【作者】 李珍；

【导师】 何维；

【作者基本信息】 北京协和医学院 ， 免疫学， 2012， 博士

【摘要】 人类获得性免疫缺陷综合症(Acquired Immune Deficiency Syndrome, AIDS)是由人类免疫缺陷病毒(Human Immunodeficiency Virus, HIV)感染所引起的以免疫系统缺陷、机会性感染、恶性肿瘤等为临床特征的一种传染性疾病。慢性免疫活化(Chronic immune activation)是HIV感染及其致病过程中的主要特点。HIV感染后,活化的免疫细胞的比例增高,同时表面表达活化、增殖和凋亡标记。免疫活化的水平比血浆病毒载量能更好地预测HIV感染的疾病进展。γδT细胞是一种固有免疫细胞,以非主要组织相容性复合物限制性的方式识别抗原、通过自然杀伤样的杀伤机制或分泌抗病毒因子杀伤肿瘤细胞或病毒感染的细胞,且具有免疫调节和抗原提呈作用,这些特点决定了γ6T细胞在HIV感染中发挥重要的作用。然而,HIV感染后γδ T细胞的活化状态与成因以及γδ T细胞活化与其数量、功能变化之间的相关性迄今尚未阐明。这是本文试图探讨的第一个科学问题。微小RNA (microRNA, miRNA)是一类长度为20-22个核苷酸,高度保守的非编码核糖核酸(Ribonucleic acid, RNA)分子,能够与靶基因的3’非翻译区(Untranslated region, UTR)结合,抑制目的基因的转录,或降解目的基因信使RNA(message, mRNA),从而对基因进行转录后调控。近来研究表明,miRNA在免疫系统中发挥重要作用,参与免疫细胞的分化发育、调节免疫细胞的功能等。然而,目前尚缺乏对γδ T细胞特异性miRNA的研究。尤其在HIV感染过程中,miRNA对γδ T细胞的调节作用尚未明确。因此,本文的第二个科学问题将集中在HIV相关γδ T细胞特异性microRNA的研究上。本研究分为两部分,第一部分主要检测HIV感染者在高效活化的抗逆转录病毒治疗(Highly activate anti-retroviral therapy, HAART)前后γδ T细胞活化状态、数量和功能的变化,并对其影响因素进行研究；第二部分是对HIV感染相关的γδ T细胞特异性miRNA进行筛选,旨在为HIV感染过程中γδ T细胞的功能研究提供新线索。围绕第一个科学问题,我们开展了第一部分工作。首先以74例HIV感染者包括46例未经HAART治疗和28例经HAART治疗的HIV感染者和21名健康对照为研究对象。流式细胞术检测外周血单个核细胞(Peripheral Blood Mononuclear Cells,PBMCs)中γ6T细胞活化标志CD38和人类白细胞抗原-DR(Human leukocyte antigen DR, HLA-DR)的表达。结果显示,HIV感染者γδT细胞CD38和HLA-DR的表达水平显著高于健康对照,提示HIV感染者γδT细胞处于高度活化状态,而HAART治疗不能有效降低γδ T细胞的活化水平。同时,本研究还发现活化γδ T细胞的比例与CD4+T细胞的数量无相关性,而与HIV感染者(CD4+T细胞>350个/μl)血浆病毒载量呈明显正相关。利用酶联免疫吸附试验(Enzyme-linked Immunoabsobent Assay, ELISA)检测发现,HIV感染者血浆中微生物转位产物如脂多糖(lipopolysaccharide, LPS)的含量与健康对照相比无明显增加。且活化的γδ T细胞的比例与血浆LPS的水平无相关性,提示微生物转位不是引起HIV感染者γδ T细胞的高度活化的主要原因。随后,我们运用流式细胞术,发现HIV感染者γδ T细胞的总数与健康对照没有显著差异。γδT细胞亚群的比例分析发现,HIV感染者Vδ1T细胞的比例增加,而Vδ2T细胞的比例减少,且HAART治疗不能改善这种趋势。此外,与健康对照相比,HIV感染者中心记忆(central memoryTCM,CD27+CD45RA-)和效应记忆(effector memoryTEM,CD2TCD45RA-)γδ T细胞的比例均降低。