【作者】 王会芳；

【导师】 曾庆磊；

【作者基本信息】 郑州大学 ， 内科学（感染）， 2025， 博士

【摘要】 研究背景自1981年首例艾滋病(HIV/AIDS)病例报道以来,HIV/AIDS逐渐在全球范围内持续流行。根据世界卫生组织(WHO)统计数据显示,截至2023年底,全球现存HIV感染者约有3990万例,当年新增死亡病例约63万。值得注意的是,我国法定传染病疫情监测数据显示,艾滋病的死亡率和死亡人数已连续多年位居传染病首位。迄今为止,HIV/AIDS仍无法被彻底治愈,艾滋病依然是21世纪最具威胁性的全球性公共卫生危机之一。目前,已有30多种抗逆转录病毒药物成功研发并获批,联合疗法已将艾滋病从一种致死性疾病转变为慢性可控的疾病。最新研究表明,尽管有效的抗逆转录病毒治疗(ART)显著延长了HIV感染者(PLWH)的总体预期寿命,使其接近健康人群的水平。然而,PLWH的无合并症预期寿命仍显著低于健康人群,这主要归因于高风险的慢性合并症,如心脑血管疾病、神经认知功能障碍、代谢综合征等衰老相关疾病。在衰老相关疾病的机制研究中,随年龄增长而持续累积的系统性炎症是核心问题,这一现象被称为“炎性衰老”。HIV感染后,肠道屏障的破坏、菌群异位、巨细胞病毒/EB病毒(CMV/EBV)等共感染、潜伏病毒再激活以及抗病毒药物毒性的积累,共同加剧了炎性衰老的进程。然而,其中潜在的免疫学机制仍缺乏深入研究。HIV感染后,免疫系统的破坏主要表现为CD4+T细胞数量的持续下降及功能的耗竭,而CD8+T细胞则由于持续的免疫激活则处于异常高位水平。尽管有效的ART可将CD4+T细胞恢复至接近健康水平,但其对CD8+T细胞数量和功能的恢复作用非常有限。研究发现CD8+T细胞的持续克隆扩增与慢性炎症及非AIDS相关事件的高风险均密切相关,但其具体的作用机制尚未阐明。研究目的本研究旨在:(1)系统解析PLWH不同疾病阶段T细胞库紊乱的动态变化特征;(2)鉴定持续克隆扩增T细胞的表型特征;(3)揭示其在驱动PLWH炎性衰老中的作用机制,为PLWH过早衰老的干预提供新的理论依据。研究方法采用多组学整合分析策略:(1)大规模队列研究,纳入228例PLWH和257例健康对照(HC),通过TCRβ测序评估克隆扩增与多样性的变化特征,并构建基于TCR指标的生物学年龄预测模型;(2)单细胞多组学分析:分析9例PLWH及4例HC的单细胞RNA联合TCR测序数据,筛选表征持续克隆扩增T细胞亚群的特征基因,并通过流式细胞术探究其表型特征;(3)通过流式细胞术、高通量多因子检测、数字Western blot及体外共培养实验,建立了克隆扩增T细胞与持续炎症之间的联系,并初步探索了克隆扩增T细胞亚群驱动炎性衰老的免疫学机制。研究结果1、TCRβ库的动态特征:与HC相比,PLWH组中TCRβ库的克隆扩增水平显著增高、多样性显著降低,即使经过长期抗逆转病毒治疗也无法恢复到HC水平。克隆扩增的程度与治疗持续时间、病毒学反应速度和免疫重建结局均显著相关。基于TCR克隆性及多样性指标的生物学年龄预测模型显示,HC表现为非线性衰老模式,分别在28岁和54岁出现两个衰老高峰;而PLWH表现出持续的衰老加速轨迹,在年轻患者中尤为显著,无明显的衰老高峰。2、克隆扩增细胞亚群鉴定:通过单细胞RNA联合TCR测序数据分析,CD8+T细胞的克隆扩增水平显著高于CD4+T细胞。对克隆扩增频率相关基因的筛选及验证,发现颗粒溶素(GNLY)是与克隆扩增频率相关性最高的基因,在流式细胞术中具有较好的分群特点,并与其他细胞毒标志物具有高度共表达的特点。相对于GNLY-CD8+T细胞在ART后的克隆收缩,GNLY+CD8+T细胞在ART后仍然稳定克隆扩增。此外,与GNLY-CD8+T细胞相比,GNLY+CD8+T细胞具有CD27、CD28、PD-1低表达,CD57、GZMB、Perforin、CX3CR1、T-bet、NKG2D、KLRG1高表达的表型特点。3、促炎机制探索:IL-15(50 ng/m L,48h)可在体外显著刺激GNLY+CD8+T细胞的频率升高、HLA-DR、GZMB及Perforin的表达上调。GNLY+CD8+T细胞的频率和绝对数与炎症因子(IP-10、MIG)和肠道损伤标志物(Zounlin、PGRPS、s CD14)显著正相关。CD8+T细胞中的GNLY以促炎型的15 kDa亚型为主,富含GNLY的细胞上清能够诱导单核细胞分泌IP-10、MIG,诱导肠上皮细胞的Zounlin表达升高,紧密连接蛋白Claudin、ZO-1表达下调,抗-GNLY抗体能够部分逆转这一效应。研究结论本研究结果表明,HIV感染导致TCRβ库的持续克隆扩增及PLWH的持续衰老加速;GNLY+CD8+T细胞是PLWH中持续克隆扩增的主要细胞群,具有诱导单核细胞激活及破坏肠上皮细胞完整性的效应,在PLWH的炎性衰老过程中具有重要作用。这些结果为深入理解HIV相关免疫衰老的病理机制提供了新的视角,并为开发针对炎性衰老的干预策略构建了的理论基础。第一部分HIV感染者TCRβ库的动态变化特征目的本部分旨在阐明HIV感染对TCRβ库的影响,解析PLWH不同治疗阶段TCRβ库的动态演变特征,并构建基于TCR指标的生物学年龄模型以评估PLWH的衰老特征。方法本部分采用横断面队列研究,纳入228例PLWH(包括未治疗组、ART<24m即治疗波动期患者、ART>24m即治疗稳定期患者)及年龄、性别匹配的健康对照组(n=257)。基于高通量测序技术分析外周血TCRβ克隆组成,分析HIV感染对TCRβ库克隆性及多样性的影响;根据ART时长、病毒学应答速度(在ART启动后3个月内病毒载量低于检测下限为快速应答组)及免疫重建结局(外周血CD4+T细胞数目>500个/μL为免疫应答良好组)进行分层比较,进一步对比分析TCRβ库的动态变化特征及其影响因素;分别利用TCRβ库的克隆性及多样性指标构建生物学年龄预测模型,对比分析HC及PLWH的衰老特征。结果1.与HC相比,PLWH的TCRβ库表现出克隆性升高和多样性下降的特征;随着ART时间的延长,PLWH的克隆扩增水平逐渐下降,多样性逐步升高,但即使经过长期抗病毒治疗(>24m)到达病情稳定期,其克隆扩增水平仍高于HC,多样性水平仍低于HC。2.病毒学慢速应答组的TCRβ克隆扩增水平显著高于快速应答组,免疫应答不良组的TCRβ克隆扩增水平要显著高于免疫应答良好组,TCRβ库的多样性则呈相反趋势。PLWH中克隆扩增的水平与治疗持续时间、CD4+T细胞计数、CD4/CD8比值呈显著负相关,与CD8+T细胞计数呈显著正相关。3.基于TCR克隆性及多样性指标的生物学年龄预测模型显示,HC表现为非线性衰老模式,分别在28岁和54岁出现两个衰老高峰;而PLWH表现出持续的衰老加速轨迹,在年轻患者中尤为显著,且无明显的衰老高峰。结论HIV感染导致TCRβ库持续性的克隆水平显著升高及多样性显著降低,其动态演变与病毒抑制效率及免疫重建结局显著相关。基于TCRβ库指标的生物学年龄模型提示,PLWH表现出明显的衰老加速现象,在年轻患者中尤为显著。为HIV感染加剧炎性衰老的相关研究提供了新的生物学指标。