Study On The Correlation Between The Numberand Functional Alterations Of Naive And Memory CD8+T Cells Subsets And Disease Progressions In HIV/AIDS Chinese Patients

ObjectiveCD8+T cells play a crucial role in the course of HIV infection.Naive CD8+T cells have not encountered antigens,so they are critical to the immune responses against new antigens,because if stimulated,they will activate,proliferate and differentiate into cytotoxic T cell rapidly.Memory CD8+T cells are important for the rapid secondary immunity response and maintaining the balance of organism immunity. According to the different phenotypes,memory CD8+T cells in circulation are further subdivided into three subsets,central memory(TCM)and two effector memory (TEM)T cells(TEM-1 and TEM-2).If stimulated,TCMs would be absent of effective function,but coud urge DCs to present antigens,proliferate themselves quicker,then differentiate into effector T cells.TEMs could excrete effector cytokine efficiently, such as IL-4,IFN-γ,TNF-αand porforin by which could exert cytotoxic function and kill target cells quickly to mediate inflammatory reactions.As reported,CD8+TEM-2 cells express largest amount of porforin and could proliferate.At present,it is still poorly understood about the dynamic development and the immune functional mechanism of naive and memory CD8+T cells,and there is still no related report on HIV-1-infected individuals and HAART-treated patients in China.This study is mainly about the number alterations,the activation,proliferation and the expression of co-receptors of both naive and memory CD8+T cells and their subsets,in addition,we investigated the correlation between the indexes and disease progressions.Material and MethodsStudy subjects and specimensA total of 58 treatment naive HIV-infected patients from Liaoning,Jilin and Henan provinces of China were enrolled(28 males,30 females;median age 40 years, range 30-68 years).HIV-infected subjects were classified into 3 stages.The first stage was long-term non-progressors(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 18 LTNPs,20 HIV and 20 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.20 seronegative subjects that never exposed to HIV-1 were selected as normal controls and there were 10 males and 10 females.Blood was drawn by venipuncture from each subject in EDTA tubes(Becton Dickinson)for FACS analysis and the detection of viral load.·CD4~+ T cell absolute countCD4~+ T cell absolute counts were enumerated in 50μl freshly obtained EDTA-anticoagulated whole blood using 20μl directly labeled monoclonal antibodies (TriTEST:CD4-FITC/CD8-PE/CD3-PerCP)according to the "lyse-no-wash" procedure.All samples were acquired with a four-color FACSCalibur(Becton Dickinson)and analyzed using MultiSET softwares.·Viral load Plasma viral load was determined with RT-PCR detecting HIV-1 RNA(Roche Amplicor Monitor Standard Assay,Roche Diagnostics,Branchburg,NJ).All undetectable values(below 400 copies)were assigned a value of 399.·Phenotyping of naive,memory CD8+T lymphocytes100μl whole blood were incubated with the following monoclonal antibody: CD28-FITC/CD27-PE/CD8-PerCP/CD45RA-APC/CCR7-PE-Cy7/CD4-APC-Cy7, and their isotype controls 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 naive and memory CD8+T T cells were gated according CD45RA,CCR7 and CD28 expression on T lymphocytes and the percentage of naive,memory CD8+T T cells were determined.Phenotypes of naive, memory CD8+T T cells are known as following:Naive CD8+T cells:CD8+CD45RA+CCR7+CD28+;memory CD8+T cells: central memory(CD8+CD45RA-CCR7+CD28+)and effector memory cells (CD45RA-CCR7-CD28+/CD45RA-CCR7-CD28- and CD45RA+CCR7-CD28-).