Characterization Of The Phenotypes And Function Of γδT Cells In HIV-infected Individuals And The Identification Of HIV-associated γδ T Cell Specific MicroRNAs

Human acquired immune deficiency syndrome (AIDS) is an infectious disease, which is caused by human immunodeficiency virus (HIV) and characterized by immune system defects, opportunistic infections and malignancies. Chronic immune activation is a key characteristic of HIV infection and pathogenesis. The HIV-associated immune activation is typically characterized by a high frequency of activated immune cells, expressing markers of activation, proliferation, and apoptosis. The levels of chronic immune activation present in HIV-infected individuals could provide a better prediction of the progression and prognosis of AIDS than plasma viral load. γδ T cells, a crucial population of innate immune cells, recognize antigens in a non-major histocompatibility complex (MHC) restrict pattern and kill the infected cells or tumor cells in a natural killer-like (NK-like) manner or through the release of anti-viral factors. In addition, γδ T cells play important roles in immune regulation and antigen presentation. γδ T cells serve as a linker between the innate immune and the adaptive immune. All these features suggest that γδ T cells play crucial roles in HIV infection. However, the properties of γδ T cells in HIV-infected individuals have not been fully characterized. It is the first scientific question we want to discuss.MicroRNAs (miRNAs) are20-22nucleotide length non-coding ribonucleic acid (RNA) molecules. MiRNAs control the gene expression through posttranscriptional regulation by binding to the3'-untranslated region (UTR), thereby repressing the translation or inducing message RNA (mRNA) degradation. Recently, several studies demonstrated that miRNAs play very important roles in immune system, including the differentiation of immune cells or the functions of immune cells. However, there are few researches on miRNAs specifically related to γδ T cells. The profile of miRNAs of γδ T cells from HIV infected individuals is still unrevealed. Therefore, the second scientific question we will focus on the research on HIV-associated γδ T cell specific microRNAs.This study contains two parts. The first part focused on examining the activation status of γδ T cells, frequencies of γδ T cells, and the potential factors related to these characteristics. In the second part, we performed a screen of miRNAs specifically related to the HIV infection-associated γδ T cell.In part one,74HIV-infected individuals including46HIV-infected individuals without highly activate anti-retroviral therapy (HAART) and28HIV-infected individuals with HAART treatment and21healthy controls were recruited. Flow cytometry was performed to examine the activation markers CD38and Human Leukocyte Antigen-DR (HLADR) on γδ T cells from HIV-infected patients and healthy controls. The result showed that the frequencies of CD38+and/or HLADR+γδ T cells were significantly elevated in HIV-infected individuals compared to healthy controls. HAART treatment could not reduce the level of γδ T cell activation. Activated γδ T cells were positively correlated to the plasma viral load in HIV-infected individuals with CD4+T cells more than350cells/μl, but not to CD4+T cell count. The levels of microbial translocation product lipopolysaccharide (LPS) in the plasma of HIV-infected patients and healthy controls were measured by enzyme-linked immunoabsobent assay (ELISA). No significant difference was found in levels of plasma LPS between HIV-infected patients and healthy controls, indicating that microbial translocation is not the cause of γδ T cells activation in HIV-infected individuals.Next, the frequencies of γδ T cells in HIV-infected individuals were assessed by using flow cytometry. No change was observed in the total cell numbers of γδ T cells between the healthy controls and HIV-infected individuals. However, the frequency of Vδ1T cells was significantly increased in HIV-infected individuals when compared to that of the healthy control. In contrast, the frequency of Vδ2T cells was decreased in HIV-infected individuals. HAART treatment cannot restore this bias. In addition, compared to healthy controls, the frequencies of central memory γδ T cells (TCM, CD27+CD45RA-) and effector memory γδ T cells (TEM, CD27-CD45RA-) were