Study On Activation Of T Lymphocyte And The Correlation Of Activation And Coreceptor Expression Of HIV-1 Infected Patients

IntroductionIn Human Immunodeficiency Virus - 1 ( HIV - 1) infection, the antigenic stimulation by HIV or other infections can cause T cell activation, which will attribute to the serious dysfunction of immune system. T cell activation provides an environment in vivo more conductive to enhance viral replication and it can also result in functional an-ergy, because preactivated T cells are unable to respond to cognate antigen. HIV -1 can use a number of corecptors to enter target cells in addition to CD4 receptor. The coreceptor CCR5 is expressed predominantly on activated memory cells and CXCR4 is expressed on un-activated phenotype. Since HIV - 1 can use different coreceptors at different stages and cellular activation is critical for productive infection in vivo, it can be postulated that the targets for HIV -1 infection must express an appropriate coreceptors and are activated. The activation of immune system will cause an alter of activation markers in circulating T cells and the increase of HLA - DR expression is considered as one of the most important markers of T cell activation.Although many evidence have been shown about the important role of T cell activation in HIV -1 infection and disease progression, there are no data about the T cell activation in HIV -1 infected Chi-nese. Because there are many differences between European people and Chinese, some of their immune markers are verified. We took the research of T cell activation and the correlation between activation and coreceptor expression about the HIV - 1 infected patients of our country to investigate the role of this marker in the evaluation of the immune status of them.Materials and methods1. Study objects24 HIV - 1 infected patients were selected from the AIDS Research Lab of the first affiliated hospital of China Medical University, age arrange 19 -41 years old. Most of them were injecting drug users. WB test was used to confirm the HIV -1 infection. 13 health persons who never exposed to HIV -1 were selected as normal controls. Blood was drawn by venipuncture from each subject in EDTA tubes for FACS analysis.2. Equipment and reagentsMost of the equipment and reagents were obtained from BECTON DICKINSON company, including flowcytometer ( FACSCalibur) ; 10ml EDTA venipuncture vials; FACS lysing solution and the following mAbs: TriTEST CD4/CD8/CD3 reagent,anti - CD4, anti - CDS (FITC conjugated) , anti - CCR5, anti - CXCR4 (PE conjugated) and anti - HLA - DR ( PerCP conjugated).3. CD4 absolute countThe 20ul TriTEST regents CD4/CD8/CD3 was added to the CD4 absolute vial and 50ul whole blood was added then. After a 15 - min incubation at room temperature, the erythrocytes were lysed usingFACS lysing solution. After another 15 - min incubation at room temperature, the vials were taken to perform FACS analysis by using MULTISET software. The absolute count and the percentage of CD4 + , CDS + and CDS + T cells were calculated automatically.4. Whole blood surface staining and FACS analysisFor each HIV - 1 infected patient, we used the following Ab combinations: CD4/CCR5/HLA - DR; CD4/CXCR4/HLA - DR; CD8/CCR5/HLA - DR; CD8/CXCR4/HLA - DR; no Abs and CD4 FITC. 80ul whole blood was added and the 6ul of each Abs were added as the combinations shown above. After a 30 - min incubation at room temperature, the erythrocytes were lysed using 2ml RBC lysing solution . The cells were washed twice with PBS and resuspended in 0.5ml PBS solution consist of 0. 5% polyformaldehyde. There color flow - cytometric analysis was performed and FAGS CELLQUEST software was used to make analysis. For subset analysis, lymphocyte were gated by forward scatter and side scatter, then CD4 + or CDS + T cells were gated and the HLA - DR expression of CD4 + , CD8 + T cells or CD4 + ,CD8 + T cells expressed CCR5 or CXCR4 were calculated.5. Statistical analysisSPSS 10. 0 software was used to make the Statistical analysis. Geometric means were determined for log - distributed variables. Comparisons of means of different gro...