·Expression of activation and coreceptors on naive,memory CD8+T lymphocytes100μl whole blood were incubated with the following monoclonal antibody: CD28-FITC/CCR5-PE/CD8-PerCP/CD45RA-APC/CCR7-PE-Cy7/CD4-APC-Cy7, CD28-FITC/CXCR4-PE/CD8-PerCP/CD45RA-APC/CCR7-PE-Cy7/CD4-APC-Cy7,C D28-FITC/CD38-PE/CD8-PerCP/CD45RA-APC/CCR7-PE-Cy7/CD4-APC-Cy7, and their isotype controls 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 total CD8+,naive and memory CDS+T T cells were gated and the percentage of CCR5,CXCR4,CD38 expression of naive and memory CD8+T T cells were determined.All monoclonal antibodies were purchased from Becton Dickinson. ·Expression of ki67 in CD4+ and CD8+T lymphocytesPBMCs were obtained from HIV-1 infected individuals and healthy controls by Ficoll-Hypaque density gradient centrifugation.(Ficoll-Hypaque;Amersham Biosciences,Uppsala,Sweden).100μl of prepared cells(1x106)were first stained with 10μl anti-CD28-FITC,anti-CD8-PerCP,anti-CD45RA-APC,anti-CCR7-PE-cy7和anti-CD4-APC-Cy7(BD Biosciences)for 25 min at 4℃.The cells were washed in cold PBS and 500μl Fixation/Permeabilization solution was added to each sample and incubated at room temperature for 20 minutes in the dark.Then 2ml BD Perm/Wash buffer were added into the cells and incubated at room temperature for 10 minutes in the dark.After the cells were washed anti-ki67-PE and isotypes IgG1-PE was added and incubate at room temperature for 30 minutes in the dark.The cells were washed twice with 2ml BD Perm/Wash buffer and fixed in 1%polyformaldehyde.Cells were analyzed on a FACSAria(Becton Dickinson)with FACSDiva software.T lymphocytes were identified by gating on forward scatter and side scatter,then total CD4+,CD8 and naive,memory CD4+,CD8+T cells were gated and the percentage of ki67 were determined.Results1.The number alterations of naive and memory CD8+T lymphocytes and their correlation to the progression of disease in HIV/AIDS patientsIn the LT-NP,HIV,and AIDS groups the percentages and absolute numbers of naive CD8+T cells significantly decreased.The numbers of naive CD8+T cells in AIDS and HIV groups were notably lower than that of LT-NP and healthy control groups(P＜0.05).There was no significant difference between the LT-NP group and control group.The number memory CD8+T lymphocytes significantly decreased in LT-NP,HIV,and AIDS groups(P＜0.05).Of subsets,the number of CD8+TEM-1 increased in the same order as above,and the AIDS group was notably higher than the LT-NP group(P＜0.05).The absolute number of CD8+TEM-2 in the AIDS group was significantly lower than the LTNP group(P＜0.05).Comparing with healthy control, the number of CD8+TEM-1 in the HIV group,the percentage of CD8+TEM-1 in the AIDS group,the number of CD8+TEM-2 in the LTNP group significantly increased(P＜0.05),the percentage of CD8+TEM-2 in the AIDS group significantly decreased(P＜0.01).Naive CD8+T cell was in positive correlation with CD4+T cell number(P＜0.01) and in negative correlation with viral load(P＜0.01).The number of CD8+T TCM and TEM-2 were in positive correlation with CD4+T cell number(P＜0.01),but no relationship was observed with HIV viral load.2.The impact of HAART on the naive and memory CD8+T lymphocytes subsetsAfter the initiation of HAART,the percentages and absolute numbers of naive and TEM-2 CD8+T cells significantly increased(P＜0.01),and the percentage of TEM-1 CD8+T cells notably decreased(P＜0.01).3.The expression of co-receptor CCR5 and CXCR4 on the naive and memory CD8+T cells and their correlation to the progression of disease in HIV/AIDS patientsThe expression of CCR5 on the CD8+T cells increased in the order of the LTNP, healthy control,HIV and AIDS groups,and that of the LTNP group was significantly lower than the HIV and AIDS groups(P＜0.01).Comparing with healthy control group,the expression of CCR5 on naive and TEM-2 CD8+T cells in the HIV and AIDS