【作者】 钱莉；

【导师】 曹雪涛；

【作者基本信息】 第二军医大学 ， 免疫学， 2009， 博士

【摘要】 树突状细胞（Dendritic cell, DC）是目前已知的体内功能最强大的专职抗原提呈细胞,能够整合一系列的外来病原体信号并将其传递给淋巴细胞,启动合适的免疫应答反应。未成熟DC在微生物感染或移植后识别、吞噬外源性抗原,可快速成熟。成熟DC的吞噬能力减弱,但细胞表面共刺激分子和粘附分子的表达上调,并分泌促炎症细胞因子,启动初始的T细胞介导的免疫应答反应。近年来,越来越多的研究表明DC是一种异质性的细胞群体,分布于不同的解剖部位,含有不同的细胞亚群,处于不同的成熟阶段,表达不同的表型和细胞因子,其功能也是多样性的。其中,具有负向免疫调控作用的DC亚群的发现及其功能特点与作用机制的研究是该领域近年来的重要进展之一,此类具有负向免疫调控作用的DC亚群被称为调节性DC （Regulatroy DC）。目前认为,调节性DC主要通过选择性的诱导Th2型的免疫应答或促使初始CD4⁺和CD8⁺ T细胞分化为分泌IL-10的调节性T细胞来负向调控免疫应答。在前期的研究中,我们发现以往被认为终末分化细胞的成熟DC,与脾基质细胞共培养后能够进一步增殖并分化为一类能够通过分泌NO而抑制T细胞增殖的新型调节性树突状细胞,我们将之命名为“分化样树突状细胞”（Differentiated dendritic cell,diffDC）,从而提出了成熟DC并非均为终末分化细胞的观点,其在完成了抗原递呈任务之后,在次级淋巴器官微环境的作用下,仍能够进一步增殖分化,从而被赋予了新的生命与功能。我们进一步对于diffDC这一新型调节性DC亚群的性质与功能特点进行了研究,发现了ERK的高度磷酸化和p38的低活化与diffDC的独特细胞因子谱（高表达IL-10而低表达IL-12）的表达密切相关。在不同的生理或病理情况下,diffDC与周围很多免疫细胞存在相互作用,彼此精密调节,例如可以通过分泌IL-10活化NK细胞而导致自身的杀伤,并通过分泌IP-10选择性地趋化Th1细胞并抑制其增殖,从而维持机体的稳态。而B细胞是免疫系统中的一种主要细胞,能够通过产生抗体在体液免疫中发挥着中心作用,也可以作为抗原提呈细胞,通过产生细胞因子,提供抗原信息和共刺激信号以促进CD4⁺ T细胞向Th1还是Th2分化,来调节CD4⁺ T细胞对外来或自身抗原的免疫反应。在脾脏淋巴细胞中B细胞约占50%,在免疫反应的后期,脾脏中的具有独特调节功能的diffDC有可能会与B细胞相遇。那么diffDC能否作用于B细胞,调控B细胞的功能?如果具备此功能,其生物学意义及其相关的作用机制又是什么呢?鉴于目前尚未见调节性DC参与B细胞功能调控的报道,我们在本研究中分为两部分内容,分别围绕调节性DC对B细胞的调控作用及其相关的机制进行了探讨。一、调节性树突状细胞诱导高分泌IL-10的新型调节性B细胞的产生在本部分实验中,我们研究了调节性DC（diffDC）对B细胞功能的调控作用。首先,我们将diffDC在体外与脾脏来源的B细胞共培养,观察共培养后,B细胞的增殖、抗体分泌和凋亡情况。结果表明,diffDC不能引起B细胞凋亡,对B细胞的增殖也没有明显影响。与imDC和maDC一样,diffDCs也能诱导B细胞分泌IgM和IgG,这与原来文献报道DCs可诱导B细胞合成IgM和IgG是一致的。我们又进一步检测了diffDC对B细胞分泌细胞因子谱的影响。结果发现,diffDCs不影响B细胞分泌IL-12、IFN-γ、IL-6、IP-10、PGE2和TGF-β等细胞因子,但是让我们感兴趣的是,diffDC和B细胞共培养后产生的IL-10水平显著高于imDC或maDC与B细胞共培养对照组。diffDC与B细胞按不同的比例共培养均能诱导高水平的IL-10分泌,其中diffDC和B共培养比例为1:5的情况下,所诱导产生的IL-10水平最高,1:10的共培养比例次之。由于1:10的diffDC与B细胞共培养比例更接近于体内DC和B细胞的比例,因此,我们在随后的实验体系中均选择了这一比例进行DC和B细胞的共培养。diffDC和B细胞共培养后24小时IL-10的分泌就出现显著升高,48小时达到高峰。胞内染色流式检测确证IL-10主要来源于B细胞。以上结果提示我们,diffDC有可能作用于B细胞,诱导B细胞向高分泌IL-10的调节性B细胞分化。