新型B细胞亚群的发现及其免疫调控功能与机制的研究
摘要
近年来,人们对于免疫细胞亚群及其功能的认识大为拓展,特别是对于T细胞亚群的研究有了许多新的进展,除了以往广泛公认的Th1、Th2之外,近年来对于调节性T细胞(Treg)、Thl7、Th9, Tfh等T细胞亚群的进一步区分与功能特征的研究以及与临床疾病的联系,使得人们对于免疫应答与免疫调控的细胞与分子机制研究有了更深入的认识。此外,对于巨噬细胞和树突状细胞(DC)的亚群、特别是M2和调节性DC亚群的研究也有了新的进展。但是,目前对于机体体液免疫中最关键的免疫细胞.---B细胞(B lymphocytes)的亚群及其相应的功能特征的研究相比而言比较少。过去认为B细胞通过分泌抗体,在体液免疫系统中发挥“指挥家”的作用；B细胞还是一类重要的抗原提呈细胞,参与对抗原的摄取、加工、提呈,在启动获得性免疫应答中起重要作用；此外,B细胞还可以通过分泌多种细胞因子和某些趋化因子以及表达某些重要膜分子,参与免疫应答的调控。可见,B细胞具有功能多样性,通过进一步研究B细胞的亚群组成及其功能特征,将有助于全面认识B细胞的功能和作用,从而对免疫应答与调控的方式与机制增添新的学术观点。
根据表面分子、定位和功能特点的不同,B细胞被分为B-1细胞和B-2细胞。B-2细胞即为传统意义的B细胞。而B-1细胞主要在胚胎的胎肝、网膜中发育,分布于腹腔、胸腔和肠壁固有层中,又分为B-1a细胞(CD11b+CD5+)和B-1b细胞(CDllb+CD5-)。B-1细胞在IL-10帮助下保持自我更新能力,通过识别某些细菌表面共有的多糖抗原和一些变形的自身抗原,B-1a细胞及其分泌的自身抗体在幼儿的抗感染免疫中提供有效保护,而B-lb细胞在感染时则能对多糖和其他非T细胞依赖性的抗原产生长效的抗体应答。近年来,人们发现了一类可以分泌IL-10的B细胞,根据其功能而将之称为调节性B细胞(regulatory B cell),作为一类新的B细胞亚群,其能够调节机体免疫应答,大量的基础研究和临床证据表明,B细胞的功能具有明显的异质性,提示B细胞在免疫应答被激活后可能分化为多个不同的功能亚群参与并调控免疫应答,参与肿瘤、自身免疫性疾病等疾病的发生发展过程,因此,有关新的B细胞亚群的发现与功能分析成为近年来免疫学研究的热点之一。
由于天然免疫系统是机体抵御病原体入侵的第一道屏障,近年来已成为免疫学研究的重要领域,天然免疫应答由NK细胞、巨噬细胞、嗜酸性粒细胞、中性粒细胞和肥大细胞等天然免疫细胞介导,通过模式识别受体识别细菌、真菌及病毒成分并活化,引发下游的多种免疫效应,但B细胞在其中的作用如何尚不明确。但一方面,B细胞能够不依赖BCR信号介导而被活化；另一方面,由于B细胞能表达多种Toll样受体(如TLR2、TLR3、TLR4、TLR5、TLR7、TLR9)和其他的模式识别受体识别,那么病原体感染后微环境中存在的多种TLR配体及模式识别配体有可能能够通过与B细胞上的受体交联,激发下游的信号传递,从而为B细胞参与天然免疫反应以及随后调控免疫反应奠定了基础。
考虑到B细胞组成的异质性以及B细胞参与天然免疫应答和调控获得性免疫应答的多样性功能特点,我们在本研究中分为三部分,研究了病原体感染或者病原体成分激活天然免疫反应之后B细胞的亚群组成,以及我们所发现和鉴定的新型B细胞亚群的功能特点与相关作用机制,还对B细胞发育和功能分化的转录因子调控机制进行了初步探讨,以期为B细胞参与免疫应答调控及其作用机制,以及其功能分化的分子机制增添新的认识。
一、CD19+DX5+B细胞亚群(NKB)的发现及其参与天然免疫、
调控获得性免疫的作用与相关机制
为了探索B细胞在病原体感染后机体免疫应答中的作用,我们对李斯特菌、大肠杆菌、水泡性口炎病毒(Vesicular stomatitis virus, VSV)感染的小鼠模型及TLR配体LPS、CpG-ODN、poly I:C注射的小鼠炎症模型的各脏器B细胞采用流式细胞仪分析,发现了一群由病原体感染和TLR配体诱导增加的并广泛分布于骨髓、脾脏、淋巴结组织中CD19+DX5+新型细胞亚群。为了进一步探索其特征,我们动态检测了其产生过程,发现脾脏中CD19+DX5+双阳性细胞在细菌感染后第3天数量开始显著上升,到第7天达到峰值,其后逐渐下降,直至感染后第17-21天恢复至初始水平；而在LPS、CpG-ODN、polyI:C体内注射所诱导的小鼠模型中,CD19+DX5+细胞数量于刺激后第5天达高峰,随后下降；结果表明,该细胞亚群可被病原体感染或者病原体组分刺激所诱导产生,提示其可能会参与或者调控机体抗感染的天然免疫及获得性免疫应答。
流式分析发现CD19+DX5+双阳性细胞表面表达NK及B细胞的相关表面标志,包括mIgD、mIgM、B220、IA-e、CD40、CD 154、CD86、Ly49A、Ly49C/I、Ly49D、NKG2D、NKG2A/C/E、CD43、CD25、CD122、CD 11b等。电镜下CD19+DX5+双阳性细胞染色质疏松,胞浆富含线粒体和高尔基体。胞内染色及ELISA结果显示其分泌IFN-γ、IL-1α、IL-1β、IL-6、Granzyme-B,而不分泌Th2型细胞因子IL-4和IL-10。此外,由于该细胞具有B细胞的表面标志,我们检测其免疫球蛋白分泌,发现该细胞在LPS刺激后可以分泌IgG2a、IgG2b、IgG3、IgM,采用李斯特菌感染B细胞缺陷Btk-/-小鼠及NK细胞剔除小鼠证明体内诱导产生的该细胞亚群来源于B细胞而非NK细胞,由此推测CD19+DX5+细胞是一个具有NK样表型和分泌细胞因子特征的B淋巴细胞亚群(我们简称NKB)。
我们在体外将DX5-CD19+B细胞分别与纯化的CD4+或CD8+T细胞、NK细胞、树突状细胞、巨噬细胞进行共培养,并加入LPS刺激,发现树突状细胞可以诱导DX5+CD19+NKB细胞的产生。为进一步明确其产生机制,我们在共培养体系中分别采用0.4μM transwell或者加入抗IL-6、IL-1β、IL-12、CD40、CD40L的单克隆抗体,发现树突状细胞可以通过CD40-CD40L途径诱导NKB细胞产生,且其分泌的IL-6和IL-1β对这一过程有促进作用,李斯特菌感染的CD 11 c-DTR、Cd40-/-、Cd401-/小鼠体内实验结果进一步证实了一旦缺失了树突状细胞、CD40-CD40L相互作用,NKB细胞将不能被诱导产生；而在李斯特菌感染仅能在I11r-/-及I16-/-小鼠体内诱导产生少量NKB细胞。证明了NKB的产生需要树突状细胞通过CD40-CD40L与B细胞前体相互作用,IL-6和IL-1β对这一诱导过程有促进作用。
接下来我们研究了NKB细胞在细菌感染天然免疫应答的作用,发现NKB细胞通过分泌的IFN-γ活化巨噬细胞,可以增强其对胞内菌的杀伤,抑制李斯特菌在胞内的数量增长；体内回输NKB细胞也显著降低李斯特菌感染早期的肝脾细菌负荷并上调血清IFN-γ水平,但回输Ifnγ-/-NKB细胞则无类似效应。说明NKB细胞能通过其分泌的IFN-γ激活巨噬细胞,参与细菌感染早期的天然免疫应答反应。
由于NKB细胞数量高峰出现于感染第7天,因此我们对C57BL／6小鼠回输CD45.1+OT-I CD8+T细胞,然后感染能分泌OVA的李斯特菌株(LM-OVA),旨在研究NKB细胞在获得性免疫应答的作用。结果发现,与感染后第7天的NKB细胞共培养可以促进李斯特菌感染小鼠抗原特异性CD8+T细胞的凋亡。进一步研究发现NKB细胞来源的IFN-γ能上调其本身表达FasL及OT-I CD8+T细胞Fas的表达,旦阻断IFN-γ或Fas-FasL信号,则NKB细胞诱导CD8+T细胞的凋亡比例降低。我们在LM-OVA感染Btk-／-小鼠前后分别回输CD45.1+OT-I CD8+T细胞与NKB细胞、NK细胞或B细胞,发现细菌感染7天后NKB细胞回输组CD8+T细胞数量较与NK细胞或B细胞回输组显著下降,但CD8+T细胞表面活化标志CD44.CD62L.CD69较对照组表达无显著变化。采用CFSE标记的OT-I CD8+T细胞与NKB细胞共同回输并检测李斯特菌感染3天后T细胞增殖,结果表明OT-I CD8+T细胞增殖并无差异,结果表明,NKB细胞在体内不显著影响抗原特异性CD8+T细胞产生。若采用Ifnγ-/-或Fasl-/-小鼠NKB细胞与OT-ⅠCD8+T细胞共同回输,对OT-IT细胞的数量及活化并无影响。因此,于感染第7天大量诱导产生的NKB细胞通过IFN-γ和Fas-FasL通路可以促进抗原特异性CD8+T细胞凋亡,使得抗原特异性获得性免疫应答反应受到适度的负相调控,从而利于维持免疫应答的稳定性。可见,我们发现的这群由病原体感染诱导产生的CD19+DX5+NKB细胞在天然免疫应答的早期通过IFN-γ激活巨噬细胞而起促进作用,发挥了NK细胞样的天然免疫效应细胞的作用；但在获得性免疫应答的晚期大量产生之后,其通过IFN-γ和Fas-FasL通路诱导抗原特异性CD8+T细胞凋亡而对免疫应答反应起负相调控作用,发挥了调节性B细胞样的作用,表明该新型B细胞亚群具有独特的双重免疫功能,即被病原体感染诱导产生之后反馈促进天然免疫诱导以利于机体清除病原体,但另外一方面,通过负相调控抗原特异性CD8+T细胞应答反应以利于免疫应答处于平衡状态,从而避免免疫应答过度活化之后造成机体病理学损害。
二、CD19+PDCA-1+B细胞亚群的发现及其参与天然免疫功能
与机制的研究
我们在实验中发现Btk-／-小鼠对李斯特菌感染相比野生型小鼠具有较高易感性,在感染早期的肝脏和脾脏细菌增殖更活跃,且血清IFN-γ水平低下,提示B细胞在抗御李斯特菌感染中起重要作用。为了明确B细胞在抗感染天然免疫应答中的功能,我们在李斯特菌及大肠杆菌体内感染48小时后的C57BL／6小鼠脾脏细胞的B细胞及其亚群进行了分析,发现了一群广泛分布于骨髓、脾脏、淋巴结的CD19+PDCA-1+双阳性细胞,在感染后48小时达高峰,随后逐渐降低,至第八天达到感染前水平,提示该细胞可能直接参与机体的天然免疫应答过程。
共聚焦显微镜及H-E染色后观察结果显示,CD19+PDCA-1+双阳性细胞直径6-8μM,呈圆形,表面光滑,细胞质稀少,细胞核致密,具有典型的B淋巴细胞形态特征；膜表面表达mIgM.mIgD.B220.CD23等B细胞特异性分子；胞内表达Thl型细胞因子IL-12p40、TNF-α、IL-1α、IL-6,并在HKLM刺激后高分泌IFN-α。综合以上特征,我们认为CD19+PDCA-1+双阳性细胞是一群独特的具有Ⅰ型干扰素分泌能力的新型B淋巴细胞亚群。
