背景：截至目前，二十年代初期爆发的新型冠状病毒（SARS-CoV-2）大流行是百年以来最大的公卫危机事件，给全球卫生医疗系统造成了巨大冲击，更给人类健康带来了极大的威胁和挑战。随着全球化发展和世界变局，后疫情时代呼吸道疾病传播的预防不但会是公卫未来工作的重点，还需要更有效的防御手段。这次新冠大流行掀起了全球研发疫苗（vaccine）的热潮，各种新型疫苗层出不穷，毋庸置疑在疫情防控中发挥了关键作用。然而接下来，如何激发和拥有持久的免疫记忆（long-term immune memory）以应对病毒长期存在的威胁正在成为人们最为关切的问题。

     疫苗接种能激活T细胞反应，包括CD8⁺ T细胞介导的抗原特异性免疫反应，在机体抵御病原微生物入侵中发挥关键作用。CD8⁺ T细胞是免疫记忆形成的核心，CD8⁺ T细胞遇到抗原刺激时增殖分化为中央记忆细胞（central memory t cell, Tcm）、效应记忆细胞（effector memory t cell, Tem）以及组织驻留记忆细胞（tissue resident memory t cell, Trm），介导更快速的病原体清除过程。因此，如何让机体产生更多记忆T细胞是疫苗设计的重点。药用植物所含活性成分很多兼具安全、有效与毒副反应少的特点，其中不乏促进和调控免疫颇佳的天然化合物（natural compound），是探寻治疗免疫相关疾病潜在药物的主要来源，也是发现新型疫苗佐剂的一个重要起点，因此利用好这些天然产物（natural product）以助力疫苗开发具有广阔发展空间。我们前期发现具有免疫调节作用的小檗碱（berberine, BBR）在CD8⁺ T细胞活分化（activation and differentiation）过程中发挥质变调节作用，表现为CD62L、CD44表达双上调，Tem/Teff增殖显著受抑，Tcm:Tem/Teff比例反转，Tcm分化质量提升，免疫记忆期应答反应获得增强。

      目的：本论文拟加强BBR促进CD8⁺ Tcm分化特点与作用的研究，包括CD69表达上调和BBR记忆期免疫保护力提升的确认；并且进一步研究代表性天然化合物调节T细胞记忆分化（memory differentiation）的作用与机制特点，结合疫苗接种策略探讨天然产物免疫记忆增效的可能性。

       方法：本论文继续采用由T细胞受体共刺激（T-cell receptor and co-stimulation）激发、天然产物分别主导和调节T细胞活分化的体外培养体系，体外结合天然化合物或植物粗提物刺激小鼠脾淋巴细胞活化，培养72 h后使用流式细胞术检测相关表面标志物表达水平。体内实验则口服或腹腔注射给予天然化合物，同时进行初次和加强免疫，测定记忆期抗原特异性T细胞在组织的分布或攻击后免疫保护力。利用高纯度分选获取抗原特异性Tcm细胞，经过继转移验证天然化合物调节的Tcm所具有的免疫保护功能。

       结果：发现BBR作用下，Tcm标志膜蛋白极化特征以及CD4⁺ T细胞非依赖性Tcm分化机制特点。鉴于免疫调节化合物姜黄素（curcumin, CCM）和二甲双胍（metformin, MTF）与BBR作用互异，是下一步探索的重要启发，本研究就两化合物的作用特征又加以勾勒，发现CCM 不仅反转Tcm:Tem/Teff比例，且促进两亚型增殖，在上调CD62L的同时并不改变CD44和CD69表达水平；而MTF则主要促进Tem/Teff增殖，Tcm增量不显著，包括CD44、CD62L和CD69表达水平也均无变化。由此提示，天然产物在促进T细胞记忆分化上呈现多样化作用特征。

       为了佐证上述三个天然产物调节T细胞分化的差异化作用，也为了加深对天然产物免疫活性作用的认识，本研究又拓展性探索了其他免疫调节或增强化合物，包括丹参酮I（tanshinone I, TSN1）、双氢青蒿素（artenimol, ATN）、天麻素（gastrodin，GST）、三七皂苷R1（notoginsenoside R1, NGR1）以及几种代表性南药粗提物（southern medicinal plant extracts）。ATN、GST和NGR1及南药粗提物均未引起T细胞各亚型分化的改变，而TSN1则显示不亚于BBR的促CD8⁺ Tcm分化作用，但上调CD62L表达幅度不显著，抑制Tem/Teff增殖程度也不明显，由此Tcm:Tem/Teff比例未发生反转。当剂量低至5倍时，TSN1反而呈现较强的促Tem/Teff增殖作用；此时Tem/Teff与Tcm几乎同比增长，并维持原有少Tcm、多Tem/Teff的比例。为证明TSN1的分化调节作用有助于增强疫苗诱导的免疫记忆反应，采用了蛋白疫苗皮下注射联合TSN1腹腔给药的接种策略免疫小鼠，发现血循环和肺内抗原特异性细胞占比显著升高，但脾内同类细胞却无明显改变；提示TSN1促进记忆T细胞在血液系统的循环和向肺组织的归巢，相较于常规疫苗接种，可能赋予全身和外周组织更强的T细胞免疫记忆。