reduced in HIV-infected individuals. However, the frequency of terminally differentiated (TEMRA, CD27-CD45RA+)γδ T cells was significantly increased in HIV-infected individuals. Compared to healthy donors, the expression levels of CD38on TNaive, TCM, TEM and TEMRA γδ T cells were significantly elevated in HIV-infected individuals. Correlation analysis showed that the frequencies of Tcm and TEM cells were negatively correlated to that of CD38+γδ T cells, but the frequencies of TEMRA γδ T cells were positively correlated to that of CD38+γδ T cells. These results taken together indicate that the activation of γδ T cells might lead to the decrease of memory γδ T cells. We also found that the frequency of interleukin (IL)-17+(IL-17+)γδ T cells was increased in HIV-infected patients without HAART treatment compared to the healthy controls, but the frequency of interferon (IFN)-γ+γδ T cells were decreased. HAART treatment could reduce the frequency of IL-17+γδ T cells, but it had little effect on IFN-γ+γδ T cells.In addition, lactate dehydrogenase (LDH) assay was performed to assess the cytotoxicity of γδ T cells. Compared to healthy controls, the cytotoxicity of y8T cells to Daudi cells was reduced in HIV-infected patients and with the disease progression the cytotoxicity was lower. Intriguingly, γδ T cells from HIV-infected individuals could kill the autologous HIV-infected and uninfected CD4+T cells, indicating that activated γδ T cells might contribute to the depletion of CD4+T cells.To investigate the possible mechanisms responsible for the reduction of cytotoxicity of γδ T cells in HIV-infected individuals, the levels of soluble MHC class I chain-related A/B proteins (sMICA/B) were measured by using ELISA. The result showed that the levels of sMICA/B in the plasma of HIV-infected individuals were significantly elevated when compared to healthy controls. Moreover, the increased levels of sMICA/B were accompanied with the increase of plasma viral load. These results suggest that the elevated level of sMICA/B may be an important cause for the lower cytotoxicity of γδ T cells in HIV-infected individuals.Finally, we also found that the level of IL-23in the plasma of HIV-infected individuals were lower than that in the healthy controls. The expression of cytotoxicity molecules such as perforin and granzymeB in γδ T cells were reduced in HIV-infected individuals. The administration of high dose of IL-23could increase the expression of these molecules and enhance the cytotoxicity of γδ T cells in HIV-infected individuals in vitro. But it had little effect on the cytotoxicity of y8T cells in healthy controls. These results suggest that IL-23might be a potential novel candidate for HIV/AIDS treatment.In the second part, we look forward to find some miRNAs which specifically expressed by γδ T cells and associated with HIV infection. Quantitative real-time PCR (qRT-PCR) was used to screen the miRNAs specifically related to HIV infection-associated γδ T cell. First, the expression profiles of347miRNAs were detected in both γδ T cells and αβ T cells from3healthy donors.13miRNAs differentially expressed by γδ T cells were identified. The expression profiles of these13miRNAs combined with11miRNAs from mouse y8T cells were further assessed in20 healthy donors.14miRNAs showed differentially expression in γδ T cells. By comparison of the expression profiles of these14miRNAs in γδ T cells from healthy controls and HIV-infected individuals,11miRNAs specifically related HIV infection-associated γδ T cells were identified.In conclusion, our findings in this study include:(1)γδ T cells were hyperactivated in HIV-infected individuals and HAART treatment cannot reduce the activation status of γδ T cells;(2) Microbial translocation is not the major cause for γδ T cells activation;(3) Memory γδ T cells were reduced in HIV-infected individuals;(4) To our knowledge, it's the first time to report that γδ T cells from HIV-infected individuals could kill the autologous HIV-infected or uninfected CD4+T cells, and high doses of IL-23could increase the cytotoxicity of γδ T cells from HIV-infected individuals, which provide a new strategy for the clinical treatment of HIV/AIDS;(5) It is the first time to report the y8T cells specific miRNAs in HIV infection, which provide a new direction for better understanding the roles of γδ T cells in HIV infection.