明书进行。结果用pg／ml表示，最低检测范围是5pg／ml。
7、CNAR功能分析：
将收集5次的上清p24值用Excel绘制折线图，应用Origin7.0软件计算曲线下面积，根据下列公式计算出CNAR功能。
CNAR功能％=(对照组面积-实验组面积)/对照组面积×100％
8、统计学分析：
用SPSS11.5统计软件包进行统计学分析，数据经转换后为正态分布。用方差分析(one-way ANOVA)进行不同组间的均值比较。用直线相关来比较CNAR功能与CD4+T淋巴细胞绝对值及HIV-1 RNA病毒载量的相关性。P＜0.05认为有统计学差异。
结果
1、CD4+T／CD8+T淋巴细胞的纯化
用免疫磁珠法纯化CD4+／CD8+T淋巴细胞，细胞纯度大于95％。
2、HIV感染LTNP的CNAR功能
LTNP组的CNAR功能高于HIV组、健康对照组和AIDS组(P＜0.01)。
3、CNAR功能与CD4+T淋巴细胞绝对计数和病毒载量相关性52例HIV感染者的CNAR功能与CD4+T细胞绝对计数呈显著正相关(r=0.2，P=0.022)，与病毒载量不呈显著负相关(r=-0.144，P=0.308)。
4、IL-15对疾病不同进展期CNAR功能的影响
经独立样本T检验分析，加入IL-15组和未加入IL-15组CNAR功能存在著性差异(P＜0.05)，加入IL-15组CNAR功能高于未加入IL-15组。且增高值AIDS＞HIV＞LTNP。
结论
1．我国LTNP的CNAR功能高于HIV组、健康对照组和AIDS组(P＜0.01)。提示CNAR功能可能是我国HIV感染疾病长期不进展的保护性因素。
2．加入IL-15组CNAR功能高于未加入IL-15组，且增高值AIDS组最显著。提示IL-15可实验性应用于HIV-1的免疫治疗。
3．52例HIV感染者CNAR功能与CD4+T细胞绝对计数呈显著正相关，提示CNAR功能与HIV感染者疾病进展有关，是监测HIV感染者免疫状况、预示疾病进程的一个新指标。
Objective
The CD8+T cell immune response for human immunodeficiency virus (HIV) is divided into a cytotoxic and non-cytotoxic mechanism. CTL is the main effector cells in killing HIV. But the cell immunity mediated by CTL is not enough to protect the people. CNAR hold-back HIV infect cells and HIV copying by secreting a cell factor. The CNAR play a important role in anti-HIV infection. Recently, the studies on the CNAR from alien are always focus on the symptomless infectors, and very few on the long term non-progressors (LTNP) and the AIDS. The immune state of the LTNP are very near to the normal level, the CNAR may play an important role, but it's mechanisms are not very clear. IL-15 is a cell factor secreting by dendritic cells. Professor Levy reported that the DC could strong the CNAR of the symptomless infectors, but the effect of the IL-15 on the CNAR is not reported, This study explore the change of the CNAR and the IL-15 effect on the CNAR.We set four groups: LTNP, HIV, AIDS and Health control, and analysis the relations between the CNAR and the progression of disease.
Methods
1.Study population
All patients were from Liaoning, Jilin and Henan province in China. HIV serology was determined by ELISA (Vironostika, Organon Tednika, The Netherlands) and confirmed by Western blot (Genelab Diagnostic, Singapore) in the HIV Confirmation Laboratory at University Hospital. HIV-infected subjects were classified into 3 clinial stages. The first stage was slowly progressors (LTNPs) and included subjects with persistent CD4+T-cell counts more than 500 cells/μl, no antiretroviral therapy, and no clinical sign of disease for at least 10 years. The second stage was symptomless HIV-infected subjects and included patients Who had CD4+ T-cell counts less than 500 cells/μl, and no AIDS-defining condition without antiretroviral therapy. 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, present or previous opportunistic infections or HIV-related neoplasms. We choose 17 LTNP, 28 HIV, 7 AIDS and 8 Health control. Blood was collected in EDTA tubes (Becton Dickinson) and processed within 12 hours.
2.Detection of the TCID_(50)
Virus was lab-virus strain SF33 (donated by CDC in China). Revival MT4 cells, Cultures in 37℃5％CO_2 for 3 days. Collection the MT4, Washed with PBS twice at 300g for 10 minutes and add 600TCID_(50) SF33, blow 20 times, cultures in 37℃5％CO_2 for 90 min. Washed with PBS twice at 300g for 10 minutes. Cultured with RPMI-1640 (adding IL-2 by 100U/ml) in 37℃5％CO_2. Virus replication was assessed by measuring p24 antigen in the culture supernatants.
3.Seperate the CD4+/CD8+ T lymphocyte cell
PBMC of subjects were isolated by standard Ficoll-hypaque density centrifugation. Remove monocytes. Per 1×10~7 cells were treated with 20μl anti-CD4 immunomagenetic beads in 6℃for 15 minutes. Wash at 300g for 10 minutes. 500μl cell suspend liquor went through LS column to positive selection for CD4+T lymphocytes. Infection of C D4+T c ell with HIV-1: Cultures of C D4+ lymphocytes from ESN and control subjects were inoculated with HIV-1 of 600 TCID50 in 37℃for 4 hours. Washed with PBS twice at 300g for 10 minutes and cultured with RPMI-1640 (adding IL-2 by 100U/ml). Virus replication was assessed at regular interval over a 2-week period by measuring p24 antigen in the culture supernatants.