采用体外细胞共培养,我们证明在热灭活的李斯特菌(Heat-killed L. monocytogenes)刺激下,巨噬细胞能够诱导CD19+PDCA-1-细胞向CD19+PDCA-1+细胞分化；究其机制,我们在共培养体系中分别采用0.4μM transwell各两者隔离开,或者加入CD40、CD40L、IFN-γ阻断性抗体以及相应重组细胞因子,发现CD19+ PDCA-1+细胞诱导产生显著减少。此外,我们在李斯特菌感染的Cd40-/-、Cd401-/-小鼠体内也无法检测到CD19+ PDCA-1+细胞的产生,而在相应的Ifnγ-/-小鼠模型中也检测到了PDCA-1+ B细胞比例显著下降。结果表明,巨噬细胞诱导PDCA-1+CD19+细胞的产生是由IFN-γ和CD40-CD40L信号介导的。
根据PDCA-1+B细胞的产生特点以及B细胞缺陷的Btk-/-小鼠在早期对李斯特菌感染的高易感性,我们认为这群PDCA-1+B细胞是参与机体抗感染天然免疫应答的B细胞亚群。体外实验证实PDCA-1+B细胞对李斯特菌在巨噬细胞内增殖并无显著直接影响。已知NK细胞活化和分泌大量IFN-γ对于李斯特菌体内清除其重要作用,为了观察PDCA-1+B细胞对李斯特菌体内清除的影响,我们将其与NK细胞共培养,发现PDCA-1+B细胞能通过分泌IFN-a促进NK细胞活化,杀伤Yac-1细胞,并分泌IFN-γ。对李斯特菌感染的Btk-/-小鼠回输PDCA-1+B细胞能有效降低脏器中的细菌负荷并上调外周血IFN-γ水平；但若用纯化的PK136抗体将NK细胞剔除后,回输PDCA-1+B细胞对李斯特菌感染的Btk-/-小鼠的保护作用消失,且回输Ifnγ-/-鼠来源的NK细胞也无法恢复PDCA-1+B细胞对李斯特菌感染时对机体的保护效应。
因此，我们通过体内外实验证实,由病原体感染所诱导产生的PDCA-1+B细胞能在李斯特菌感染早期通过其分泌的IFN-a激活NK细胞并促进其IFN-γ的分泌,从而参与机体天然免疫应答,利于机体清除入侵的病原体。本研究为B细胞直接参与抗感染免疫应答提供了新的实验依据,也为全面认识B细胞亚群的非经典的生物学功能提出了新的研究方向。
三、转录因子Foxpl对B细胞功能调控作用的初步探讨
研究表明,转录因子对于免疫细胞的分化发育起决定性作用。Foxp亚家族(Foxpl,Foxp2,Foxp3)的功能及调控机制研究已成为近年来人们的研究热点。Foxpl从小鼠B淋巴瘤细胞系BCL1中被首次发现和克隆至今已逾十年,但有关它在免疫细胞发育分化过程中的作用及相关机制的研究却刚刚起步。近年来,Foxpl对B细胞、T细胞、单核巨噬细胞的发育分化乃和功能调控的作用陆续被人们提出并证实,这一系列的发现令Foxpl这一重要转录因子在免疫细胞分化发育及功能调控中的作用与机制研究越来越受到关注。
为探索B细胞及其亚群的发育及功能分化的转录因子调控机制,我们初步探讨了在LPS刺激或病原体感染后Foxpl对B细胞的功能影响。我们发现Foxpl表达广泛,在心脏、脑、肺、肝、肾、胸腺、脾、腋窝淋巴结、肠系膜淋巴结和骨髓中均有表达,其中心、脑、肺、胸腺、脾脏、腋窝和肠系膜淋巴结中Foxp1表达较高。我们进一步鉴定了本室引进的Foxp1基因敲除小鼠(Foxp1-/-)B细胞、CD4+T细胞、CD8+T细胞和巨噬细胞中Foxp1蛋白表达较野生型Foxp1+／+小鼠的相应免疫细胞的表达水平下调80%-90%。我们对Foxp1-/-小鼠的B细胞功能进行了初步分析,研究发现,无论是LPS体外刺激还是李斯特菌体内感染,Foxp1-/-B细胞的IL-10分泌均显著下调；另一方面,在LPS刺激后Foxp1-/小鼠中B细胞的炎性因子IL-6、IFN-α、IFN-γ较野生型小鼠B细胞分泌量上升,但对李斯特菌体内感染的B细胞的细胞因子mRNA表达水平进行分析,我们并未获得同样结果,IL-6、IFN-a表达较野生型小鼠并无显著变化,但IFN-γ的mRNA表达水平却显著下调。由此我们推测,Foxp1对B细胞负向免疫调控功能可能具有潜在的诱导作用；而Foxp1对B细胞炎性因子分泌的影响以及对B细胞抗感染免疫应答功能的调控作用尚不明确,需要我们设计实验进行深入探讨。
四、总结
在第一部分实验中,发现并鉴定了一群具有NK细胞样特征、能够分泌IFN-γ的DX5+ CD19+ B细胞,称为NKB细胞亚群。NKB细胞能在免疫应答早期(李斯特菌感染3天内天然免疫阶段)通过分泌IFN-γ激活巨噬细胞、促进其对胞内菌的清除,发挥了NK细胞样的功能；NKB细胞能在免疫应答晚期(李斯特菌感染7天获得性免疫阶段)通过分泌的IFN-γ上调自身FasL及抗原特异性CD8+T细胞表面Fas的表达,从而诱导CD8+T细胞凋亡,从而发挥了调节性B细胞的功能,适度控制了获得性免疫应答的强度,从而使得抗原特异性T细胞应答反应受到适度的负相调控,有助于维持免疫应答的稳定性。可见,该病原体感染诱导产生的新型NKB细胞亚群具有NK细胞的天然免疫应答效应和调节性B细胞的负相免疫调控功能,其分泌的IFN-γ在介导其双重功能中起重要的作用。新型B细胞亚群(DX5+ CD19+ NKB)的发现与初步揭示的其功能特点,为天然免疫的效应机制增添了新的学术观点,也丰富了人们对调节性B细胞的负向调控功能及机制的认识,并为获得性免疫负向调控提出了新机制。
在第二部分实验中,发现并鉴定了一群能分泌IFN-α的CD19+ PDCA-1+ B细胞,该新型B细胞亚群能通过分泌的IFN-α而激活NK细胞并促进其分泌IFN-γ,从而参与、促进了胞内菌感染早期的天然免疫应答过程。此外,我们的研究结果证明,IFN-α能正反馈调节IFN-γ信号通路,这一发现为病原体入侵机体后天然免疫应答的快速启动及蛋白调控提出了新机制,也为B细胞的天然免疫功能增添了新的认识。
在第三部分实验中,我们对B细胞及其亚群的发育及功能分化的转录因子调控机制进行了初步探讨,结果发现,在LPS体外刺激和李斯特菌体内感染后转录因子Foxp1的表达均能促进B细胞分泌IL-10,但其炎性因子分泌变化趋势并不相同,我们推测Foxp1对B细胞负向免疫调控功能可能具有潜在的诱导作用,而其对B细胞抗感染免疫应答的调控作用以及在B细胞亚群产生和功能中作用尚不明确,需要将来开展进一步实验深入探讨。
总之,深入研究B细胞及其亚群在不同感染模型中的功能及其机制将有助于我们进一步了解B细胞在机体受到外来病原体或者异源抗原刺激后免疫应答的启动、维持和调控中的作用,更加全面认识机体复杂而平衡的免疫调控网络,并为免疫细胞亚群认识的拓展及感染性疾病的免疫治疗提供新思路。
More and more new subsets of immune cells have been identified and their functions have been extensively investigated in recent years. For example, T cell subsets, such as regulatory T cells (Treg), Th17, Th9, Tfh, have been successfully identified and shown to be important in the initiation and regulation of immune response, thus contributing to the better understanding of the cellular and molecular mechanisms for the regulation of immune responses. In addition, new subsets of macrophages and dendritic cells (DC) have also been identified. However, subpopulations of B cells, the most important immune cells in humoral immunity, were poorly studied. As we know, B lymphocytes protect host from pathogens by producing antibodies, as well as opsonization, and complement fixation, thus playing critical role in humoral immune responses against pathogens. Moreover, B cells are also implicated in cellular immunity. For example, they can promote naive CD4+T cell differentiation into T helper 1(Th1) or Th2 subsets. B cells can also serve as antigen-presenting cells, providing co-stimulatory signals for T cell activation. Additionally, they are capable of producing cytokines and participate in the regulation of immune responses. Therefore, identification of new B cell subsets and their functional characteristics will contribute to the comprehensive understanding of B cells in the immune network and present new concepts of underlying mechanisms of immune response and immune regulation.