第二部分GNLY+CD8+T细胞是HIV感染者中持续克隆扩增的关键细胞亚群目的本部分旨在鉴定PLWH中持续克隆扩增的关键细胞亚群,并解析其表型特征。方法本部分采用整合单细胞多组学与流式验证策略,研究纳入了包含HC(n=4)、PLWH(n=9)的单细胞RNA联合TCR测序数据,通过数据质控、标准化和数据合并等处理步骤,对比CD4+T细胞与CD8+T细胞的克隆扩增程度,明确驱动克隆扩增的关键淋巴细胞亚群。基于相关性分析,筛选出与克隆扩增频率高度相关的候选基因簇,并对比不同基因簇在ART后的克隆维持能力。随后,对候选基因进行排序,对比其表征克隆扩增细胞群的特异性及敏感性,进一步通过多色流式在蛋白层面验证,确定持续克隆扩增细胞群的特征基因。最终,以持续克隆扩增细胞群为对象,利用单细胞测序数据和多色流式在转录组和蛋白层面联合解析其表型特征。结果1.CD8+T细胞的克隆扩增程度显著高于CD4+T细胞。2.GNLY是与CD8+T细胞克隆扩增相关性最高的候选基因,具有表征克隆扩增细胞群较高的特异性和敏感性。相对于GNLY-CD8+T细胞在ART后的克隆收缩,GNLY+CD8+T细胞在ART后表现出持续克隆扩增的特点。3.相对于GNLY-CD8+T细胞,GNLY+CD8+T细胞的CD27、CD28、PD-1表达下调,CD57、GZMB、Perforin、CX3CR1、T-bet、NKG2D及KLRG1的表达水平显著升高。结论T细胞的克隆扩增主要由CD8+T细胞驱动。GNLY+CD8+T细胞在PLWH中表现出显著的克隆扩增和维持能力,具有复制性衰老、高细胞毒、低耗竭及NK受体高表达等特点,提示该亚群在PLWH持续慢性炎症微环境中具有重要的适应性存活机制及病理性意义。第三部分GNLY+CD8+T细胞克隆扩增及促炎机制研究目的本部分旨在探索GNLY+CD8+T细胞持续克隆扩增的上游调控机制,并阐明其驱动炎性衰老的免疫学机制。方法本部分采用单细胞数据分析联合功能实验的策略,首先对比GNLY+CD8+T细胞亚群在ART前后,其TCR依赖性和旁观者激活信号通路的动态变化;利用流式高通量多因子检测、ELISA和流式细胞术检测了与旁观者激活通路相关的血浆细胞因子水平及其相应受体的表达情况;随后,通过体外多种细胞因子(IL-12、IL-15、IL-18)及CD3/28刺激实验,模拟并对比不同刺激信号对其增殖、活化等表型的影响。采用流式细胞术联合数字Western blot技术,鉴定了CD8+T细胞中GNLY的具体亚型,并分析了其与多种炎症因子的相关性。最终,将IL-15刺激后的CD8+T细胞上清与THP-1单核细胞及NCM460肠上皮细胞共培养48小时,通过抗GNLY抗体阻断实验,评估GNLY激活单核细胞和破坏肠上皮细胞的直接效应。结果1.GNLY+CD8+T细胞的在旁观者激活通路信号在ART后持续增强,IL-15(50 ng/m L,48h)可显著刺激GNLY+CD8+T细胞的频率升高、HLA-DR、GZMB及Perforin的表达上调。2.CD8+T细胞中以促炎型15 kDa亚型为主,GNLY+CD8+T细胞的频率和绝对计数与血浆中IP-10、MIG、Zonulin、PGRP-S和s CD14的水平呈显著正相关。3.富含GNLY的细胞上清可以显著诱导单核细胞分泌IP-10、MIG,诱导肠上皮细胞的Zounlin表达升高,紧密连接蛋白Claudin、ZO-1表达下调,抗-GNLY抗体可以部分逆转这一效应。结论IL-15介导的旁观者激活通路驱动GNLY+CD8+T细胞的克隆扩增和活化,其分泌的15kDa的GNLY直接激活单核细胞并破坏肠上皮细胞的完整性,共同加剧了PLWH的炎性衰老。更多还原

【Abstract】 Research BackgroundSince the first case of HIV/AIDS was reported in 1981,the disease has gradually spread globally.According to data from the World Health Organization(WHO),as of the end of 2023,there were approximately 39.9 million people living with HIV(PLWH)worldwide,with about 630,000 deaths from the disease in that year.Notably,data from China’s notifiable infectious disease surveillance system indicate that AIDS has had the highest mortality rate and number of deaths among infectious diseases for several consecutive years.To date,HIV/AIDS remains incurable and continues to be one of the most threatening global public health crises of the 21st century.Currently,more than 30 antiretroviral drugs have been successfully developed and approved,and combination therapy has transformed AIDS from a fatal disease into a chronic,manageable condition.Recent studies show that although effective antiretroviral therapy(ART)significantly extends the overall life expectancy of PLWH to levels close to those of healthy individuals,their morbidity-free life expectancy remains significantly lower than that of healthy people.This is primarily due to the high risk of chronic comorbidities(such as chronic liver disease,chronic kidney disease,chronic lung disease,diabetes,cancer,and cardiovascular disease)that lead to premature aging in PLWH.In the study of aging-related diseases,the continuous accumulation of systemic