| IntroductionHIV, namely human immunodeficiency virus, is the pathogen of acquired immunodeficiency syndrom( AIDS) , mainly attack CD4~+ T lymphocytes, the patient finally die of various opportunitic infections and cancer, there were no effective methods to cure or to prevent AIDS.Host can' t clear virus completely after they were infected by HIV, and progressed to AIDS after 8-10 years if they had not received antiretroviral therapy. it's reported that about 5 percent of HIV infected subjects though they were infected over ten years and not received HAART ( highly active antiretroviral therapy) , their CD4~+ T cell count were nomal, their plasma viral load kept low level, they were called long term non - progressor ( LTNP). What mechanisms made these subjects keep long term nonprogression? CTL ( cytotoxic T lymphocytes) is a kind of immune effector cell that can specifially kill virus infected cells and tumor cells, its demonstrated that HIV specific CTL response was deficient in rapid progressor and their viral load were high, but in LTNP, HIV specific CTL response were strong and their viral load were low, so CTL is an important protective immune response. Though HIV specific CTL response is so strong, it can't eradiate HIV finally, suggest it's founction was injured, the mechanisms about it and the evaluate parameters were not established, there were also few report about the effect of HAART on it. HIV specific CD4~+ T helper cell can surpport effective CTL response, it can recognize virus protein and peptides, inhibite HIV replication through inducing and sustainning CD8~+ T cell response,T helper cell was shown to play an protective role in AIDS progres-sion, and play a central role in immune response, but study about the number and founction of HIV specific CD4+ T helper cell was rare. Natural immune response is the first line to defend outside pathogen, NK( natural killer cell)is important in natural immune response, however, there were no report about the founction alteration and the effect of antiretroviral therapy on NK cell. Cytokine involved in nomal immune response and homeostatic regulation, there were large number of papers about cytokine malregulation in HIV infection, this play an important role in HIV immunopathogenesis, IL -7 is one of it. Recently, it's reported that increased circulating levels of IL - 7 are strongly associated with CD4 + T lymphopenia in HIV disease and elevated IL - 7 levels in plasma may be a marker of SI variants, so plasmic IL - 7 level may be a marker of disease progression. Nevertheless, this hypothese needs further confirmation, there were no related study in our country yet.The immunological characters of Chinese is different from that of the west, making further research about these immunological characters will have important reference value on understanding host immune status, judging disease prognosis , evaluating anti - virus effects and choosing index for immune reconstitu-tion or vaccine research for Chinese HIV/AIDS patients. In this study, using flow cytometry intracellular staining, surface staining methods and ELISA methods, we detected proliferative responses ofCD4 + T, CD8 + T lymphocytes, and perform expression in NK cells and CD8 + T lymphocytes, and plasma IL - 7 levels in plasma of Chinese HIV/AIDS patients, and then we investigated correlations between these parameters and disease progression as well as immune founc-tions.Material and Methods1. Study subjectsStudy population came from China Medical University AIDS research center confirming laboratory and Disease Prevention and Control Center of IiaoNing province, Jilin province, HeiLongJiang province confirming laboratory, all subjects were confirmed positive by Western blot . HIV - seronegative controls wererandomly selected from healthy population with nomal blood routine test, liver and renal founction.2. Determination of Lymphocyte Counts and Corresponding RatioPipette 20uJ of TriTEST CD4FITC/CD8PE/CD3PerCP or CD3FITC/CD56 CD16PE/CD45 PerCP regent and add 5Ojxl anti - coagulated whole blood into the bottom of the TruCOUNT Tube using