TLRs Expression And Immune Function In CD8⁺T Cells In HIV-1 Infection

?Background? Acquired immunodeficiency syndrome(AIDS) is a chronic infectious disease caused by human immunodeficiency virus(HIV). CD8⁺ T cells play a crucial role in the control of HIV-1 viremia. However, the underlying mechanisms are not fully understood. Toll-like receptors(TLRs), which can link innate immune responses and adaptive immune responses, activate signal transduction pathway in cells and lead to cytokines secretion. This is considered as the first barrier of host to defense the invading pathogens. Previous studies showed that TLRs are significantly related to HIV-1 infection. However, no studied are available regarding expression of TLRs on CD8⁺ T cells in HIV-1 infection. In this study, we examined the expression of TLR4,TLR5 and TLR7 on CD8⁺ T cells from 31 HIV-1 infected individuals and 20 healthy controls. TLRs expressed abnormally were screened out and follow-up verification and functional studies were carried out to elucidate the role of TLRs on CD8⁺ T cells in HIV-1 infection, and provide solid experimental basis for the development of novel targets for the design of vaccine and medicine. ?Objectives? 1. To screen out the TLRs expressed abnormally on CD8⁺T cells in HIV-1 infection; 2. To analyze the relationships between TLRs expression and CD4+T cells counts, HIV plasma viral loads and HAART; 3. To explore the function of TLR ligand on activation of CD8⁺ T cells. ?Methods? 1. Human CD8⁺ T cell Enrichment Cocktail was used for the purifying of CD8⁺ T cells.The expression of TLRs was analyzed by real-time PCR and flow cytometry; 2. CD4+ T cell counts and HIV viral load were determined by real-time PCR and flow cytometry. Statistic analyses were explored to value the correlation of TLRs expression and CD4+ T cells counts, HIV viral loads and HAART; 3. Fresh PBMCs were separated from whole blood by Ficoll-Hypaque (Sigma) density gradient centrifugation. The expression of CD69 after stimulation of CD8⁺ T cells with TLR7 ligand(0.5, 1 and 5?g/ml)for 6hr was analyzed by flow cytometry; 4. To explore the cytokine(IL-2,IFN-?) secretion by CD8⁺ T cells, ICS was used after stimulation with TLR7 ligand(0.5, 1 and 5?g/ml)for 6hr and 20hr. ?Results? 1. To examine the expression levels of TLR4, TLR5 and TLR7 during HIV-1 infection, we sorted CD8⁺ T cells from HIV-1 infected individuals and healthy volunteers and evaluated TLR-specific mRNA in CD8⁺ T cells by QRT-PCR. We observed that TLR4, TLR5 and TLR7 mRNA were detected across both populations. Compared with 20 healthy volunteers, 31 HIV-1 infected individuals had significantly increased TLR7 mRNA expression in freshly isolated CD8⁺ T cell(P<0.05), whereas no significant differences were observed for TLR4 and TLR5 expression(P>0.05). To further confirm the enhanced expression of TLR7 in CD8⁺ T cells in HIV-1 infected individuals, we examined the TLR7 expression at protein level in CD8⁺ T cells by flow cytometry. 31 HIV-1 infected individuals had significantly increased TLR7 protein expression in freshly isolated CD8⁺ T cells compared with 20 healthy volunteers(P<0.05). We found no statistically significant correlations between peripheral CD4+ T cell counts or plasma HIV RNA levels and expression of TLR4, TLR5 and TLR7. Furthermore, when dividing patients into groups according to receipt of antiretroviral therapy, CD4+ T cell count(>200cells/?l or <200cells/?l), and HIV RNA level(>105copies/ml or <105copies/ml), no differences in TLR4, TLR5 and TLR7 expression were observed. 2. To assess whether treatment with TLR7 ligand can increase immune activation of CD8⁺ T cells in HIV-1 infected individuals, we analyzed CD69 expression on purified CD8⁺ T cells, which is considered as early activation state marker after exposure to tested stimuli. Although following stimulation of CD8⁺ T cells with TLR7 ligand, CD69 expression on CD8⁺ T cells was significantly increased in HIV-1 infected individuals and healthy group at concentration of 0.5, 1 and 5?g/ml, the increase of CD69 was more significant in HIV-1 infected group(P<0.05). Moreover, CD69 expression was dose dependent and maximal at concentrations of 0.5?g/ml. In order to further assess whether the activation of CD8⁺ T cells by CL097 depended on the presence of accessory cells, we evaluated the CD69 expression of CD8⁺ T cells from HIV-1 infected individuals following stimulation of PBMC with CL097. As expected, treatment of PBMC with CL097 resulted in a significant increase in CD69 expression. However, no significant difference between PBMC and highly purified CD8⁺ T cells response to CL097 were seen in the amounts of CD69(P>0.05). 3. To examine whether the differences in TLR7 expression could have functional consequences, we analyzed the effect of CL097 at concentration of 0.5, 1 and 5?g/ml on IL-2 and IFN-?production by CD8⁺T cells from HIV-1 infected individuals and healthy control subjects. After 6hr and 20hr incubation with CL097, purified CD8⁺ T cells from two groups did not produce cytokines (IL-2 and IFN-?) significantly as assessed by intracellular cytokine staining. We subsequently studied cytokine-secretion of CD8⁺ T cells following stimulation of PBMC with TLR7 ligand at concentrations of 0.5, 1 and 5?g/ml. After 6hr incubation, CD8⁺ T cells from HIV-1 infected individuals did not produce IFN-?and IL-2. However, we observed that TLR7 ligand up-regulated IFN-?production by CD8⁺T cells and failed to modulate IL-2 production after 20hr incubation. The effects of TLR7 ligand on IFN-?production was dose dependent and maximal at concentration of 1?g/ml. ?Conclusions? 1. CD8⁺ T cells could express TLR4, TLR5 and TLR7. TLR7 expression in freshly isolated CD8⁺ T cells was significantly increased in HIV-1 infected individuals compared with healthy volunteers, which suggests that TLR7 may relate to HIV-1 infection. 2. TLR7 ligand can change the activation status of CD8⁺T cells from HIV-1 infected individuals. 3. TLR7 ligand can induce significant IFN-?secretion in CD8⁺ T cells in an accessory cell-dependent manner during HIV-1 infection.