4.Coculture the CD4+ T and CD8+T lymphocyte
Adjust the CD4+ T and the CD8+ T density to 1×10~6cells/ml, add the cells into the 48 bore plate, CD8:CD4=2:1,1:1,0.5:1, collect the supernatant every 3 days.
5.p24 detection:
p24 antigen was detected by Biomerieux kit according to instruction.
6.Statistical analysis
SPSS 11.5 software package was used for the statistical analysis. Geometric means were determined for log-distributed variables. The significance of the differences in CNAR in patients and controls was evaluated with one-way ANOVA. A P value of 0.05 or less was considered to represent significance.
Results
1.The purity of the CD4+/CD8+ T lymphocyte cell
Purify the CD4+/CD8+ T: The purity of the cells is more than 95％.
2. The CNAR function in the LTNP
Our research finding the function of CNAR in the LTNP is stronger than the HIV, Health control and the AIDS.
3. Add IL-15, Inhibition ratio changes in different disease progression
We analysis the data by single sample T-test, there is a significant difference between the IL-15 group and the no-IL-15 group (P＜0.05). Further more, AIDS＞HIV＞LTNP
4. CD4+ T true count and the inhibition ratio
We found that following the descend of the CD4+ T true count, the function of CNAR significant decreased. A statistically significant positive correlation was found between the 2 parameters (r=0.2, P＜0.05).
Conclusion
1.The function of the CNAR in the LTNP is stronger than the HIV, Health control and the AIDS. It indicated that the CNAR can protect the disease non-progression.
2.There is a significant difference between the IL-15 group and the no-IL-15 group (P＜0.05). Further more, AIDS＞HIV＞LTNP. It indicate that the IL-15 could stronger CNAR, and flowing disease progression, the enhancement property is more significant. The AIDS is the highest.
3.We found that following the descend of the CD4+ T true count, the CNAR significant decreased. It indicate that there is relationship between the CD4+ true count and the function of CNAR.
CD8+T淋巴细胞在机体抗HIV-1感染的免疫反应中具有细胞毒性和非细胞毒性的免疫应答(CD8+T cell non-cytotoxic anti-virus responses，CNAR)。细胞毒性T淋巴细胞(Cytotoxic T lymphocyte，CTL)是细胞免疫反应中杀灭HIV的主要效应细胞，但是单纯靠CTL介导的细胞免疫作用不足以保护机体免受HIV感染。CNAR功能通过分泌抗病毒因子抑制HIV感染细胞及HIV复制，在抗HIV感染中具有重要作用。近年来，国外关于CNAR功能的研究主要以无症状感染者为研究对象，对疾病长期不进展者(Long term non-progressors，LTNP)和艾滋病期的患者研究较少。LTNP是HIV-1感染超过10年，未经任何抗病毒治疗，CD4+T淋巴细胞数大于或等于500x10~6个／l，未出现艾滋病指征性症状的患者，疾病呈现长期不进展状态。CNAR功能可能在维持LTNP较好的免疫功能中发挥了重要作用，但其机制尚未明确。IL-15是一种多功能的细胞因子，能够促进体内NK细胞和记忆性T淋巴细胞分化和增殖，在机体抗病毒感染中发挥重要作用。国外研究显示IL-15能够增强无症状感染者CNAR功能，但CNAR功能在LTNP中的变化以及是否与IL-15相关国内外均未见报道。本研究以中国HIV感染LTNP为对象，研究其外周血中CD8+T淋巴细胞CNAR功能的变化及IL-15对CNAR功能的影响，并与HIV组、AIDS组及健康对照组进行比较，探讨其与疾病进展的关系。
方法
1、研究对象：
由中国医科大学艾滋病研究所、辽宁省疾病预防与控制中心以及吉林省疾病预防与控制中心、河南省疾病预防与控制中心艾滋病确认实验室经Western blot试验确认为HIV阳性标本52例，所有标本均在未接受抗病毒治疗前采集。根据HIV感染者CD4+T细胞数量和感染时间进行分组。LTNP组：CD4+T淋巴细胞≥500个／μl，HIV感染10年以上，未经抗病毒治疗，无艾滋病指征性症状；HIV组：CD4+T淋巴细胞介于200-500个／μl之间，无艾滋病指征性症状；AIDS组：CD4+T淋巴细胞＜200个／μl或出现艾滋病指征性症状。其中LTNP组17例，HIV组28例，AIDS组7例，健康对照组8例。每例采集EDTA抗凝全血9ml。
2、T淋巴细胞绝对计数：