reverse pipetting and incubate for 15 minutes in dark at room temperature, add 450|xll x FACS lysing solution to the tube, incubate for 15 min in the dark at room temperature and analyze the samples on the flow cytometer, using FACS MULTISET software to acquire T lymphocyte count, NK cell count and corresponding ratios.3. Determination of viral load.Plasma HIV RNA levels were determined using a commercial assay (ROCHE COBAS AMPLICOR SYSTERM) according to the manufacture s instructions , undetectable levels of RNA in plasma were considered equivalent to 400 copies/ml.4. Intracellular Stain AssayWhole blood was collected into EDTA - anticoagulant vacutainer by veni-puncture and processed within 8 hour, detection of intracellular perform in CD8 + Tcells and NK cells was performed according to portales 's method and modified by us. Brifly, aliquot lOOfxl whole blood into flow cytometric tubes, add 2ml 1 x FACS lysing solution, vortex, incubate lOmin at room temperature in the dark for 10 min, spin 5min at 500 x g, wash once with stain buffer, fix and permeabilize cells by adding 500|xl BD cytofix/cytoperm solution, vortex and incubate at room temperature in the dark for 20 min, spin 5min at 500 X g, wash one time by adding 2ml BD cytoperm/cytowash solution, resuspend cell pellet in lOOjjd BD cytoperm/cytowash solution, stain cells with surface and intracellular monoclone antibody at saturating concentration for 30 min, that' sCD3FITC/perforin - PE/CD8PerCP, CD3nTC/perforin - PE /CD16PerCP, CD56FITC/perforin-PE/CD3PerCP, each patient with an unstain control, after further washing BD cytoperm/cytowash solution, cells were re - suspended in 1 %paraformaldehyde -PBS, and ready for flow cytometric analysis.5. T lymphocyte proliferation.Fresh PBMC( 106)from healthy donors were cultured with different antigens as follows: medium control, phytohemagglutinin ( PHA) (4ng/ml) plus IL - 2 (4ng/ml) as a positive control, IL - 7 (lOOng/ml) , IL - 7 ( lOng/ml) , IL - 7 (lng/ml) ,the cultures were maintained at 371 in a 5% CO2 incubator for 5 days,l hour before culture was over, 10uJ(lmM)BrdU was added until culture was over,100ui cells were collected in flow tubes, wash once; then stain with 6uJCD3 - PerCP,CD4 - PE monoclonal antibodies for 15 min in dark, wash once ; lOOui eytofix/cytoperm were added for 10 min at room temperature in dark, wash once; the cells were response with lOOul cytoperm plus for 10 min at room temperature in dark; then 100ui diluted Dnase were added in 37^for lhour, wash once, 50|xlcytoperm/cytowash resuspend cells, ljjd BrdU - FITC antibody stain cells for 20 min in dark, wash once, and analyzed in a FACScali-bur flow cytometer( BD).6. CXCR4 expression.Fresh PBMC from healthy donors were cultured with IL - 7 at different concentrations or medium alone as negative control. After 5 days of incubation, PBMC were collected , aliquots of 50jjl1 PBMC were stained with monoclonal antibodies CD4 -PerCP and CXCR4 - PE(BD)for 15 min, and then the samples were washed twice in phosphate - buffered saline ( PBS) , resuspend in 1 % formaldehyde/PBS , and analyzed in a FACScalibur flow cytometer.7. Detection of IL -7levels in plasma.Plasma IL - 7 levels were determined by an ultra - sensitive commercial enzyme - linked immunosordent assay ( ELISA) ( DIACLONE ) according to the manufacturer instructions.8. Viral isolation and phenotype in MT -2 cells.Virus were isolated from HIV positive individuals with micro - separation of the whole blood, briefly, diluted whole blood from HIV/AIDS patients were co -cultured with PBMC(106/ml)from healthy donors pre -stimulated with PHA (3jxg/ml) , viral replication was quantified by evaluation of antigen P24 production in co - culture supernatants , using a commercial ELISA kit ( Akzo - Nobel) , the isolated virus were then collected ,and SI and NSI phenotype was determined in MT - 2 cells as was previously described, for simplicity, individualsfrom whom SI or NSI variants were isolated are refer to hereafter as SI or NSI group respectively.9. HAART regimen:patients with no obvious liver and renal function abnormality were chosen