groups notably increased(P＜0.05);the expression of CCR5 on TCM and TEM-1 CD8+T cells in the LTNP group significantly deceased(P＜0.05).In the LT-NP,HIV,and AIDS groups there is a increased tendency in the expression of CXCR4 on naive CD8+T cells.The expression level of CXCR4 on TCM-1 and TEM-1 CD8+T cells in the AIDS group was significantly higher than that of the LTNP group(P＜0.05).Except the TEM-1 subset,the expression of CCR5 on other CD8+T cell subsets was in inverse correlation with CD4+T absolute number(P＜0.05).The level of CXCR4 on TCM CD8+T cell was in positive correlation with CD4+T cell number(P＜0.05),and in inverse correlation with viral load(P＜0.05).The absolute number of naive CD8+T cell was in inverse correlation with their level of CCR5 expression(P＜0.01).The number of TEM-2 CD8+T cell was in negative correlation with the their lewls of CCR5 and CXCR4(P＜0.05).There were no correlation observed between the other subsets and receptors.4.The ki67 levels,activation of T lymphocytes subsets and their correlation to the progression of disease in HIV/AIDS patientsIn the LT-NP,HIV,and AIDS groups the ki67 levels in CD4+ and CD8+T lymphocytes and their subsets significantly increased(P＜0.05),and no statistical differences were observed between that of the LTNP and healthy control group.In the respect of the subset distribution,the number of naive T cell increased 2-4 folds,and the number of memory increase 3-5 folds.In the LT-NP,HIV,and AIDS groups,the levels of CD38 on CD4+ and CD8+T cell increased gradually.Except the naive and TEM-2 CDS+T subsets,the expression of CD38 on other subsets increased,and that of the AIDS group was significantly higher than the LTNP and healthy control groups(P＜0.05=.The levels of intra-cellular ki67 on CD4+ and CD8+T cells were in negative correlation with CD4+T cell number(P＜0.01=,and in positive correlation with the level of CD38(P＜0.05=.The level of intra-cellular ki67 on CD4+T cells was in positive correlation with that of CD8+T cells(P＜0.01).Conclusion1.In HIV-1 infected Chinese,the frequency of naive CD8+T cells continuously decreased in the LTNP,HIV,and AIDS group and correlated positive with disease progression;the frequency of memory CD8+T cells,expecially TEM-1 CD8+T cells continuously increased,but TEM-2 CD8+T cells significantly decreased. 2.In HIV-1 infected long-termnonprogress Chinese,the frequency of memory CD8+T cells,expecially TEM-2 CD8+T cells significantly increased,which indicates TEM-2 CD8+T cells may be one of the protective factors to delay the disease progression of HIV infection.3.After the initiation of HAART,the frequency of naive CD8+T cells,TCM and TEM-2 CD8+ T cells significantly increased,which indicates HAART may improve the immune status of HIV infected Chinese.4.In HIV-1 infected Chinese,the expression of CCR5 on naive and TEM-2 CD8+T cells significantly increased and correlated positive with disease progression, correlated negative with the number of naive and TEM-2 CD8+T cells.The expression of CXCR4 on naive T cells significantly decreased,but there is a increased tendency in the expression of CXCR4 on naive CD8+T cells.5.The level of the expression of CCR5 on CD8+T-cell subsets is siginificantly lower in LTNP than that in the HIV and AIDS group,even lower than that in healthy control group.The results indicates the lower level of CCR5 on CD8+T cells may delay the disease progression of HIV infection.6.The ki67 levels in CD4+ and CD8+T lymphocytes and their subsets significantly increased,and correlated positive with CD4+T absolute number and the activating levels of T lymphocytes.The results indicates the proliferation of T cells which associated with activation correlated positive with disease progression of HIV infected Chinese.