将20μlCD4／CD8／CD3 TriTEST试剂加入TruCOUNT管中，加入50μlEDTA抗凝血，室温避光15min，加入免洗溶血450μl，室温避光15min，用FACS MULTISET软件检测并进行自动分析，计算CD4+、CD8+、CD3+T淋巴细胞绝对值及相应比值等。
3、病毒株TCID_(50)检测：
HIV病毒株为中国CDC参比实验室惠赠的实验室毒株SF33。提取健康人外周血单个核细胞(Peripheral blood monouclear cells，PBMC)，PHA刺激3天，用RPMI-1640(含100U／ml IL-2)重悬，调整浓度为4×10~6个／ml，放入37℃、5％CO_2孵箱中待用。在96孔板中部选12孔，每孔加入180μl RPMI-1640，PBMC 50μl。每孔加入200μl病毒，在板上做4倍系列稀释。3天后换液，第7天测p24，计算TCID_(50)。
4、CD4+T／CD8+T淋巴细胞的分选：
用密度梯度离心法提取8例健康对照和52例HIV感染者的PBMC。采用德国Miltenyi公司的MACS免疫磁珠分选装置，严格按照说明书进行操作。每1×10~7个细胞加20μl抗CD4或抗CD8免疫磁珠，10℃结合25min。300g离心μlin，500μ1缓冲液重悬细胞，经LS柱子进行阳性分选。取少量分选后细胞加入6μlCD4-FITC、6μl CD8-PE及6μl CD3-PerCP，用流式细胞仪Cellquest软件检测分选细胞纯度。
5、T淋巴细胞的体外培养：
用37℃水浴复苏病毒。准备15ml离心管，每管加入5×10~5个CD4+T细胞和600TCID_(50)的病毒，并用1ml吸管轻轻吹打20次，混匀后37℃、5％CO_2孵箱共培养3h。300g10min离心两次，洗去游离的病毒，向每管加入0.5ml RPMI-1640 (IL-2100U／ml)，重悬使细胞浓度为1×10~6个／ml。将预刺激72h的CD8+T细胞重悬，调整浓度到1×10~6个／ml。将CD8+T细胞和CD4+T细胞以2：1、1：1、0.5：1的比例加入到48孔板中，固定CD4+T细胞的数量为0.4×10~6个／孔，每个浓度设2个复孔，同时设定2个阴性对照。调整每孔的液体总量为1.2ml。37℃、5％CO_2孵育，每3天换液收集上清600μl，同时补充600μl RPMI-1640，培养至15天。在实验孔中加入IL-15，浓度为300ng／ml，对照孔中只加入细胞，培养15天。
6、p24检测：
采用Biomerieux公司的p24检测试剂盒，严格按照操作说明书进行。结果用pg／ml表示，最低检测范围是5pg／ml。
7、CNAR功能分析：
将收集5次的上清p24值用Excel绘制折线图，应用Origin7.0软件计算曲线下面积，根据下列公式计算出CNAR功能。
CNAR功能％=(对照组面积-实验组面积)/对照组面积×100％
8、统计学分析：
用SPSS11.5统计软件包进行统计学分析，数据经转换后为正态分布。用方差分析(one-way ANOVA)进行不同组间的均值比较。用直线相关来比较CNAR功能与CD4+T淋巴细胞绝对值及HIV-1 RNA病毒载量的相关性。P＜0.05认为有统计学差异。
结果
1、CD4+T／CD8+T淋巴细胞的纯化
用免疫磁珠法纯化CD4+／CD8+T淋巴细胞，细胞纯度大于95％。
2、HIV感染LTNP的CNAR功能
LTNP组的CNAR功能高于HIV组、健康对照组和AIDS组(P＜0.01)。
3、CNAR功能与CD4+T淋巴细胞绝对计数和病毒载量相关性52例HIV感染者的CNAR功能与CD4+T细胞绝对计数呈显著正相关(r=0.2，P=0.022)，与病毒载量不呈显著负相关(r=-0.144，P=0.308)。
4、IL-15对疾病不同进展期CNAR功能的影响
经独立样本T检验分析，加入IL-15组和未加入IL-15组CNAR功能存在著性差异(P＜0.05)，加入IL-15组CNAR功能高于未加入IL-15组。且增高值AIDS＞HIV＞LTNP。
结论
1．我国LTNP的CNAR功能高于HIV组、健康对照组和AIDS组(P＜0.01)。提示CNAR功能可能是我国HIV感染疾病长期不进展的保护性因素。
2．加入IL-15组CNAR功能高于未加入IL-15组，且增高值AIDS组最显著。提示IL-15可实验性应用于HIV-1的免疫治疗。
3．52例HIV感染者CNAR功能与CD4+T细胞绝对计数呈显著正相关，提示CNAR功能与HIV感染者疾病进展有关，是监测HIV感染者免疫状况、预示疾病进程的一个新指标。
Objective
The CD8+T cell immune response for human immunodeficiency virus (HIV) is divided into a cytotoxic and non-cytotoxic mechanism. CTL is the main effector cells in killing HIV. But the cell immunity mediated by CTL is not enough to protect the people. CNAR hold-back HIV infect cells and HIV copying by secreting a cell factor. The CNAR play a important role in anti-HIV infection. Recently, the studies on the CNAR from alien are always focus on the symptomless infectors, and very few on the long term non-progressors (LTNP) and the AIDS. The immune state of the LTNP are very near to the normal level, the CNAR may play an important role, but it's mechanisms are not very clear. IL-15 is a cell factor secreting by dendritic cells. Professor Levy reported that the DC could strong the CNAR of the symptomless infectors, but the effect of the IL-15 on the CNAR is not reported, This study explore the change of the CNAR and the IL-15 effect on the CNAR.We set four groups: LTNP, HIV, AIDS and Health control, and analysis the relations between the CNAR and the progression of disease.