而终末分化(terminally differentiatedTEMRA,CD2TCD45RA+)γδ T细胞的比例显著增加。TNaive、TCM、TEM和TEMRAγδ T细胞表面CD38的表达显著高于健康对照。相关性分析显示,TCM和TEMγδ T细胞的比例与CD38+γδ T细胞的比例呈负相关,而TEMRAγδ T细胞的比例与CD38+γδ T细胞的比例呈显著正相关。上述结果提示,HIV感染者记忆γδT细胞数量的下降可能与γδ T细胞活化有关。流式细胞内染色发现,与健康对照相比,未治疗的HIV感染者分泌白细胞介素(Interleukin, IL)-17的γδ T细胞比例明显增加,而分泌干扰素(interferon, IFN)-γ的γδT细胞的比例减少。HAART治疗使分泌IL-17的γδ T细胞的比例降低,但是对分泌IFN-y的γδ T细胞的比例没有影响。此外,本研究对HIV感染者γδ T细胞的功能进行分析。利用乳酸脱氢酶(Lactate dehydrogenase, LDH)法检测HIV感染者γδ T细胞的细胞毒活性。结果显示,与健康对照相比,HIV感染者γδ T细胞对Daudi细胞的杀伤能力降低。随着疾病的进展,γδ T细胞的杀伤能力下降。而HIV感染者γδ T细胞能够杀伤自体HIV感染和未感染的CD4+T细胞。这一结果提示,γδ T细胞活化有可能引起CD4+T细胞的耗竭。为探讨HIV感染者γδT细胞杀伤能力下降的潜在机制,我们采用ELISA法检测血浆中可溶性MHC Ⅰ类链相关基因A/B编码蛋白(soluble MHC class Ⅰ chain-related A/B, sMICA/B)的含量。结果显示,HIV感染者血浆中sMICA/B的含量显著高于健康对照,且sMICA/B的含量随着病毒载量的增加而增加。提示,sMICA/B含量的增加可能与γδ T细胞的杀伤能力下降有关。最后,本研究采用ELISA检测发现,HIV感染者血浆中IL-23的含量较健康对照降低。流式细胞内染色发现,HIV感染者γδ T细胞细胞毒性分子穿孔素和颗粒酶B等的表达较健康对照降低。而高剂量(20ng/ml)IL-23可促进HIV感染者γδ T细胞表达这些细胞毒性分子。同时,高剂量IL-23能增强HIV感染者γδ T细胞对Daudi细胞的杀伤能力,但对健康对照γδ T细胞的杀伤能力无影响。上述结果提示,IL-23可能成为辅助HIV/AIDS临床免疫治疗的候选因子。本研究第二部分工作主要围绕第二个科学问题,希望能够发现HIV相关γδ T细胞特异性miRNA。我们采用实时荧光定量PCR (Quantitative real-time PCR, qRT-PCR)技术对HIV感染相关的γδ T细胞特异性miRNA进行筛选。首先,检测3例健康对照αβT细胞和γδ T细胞347条T细胞相关的miRNA的表达。获得13条差异表达的γδ T细胞miRNA。进而,扩大健康对照样本量(20例),对上述13条差异表达的人γδT细胞miRNA和小鼠γδ T细胞特异表达的11条miRNA进行第二轮筛选,共获得14条γδT细胞特异性miRNA。最后比较了HIV感染不同病程阶段和健康对照γδ T细胞14条miRNA的表达差异,获得11条差异表达的HIV相关γδT细胞特异性miRNA。综上所述,本研究的主要结论与创新点体现在：(1)HIV感染者γδ T细胞高度活化,而HAART治疗不能完全降低γδ T细胞的活化水平。(2)微生物转位并非引起HIV感染者γδ T细胞活化的原因。(3)HIV感染者记忆γδ T细胞数量的下降。(4)首次报道HIV感染者γδT细胞能够杀伤自体HIV感染的CD4+T细胞,且高剂量IL-23可增强HIV感染者γδ T细胞的杀伤能力,为HIV/AIDS的临床免疫治疗提供了新的思路。(5)首次筛选出HIV相关的γδ T细胞特异性miRNA,为进一步研究γδ T细胞在抗HIV感染中的作用展示了新的方向。更多还原

【Abstract】 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.更多还原