inflammation with age is a core issue,known as"inflammaging."After HIV infection,the disruption of the intestinal barrier,microbial translocation,co-infections with CMV/EBV,reactivation of latent viruses,and the accumulation of antiviral drug toxicity all contribute to the acceleration of inflamm-aging.However,the underlying immunological mechanisms remain poorly understood.HIV infection primarily disrupts the immune system by causing a continuous decline in CD4+T cell numbers and functional exhaustion,while CD8+T cells remain at abnormally high levels due to persistent immune activation.Although effective ART can restore CD4+T cells to near-normal levels,its effects on CD8+T cell numbers and function are very limited.Studies have shown that the continuous clonal expansion of CD8+T cells is closely related to chronic inflammation and the high risk of non-AIDS-related events,but the specific mechanisms remain unclear.Research ObjectiveThis study aims to:(1)systematically analyze the dynamic evolution of T cell repertoire disorders in different disease stages of PLWH;(2)identify the phenotypic characteristics of persistently clonally expanded T cells;and(3)elucidate their role in driving inflammaging in PLWH,providing new theoretical basis for interventions targeting premature aging in PLWH.Research MethodsA multi-omics integrative analysis strategy was employed:(1)A large-scale cohort study was conducted,including 228 PLWH and 257 healthy controls(HC).TCRβsequencing was used to assess clonal expansion and diversity,and a biological age prediction model based on TCR indicators was constructed.(2)Single-cell multi-omics analysis:Single-cell RNA sequencing combined with TCR sequencing was performed on samples from 9 PLWH and 4 HC to identify characteristic genes of persistently clonally expanded T cell subsets,which were further validated by flow cytometry.(3)Flow cytometry,high-throughput multiplex detection,digital Western blot,and in vitro co-culture experiments were used to establish the link between clonally expanded T cells and chronic inflammation,and to preliminarily explore the immunological mechanisms by which clonally expanded T cell subsets drive inflammaging.Research Results1.Dynamic features of the TCRβrepertoire:Compared with HC,PLWH exhibited significantly higher clonal expansion and lower diversity in the TCRβrepertoire,which could not be restored to HC levels even after long-term antiretroviral therapy.The degree of clonal expansion was significantly associated with treatment duration,virological response rate,and immune reconstitution outcomes.The biological age prediction model based on TCR clonality and diversity that showed HC exhibited a non-linear aging pattern with two aging peaks at 28 and 54 years of age,while PLWH showed a continuous accelerated aging trajectory,especially in younger patients,without distinct aging peaks.2.Identification of Clonally Expanded Cell Subsets:Through single-cell RNA sequencing combined with TCR