B lymphocytes are divided into B-1 and B-2 B cells according to the different membrane molecules, distribution and function. B-1 B cells, which are characterized by their unique phenotype of sIgMhisIgDloCDllb+, constitute most of the B cells existing in the peritoneal and pleural cavities in mice. CD5+ B-1a B cells recognize high-molecular-weight polymeric antigens and produce'natural'antibodies which are critical in the early responses to encapsulated extracellular bacteria, whereas CD5- B-1b B cells produce antibodies required for long-lasting protective immunity to pathogens such as Streptococcus pneumoniae. Conventional B-2 cells function as antibody-producing cells or antigen-presenting cells, promote T helper cell differentiation and provide co-stimulatory signals for T cell activation and thus are implicated in the humoral and cellular immunity. Recently, a subset of CDldhiCD5+CD19hi B10 cells have been identified and proved to negatively regulate T cell-mediated inflammatory responses through IL-10 secretion. Massive evidences from clinical observations and basic researches prove that B cells are of great heterogeneity, indicating.they may differentiate into different subsets with various activities under different physiological and pathological conditions. So, identification and functional analysis of new subsets of B cells are one of challenging and hot topics in the froniters of immunology nowadays.
Innate immune response is the first line of immune defense against pathogens. Well-established components of the innate immune system include natural killer (NK) cells, macrophages, eosinophils, neutrophils and mast cells. A recent study in teleost fish demonstrated that B cells have potent phagocytic activities. Activation of B cells by a wide range of stimuli independently of the B cell receptor (BCR) also suggests that B cells may play a role in innate immunity. Components of bacteria, fungi and virus selectively activate Toll-like receptors (TLR), leading to the shared and unique responses in innate immune cells. So, TLRs (e.g., TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9), which are widely expressed on B cells, may facilitate B cells to sense and respond to microbial antigens, thus proposing a possibility that B cells may participate in innate immune responses aganist pathogenic infections.
Considering the heterogeneity and multiple functions of B cells in innate and adaptive immune responses, we, in the following three parts of our work, analyzed the composition of B cells in the mie after microbial infection and different inflammatory stimulation, investigated the functions and the underlying mechanisms of new B cell subsets we identified, as well as studied the transcriptional regulation of B cell function. The primary aim of this study is to identify new subsets of B cells and investigate the role of the B cell subsets in the innate immunity and regulation of adaptive immune response.
1. Identification and functional characteristics of CD19+DX5+ B cells
To study the role of B cells in the anti-microbial immune responses, we analyzed the B cell subpopulations of different lymph organs from the mice infected with L. monocytogene-, E. coli-or VSV, as well as challenged with LPS-, CpG-ODN-or poly I:C. We found one new subset of B cells with unique phenotype (CD19+DX5+) in the bone marrow, spleen and mesenteric lymph nodes. Then we analyzed the number and proportion of these CD19+ DX5+ cells generated in the infected mice in the following 21 days and found that CD19+ DX5+ cells in the spleen started to increase on the third day post-infection, and reached the peak on the seventh day, then drop gradually and back to the initial number during the 17-21 days post-infection. Besides, number of CD19+DX5+ cells in the stimulus-injected mice increased to the peak five days post-infection and decreased gradually. The data indicated that pathogens and their components could induce the in vivo generation of CD19+DX5+ cells, which may be involved in the regulation of innate and adaptive immune responses against microbial infection.
To further confirm the characteristics of CD19+DX5+ cells, we observed their morphology, and analyzed their phenotype, cytokine profiles. We found that CD19+DX5+ cells had smooth compact nuclei and rare cytoplasm, expressed markers of both NK cells and B cells, such as mIgD, mIgM, B220, IA-e, CD40, CD 154, CD86, Ly49A, Ly49C/I, Ly49D, NKG2D, NKG2A/C/E, CD43, CD25, CD122, CD11b. Thus we named these CD19+DX5+ cells as NKB cells. By intracellular staining and ELISA, we confirmed that NKB cells expressed IFN-γ, IL-1α, IL-1β, IL-6 and Granzyme-B, but not IL-4 or IL-10. In addition, LPS-stimulated NKB cells secreted IgG2a, IgG2b, IgG3, IgM. Importantly, CD19+DX5+ cells could not be inducible generated in Btk-/- mice but could be generated in NK cell-depleted mice, indicating these CD19+DX5+ cells are derived from B cells but not from NK cells. Therefore, we identified one new subset of B cells with NK-like phenotype and secretion of IFN-y and other Thl cytokines.
But how are NKB cells inducibly generated? What are the progenitors of NKB cells? By using co-culture system of CD19+DX5- cells with different kinds of lymphocytes and immune cells, we found that dendritic cells (DC) could induce the differentiation of NKB cells in vitro in the presence of the stimulation with Heat-killed L. monocytogenes (HKLM). To understand the mechanism of NKB cell generation, we used 0.4μM transwell system or added neutralizing antibodies against IL-6, IL-1β, IL-12, CD40, CD40L in the coculture system, and found that DC induced NKB differentiation through CD40-CD40L pathway, while IL-6 and IL-1βproduced by DC could promote the generation of NKB cells. We also confirmed this conclusion in the CD11c-DTR, Cd40-/-, Cd40l-/-, Il1r-/- and Il6-/- mice with L.monocytogenes, in which NKB cells could not be efficiently generated.
Then we investigated the functions of NKB cells in innate defense against listeria, and found that IFN-γproduced by NKB cells could activate macrophages and subsequently promote bacterial clearance. Adoptively transfer of NKB cells from wild type mice but not from Ifnγ-/- mice significantly reduced the bacterial burden in the liver and spleen of the infected mice, with increased serum IFN-y level. So, upon bacterial infection of the mice, NKB cells produced IFN-y to activate macrophages and contributed to the clearance of listeria, suggesting that NKB cells, just like the NK cells, can participate in the innate responses during the early period of listeria infection.
Considering that the number of NKB cells could be induced to reach the peak on the seventh day post-infection, we adoptively transferred CD45.1+ OT-I CD8+T cells into C57BL/6 mice before LM-OVA infection, then purified OT-I T cells and NKB cells from the mice infected with listeria for seven days (LM-7d) and investigated the role of NKB cells in adaptive immune responses. Data showed that NKB cells could induce apoptosis of antigen-specific OT-I CD8+T cells derived from listeiria-infected mice on day 7. Then we went further to study the underlying mechanisms and found that IFN-y produced by NKB cells could upregulate FasL expression on NKB cells and Fas. expression on CD8+T cells in the NKB-CD8+T co-culture system in vitro. Once signals of IFN-y or Fas-FasL were blocked, the apoptosis of antigen-specific OT-I CD8+T cells induced by NKB cells significantly reduced. The data demonstrate that NKB cells can induce the apoptosis of antigen-specific CD8 T cells through IFN-y and FasL-Fas pathway at the late stage of immune response, thus controlling the immune response in an approariate state. So, in other side, NKB cells may exert their immunoregulatory function in the regulation of adaptive immune response, just like regulatory B cells.
Next, the in vivo experiments were designed as follows. We adoptively transferred CD45.1+OT-I CD8+T and LM-7d NKB cells, NK cell or B cells into Btk-/- mice before or after LM-OVA infection. Compared to the control groups with NK cell or B cell transfer, the mice transferred with NKB cells showed decreased numbers of OT-I CD8+T cells seven days post-infection but the expression of CD44, CD62L and CD69 on CD8+T cells remained unchanged. However, the expansion of OT-I CD8+T cells after NKB cell transfer was comparable to that in the control groups, indicating that NKB cells did not affect the generation of antigen-specific CD8+T cells. In addition, adoptive transfer of Ifnγ-/- or Fasl-/- NKB cells had no effect on the number and activation of OT-I CD8+T cells. In brief, NKB cells, generated from the mice infected with listeria on day 7, can induce the apoptosis of antigen-specific CD8+ T cells through IFN-y and Fas-FasL pathway, thus negatively regulating the antigen-specific T cell adaptive immunity appropriately and stabilizing the immune responses once the pathogen infection is cleared.