Methods
1.Study population
All patients were from Liaoning, Jilin and Henan province in China. HIV serology was determined by ELISA (Vironostika, Organon Tednika, The Netherlands) and confirmed by Western blot (Genelab Diagnostic, Singapore) in the HIV Confirmation Laboratory at University Hospital. HIV-infected subjects were classified into 3 clinial stages. The first stage was slowly progressors (LTNPs) and included subjects with persistent CD4+T-cell counts more than 500 cells/μl, no antiretroviral therapy, and no clinical sign of disease for at least 10 years. The second stage was symptomless HIV-infected subjects and included patients Who had CD4+ T-cell counts less than 500 cells/μl, and no AIDS-defining condition without antiretroviral therapy. 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, present or previous opportunistic infections or HIV-related neoplasms. We choose 17 LTNP, 28 HIV, 7 AIDS and 8 Health control. Blood was collected in EDTA tubes (Becton Dickinson) and processed within 12 hours.
2.Detection of the TCID_(50)
Virus was lab-virus strain SF33 (donated by CDC in China). Revival MT4 cells, Cultures in 37℃5％CO_2 for 3 days. Collection the MT4, Washed with PBS twice at 300g for 10 minutes and add 600TCID_(50) SF33, blow 20 times, cultures in 37℃5％CO_2 for 90 min. Washed with PBS twice at 300g for 10 minutes. Cultured with RPMI-1640 (adding IL-2 by 100U/ml) in 37℃5％CO_2. Virus replication was assessed by measuring p24 antigen in the culture supernatants.
3.Seperate the CD4+/CD8+ T lymphocyte cell
PBMC of subjects were isolated by standard Ficoll-hypaque density centrifugation. Remove monocytes. Per 1×10~7 cells were treated with 20μl anti-CD4 immunomagenetic beads in 6℃for 15 minutes. Wash at 300g for 10 minutes. 500μl cell suspend liquor went through LS column to positive selection for CD4+T lymphocytes. Infection of C D4+T c ell with HIV-1: Cultures of C D4+ lymphocytes from ESN and control subjects were inoculated with HIV-1 of 600 TCID50 in 37℃for 4 hours. Washed with PBS twice at 300g for 10 minutes and cultured with RPMI-1640 (adding IL-2 by 100U/ml). Virus replication was assessed at regular interval over a 2-week period by measuring p24 antigen in the culture supernatants.
4.Coculture the CD4+ T and CD8+T lymphocyte
Adjust the CD4+ T and the CD8+ T density to 1×10~6cells/ml, add the cells into the 48 bore plate, CD8:CD4=2:1,1:1,0.5:1, collect the supernatant every 3 days.
5.p24 detection:
p24 antigen was detected by Biomerieux kit according to instruction.
6.Statistical analysis
SPSS 11.5 software package was used for the statistical analysis. Geometric means were determined for log-distributed variables. The significance of the differences in CNAR in patients and controls was evaluated with one-way ANOVA. A P value of 0.05 or less was considered to represent significance.
Results
1.The purity of the CD4+/CD8+ T lymphocyte cell
Purify the CD4+/CD8+ T: The purity of the cells is more than 95％.
2. The CNAR function in the LTNP
Our research finding the function of CNAR in the LTNP is stronger than the HIV, Health control and the AIDS.
3. Add IL-15, Inhibition ratio changes in different disease progression
We analysis the data by single sample T-test, there is a significant difference between the IL-15 group and the no-IL-15 group (P＜0.05). Further more, AIDS＞HIV＞LTNP
4. CD4+ T true count and the inhibition ratio
We found that following the descend of the CD4+ T true count, the function of CNAR significant decreased. A statistically significant positive correlation was found between the 2 parameters (r=0.2, P＜0.05).
Conclusion
1.The function of the CNAR in the LTNP is stronger than the HIV, Health control and the AIDS. It indicated that the CNAR can protect the disease non-progression.
2.There is a significant difference between the IL-15 group and the no-IL-15 group (P＜0.05). Further more, AIDS＞HIV＞LTNP. It indicate that the IL-15 could stronger CNAR, and flowing disease progression, the enhancement property is more significant. The AIDS is the highest.
3.We found that following the descend of the CD4+ T true count, the CNAR significant decreased. It indicate that there is relationship between the CD4+ true count and the function of CNAR.