according to the " standard therapy management" issued by the Ministry of Health and treated with antiviral drugs: Indinavir. a kind of proteinase inhibitor, taken orally lOOOmg every 8 hours; Efavirenz, a kind of non - nucleoside reverse transcriptase inhibitor, taken orally 600mg daily.Results1. Perforin expression in NK cells and CD8 +T cells in untreated HIV - infected individuals and HAART - treated HIV - infected individualsWe performed an intracellular stain to evaluate circulating perforin expression of a corhort of 31 untreated HIV - infected individuals at different stage of disease and 17 HAART - treated HIV/AIDS patients as well as 15 healthy control , their CD4 + T cell count were low and CD8 + T cell count were high as compared with health control, perforin expressed NK cells were lower than that of health control, however, the percentage of perforin expression in CD8 + T cells were higher than that of health control. In HAART - treated group, CD3V CD16+ and CD56VCD3" NK cells expressed significant high level of perforin in this group than untreated group, when compared with health control, the difference was not significant, however, perforin expression in CD8 + T cell in this group were marginally lower than HIV infected group, the difference was not significant,2. Correlated factors influence perforin expression in CD8 + Tcells and NK cells in HIV - infected individualsWe studied the correlations between perforin expression of NK cells, CD8+ T cell and CD4 + T cell count, CD8 + T cell count, plasma viral load, NK cell count, NK cell percent in HIV - infected group. Our results showed that perforin expression in the two subsets of NK cells correlated significantly with NK cell count and NK cell percent( P <0.01 ,P <0.05) , but not correlated with CD4 +Tcell count or CD8 +T cell count or viral load ; perform expression in CD8 +T cell correlated significantly with CD8 + T cell count ( P < 0. 01, P < 0. 01) , but not correlated with CD4 +T cell count or viral load or NK cell count or NK cell percent.3. HIV - 1 Gag - specific T lymphocyte proliferative responses in HIV -1 infected patientsWe detected HIV - 1 Gag - specific T lymphocyte proliferative responses by BrdU incorporated intracellular staining method, the results showed that HIV - 1 gag specific CD4 + T lymphocytes proliferative responses in health control group, asymptomatic infected group and AIDS group decrease one by one, and that of asymptomatic infected group and AIDS group were significantly lower than that of health control group (P<0. 001,P<0.001), that of AIDS group were significantly lower than that of asymptomatic infected group ( P < 0. 01 ) ; HIV - 1 gag specific CD8 + T lymphocytes proliferative responses in health control group, a-symptomatic infected group and AIDS group increase in order, the differences between them were significant(P<0. 001;P<0. 001) except for HIV - 1 a-symptomatic infected group and health control group(P >0. 05) .4. Correlation between T lymphocyte activate status and proliferative responsesThe mean CD38 expression on CD8+ T lymphocytes were 43. 31 % in HIV infected patients, and that of health control were 25. 92% , the activate status of CD8+T cells were significantly higher than that of health control group (P <0. 001) ,but the activate status of CD4 + T cells were not significantly different from that of health control group( P > 0.05). we study the correlation between HIV -1 Gag-specific CD4+T,CD8+T lymphocytes proliferative responses with CD38 expression on CD8 + T cells, the results showed that HIV - 1 Gag - specific CD4+T lymphocytes proliferative responses were inversey correlated with CD38 expression on CD8 + T ceUs ( r = - 0. 416; P < 0. 01) , HIV - 1 Gag - specific CD8 + T lymphocytes proliferative responses were directly correlated with CD38 expression on CD8 + T cells( r = 0. 594;P <0.01).5. Correlation between T lymphocytes proliferative responses and disease progressionUsing CD4 + absolute count and plasma virus load as disease progression index , we study the correlation between T lymphocytes proliferative responses and disease progression, the results showed that HIV - 1 Gag - specific CD4 +T lymphocytes proliferative responses were directly correlated with CD4* T count(r = 0. 