2. Identification and functional characteristics of CD19+PDCA-1+B cells involved in the innate immune responses
Btk-/- mice were found to be more susceptible to L. monocyto genes infection, showing heavier bacterial burden and lower serum level compared to wild type mice. The data indicate that B cells are required for the immune response against L. monocytogenes infection. To investigate the role of B cells in innate immune responses, we analyzed the populations of splenic B cells from L. monocytogene- or E. coli-infected mice, and found one new subset of B cells with unique phenotype (CD19+PDCA-1+), which were inducibly generated in vivo, and existed in the bone marrow, spleen and mesenteric lymph nodes. Number of CD19+PDCA-1+cells in the spleen increased quickly and reached the peak 48 hours post-infection, then droped gradually and back to the initial number 8 days post-infection. The data indicated that CD19+PDCA-1+cells may be involved in the innate immune responses against microbial infection.
To further confirm the characteristics of CD19+PDCA-1+cells, we observed their morphology, analyzed their phenotype and cytokine profiles. We found that CD19+PDCA-1+cells of diameter 6-8μM were morphologically similar to B cells with smooth compact nuclei and rare cytoplasm. The cells expressed membrane markers of B cells, such as mIgM、mIgD、B220、CD23. Intracellular staining showed that CD19+PDCA-1+ cells were positive for IL-6, IL12p40, TNF-a and IL-la intracellular expressions. ELISA results demonstrated higher concentration of IFN-a and IL-6 was found in the supernatants of PDCA-1+ B cells stimulated with HKLM. So, CD19+PDCA-1+ B cells were proved to be a new unique B cell subset with preferentially production of typeⅠIFN.
We went further to investigate how these CD19+PDCA-1+ B cells were inducibly generated? HKLM stimulation alone failed to convert PDCA-1- B cells from wild-type mice into PDCA-1+ cells. HKLM stimulation generated PDCA-1+ B cells in co-culture of PDCA-1- B cells with macrophages, but not with NK cells, CD4+or CD8+T cells. Physical separation of macrophages from PDCA-1- B cells using a transwell system dramatically reduced the number of PDCA-1+ B cells converted. So, macrophages are required for the generation of PDCA-1+ B cells. What are the molecular mechanisms by which macrophages induce the generation of PDCA-1+ B cells? The generation of PDCA-1+ B cells induced by HKLM in macrophage/PDCA-1-B-cell co-culture was significantly reduced by neutralizing anti-CD40 or anti-CD40L antibody. PDCA-1- B cells from Cd40l-/- mice were unable to switch to the PDCA-1+ phenotype upon HKLM stimulation when co-cultured with macrophages. Also, macrophages from Cd40-/- mice did not induce the generation of PDCA-1+ B cells when cultured with wild type PDCA-1- B cells. Inclusion of neutralization IFN-y antibody in the macrophage-PDCA-1--B cell co-culture significantly reduced the ratio of PDCA-1+ B cells. L. monocytogenes infection of the Ifny-/- mice failed to induce generation of PDCA-1+ B cells. In vivo experiments in Cd40-/-or Cd40l-/- mice failed to demonstrate induction of PDCA-1+ B cell generation after listeria infection. Co-culture with macrophages isolated from Ifnγ-/- mice did not switch PDCA-1-B cells to the PDCA-1+ phenotype. Therefore, macrophages contribute to the generation of PDCA-1+ B cells though CD40-CD40L ligation and IFN-y signaling.
We found that PDCA-1+ B cells facilitated NK cells to produce IFN-y though IFN-a. Deletion of NK cells in L. monocytogenes-infected Btk-/- mice attenuated the protective effects of adoptive transfer of PDCA-1+ B cells as well as the IFN-y response. Adoptive transfer of NK cells from wild-type mice, but not Ifnγ-/- NK cells from Ifnγ-/- mice into NK cell-deleted Btk-/- mice restored the protective action of PDCA-1+ B cells adoptively transferred and the IFN-y production. So, the data suggested that PDCA-1+ B cells facilitate NK cells to produce IFN-y though IFN-a, and IFN-y production by NK cells plays important role in innate protective function of PDCA-1+ B cells against L. monocytogenes infection in vivo. The existence of PDCA-1+ B cells explains more effective anti-listeria responses in WT mice compared to B cell deficient mice. Meanwhile, NK depleted mice also showed heavy bacterial burden and little IFN-y level in sera, which were not recovered after PDCA-1+ B cell/and afterward IFN-γ-/- NK transfusion, indicating that PDCA-1+ B cells could not perform their function without IFN-y-producing NK cell involvement. Therefore, these IFN-a-producing B cells with unique phenotype of PDCA-1+ have been confirmed for the first time as a novel B cell subset inducing an effective responses during the innate defense against L. monocytogenes.
In sum, the results from the current study demonstrated that a novel subset of PDCA-1+IFN-a-producing B cells could trigger IFN-γresponse by NK cells, and by doing so, participate in the early innate immune responses to intracellular bacterial infection. Our results also stand as a proof of the positive feedback of IFN-αand IFN-γsignaling which provide a new mechanistic explanation for the rapid start-up of innate responses to pathogen invasion. Moreover, the identification and functional characterization of the IFN-a-producing PDCA-1+ B cell subset in this study may lead to further research of this B subset in humans that may similarly promote innate defense after bacterial infection. In brief, further defining the role and mechanism of PDCA-1+CD19+ cells in different infective diseases in vivo may provide new insights in innate function of B cells and find therapeutic approaches for treating infections in addition to previous ones.
3. Functional regulation of B cells by transcriptional factor Foxpl
It is well known that transcriptional factors paly critical roles in determining the differentiation and regulating function of immune cells. The subfamily members of Foxp (Foxp1, Foxp2, Foxp3) have attracted much attention in recent years in the field of immunology and cell biology. Altough discovered abd cloned from mouse B lymphoma cell line BCL110 years ago, few studies have been reported for the roel of Foxpl in the differentiation and functiona regulation of immune cells, especially B cells. To study the molecular mechanism of differentiation and function of B cells, we investigated regulation of B cell function by Foxpl. First, we confirmed Foxpl was widely expressed in the tissues, including heart, brain, lung, thymus, spleen, axillary and mesenteric lymph nodes. Then we found that Foxpl was expressed in B cells, CD4+T cells, CD8+T cells and macrophages. Furthermore, we confirmed that Foxpl expression in the B cells of Foxpl mutant mice decreased signicantly as compared to that of wild-type mice, identifying the mutant mice with Foxpl gene knockdown. B cells from Foxpl knockdown mice produced less IL-10 in vitro than B cells from wild type mice after LPS stimulation. Also, B cells from Foxpl knockdown mice produced less IL-10 than B cells from wild type mice after listeria infection. After LPS stimulation, the mRNA expression of inflammatory factors, such as IL-6, IFN-a, IFN-y, was upregulated. But IFN-γexpression in B cells from Foxpl knockdown mice were downregulated, while expression of IL-6 and IFN-a had no significant difference between mutant and wild type mice after liseria infection. Therefore, we suggest that Foxpl may facilitate the regulatory function of B cells, but how it regulate the inflammatory cytokine production and what role it plays in the innate function of B cells remain unclear, which need to be investigated in the future.
4. Summary
In PartⅠof our study, we have identified a new B cell subset (DX5+CD19+) with NK-like phenotype and cytokine profiles and named them as NKB cells. DX5+CD19+NKB cells have been found to produce IFN-γto activate macrophages and promote the clearance of intracellular bacteria in early period of listeria infection, indicating NKB cells could participate in the innate immune responses. Interestingly, we found large amounts of NKB cells generated in the late period of immune response (7 days after listeria infection), and NKB cells could upregulate FasL expression on themselves and Fas expression on antigen-specific CD8+ T cells by IFN-γand consequently induce apoptosis of antigen-specific CD8+ T cells. The dat suggest that NKB cells may negatively regulate the antigen-specific T cell adaptive immunity in the late period of immune response against pathogen and stabilizing the immune responses appropriately. Therefore, identification and functional chracterististics of DX5+CD19+ NKB cells provide new insights in innate responses, contribute to the better understanding of regulatory function of B cells, and outlines a new manner of negative regulation of adaptive T cell immunity by B cells.
In PartⅡof our study, we identified a new subset of PDCA-1+CD19+ B cells inducibly generated in the mice infected with bacteria L. monocytogenes and E. coli. PDCA-1+CD19+ B cells secreted a large amount of IFN-αand thus facilitated IFN-γproduction and cytotoxicity function of NK cells via IFN-α. B cell-deficient Btk-/- mice were incapable of producing PDCA-1+CD19+ B cells, and more sensitive to L. monocytogenes infection. Adoptive transfer of PDCA-1+CD19+B cells to Btk-/- mice normalized their resistance to L. monocytogenes infection. Furthermore, we found that macrophages were essential for the inducible generation of PDCA-1+CD19+ B cells via CD40-CD40L ligation and IFN-γ. Therefore, we identify a new subset of PDCA-1+CD19+ B cells during the bacterial infection, which can enhance innate immune responses against bacterial infection by activating NK cells via secretion of IFN-α.