引文
1 Borrow P,Lewicki H,Hahn B,et al.Virus-specific CD8+ cytotoxic T-1ymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.J.Virol.1994;68:6103-6110．
2 Carmichael A,Jin X,Sissons P,et al.Quantitative analysis of the human immunodeficiency virus type 1 (HIV-1) -specific cytotoxic T lymphocyte (CTL) response at different stages of HIV-1 infection:differential CTL responses to HIV-1 and Epstein-Barr virus in late disease.J.Exp.Med.1993;177:249-256．
3 Ferbas J,Walker CM,Moody,et al.Perspectives on the role of CD8+ cell suppressor factors and cytotoxic T 1ymphocytes during HIV infection.AIDS Res Hum Retroviruses.1998;14:S153-160．
4 Cocchif A,Devico AL,Sissons P,et al.I dentification of R ANTES,MIP-1 alpha,and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells.Science.1995;270:1811-1815．
5 Mackewicz CE,Blackbourn DJ,Levy JA,et al.CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription.Proc.Natl.Acad.Sci.1995;92:2308-2312．
6 Mackewicz CE,Patterson BK,Lee SA,et al.CD8(+) cell noncytotoxic anti-human immunodeficiency virus response inhibits expression of viral RNA but not reverse transcription or provirus integration.J.Gen Virol.2000;81:1261-1264．
7 Watari H,Nozawa R,Tokunaga A,et al.Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF.Proc.Natc.Acad.Sci.2000;97:3503-3508．
8 Walker CM,Moody DJ,Stites DP,et al.CD 8+ lymphocytes can control HIV infection in vitro by suppressing virus replication.Science.1986;234:1563-1566．
9 Levy JA,Mackewicz CE,Barker E,et al.Controlling HIV pathogenesis:the role of the noncytotoxic anti-HIV response of CD8+ T cells.Immunol Today.1996;17:217-24．
10 Walker CM,Moody DJ,Stites DP,et al.Non-cytolytic control of HIV replication by CD8+ T cells. Semin Immunol. 1993; 5: 195-201.
11 Barker TD, Weissman D, Daucher DA, et al. Identification of multiple and Distinct CD8+ T cell suppressor activities: dichotomy between infected and uninfected individuals, evolution with progression of disease, and sensitivity to gamma irradiation. J. Immunol. 1996; 156: 4476-4483.
12 Barker E, Mackewicz CE, Reyes-Teran G, et al.Virological and immunological features of long-term human immunodeficiency vires-infected individuals who have remained asymptomatic compared with those who have progressed to acquired immunodeficiency syndrome. Blood. 1998; 92: 3105-3114.
13 Ahrnad A, Banapour B, Levy JA, et al. IL-15 and HIV infection:lessons for immunotherapy and vaccination. Curr HIV res. 2005; 3: 261-270.
14 Vella C, Daniels RS, Reyes-Teran G, et al. CD8+T cell mediated Non cytolytic Suppression of Human Immunodeficiency Viruses. Curr Drug Targets Infect Disord. 2003; 3: 97-113.
15 Hoffman AD, Banapour B, Levy JA, et al. Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection invirions.Virology. 1985; 147: 326-335.
16 Lifson AR, Rutherford GW, Jaffe HW, et al. The natural history of human HIV infection. J. Infect Dis. 1988; 158: 1360-1367.
17 Phair 3P, Brien HW, Daniels RS, et al. Variations in the natural histOTii of HW mfection. AIDS Res Human Retrovir. 1994; 10: 883-885.
18 Mulherin SA, Brien TR, Isoannidis JP, et al. Effect of CCRS-A32 and CCR2-64I alleles on HIV-1 disease progression:the protection varies with duration of infection. AIDS. 2003; 17: 377-387.
19 王树祥 尚红 韩晓旭 等。中国HIV-1感染疾病长期不进展者CCR2264I、SDF123和CCR5A32的基因变异研究。Chinese J Exp Clin Virol．2006；20：16—19．
20 Gulzar N, Copeland KF, Brien TR, et al. CDS+ T-Cells: Function and Response to HIV Infection. Curr HIV Res. 2004; 2: 23-37.