435; P < 0. 01 ) , and inversely correlated with plasma viral load( r = - 0. 486;P <0.05) , furthermore, HIV - 1 Gag - specific CD4 + T lymphocytes proliferative responses were not correlated with HIV - 1 Gag - specific CD8 + T lymphocytes proliferative responses( r = -0. 144;P =0.415).6. IL - 7 enhanced T - lymphocyte proliferation and CXCR4 expression in vitroIL -7 induced T cells proliferation at Ing/ml and lOng/ml, but lOOng /ml suppressed T cell proliferation, they were all lower than PHA plus IL -2. IL -7 up - regulated the expression of the CXCR4 receptor in a dose - dependent manner, although IL - 7 caused the greatest up - regulation of CXCR4 expression at concentration of lOOng/ml. Ing/ml was sufficient to up -regulation CX-CR4 expression in vitro. However, CD4 expression was not modified by IL -7 (data not shown ) .7. IL - 7 levels in Chinese HIV/ AIDS patients and as a marker of disease progressionIL - 7 levels in Chinese HIV/ AIDS patients and healthy controls were analyzed in a cross - sectional study, the HIV/AIDS group (mean 7.09 ± 3. 51 pg/ ml)had significantly (P <0. 05) higher levels of IL -7 than the healthy control group (mean 3. 67 ±0. 73 pg/ml). We have found a clear negative correlation (r = - 0. 709; P < 0. 01) between IL - 7 levels and absolute CD4+ T ceU counts,a positive correlation (r = 0. 35 , P < 0. 05) was found between IL - 7 levels and viral load in Chinese HIV/AIDS patients,but IL -7 were not correlated with absolute CD8+T cell counts (r = -0.214; P=0.09) ,IL-7 levels in AIDS group(10. 08 ±3. 71 pg/ml) were significantly higher than that of CI group (5.89±2.08pg/ml) (p<0.05) and LTNP group(4.46 ± 1.58 pg/ml) (P < 0.05) ,IL -7 levels in CI group were higher than that of LTNP group, but the difference was not significant.8. Correlation between IL -7 and HIV phenotypeThe CD4 + T ceils, CD8 + T cells and viral load were significantly different between NSI group and SI group, suggest SI group represent higher disease stages, but IL -7 levels were not significantly different between the two groups.Conclusion1. Perform expression in NK cells were lower than that of health control in Chinese HIV/AIDS patients, it's level elevated in HAART group, and significantly correlated with NK absolute count and NK percent, this suggest that founction of NK cells was injured in HIV infection, it can be restored by antiret-roviral therapy.2 perform expression in CD8 + T lymphocytes were higher than that of health control in Chinese HIV/AIDS patients, its level decreased by HAART, and directly correlated with CD8 T Tcell count, this suggest that CD8 * T lymphocytes fouction was increased in HIV infection.3. HIV - 1 Gag - specific CD4 + T lymphocytes proliferative responses were significantly lower thant that of health control in Chinese HIV/AIDS patients, and directly correlated with CD4 + T count, inversely correlated with plasma viral load and inversey correlated with CD38 expression on CD8^T cells, suggest that HIV - 1 Gag - specific CD4 + T lymphocytes proliferative responses were decreased in Chinese HIV/AIDS patients, the percent can be regarded as as a marker of the immune fonction and disease progression for Chinese HIV/AIDS patients.4. HIV - 1 Gag - specific CD8 + T lymphocytes proliferative responses were significantly higher than that of health control in Chinese HIV/AIDS patients, and inversely correlated with CD4 + T count, directly correlated with CD38 expression on CD8 +T cells, suggest that HIV - 1 Gag - specific CD8 +T lymphocytes proliferative response in Chinese HIV/AIDS patients was increased.5. Plasma IL -7 levels in Chinese HIV/AIDS patients were elevated, it's levels inversely correlated with CD4 + T lymphocyte counts, and directly correlated with viral load, rhIL -7 enhanced T -lymphocyte proliferation and CXCR4 expression in vitro, this suggest IL - 7 level may be regarded as a marker of HIVdisease progression.
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