In PartⅢof our study, we studied the transcriptional regulation of differentiation and function of B cells, and found that less IL-10 was produced by B cells from Foxp1 knockdown mice after LPS stimulation and listeria infection. The premilinary data suggest that Foxpl may facilitate the regulatory function of B cells, but how it regulates the inflammatory cytokine production and the innate function of B cells remain unclear to date.
In brief, defining the roles and the underlying mechanisms of B cell subsets in the innate and adaptive immune response against pathogens may provide new insights in the initiation, maintenance and modulation of immune response, broaden our understanding of immune regulation network, and may be helpful to the design of new approaches to the immunotherapy of infectious diseases.
根据表面分子、定位和功能特点的不同,B细胞被分为B-1细胞和B-2细胞。B-2细胞即为传统意义的B细胞。而B-1细胞主要在胚胎的胎肝、网膜中发育,分布于腹腔、胸腔和肠壁固有层中,又分为B-1a细胞(CD11b+CD5+)和B-1b细胞(CDllb+CD5-)。B-1细胞在IL-10帮助下保持自我更新能力,通过识别某些细菌表面共有的多糖抗原和一些变形的自身抗原,B-1a细胞及其分泌的自身抗体在幼儿的抗感染免疫中提供有效保护,而B-lb细胞在感染时则能对多糖和其他非T细胞依赖性的抗原产生长效的抗体应答。近年来,人们发现了一类可以分泌IL-10的B细胞,根据其功能而将之称为调节性B细胞(regulatory B cell),作为一类新的B细胞亚群,其能够调节机体免疫应答,大量的基础研究和临床证据表明,B细胞的功能具有明显的异质性,提示B细胞在免疫应答被激活后可能分化为多个不同的功能亚群参与并调控免疫应答,参与肿瘤、自身免疫性疾病等疾病的发生发展过程,因此,有关新的B细胞亚群的发现与功能分析成为近年来免疫学研究的热点之一。
由于天然免疫系统是机体抵御病原体入侵的第一道屏障,近年来已成为免疫学研究的重要领域,天然免疫应答由NK细胞、巨噬细胞、嗜酸性粒细胞、中性粒细胞和肥大细胞等天然免疫细胞介导,通过模式识别受体识别细菌、真菌及病毒成分并活化,引发下游的多种免疫效应,但B细胞在其中的作用如何尚不明确。但一方面,B细胞能够不依赖BCR信号介导而被活化；另一方面,由于B细胞能表达多种Toll样受体(如TLR2、TLR3、TLR4、TLR5、TLR7、TLR9)和其他的模式识别受体识别,那么病原体感染后微环境中存在的多种TLR配体及模式识别配体有可能能够通过与B细胞上的受体交联,激发下游的信号传递,从而为B细胞参与天然免疫反应以及随后调控免疫反应奠定了基础。
考虑到B细胞组成的异质性以及B细胞参与天然免疫应答和调控获得性免疫应答的多样性功能特点,我们在本研究中分为三部分,研究了病原体感染或者病原体成分激活天然免疫反应之后B细胞的亚群组成,以及我们所发现和鉴定的新型B细胞亚群的功能特点与相关作用机制,还对B细胞发育和功能分化的转录因子调控机制进行了初步探讨,以期为B细胞参与免疫应答调控及其作用机制,以及其功能分化的分子机制增添新的认识。
一、CD19+DX5+B细胞亚群(NKB)的发现及其参与天然免疫、
调控获得性免疫的作用与相关机制
为了探索B细胞在病原体感染后机体免疫应答中的作用,我们对李斯特菌、大肠杆菌、水泡性口炎病毒(Vesicular stomatitis virus, VSV)感染的小鼠模型及TLR配体LPS、CpG-ODN、poly I:C注射的小鼠炎症模型的各脏器B细胞采用流式细胞仪分析,发现了一群由病原体感染和TLR配体诱导增加的并广泛分布于骨髓、脾脏、淋巴结组织中CD19+DX5+新型细胞亚群。为了进一步探索其特征,我们动态检测了其产生过程,发现脾脏中CD19+DX5+双阳性细胞在细菌感染后第3天数量开始显著上升,到第7天达到峰值,其后逐渐下降,直至感染后第17-21天恢复至初始水平；而在LPS、CpG-ODN、polyI:C体内注射所诱导的小鼠模型中,CD19+DX5+细胞数量于刺激后第5天达高峰,随后下降；结果表明,该细胞亚群可被病原体感染或者病原体组分刺激所诱导产生,提示其可能会参与或者调控机体抗感染的天然免疫及获得性免疫应答。
流式分析发现CD19+DX5+双阳性细胞表面表达NK及B细胞的相关表面标志,包括mIgD、mIgM、B220、IA-e、CD40、CD 154、CD86、Ly49A、Ly49C/I、Ly49D、NKG2D、NKG2A/C/E、CD43、CD25、CD122、CD 11b等。电镜下CD19+DX5+双阳性细胞染色质疏松,胞浆富含线粒体和高尔基体。胞内染色及ELISA结果显示其分泌IFN-γ、IL-1α、IL-1β、IL-6、Granzyme-B,而不分泌Th2型细胞因子IL-4和IL-10。此外,由于该细胞具有B细胞的表面标志,我们检测其免疫球蛋白分泌,发现该细胞在LPS刺激后可以分泌IgG2a、IgG2b、IgG3、IgM,采用李斯特菌感染B细胞缺陷Btk-/-小鼠及NK细胞剔除小鼠证明体内诱导产生的该细胞亚群来源于B细胞而非NK细胞,由此推测CD19+DX5+细胞是一个具有NK样表型和分泌细胞因子特征的B淋巴细胞亚群(我们简称NKB)。
我们在体外将DX5-CD19+B细胞分别与纯化的CD4+或CD8+T细胞、NK细胞、树突状细胞、巨噬细胞进行共培养,并加入LPS刺激,发现树突状细胞可以诱导DX5+CD19+NKB细胞的产生。为进一步明确其产生机制,我们在共培养体系中分别采用0.4μM transwell或者加入抗IL-6、IL-1β、IL-12、CD40、CD40L的单克隆抗体,发现树突状细胞可以通过CD40-CD40L途径诱导NKB细胞产生,且其分泌的IL-6和IL-1β对这一过程有促进作用,李斯特菌感染的CD 11 c-DTR、Cd40-/-、Cd401-/小鼠体内实验结果进一步证实了一旦缺失了树突状细胞、CD40-CD40L相互作用,NKB细胞将不能被诱导产生；而在李斯特菌感染仅能在I11r-/-及I16-/-小鼠体内诱导产生少量NKB细胞。证明了NKB的产生需要树突状细胞通过CD40-CD40L与B细胞前体相互作用,IL-6和IL-1β对这一诱导过程有促进作用。
接下来我们研究了NKB细胞在细菌感染天然免疫应答的作用,发现NKB细胞通过分泌的IFN-γ活化巨噬细胞,可以增强其对胞内菌的杀伤,抑制李斯特菌在胞内的数量增长；体内回输NKB细胞也显著降低李斯特菌感染早期的肝脾细菌负荷并上调血清IFN-γ水平,但回输Ifnγ-/-NKB细胞则无类似效应。说明NKB细胞能通过其分泌的IFN-γ激活巨噬细胞,参与细菌感染早期的天然免疫应答反应。
由于NKB细胞数量高峰出现于感染第7天,因此我们对C57BL／6小鼠回输CD45.1+OT-I CD8+T细胞,然后感染能分泌OVA的李斯特菌株(LM-OVA),旨在研究NKB细胞在获得性免疫应答的作用。结果发现,与感染后第7天的NKB细胞共培养可以促进李斯特菌感染小鼠抗原特异性CD8+T细胞的凋亡。进一步研究发现NKB细胞来源的IFN-γ能上调其本身表达FasL及OT-I CD8+T细胞Fas的表达,旦阻断IFN-γ或Fas-FasL信号,则NKB细胞诱导CD8+T细胞的凋亡比例降低。我们在LM-OVA感染Btk-／-小鼠前后分别回输CD45.1+OT-I CD8+T细胞与NKB细胞、NK细胞或B细胞,发现细菌感染7天后NKB细胞回输组CD8+T细胞数量较与NK细胞或B细胞回输组显著下降,但CD8+T细胞表面活化标志CD44.CD62L.CD69较对照组表达无显著变化。采用CFSE标记的OT-I CD8+T细胞与NKB细胞共同回输并检测李斯特菌感染3天后T细胞增殖,结果表明OT-I CD8+T细胞增殖并无差异,结果表明,NKB细胞在体内不显著影响抗原特异性CD8+T细胞产生。若采用Ifnγ-/-或Fasl-/-小鼠NKB细胞与OT-ⅠCD8+T细胞共同回输,对OT-IT细胞的数量及活化并无影响。因此,于感染第7天大量诱导产生的NKB细胞通过IFN-γ和Fas-FasL通路可以促进抗原特异性CD8+T细胞凋亡,使得抗原特异性获得性免疫应答反应受到适度的负相调控,从而利于维持免疫应答的稳定性。可见,我们发现的这群由病原体感染诱导产生的CD19+DX5+NKB细胞在天然免疫应答的早期通过IFN-γ激活巨噬细胞而起促进作用,发挥了NK细胞样的天然免疫效应细胞的作用；但在获得性免疫应答的晚期大量产生之后,其通过IFN-γ和Fas-FasL通路诱导抗原特异性CD8+T细胞凋亡而对免疫应答反应起负相调控作用,发挥了调节性B细胞样的作用,表明该新型B细胞亚群具有独特的双重免疫功能,即被病原体感染诱导产生之后反馈促进天然免疫诱导以利于机体清除病原体,但另外一方面,通过负相调控抗原特异性CD8+T细胞应答反应以利于免疫应答处于平衡状态,从而避免免疫应答过度活化之后造成机体病理学损害。
二、CD19+PDCA-1+B细胞亚群的发现及其参与天然免疫功能
与机制的研究
我们在实验中发现Btk-／-小鼠对李斯特菌感染相比野生型小鼠具有较高易感性,在感染早期的肝脏和脾脏细菌增殖更活跃,且血清IFN-γ水平低下,提示B细胞在抗御李斯特菌感染中起重要作用。为了明确B细胞在抗感染天然免疫应答中的功能,我们在李斯特菌及大肠杆菌体内感染48小时后的C57BL／6小鼠脾脏细胞的B细胞及其亚群进行了分析,发现了一群广泛分布于骨髓、脾脏、淋巴结的CD19+PDCA-1+双阳性细胞,在感染后48小时达高峰,随后逐渐降低,至第八天达到感染前水平,提示该细胞可能直接参与机体的天然免疫应答过程。