sequencing,CD8+T cells were found to have significantly higher clonal expansion levels than CD4+T cells.GNLY was identified as the gene most highly correlated with clonal expansion frequency,with good clustering characteristics in flow cytometry and high co-expression with other cytotoxic markers.Unlike GNLY-CD8+T cells,GNLY+CD8+T cells continued to expand clonally after ART.Additionally,GNLY+CD8+T cells exhibited a phenotype characterized by low expression of CD27,CD28,and PD-1,and high expression of CD57,GZMB,Perforin,CX3CR1,T-bet,NKG2D,and KLRG1.3.Exploration of Pro-inflammatory Mechanisms:IL-15(50 ng/m L,48h) significantly increased the frequency of GNLY+CD8+T cells and upregulated the expression of HLA-DR,GZMB,and Perforin in vitro.The frequency and absolute number of GNLY+CD8+T cells were positively correlated with inflammatory cytokines(IP-10,MIG)and intestinal damage markers(Zonulin,PGRPS,s CD14).The GNLY in CD8+T cells was predominantly the pro-inflammatory 15 k Da isoform.Supernatants rich in GNLY could induce IP-10 and MIG secretion from monocytes and increase Zonulin expression in intestinal epithelial cells,while downregulating tight junction proteins Claudin and ZO-1.These effects could be partially reversed by anti-GNLY antibodies.Research ConclusionsOur study demonstrates that HIV infection leads to persistent clonal expansion of the TCRβrepertoire and accelerated aging in PLWH.GNLY+CD8+T cells represent the key subset of persistently clonally expanded cells in PLWH,with the capacity to activate monocytes and disrupt intestinal epithelial cell integrity,playing a significant role in the inflammaging process of PLWH.These findings provide new insights into the pathogenesis of HIV-related immune aging and lay the theoretical foundation for developing interventions targeting inflammaging.Part Ⅰ Dynamic Characteristics of TCRβ Repertoire in PLWHObjectiveThis part aims to elucidate the impact of HIV infection on the TCRβ,repertoire analyze the dynamic evolution of the TCRβrepertoire in different treatment stages of PLWH,and construct a biological age model based on TCR indicators to assess aging characteristics in PLWH.MethodsA cross-sectional cohort study was conducted,including 228 PLWH(untreated group,ART<24 months group,and ART>24 months group)and 257 age-and sex-matched healthy controls.High-throughput sequencing was used to analyze the clonal composition of peripheral blood TCRβ,and the impact of HIV infection on TCRβclonality and diversity was assessed.Stratified comparisons were made based on ART duration,virological response rate(viral load below the detection limit within3 months of ART initiation defined as rapid responders),and immune reconstitution outcomes(peripheral blood CD4+T cell count>500 cells/μL defined as good immune responders).Biological age prediction models were constructed using TCRβclonality and diversity indicators,and aging characteristics were compared between HC and PLWH.Results1.Compared with HC,PLWH exhibited