The Study On The CD8+T Lymphocyte Non-cytotoxic Response In The Long-term Non-progressors Of HIV Infected Individuals

ObjectiveThe 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.Methods1.Study populationAll 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 TCID₅₀Virus was lab-virus strain SF33 (donated by CDC in China). Revival MT4 cells, Cultures in 37℃5ï¼…CO₂ for 3 days. Collection the MT4, Washed with PBS twice at 300g for 10 minutes and add 600TCID₅₀ SF33, blow 20 times, cultures in 37℃5ï¼…CO₂ for 90 min. Washed with PBS twice at 300g for 10 minutes. Cultured with RPMI-1640 (adding IL-2 by 100U/ml) in 37℃5ï¼…CO₂. Virus replication was assessed by measuring p24 antigen in the culture supernatants.3.Seperate the CD4+/CD8+ T lymphocyte cellPBMC of subjects were isolated by standard Ficoll-hypaque density centrifugation. Remove monocytes. Per 1×10⁷ 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 lymphocyteAdjust the CD4+ T and the CD8+ T density to 1×10⁶cells/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 analysisSPSS 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.Results1.The purity of the CD4+/CD8+ T lymphocyte cellPurify the CD4+/CD8+ T: The purity of the cells is more than 95ï¼….2. The CNAR function in the LTNPOur 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 progressionWe 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＞LTNP4. CD4+ T true count and the inhibition ratioWe 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).Conclusion1.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.