       结论：综上所述，本论文阐述了BBR改善Tcm分化质量、增强免疫记忆形成的更多作用特征与机制，加深了对天然产物免疫调节作用的认识。在此认识基础上，表型筛选了多个其他代表性天然化合物；其中发现TSN1既能提高Tcm分化质量又不过分抑制Tem生成，故在维持原有细胞体量基础上体现免疫调节效应。这一新型作用特点或与赋能T细胞循环和向外周组织归巢存在一定因果关系，由此可能提高记忆性免疫保护力，皆有待于未来工作中深入探索，并能助力天然产物潜在药效的挖掘与开发利用。

The 2019 coronavirus infectious disease (COVID-19) pandemic was the biggest public health crisis ever in the world in a century, and it has had a huge impact on the global healthcare system and the existing research priorities. The persistent globalization and world changes in the post-pandemic era call for more efficacious measures to prevent or limit the spread of respiratory infectious diseases. Despite the efficacy of several COVID-19 vaccines in preventing spread of the disease during the pandemic, people have been deeply concerned that the lack of long-lasting immunological memory may still leave them susceptible to the virus and have an increased chance of infection in the next outbreak.

It is well established that vaccines targeting a pathogen-specific antigen are capable of eliciting T-cell responses to the antigen through CD8⁺ T cells, thereby playing a pivotal role in the body's defense against the invasion of pathogenic microorganisms. CD8⁺ T cells are central to the formation and sustentation of the immunological memory. After activation, memory CD8⁺ T cells are generated and can be subdivided into central memory (Tcm) and effector memory (Tem) cells, which circulate through the lymphoid and tissues, respectively, and tissue-resident memory (Trm) cells, which become established in the peripheral tissue and basically are non-circulating cells. These memory cells play a crucial role in the secondary immune response, expediting the elimination of the pathogen through the recognition of the cognate antigen. Therefore, the primary objective of vaccine design is to generate adequate memory T cells. A variety of bioactive ingredients found in medicinal plants are effective immunomodulatory agents that are usually safe to use. They may also have potential for development as vaccine adjuvants to promote memory formation of T cells.

In this study, naïve T cells were activated through stimulation of T-cell receptors and costimulatory molecules. Representative natural compounds were used as a T-cell differentiation modulator in order to investigate memory differentiation and its underlying cellular and molecular mechanisms, as well as the efficacy of natural product-mediated T-cell memory in providing immunological protection to mice. Our previous study revealed that CD8⁺ T cells stimulated with the immunomodulatory compound berberine (BBR) underwent a qualitative change in their differentiation into memory phenotypes, characterized by an upregulation of both CD62L and CD44, a significant reduction in Tem/Teff growth, and a reversed ratio of Tcm to Tem/Teff, resulting in a more effecient formation of Tcm and a heightened overall memory response. These findings prompted further exploration in this current thesis to further characterize the role of BBR in the formation of high-quality CD8⁺ Tcm, including upregulated CD69 expression, and generation of enhanced memory for immune protection. The polarization of Tcm biomarker membrane markers and a CD4⁺ T cell-independent mechanism of Tcm formation were also investigated. Curcumin (CCM) and metformin (MTF) were characterized as well with respect to their role in mediating T-cell differentiation in the present study. CCM not only reversed the Tcm-to-Tem/Teff ratio but also stimulated both subsets to expand. However, in contrast to BBR, there was no upregulation of CD44 and CD69 exception of CD62L. MTF was found to enhance Tem/Teff expansion while Tcm remained unchanged in terms of expression levels of CD44, CD62L, and CD69.

In order to corroborate the effects of the three aforementioned natural compounds and improve our knowledge of natural product-related immunological activities, the current study extended this line of research by including tanshinone I (TSN1), artemisinin (ATN), notoginsenoside R1 (NGR1), gastrodin (GST) and a number of representative southern medicinal plant extracts. ATN, GST, NGR1 and southern medicinal plant extracts did not have any detectable effects on T-cell differentiation. However, it was found that TSN1 exerted proliferative effects on CD8⁺ Tcm with an upregulated CD62L level slightly lower than that induced by BBR. The ratio of Tcm to Tem/Teff was close to one but it was not reversed. When the dose was lowered by 5 fold, both Tcm and Tem/Teff proliferated simultaneously, keeping the original Tcm-to-Tem/Teff ratio almost unchanged. To demonstrate that TSN1 would facilitate a vaccine-induced memory response, a protein-based vaccine and TSN1 were administered by subcutaneous and intraperitoneal injections, respectively. This vaccination strategy led to an increased proportion of antigen-specific CD8⁺ T cells in both the blood stream and lung but not in the spleen, suggesting that vaccination with TSN1 might confer enhanced systemic and peripheral T-cell memory responses compared to conventional vaccination.

To summarize, this thesis seeks to provide further insights into the significant role of BBR in the differentiation of Tcm and enhanced establishment of immunological memory, thus strengthening our understanding of natural product-associated immunomodulatory effects. Based on this understanding, phenotypic screening has been performed in order to identify additional natural compounds, among which TSN1 can be optimized to elicit high quality of Tcm without unduly inhibiting Tem to develop. TSN1 is able to maintain the T cell population size by doing so while modulating the immune system. It is possible that such novel roles of TSN1 may relate to the ability of T cells empowered to circulate in the blood as well as home to the periphery, hence improving immunological memory from there to provide sustained protection, which holds potentials for further exploration. It is believed that this work will contribute to the exploration and advancement of the undiscovered efficacy of natural products.