21 Mackewicz CE,Ortega HW,Levy JA,et al.CD8+ Cell Anti-HIV Activity correlates with the Clinical State of the Infected Individual.J.Clin Invest.1991;87:1462-1466．
22 Sharon A,Stranford JS,Levy JA,et al.Lack of infection in HIV exposed individuals is associated with a strong CD8+ cell noncytotoxic anti-HIV response.Immunology.1999;96:1030-1035．
23 Castelli K,Deeks H,Levy JA,et al.Relation-ship of CD8+ T cell non-cytotoxic anti-HIV response to CD4+ T cell number in untreated asymptomatic HIV-infected individuals.Blood.2002;99:4225-4229．
24 Castelli J,Tomas EK,Gilliet M,et al.Mature dendritic cells can enhance CD8+ cell non-cytotoxic anti-HIV responses:the role of IL-15． Blood.2004;103:2699-2704．
1 Borrow P,Lewicki H,Hahn B,et al.Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.J.Virol.1994;68:6103-6110．
2 Carmichael A,Jin X,Sissons P,et al.Quantitative analysis of the human immunodeficiency virus type 1 (HIV-1) -specific cytotoxic T lymphocyte (CTL) response at different stages of HIV-1 infection:differential CTL responses to HIV-1 and Epstein-Barr virus in late disease.J.ExpMed.1993;177:249-256．
3 Ferbas J,Blackbourn DJ,Levy JA,et al.Perspectives on the role of CD8+ cell suppressor factors and cytotoxic T lymphocytes during HIV infection.AIDS Res Hum Retroviruses.1998;14:S153-160．
4 Cocchif A,Devico AL,Lee SA,et al.Identification of RANTES,MIP-1 alpha,and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells.Science.1995;270:1811-1815．
5 Mackewicz CE,Blackbourn DJ,Levy JA,et al.CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription.Proc Natl Acad Sci.1995;92:2308-2312．
6 Mackewicz CE,Patterson BK,Lee SA,et al.CD8(+) cell noncytotoxic anti-human immunodeficiency virus response inhibits expression of viral RNA but not reverse transcription or provirus integration.J.Gen Virol.2000;81:1261-1264．
7 Watari H,Nozawa R,Tokunaga A,et al.Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF.Proc Natc Acad Sci.2000;97:3503-3508．
8 Rodes B,Toro C,Paxinos E,et al.Differences in disease progression in a cohort of long-term non-progressors after more than 16 years of HIV-1 infection.AIDS.2004;18:1109-1116．
9 Walker CM,Erickson AL,Levy JA,et al.Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic Mechanism. J.Virol.1991;65:5921-5927．
10 Hess C,Altfeld M,Thomas SY,et al.HIV-l specific CD8+ T cells with an effector phenotype and control of viral replication.Lancet.2004;363:863-866．
11 Borgne SL,Nozawa R,Tokunaga A,et al.CD8(+)-Cell antiviral factor activity is not restricted to human immunodeficiency virus (HIV)-specific T cells and can block HIV replication after initiation of reverse transcription.J.Virol.2000;74:4456-4464．
12 Wang L,Nozawa R,Tokunaga A,et al.Modulation of HIV-1 replication by a novel RhoA effector activity.J.Immunol.2000;164:5369-5374．
13 Diaz LS,Foster H,Stone MR,et al.Differential gene expression in CD8+ cells exhibiting noncytotoxic anti-HIV activity.Virology.2003;311:400-409．
14 Castelli J,Elaine K,Thomas,et al.Mature dendritic cells can enhance CD8+ cell non-Cytotoxic anti-HIV responses:the role of IL-15． Blood.2004;103:2699-2704．
15 Pal R,Garzino-Demo,AMarkham PD,et al.Inhibition of HIV-1 infection by the beta-chemokine MDC.Science.1997;278:695-698．
16 Patterson BK,Behbahani H,Kabat WJ,et al.Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women.J.Gin Invest.2001;107:287-294．
17 Wollman EE,Aoriol L,Rimsky L,et al.Cloning and expression of a cDNA for human thioredoxin.J.Biol chem.1988;263:15506-15512．
18 Yodoi J,Tursz T,Thomas SY,et al.Agrowth-promoting factor derived from adult T cell leukemia and homologous to thioredoxin:involvement in lymphocyte immortalization by HTLV-I and EBV.Adr Cancer Res.1991;57:381-411．
19 Rubartelli A,Bajetto A,Allavena G,et al.Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway.J.Biol Chem.1992;267:24161-24164．
20 Chen CH,Weinhold KJ,Bartlett JA,et al.CD8+ T lymphocyte-mediated inhibition of HIV-1 long terminal repeat transcription:a novel antiviral mechanism.AIDS Res Hum Retroviruses.1993;9:1079-1086．
21 Bovolenta CA,Nozawa R,Tokunaga A,et al.Selective defect of IFN-gamma-but not of IFN-alpha-induced JAK/STAT pathway in a subset of U937 clones prevents the antiretroviral effect of IFN-gamma against HIV-1． J.Immunol.1999;162:323-330．
22 Killian MS,Mackewicz CE,Levy JA,et al.A screening assay for detecting CD8+ cell non-cytotoxic anti-HIV responses.J.Immunol Methods.2005;304:137-150．