共聚焦显微镜及H-E染色后观察结果显示,CD19+PDCA-1+双阳性细胞直径6-8μM,呈圆形,表面光滑,细胞质稀少,细胞核致密,具有典型的B淋巴细胞形态特征；膜表面表达mIgM.mIgD.B220.CD23等B细胞特异性分子；胞内表达Thl型细胞因子IL-12p40、TNF-α、IL-1α、IL-6,并在HKLM刺激后高分泌IFN-α。综合以上特征,我们认为CD19+PDCA-1+双阳性细胞是一群独特的具有Ⅰ型干扰素分泌能力的新型B淋巴细胞亚群。
采用体外细胞共培养,我们证明在热灭活的李斯特菌(Heat-killed L. monocytogenes)刺激下,巨噬细胞能够诱导CD19+PDCA-1-细胞向CD19+PDCA-1+细胞分化；究其机制,我们在共培养体系中分别采用0.4μM transwell各两者隔离开,或者加入CD40、CD40L、IFN-γ阻断性抗体以及相应重组细胞因子,发现CD19+ PDCA-1+细胞诱导产生显著减少。此外,我们在李斯特菌感染的Cd40-/-、Cd401-/-小鼠体内也无法检测到CD19+ PDCA-1+细胞的产生,而在相应的Ifnγ-/-小鼠模型中也检测到了PDCA-1+ B细胞比例显著下降。结果表明,巨噬细胞诱导PDCA-1+CD19+细胞的产生是由IFN-γ和CD40-CD40L信号介导的。
根据PDCA-1+B细胞的产生特点以及B细胞缺陷的Btk-/-小鼠在早期对李斯特菌感染的高易感性,我们认为这群PDCA-1+B细胞是参与机体抗感染天然免疫应答的B细胞亚群。体外实验证实PDCA-1+B细胞对李斯特菌在巨噬细胞内增殖并无显著直接影响。已知NK细胞活化和分泌大量IFN-γ对于李斯特菌体内清除其重要作用,为了观察PDCA-1+B细胞对李斯特菌体内清除的影响,我们将其与NK细胞共培养,发现PDCA-1+B细胞能通过分泌IFN-a促进NK细胞活化,杀伤Yac-1细胞,并分泌IFN-γ。对李斯特菌感染的Btk-/-小鼠回输PDCA-1+B细胞能有效降低脏器中的细菌负荷并上调外周血IFN-γ水平；但若用纯化的PK136抗体将NK细胞剔除后,回输PDCA-1+B细胞对李斯特菌感染的Btk-/-小鼠的保护作用消失,且回输Ifnγ-/-鼠来源的NK细胞也无法恢复PDCA-1+B细胞对李斯特菌感染时对机体的保护效应。
因此，我们通过体内外实验证实,由病原体感染所诱导产生的PDCA-1+B细胞能在李斯特菌感染早期通过其分泌的IFN-a激活NK细胞并促进其IFN-γ的分泌,从而参与机体天然免疫应答,利于机体清除入侵的病原体。本研究为B细胞直接参与抗感染免疫应答提供了新的实验依据,也为全面认识B细胞亚群的非经典的生物学功能提出了新的研究方向。
三、转录因子Foxpl对B细胞功能调控作用的初步探讨
研究表明,转录因子对于免疫细胞的分化发育起决定性作用。Foxp亚家族(Foxpl,Foxp2,Foxp3)的功能及调控机制研究已成为近年来人们的研究热点。Foxpl从小鼠B淋巴瘤细胞系BCL1中被首次发现和克隆至今已逾十年,但有关它在免疫细胞发育分化过程中的作用及相关机制的研究却刚刚起步。近年来,Foxpl对B细胞、T细胞、单核巨噬细胞的发育分化乃和功能调控的作用陆续被人们提出并证实,这一系列的发现令Foxpl这一重要转录因子在免疫细胞分化发育及功能调控中的作用与机制研究越来越受到关注。
为探索B细胞及其亚群的发育及功能分化的转录因子调控机制,我们初步探讨了在LPS刺激或病原体感染后Foxpl对B细胞的功能影响。我们发现Foxpl表达广泛,在心脏、脑、肺、肝、肾、胸腺、脾、腋窝淋巴结、肠系膜淋巴结和骨髓中均有表达,其中心、脑、肺、胸腺、脾脏、腋窝和肠系膜淋巴结中Foxp1表达较高。我们进一步鉴定了本室引进的Foxp1基因敲除小鼠(Foxp1-/-)B细胞、CD4+T细胞、CD8+T细胞和巨噬细胞中Foxp1蛋白表达较野生型Foxp1+／+小鼠的相应免疫细胞的表达水平下调80%-90%。我们对Foxp1-/-小鼠的B细胞功能进行了初步分析,研究发现,无论是LPS体外刺激还是李斯特菌体内感染,Foxp1-/-B细胞的IL-10分泌均显著下调；另一方面,在LPS刺激后Foxp1-/小鼠中B细胞的炎性因子IL-6、IFN-α、IFN-γ较野生型小鼠B细胞分泌量上升,但对李斯特菌体内感染的B细胞的细胞因子mRNA表达水平进行分析,我们并未获得同样结果,IL-6、IFN-a表达较野生型小鼠并无显著变化,但IFN-γ的mRNA表达水平却显著下调。由此我们推测,Foxp1对B细胞负向免疫调控功能可能具有潜在的诱导作用；而Foxp1对B细胞炎性因子分泌的影响以及对B细胞抗感染免疫应答功能的调控作用尚不明确,需要我们设计实验进行深入探讨。
四、总结
在第一部分实验中,发现并鉴定了一群具有NK细胞样特征、能够分泌IFN-γ的DX5+ CD19+ B细胞,称为NKB细胞亚群。NKB细胞能在免疫应答早期(李斯特菌感染3天内天然免疫阶段)通过分泌IFN-γ激活巨噬细胞、促进其对胞内菌的清除,发挥了NK细胞样的功能；NKB细胞能在免疫应答晚期(李斯特菌感染7天获得性免疫阶段)通过分泌的IFN-γ上调自身FasL及抗原特异性CD8+T细胞表面Fas的表达,从而诱导CD8+T细胞凋亡,从而发挥了调节性B细胞的功能,适度控制了获得性免疫应答的强度,从而使得抗原特异性T细胞应答反应受到适度的负相调控,有助于维持免疫应答的稳定性。可见,该病原体感染诱导产生的新型NKB细胞亚群具有NK细胞的天然免疫应答效应和调节性B细胞的负相免疫调控功能,其分泌的IFN-γ在介导其双重功能中起重要的作用。新型B细胞亚群(DX5+ CD19+ NKB)的发现与初步揭示的其功能特点,为天然免疫的效应机制增添了新的学术观点,也丰富了人们对调节性B细胞的负向调控功能及机制的认识,并为获得性免疫负向调控提出了新机制。
在第二部分实验中,发现并鉴定了一群能分泌IFN-α的CD19+ PDCA-1+ B细胞,该新型B细胞亚群能通过分泌的IFN-α而激活NK细胞并促进其分泌IFN-γ,从而参与、促进了胞内菌感染早期的天然免疫应答过程。此外,我们的研究结果证明,IFN-α能正反馈调节IFN-γ信号通路,这一发现为病原体入侵机体后天然免疫应答的快速启动及蛋白调控提出了新机制,也为B细胞的天然免疫功能增添了新的认识。
在第三部分实验中,我们对B细胞及其亚群的发育及功能分化的转录因子调控机制进行了初步探讨,结果发现,在LPS体外刺激和李斯特菌体内感染后转录因子Foxp1的表达均能促进B细胞分泌IL-10,但其炎性因子分泌变化趋势并不相同,我们推测Foxp1对B细胞负向免疫调控功能可能具有潜在的诱导作用,而其对B细胞抗感染免疫应答的调控作用以及在B细胞亚群产生和功能中作用尚不明确,需要将来开展进一步实验深入探讨。
总之,深入研究B细胞及其亚群在不同感染模型中的功能及其机制将有助于我们进一步了解B细胞在机体受到外来病原体或者异源抗原刺激后免疫应答的启动、维持和调控中的作用,更加全面认识机体复杂而平衡的免疫调控网络,并为免疫细胞亚群认识的拓展及感染性疾病的免疫治疗提供新思路。
More and more new subsets of immune cells have been identified and their functions have been extensively investigated in recent years. For example, T cell subsets, such as regulatory T cells (Treg), Th17, Th9, Tfh, have been successfully identified and shown to be important in the initiation and regulation of immune response, thus contributing to the better understanding of the cellular and molecular mechanisms for the regulation of immune responses. In addition, new subsets of macrophages and dendritic cells (DC) have also been identified. However, subpopulations of B cells, the most important immune cells in humoral immunity, were poorly studied. As we know, B lymphocytes protect host from pathogens by producing antibodies, as well as opsonization, and complement fixation, thus playing critical role in humoral immune responses against pathogens. Moreover, B cells are also implicated in cellular immunity. For example, they can promote naive CD4+T cell differentiation into T helper 1(Th1) or Th2 subsets. B cells can also serve as antigen-presenting cells, providing co-stimulatory signals for T cell activation. Additionally, they are capable of producing cytokines and participate in the regulation of immune responses. Therefore, identification of new B cell subsets and their functional characteristics will contribute to the comprehensive understanding of B cells in the immune network and present new concepts of underlying mechanisms of immune response and immune regulation.