elevated clonal expansion and reduced diversity in the TCRβrepertoire.With prolonged ART,the clonal expansion level gradually decreased,and diversity increased in PLWH,but even after long-term antiviral treatment(>24 months),the clonal expansion level remained higher than of that HC,and diversity remained lower.2.The TCRβclonal expansion level was significantly higher in the slow virological responders and poor immune responders compared with their respective counterparts.Conversely,TCRβrepertoire diversity showed the opposite trend.Clonal expansion levels in PLWH were significantly negatively correlated with treatment duration,CD4+T cell count,and CD4/CD8 ratio,and positively correlated with CD8+T cell count.3.The biological age prediction model based on TCR clonality and diversity indicators revealed a non-linear aging pattern in HC with two aging peaks at 28 and54 years of age,while PLWH showed a continuous accelerated aging trajectory,especially in younger patients,without distinct aging peaks.ConclusionsHIV infection leads to persistent and significant elevation in TCRβclonality and reduction in diversity,with dynamic evolution closely related to viral suppression efficiency and immune reconstitution outcomes.The biological age model based on TCRβrepertoire indicators indicates an accelerated aging phenomenon in PLWH,particularly in younger patients,providing new biological indicators for research on HIV-related inflammaging.Part Ⅱ GNLY+CD8+T Cells Are the Primary Clonally Expanded Population in PLWHObjectiveThis part aims to identify the key subset of persistently clonally expanded cells in PLWH and elucidate their phenotypic characteristics.MethodsAn integrative single-cell multi-omics and flow cytometry validation strategy was employed.Single-cell RNA sequencing combined with TCR sequencing was performed on samples from 4 HC and 9 PLWH.Data quality control,normalization,and merging were conducted to compare the clonal expansion levels of CD4+and CD8+T cells,identifying the key lymphocyte subset driving clonal expansion.Candidate gene clusters highly correlated with clonal expansion frequency were screened,and their clonal maintenance capacity after ART was compared.Candidate genes were ranked based on their specificity and sensitivity in characterizing clonally expanded cell populations,and further validated at the protein level using multicolor flow cytometry.The phenotypic characteristics of persistently clonally expanded cell populations were jointly analyzed at the transcriptional and protein levels using single-cell sequencing data and multicolor flow cytometry.Results1.CD8+T cells exhibited significantly higher clonal expansion levels than CD4+T cells.2.GNLY was identified as the gene most highly correlated with CD8+T cell clonal expansion,with high specificity and sensitivity in characterizing clonally expanded cell populations.Unlike GNLY-CD8+T cells,GNLY+CD8+T cells continued to expand clonally after ART.3.Compared with GNLY-CD8+T