23 Mackewicz CE,Ortega HW,Levy JA,et al.CD8+ cell anti-HIV activity correlates with the clinical state of the infected individual.J.Clin.1991;87:1462-1466．
24 Watari H,Nozawa R,Tokunaga A,et al.VCAM-1 expression on CD8+ cells correlates with enhanced anti-HIV suppressing activity.J.Immunol.2005;174:1574-1579．
25 Stranford SA,Ong JC,Martinez MB.Reduction in CD8+ cell noncytotoxic anti-HIV activity in individuals receiving highly active antiretroviral therapy during primary infection.Proc Natl Acad Sci.2001;98:597-602．
26 Mackewicz CE,Craik CS,Levy JA,et al.The CD8+ cell noncytotoxic anti-HIV response can be blocked by protease inhibitors.Proc Natl Acad Sci.2003;100:3433-3438．
27 Chang TL,Francois F,Tokunaga A,et al.CAF-mediated human immunodeficiency virus (HIV) type 1 transcriptional inhibition is distinct from alpha-defensin-1 HIV inhibition.J.Virol.2003;77:6777-6784．
28 Hecht FM,Kahn JO,Levy JA,et al.Prospects for an AIDS vaccine:encourage innate Immunity.AIDS.2004;18:2085-2086．
2 Carmichael A,Jin X,Sissons P,et al.Quantitative analysis of the human immunodeficiency virus type 1 (HIV-1) -specific cytotoxic T lymphocyte (CTL) response at different stages of HIV-1 infection:differential CTL responses to HIV-1 and Epstein-Barr virus in late disease.J.Exp.Med.1993;177:249-256．
3 Ferbas J,Walker CM,Moody,et al.Perspectives on the role of CD8+ cell suppressor factors and cytotoxic T 1ymphocytes during HIV infection.AIDS Res Hum Retroviruses.1998;14:S153-160．
4 Cocchif A,Devico AL,Sissons P,et al.I dentification of R ANTES,MIP-1 alpha,and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells.Science.1995;270:1811-1815．
5 Mackewicz CE,Blackbourn DJ,Levy JA,et al.CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription.Proc.Natl.Acad.Sci.1995;92:2308-2312．
6 Mackewicz CE,Patterson BK,Lee SA,et al.CD8(+) cell noncytotoxic anti-human immunodeficiency virus response inhibits expression of viral RNA but not reverse transcription or provirus integration.J.Gen Virol.2000;81:1261-1264．
7 Watari H,Nozawa R,Tokunaga A,et al.Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF.Proc.Natc.Acad.Sci.2000;97:3503-3508．
8 Walker CM,Moody DJ,Stites DP,et al.CD 8+ lymphocytes can control HIV infection in vitro by suppressing virus replication.Science.1986;234:1563-1566．
9 Levy JA,Mackewicz CE,Barker E,et al.Controlling HIV pathogenesis:the role of the noncytotoxic anti-HIV response of CD8+ T cells.Immunol Today.1996;17:217-24．
10 Walker CM,Moody DJ,Stites DP,et al.Non-cytolytic control of HIV replication by CD8+ T cells. Semin Immunol. 1993; 5: 195-201.
11 Barker TD, Weissman D, Daucher DA, et al. Identification of multiple and Distinct CD8+ T cell suppressor activities: dichotomy between infected and uninfected individuals, evolution with progression of disease, and sensitivity to gamma irradiation. J. Immunol. 1996; 156: 4476-4483.
12 Barker E, Mackewicz CE, Reyes-Teran G, et al.Virological and immunological features of long-term human immunodeficiency vires-infected individuals who have remained asymptomatic compared with those who have progressed to acquired immunodeficiency syndrome. Blood. 1998; 92: 3105-3114.
13 Ahrnad A, Banapour B, Levy JA, et al. IL-15 and HIV infection:lessons for immunotherapy and vaccination. Curr HIV res. 2005; 3: 261-270.
14 Vella C, Daniels RS, Reyes-Teran G, et al. CD8+T cell mediated Non cytolytic Suppression of Human Immunodeficiency Viruses. Curr Drug Targets Infect Disord. 2003; 3: 97-113.
15 Hoffman AD, Banapour B, Levy JA, et al. Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection invirions.Virology. 1985; 147: 326-335.
16 Lifson AR, Rutherford GW, Jaffe HW, et al. The natural history of human HIV infection. J. Infect Dis. 1988; 158: 1360-1367.
17 Phair 3P, Brien HW, Daniels RS, et al. Variations in the natural histOTii of HW mfection. AIDS Res Human Retrovir. 1994; 10: 883-885.
18 Mulherin SA, Brien TR, Isoannidis JP, et al. Effect of CCRS-A32 and CCR2-64I alleles on HIV-1 disease progression:the protection varies with duration of infection. AIDS. 2003; 17: 377-387.
19 王树祥 尚红 韩晓旭 等。中国HIV-1感染疾病长期不进展者CCR2264I、SDF123和CCR5A32的基因变异研究。Chinese J Exp Clin Virol．2006；20：16—19．
20 Gulzar N, Copeland KF, Brien TR, et al. CDS+ T-Cells: Function and Response to HIV Infection. Curr HIV Res. 2004; 2: 23-37.