B lymphocytes are divided into B-1 and B-2 B cells according to the different membrane molecules, distribution and function. B-1 B cells, which are characterized by their unique phenotype of sIgMhisIgDloCDllb+, constitute most of the B cells existing in the peritoneal and pleural cavities in mice. CD5+ B-1a B cells recognize high-molecular-weight polymeric antigens and produce'natural'antibodies which are critical in the early responses to encapsulated extracellular bacteria, whereas CD5- B-1b B cells produce antibodies required for long-lasting protective immunity to pathogens such as Streptococcus pneumoniae. Conventional B-2 cells function as antibody-producing cells or antigen-presenting cells, promote T helper cell differentiation and provide co-stimulatory signals for T cell activation and thus are implicated in the humoral and cellular immunity. Recently, a subset of CDldhiCD5+CD19hi B10 cells have been identified and proved to negatively regulate T cell-mediated inflammatory responses through IL-10 secretion. Massive evidences from clinical observations and basic researches prove that B cells are of great heterogeneity, indicating.they may differentiate into different subsets with various activities under different physiological and pathological conditions. So, identification and functional analysis of new subsets of B cells are one of challenging and hot topics in the froniters of immunology nowadays.
Innate immune response is the first line of immune defense against pathogens. Well-established components of the innate immune system include natural killer (NK) cells, macrophages, eosinophils, neutrophils and mast cells. A recent study in teleost fish demonstrated that B cells have potent phagocytic activities. Activation of B cells by a wide range of stimuli independently of the B cell receptor (BCR) also suggests that B cells may play a role in innate immunity. Components of bacteria, fungi and virus selectively activate Toll-like receptors (TLR), leading to the shared and unique responses in innate immune cells. So, TLRs (e.g., TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9), which are widely expressed on B cells, may facilitate B cells to sense and respond to microbial antigens, thus proposing a possibility that B cells may participate in innate immune responses aganist pathogenic infections.
Considering the heterogeneity and multiple functions of B cells in innate and adaptive immune responses, we, in the following three parts of our work, analyzed the composition of B cells in the mie after microbial infection and different inflammatory stimulation, investigated the functions and the underlying mechanisms of new B cell subsets we identified, as well as studied the transcriptional regulation of B cell function. The primary aim of this study is to identify new subsets of B cells and investigate the role of the B cell subsets in the innate immunity and regulation of adaptive immune response.
1. Identification and functional characteristics of CD19+DX5+ B cells
To study the role of B cells in the anti-microbial immune responses, we analyzed the B cell subpopulations of different lymph organs from the mice infected with L. monocytogene-, E. coli-or VSV, as well as challenged with LPS-, CpG-ODN-or poly I:C. We found one new subset of B cells with unique phenotype (CD19+DX5+) in the bone marrow, spleen and mesenteric lymph nodes. Then we analyzed the number and proportion of these CD19+ DX5+ cells generated in the infected mice in the following 21 days and found that CD19+ DX5+ cells in the spleen started to increase on the third day post-infection, and reached the peak on the seventh day, then drop gradually and back to the initial number during the 17-21 days post-infection. Besides, number of CD19+DX5+ cells in the stimulus-injected mice increased to the peak five days post-infection and decreased gradually. The data indicated that pathogens and their components could induce the in vivo generation of CD19+DX5+ cells, which may be involved in the regulation of innate and adaptive immune responses against microbial infection.
To further confirm the characteristics of CD19+DX5+ cells, we observed their morphology, and analyzed their phenotype, cytokine profiles. We found that CD19+DX5+ cells had smooth compact nuclei and rare cytoplasm, expressed markers of both NK cells and B cells, such as mIgD, mIgM, B220, IA-e, CD40, CD 154, CD86, Ly49A, Ly49C/I, Ly49D, NKG2D, NKG2A/C/E, CD43, CD25, CD122, CD11b. Thus we named these CD19+DX5+ cells as NKB cells. By intracellular staining and ELISA, we confirmed that NKB cells expressed IFN-γ, IL-1α, IL-1β, IL-6 and Granzyme-B, but not IL-4 or IL-10. In addition, LPS-stimulated NKB cells secreted IgG2a, IgG2b, IgG3, IgM. Importantly, CD19+DX5+ cells could not be inducible generated in Btk-/- mice but could be generated in NK cell-depleted mice, indicating these CD19+DX5+ cells are derived from B cells but not from NK cells. Therefore, we identified one new subset of B cells with NK-like phenotype and secretion of IFN-y and other Thl cytokines.
But how are NKB cells inducibly generated? What are the progenitors of NKB cells? By using co-culture system of CD19+DX5- cells with different kinds of lymphocytes and immune cells, we found that dendritic cells (DC) could induce the differentiation of NKB cells in vitro in the presence of the stimulation with Heat-killed L. monocytogenes (HKLM). To understand the mechanism of NKB cell generation, we used 0.4μM transwell system or added neutralizing antibodies against IL-6, IL-1β, IL-12, CD40, CD40L in the coculture system, and found that DC induced NKB differentiation through CD40-CD40L pathway, while IL-6 and IL-1βproduced by DC could promote the generation of NKB cells. We also confirmed this conclusion in the CD11c-DTR, Cd40-/-, Cd40l-/-, Il1r-/- and Il6-/- mice with L.monocytogenes, in which NKB cells could not be efficiently generated.
Then we investigated the functions of NKB cells in innate defense against listeria, and found that IFN-γproduced by NKB cells could activate macrophages and subsequently promote bacterial clearance. Adoptively transfer of NKB cells from wild type mice but not from Ifnγ-/- mice significantly reduced the bacterial burden in the liver and spleen of the infected mice, with increased serum IFN-y level. So, upon bacterial infection of the mice, NKB cells produced IFN-y to activate macrophages and contributed to the clearance of listeria, suggesting that NKB cells, just like the NK cells, can participate in the innate responses during the early period of listeria infection.
Considering that the number of NKB cells could be induced to reach the peak on the seventh day post-infection, we adoptively transferred CD45.1+ OT-I CD8+T cells into C57BL/6 mice before LM-OVA infection, then purified OT-I T cells and NKB cells from the mice infected with listeria for seven days (LM-7d) and investigated the role of NKB cells in adaptive immune responses. Data showed that NKB cells could induce apoptosis of antigen-specific OT-I CD8+T cells derived from listeiria-infected mice on day 7. Then we went further to study the underlying mechanisms and found that IFN-y produced by NKB cells could upregulate FasL expression on NKB cells and Fas. expression on CD8+T cells in the NKB-CD8+T co-culture system in vitro. Once signals of IFN-y or Fas-FasL were blocked, the apoptosis of antigen-specific OT-I CD8+T cells induced by NKB cells significantly reduced. The data demonstrate that NKB cells can induce the apoptosis of antigen-specific CD8 T cells through IFN-y and FasL-Fas pathway at the late stage of immune response, thus controlling the immune response in an approariate state. So, in other side, NKB cells may exert their immunoregulatory function in the regulation of adaptive immune response, just like regulatory B cells.
Next, the in vivo experiments were designed as follows. We adoptively transferred CD45.1+OT-I CD8+T and LM-7d NKB cells, NK cell or B cells into Btk-/- mice before or after LM-OVA infection. Compared to the control groups with NK cell or B cell transfer, the mice transferred with NKB cells showed decreased numbers of OT-I CD8+T cells seven days post-infection but the expression of CD44, CD62L and CD69 on CD8+T cells remained unchanged. However, the expansion of OT-I CD8+T cells after NKB cell transfer was comparable to that in the control groups, indicating that NKB cells did not affect the generation of antigen-specific CD8+T cells. In addition, adoptive transfer of Ifnγ-/- or Fasl-/- NKB cells had no effect on the number and activation of OT-I CD8+T cells. In brief, NKB cells, generated from the mice infected with listeria on day 7, can induce the apoptosis of antigen-specific CD8+ T cells through IFN-y and Fas-FasL pathway, thus negatively regulating the antigen-specific T cell adaptive immunity appropriately and stabilizing the immune responses once the pathogen infection is cleared.