cells,GNLY+CD8+T cells showed downregulation of CD27,CD28,and PD-1,and upregulation of CD57,GZMB,Perforin,CX3CR1,T-bet,NKG2D,and KLRG1.ConclusionsClonal expansion of T cells is primarily driven by CD8+T cells.GNLY+CD8+T cells exhibit significant clonal expansion and maintenance capacity in PLWH,characterized by replicative senescence,high cytotoxicity,low exhaustion,and high expression of NK receptors.These findings suggest the adaptive survival mechanisms and pathological significance of this subset in the chronic inflammatory microenvironment of PLWH.Part Ⅲ Mechanisms of GNLY+CD8+T Cell Clonal Expansion and Pro-inflammatory EffectsObjectiveThis part aims to explore the upstream regulatory mechanisms of persistent clonal expansion of GNLY+CD8+T cells and elucidate their immunological mechanisms in driving inflammaging.MethodsA strategy combining single-cell data analysis and functional experiments was employed.First,the dynamic changes in TCR-dependent and bystander activation signaling pathways of GNLY+CD8+T cell subsets were compared before and after ART.High-throughput multiplex detection of plasma cytokine levels and their receptor expressions related to bystander activation pathways were performed using flow cytometry,ELISA,and flow cytometry.Subsequently,in vitro stimulation experiments with various cytokines(IL-12,IL-15,IL-18)and CD3/28 were conducted to compare the effects of different stimulation signals on proliferation and activation.Flow cytometry combined with digital Western blot was used to identify the specific GNLY isoforms in CD8+T cells and analyze their correlations with multiple inflammatory cytokines.Finally,supernatants from IL-15-stimulated CD8+T cells were co-cultured with THP-1 monocytes and NCM460 intestinal epithelial cells for 48 hours,and the direct effects of GNLY on monocyte activation and intestinal epithelial cell integrity were assessed using anti-GNLY antibody blockade experiments.Results1.The bystander activation signaling pathway in GNLY+CD8+T cells was continuously enhanced after ART,and IL-15(50 ng/m L,48h)significantly increased the frequency of GNLY+CD8+T cells and upregulated the expression of HLA-DR,GZMB,and Perforin.2.The 15 k Da pro-inflammatory isoform of GNLY was predominant in CD8+T cells.The frequency and absolute count of GNLY+CD8+T cells were positively correlated with plasma levels of IP-10,MIG,Zonulin,PGRP-S,and s CD14.3.Supernatants rich in GNLY significantly induced IP-10 and MIG secretion from monocytes and increased Zonulin expression in intestinal epithelial cells,while downregulating tight junction proteins Claudin and ZO-1.These effects could be partially reversed by anti-GNLY antibodies.ConclusionsIL-15-mediated bystander activation drives the clonal expansion and activation of GNLY+CD8+T cells.The secretion of the 15 k Da pro-inflammatory isoform of GNLY directly activates monocytes and disrupts the integrity of intestinal epithelial cells,thereby exacerbating inflammaging in PLWH.更多还原