21 Mackewicz CE,Ortega HW,Levy JA,et al.CD8+ Cell Anti-HIV Activity correlates with the Clinical State of the Infected Individual.J.Clin Invest.1991;87:1462-1466．
22 Sharon A,Stranford JS,Levy JA,et al.Lack of infection in HIV exposed individuals is associated with a strong CD8+ cell noncytotoxic anti-HIV response.Immunology.1999;96:1030-1035．
23 Castelli K,Deeks H,Levy JA,et al.Relation-ship of CD8+ T cell non-cytotoxic anti-HIV response to CD4+ T cell number in untreated asymptomatic HIV-infected individuals.Blood.2002;99:4225-4229．
24 Castelli J,Tomas EK,Gilliet M,et al.Mature dendritic cells can enhance CD8+ cell non-cytotoxic anti-HIV responses:the role of IL-15． Blood.2004;103:2699-2704．
1 Borrow P,Lewicki H,Hahn B,et al.Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.J.Virol.1994;68:6103-6110．
2 Carmichael A,Jin X,Sissons P,et al.Quantitative analysis of the human immunodeficiency virus type 1 (HIV-1) -specific cytotoxic T lymphocyte (CTL) response at different stages of HIV-1 infection:differential CTL responses to HIV-1 and Epstein-Barr virus in late disease.J.ExpMed.1993;177:249-256．
3 Ferbas J,Blackbourn DJ,Levy JA,et al.Perspectives on the role of CD8+ cell suppressor factors and cytotoxic T lymphocytes during HIV infection.AIDS Res Hum Retroviruses.1998;14:S153-160．
4 Cocchif A,Devico AL,Lee SA,et al.Identification of RANTES,MIP-1 alpha,and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells.Science.1995;270:1811-1815．
5 Mackewicz CE,Blackbourn DJ,Levy JA,et al.CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription.Proc Natl Acad Sci.1995;92:2308-2312．
6 Mackewicz CE,Patterson BK,Lee SA,et al.CD8(+) cell noncytotoxic anti-human immunodeficiency virus response inhibits expression of viral RNA but not reverse transcription or provirus integration.J.Gen Virol.2000;81:1261-1264．
7 Watari H,Nozawa R,Tokunaga A,et al.Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF.Proc Natc Acad Sci.2000;97:3503-3508．
8 Rodes B,Toro C,Paxinos E,et al.Differences in disease progression in a cohort of long-term non-progressors after more than 16 years of HIV-1 infection.AIDS.2004;18:1109-1116．
9 Walker CM,Erickson AL,Levy JA,et al.Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic Mechanism. J.Virol.1991;65:5921-5927．
10 Hess C,Altfeld M,Thomas SY,et al.HIV-l specific CD8+ T cells with an effector phenotype and control of viral replication.Lancet.2004;363:863-866．
11 Borgne SL,Nozawa R,Tokunaga A,et al.CD8(+)-Cell antiviral factor activity is not restricted to human immunodeficiency virus (HIV)-specific T cells and can block HIV replication after initiation of reverse transcription.J.Virol.2000;74:4456-4464．
12 Wang L,Nozawa R,Tokunaga A,et al.Modulation of HIV-1 replication by a novel RhoA effector activity.J.Immunol.2000;164:5369-5374．
13 Diaz LS,Foster H,Stone MR,et al.Differential gene expression in CD8+ cells exhibiting noncytotoxic anti-HIV activity.Virology.2003;311:400-409．
14 Castelli J,Elaine K,Thomas,et al.Mature dendritic cells can enhance CD8+ cell non-Cytotoxic anti-HIV responses:the role of IL-15． Blood.2004;103:2699-2704．
15 Pal R,Garzino-Demo,AMarkham PD,et al.Inhibition of HIV-1 infection by the beta-chemokine MDC.Science.1997;278:695-698．
16 Patterson BK,Behbahani H,Kabat WJ,et al.Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women.J.Gin Invest.2001;107:287-294．
17 Wollman EE,Aoriol L,Rimsky L,et al.Cloning and expression of a cDNA for human thioredoxin.J.Biol chem.1988;263:15506-15512．
18 Yodoi J,Tursz T,Thomas SY,et al.Agrowth-promoting factor derived from adult T cell leukemia and homologous to thioredoxin:involvement in lymphocyte immortalization by HTLV-I and EBV.Adr Cancer Res.1991;57:381-411．
19 Rubartelli A,Bajetto A,Allavena G,et al.Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway.J.Biol Chem.1992;267:24161-24164．
20 Chen CH,Weinhold KJ,Bartlett JA,et al.CD8+ T lymphocyte-mediated inhibition of HIV-1 long terminal repeat transcription:a novel antiviral mechanism.AIDS Res Hum Retroviruses.1993;9:1079-1086．
21 Bovolenta CA,Nozawa R,Tokunaga A,et al.Selective defect of IFN-gamma-but not of IFN-alpha-induced JAK/STAT pathway in a subset of U937 clones prevents the antiretroviral effect of IFN-gamma against HIV-1． J.Immunol.1999;162:323-330．
22 Killian MS,Mackewicz CE,Levy JA,et al.A screening assay for detecting CD8+ cell non-cytotoxic anti-HIV responses.J.Immunol Methods.2005;304:137-150．
23 Mackewicz CE,Ortega HW,Levy JA,et al.CD8+ cell anti-HIV activity correlates with the clinical state of the infected individual.J.Clin.1991;87:1462-1466．
24 Watari H,Nozawa R,Tokunaga A,et al.VCAM-1 expression on CD8+ cells correlates with enhanced anti-HIV suppressing activity.J.Immunol.2005;174:1574-1579．
25 Stranford SA,Ong JC,Martinez MB.Reduction in CD8+ cell noncytotoxic anti-HIV activity in individuals receiving highly active antiretroviral therapy during primary infection.Proc Natl Acad Sci.2001;98:597-602．
26 Mackewicz CE,Craik CS,Levy JA,et al.The CD8+ cell noncytotoxic anti-HIV response can be blocked by protease inhibitors.Proc Natl Acad Sci.2003;100:3433-3438．
27 Chang TL,Francois F,Tokunaga A,et al.CAF-mediated human immunodeficiency virus (HIV) type 1 transcriptional inhibition is distinct from alpha-defensin-1 HIV inhibition.J.Virol.2003;77:6777-6784．
28 Hecht FM,Kahn JO,Levy JA,et al.Prospects for an AIDS vaccine:encourage innate Immunity.AIDS.2004;18:2085-2086．
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