2. Identification and functional characteristics of CD19+PDCA-1+B cells involved in the innate immune responses
Btk-/- mice were found to be more susceptible to L. monocyto genes infection, showing heavier bacterial burden and lower serum level compared to wild type mice. The data indicate that B cells are required for the immune response against L. monocytogenes infection. To investigate the role of B cells in innate immune responses, we analyzed the populations of splenic B cells from L. monocytogene- or E. coli-infected mice, and found one new subset of B cells with unique phenotype (CD19+PDCA-1+), which were inducibly generated in vivo, and existed in the bone marrow, spleen and mesenteric lymph nodes. Number of CD19+PDCA-1+cells in the spleen increased quickly and reached the peak 48 hours post-infection, then droped gradually and back to the initial number 8 days post-infection. The data indicated that CD19+PDCA-1+cells may be involved in the innate immune responses against microbial infection.
To further confirm the characteristics of CD19+PDCA-1+cells, we observed their morphology, analyzed their phenotype and cytokine profiles. We found that CD19+PDCA-1+cells of diameter 6-8μM were morphologically similar to B cells with smooth compact nuclei and rare cytoplasm. The cells expressed membrane markers of B cells, such as mIgM、mIgD、B220、CD23. Intracellular staining showed that CD19+PDCA-1+ cells were positive for IL-6, IL12p40, TNF-a and IL-la intracellular expressions. ELISA results demonstrated higher concentration of IFN-a and IL-6 was found in the supernatants of PDCA-1+ B cells stimulated with HKLM. So, CD19+PDCA-1+ B cells were proved to be a new unique B cell subset with preferentially production of typeⅠIFN.
We went further to investigate how these CD19+PDCA-1+ B cells were inducibly generated? HKLM stimulation alone failed to convert PDCA-1- B cells from wild-type mice into PDCA-1+ cells. HKLM stimulation generated PDCA-1+ B cells in co-culture of PDCA-1- B cells with macrophages, but not with NK cells, CD4+or CD8+T cells. Physical separation of macrophages from PDCA-1- B cells using a transwell system dramatically reduced the number of PDCA-1+ B cells converted. So, macrophages are required for the generation of PDCA-1+ B cells. What are the molecular mechanisms by which macrophages induce the generation of PDCA-1+ B cells? The generation of PDCA-1+ B cells induced by HKLM in macrophage/PDCA-1-B-cell co-culture was significantly reduced by neutralizing anti-CD40 or anti-CD40L antibody. PDCA-1- B cells from Cd40l-/- mice were unable to switch to the PDCA-1+ phenotype upon HKLM stimulation when co-cultured with macrophages. Also, macrophages from Cd40-/- mice did not induce the generation of PDCA-1+ B cells when cultured with wild type PDCA-1- B cells. Inclusion of neutralization IFN-y antibody in the macrophage-PDCA-1--B cell co-culture significantly reduced the ratio of PDCA-1+ B cells. L. monocytogenes infection of the Ifny-/- mice failed to induce generation of PDCA-1+ B cells. In vivo experiments in Cd40-/-or Cd40l-/- mice failed to demonstrate induction of PDCA-1+ B cell generation after listeria infection. Co-culture with macrophages isolated from Ifnγ-/- mice did not switch PDCA-1-B cells to the PDCA-1+ phenotype. Therefore, macrophages contribute to the generation of PDCA-1+ B cells though CD40-CD40L ligation and IFN-y signaling.
We found that PDCA-1+ B cells facilitated NK cells to produce IFN-y though IFN-a. Deletion of NK cells in L. monocytogenes-infected Btk-/- mice attenuated the protective effects of adoptive transfer of PDCA-1+ B cells as well as the IFN-y response. Adoptive transfer of NK cells from wild-type mice, but not Ifnγ-/- NK cells from Ifnγ-/- mice into NK cell-deleted Btk-/- mice restored the protective action of PDCA-1+ B cells adoptively transferred and the IFN-y production. So, the data suggested that PDCA-1+ B cells facilitate NK cells to produce IFN-y though IFN-a, and IFN-y production by NK cells plays important role in innate protective function of PDCA-1+ B cells against L. monocytogenes infection in vivo. The existence of PDCA-1+ B cells explains more effective anti-listeria responses in WT mice compared to B cell deficient mice. Meanwhile, NK depleted mice also showed heavy bacterial burden and little IFN-y level in sera, which were not recovered after PDCA-1+ B cell/and afterward IFN-γ-/- NK transfusion, indicating that PDCA-1+ B cells could not perform their function without IFN-y-producing NK cell involvement. Therefore, these IFN-a-producing B cells with unique phenotype of PDCA-1+ have been confirmed for the first time as a novel B cell subset inducing an effective responses during the innate defense against L. monocytogenes.
In sum, the results from the current study demonstrated that a novel subset of PDCA-1+IFN-a-producing B cells could trigger IFN-γresponse by NK cells, and by doing so, participate in the early innate immune responses to intracellular bacterial infection. Our results also stand as a proof of the positive feedback of IFN-αand IFN-γsignaling which provide a new mechanistic explanation for the rapid start-up of innate responses to pathogen invasion. Moreover, the identification and functional characterization of the IFN-a-producing PDCA-1+ B cell subset in this study may lead to further research of this B subset in humans that may similarly promote innate defense after bacterial infection. In brief, further defining the role and mechanism of PDCA-1+CD19+ cells in different infective diseases in vivo may provide new insights in innate function of B cells and find therapeutic approaches for treating infections in addition to previous ones.
3. Functional regulation of B cells by transcriptional factor Foxpl
It is well known that transcriptional factors paly critical roles in determining the differentiation and regulating function of immune cells. The subfamily members of Foxp (Foxp1, Foxp2, Foxp3) have attracted much attention in recent years in the field of immunology and cell biology. Altough discovered abd cloned from mouse B lymphoma cell line BCL110 years ago, few studies have been reported for the roel of Foxpl in the differentiation and functiona regulation of immune cells, especially B cells. To study the molecular mechanism of differentiation and function of B cells, we investigated regulation of B cell function by Foxpl. First, we confirmed Foxpl was widely expressed in the tissues, including heart, brain, lung, thymus, spleen, axillary and mesenteric lymph nodes. Then we found that Foxpl was expressed in B cells, CD4+T cells, CD8+T cells and macrophages. Furthermore, we confirmed that Foxpl expression in the B cells of Foxpl mutant mice decreased signicantly as compared to that of wild-type mice, identifying the mutant mice with Foxpl gene knockdown. B cells from Foxpl knockdown mice produced less IL-10 in vitro than B cells from wild type mice after LPS stimulation. Also, B cells from Foxpl knockdown mice produced less IL-10 than B cells from wild type mice after listeria infection. After LPS stimulation, the mRNA expression of inflammatory factors, such as IL-6, IFN-a, IFN-y, was upregulated. But IFN-γexpression in B cells from Foxpl knockdown mice were downregulated, while expression of IL-6 and IFN-a had no significant difference between mutant and wild type mice after liseria infection. Therefore, we suggest that Foxpl may facilitate the regulatory function of B cells, but how it regulate the inflammatory cytokine production and what role it plays in the innate function of B cells remain unclear, which need to be investigated in the future.
4. Summary
In PartⅠof our study, we have identified a new B cell subset (DX5+CD19+) with NK-like phenotype and cytokine profiles and named them as NKB cells. DX5+CD19+NKB cells have been found to produce IFN-γto activate macrophages and promote the clearance of intracellular bacteria in early period of listeria infection, indicating NKB cells could participate in the innate immune responses. Interestingly, we found large amounts of NKB cells generated in the late period of immune response (7 days after listeria infection), and NKB cells could upregulate FasL expression on themselves and Fas expression on antigen-specific CD8+ T cells by IFN-γand consequently induce apoptosis of antigen-specific CD8+ T cells. The dat suggest that NKB cells may negatively regulate the antigen-specific T cell adaptive immunity in the late period of immune response against pathogen and stabilizing the immune responses appropriately. Therefore, identification and functional chracterististics of DX5+CD19+ NKB cells provide new insights in innate responses, contribute to the better understanding of regulatory function of B cells, and outlines a new manner of negative regulation of adaptive T cell immunity by B cells.
In PartⅡof our study, we identified a new subset of PDCA-1+CD19+ B cells inducibly generated in the mice infected with bacteria L. monocytogenes and E. coli. PDCA-1+CD19+ B cells secreted a large amount of IFN-αand thus facilitated IFN-γproduction and cytotoxicity function of NK cells via IFN-α. B cell-deficient Btk-/- mice were incapable of producing PDCA-1+CD19+ B cells, and more sensitive to L. monocytogenes infection. Adoptive transfer of PDCA-1+CD19+B cells to Btk-/- mice normalized their resistance to L. monocytogenes infection. Furthermore, we found that macrophages were essential for the inducible generation of PDCA-1+CD19+ B cells via CD40-CD40L ligation and IFN-γ. Therefore, we identify a new subset of PDCA-1+CD19+ B cells during the bacterial infection, which can enhance innate immune responses against bacterial infection by activating NK cells via secretion of IFN-α.
In PartⅢof our study, we studied the transcriptional regulation of differentiation and function of B cells, and found that less IL-10 was produced by B cells from Foxp1 knockdown mice after LPS stimulation and listeria infection. The premilinary data suggest that Foxpl may facilitate the regulatory function of B cells, but how it regulates the inflammatory cytokine production and the innate function of B cells remain unclear to date.
In brief, defining the roles and the underlying mechanisms of B cell subsets in the innate and adaptive immune response against pathogens may provide new insights in the initiation, maintenance and modulation of immune response, broaden our understanding of immune regulation network, and may be helpful to the design of new approaches to the immunotherapy of infectious diseases.
引文
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