随后我们探讨了diffDC能否对B细胞的表型产生影响。我们用流式筛选了一系列的膜表面分子,最后发现diffDC诱导B细胞高表达CD19、CD16/CD32和CD62L。由于荧光标记的抗CD16/CD32单抗能识别活化型受体FcγRIII （CD16）, FcγRIIa （CD32a）和抑制型受体FcγRIIb （CD32b）,因此,我们利用流式分选出共培养体系中的CD19^hiCD16/CD32^hi的B细胞,提取其总RNA,RT-PCR的方法证实了diffDC诱导B细胞高表达的CD32分子是抑制型受体FcγRIIb。下一步我们想知道是不是diffDC所诱导的这群CD19^hiFcγRIIb^hi的B细胞产生了高水平的IL-10。我们利用流式分选出共培养体系中CD19^hiFcγRIIb^hi和CD19^loFcγRIIb^lo的B细胞,在体外分别用TLR3配体（polyI:C）、TLR4配体（LPS）、TLR9配体（CpG ODN）刺激24小时,结果发现CD19^hiFcγRIIb^hi B细胞分泌IL-10的水平显著高于CD19^loFcγRIIblo B细胞,LPS刺激可促进CD19^hiFcγRIIb^hi B细胞分泌更高水平的IL-10。综合以上的结果,我们认为diffDC诱导的高分泌IL-10的调节性B细胞具有独特的表型,即CD19^hiFcγRIIb^hi,这种表型与以往文献报道的调节性B细胞的表型均不一致,可确定其为一类新型调节性B细胞亚群。综上所述,在本部分实验中,我们发现diffDC可以诱导脾脏B细胞分化为高分泌IL-10的具有独特表型(CD19^hiFcγRIIb^hi)的新型调节性B细胞亚群。二、新型调节性B细胞的产生机制及相关功能研究在本部分实验中,我们研究了diffDC所诱导的CD19^hiFcγRIIb^hi调节性B细胞的相关功能以及difffDCs诱导其产生的机制,并在体内证实了这类CD19^hiFcγRIIb^hi调节性B细胞的存在。由于diffDC诱导的这类新型调节性B细胞亚群高表达FcγRIIb,而FcγRIIb作为重要的抑制性受体,可以介导吞噬,发挥免疫负向调节作用。因此,我们利用流式检测了diffDC所诱导的这群CD19^hiFcγRIIb^hi调节性B细胞对免疫复合物（IC）的吞噬能力。结果表明,CD19^hiFcγRIIb^hi调节性B细胞与常规脾脏B细胞相比,吞噬IC的能力明显增强。随后我们利用FcγRIIb缺陷小鼠制备调节性B细胞,结果发现,FcγRIIb缺陷后,调节性B细胞吞噬IC的能力显著下降,表明FcγRIIb受体介导了CD19^hiFcγRIIb^hi调节性B细胞对IC的吞噬。我们进一步检测了CD19^hiFcγRIIb^hi调节性B细胞对特异性抗原肽活化后的CD4+ T细胞增殖的影响。结果表明,与CD19^loFcγRIIblo B细胞相比,CD19^hiFcγRIIb^hi B细胞能够显著抑制抗原特异性T细胞的增殖反应,但不影响活化T细胞分泌IL-2和IFN-γ。以上结果表明,CD19^hiFcγRIIb^hi调节性B细胞可以通过吞噬IC以及抑制抗原特异性T细胞的增殖反应从而发挥免疫负向调控作用。为了探讨diffDCs诱导调节性B细胞的产生机制,我们利用transwell系统将diffDC与B细胞隔离培养,结果发现diffDC诱导B细胞产生IL-10既需要可溶性因子介导,也需要细胞间的直接接触。在前期的研究中,我们已证实diffDC可高分泌IL-10、IL-6、IP-10、IFN-β和NO等可溶性因子,因此,我们使用IL-10缺陷小鼠制备diffDC或者预先用中和性抗体以及NO合成酶抑制剂PBIT中和这些细胞因子和NO的效应,然后与B细胞共培养,结果发现用抗IFN-β的中和性抗体和PBIT预先处理过的diffDC与B细胞共培养后产生IL-10的水平显著降低,表明diffDC诱导调节B细胞高分泌IL-10是由diffDC所产生的IFN-β和NO所介导的。下一步我们探索了diffDC上哪些膜分子介导了调节性B细胞高分泌IL-10。由于diffDC表达CD40L和B7H1,我们将CD40缺陷的B细胞与diffDC共培养,或者在diffDC与B细胞共培养体系中加入抗B7H1的中和性抗体,结果发现diffDC与CD40缺陷小鼠共培养后产生的IL-10的水平显著降低,表明diffDC上的CD40L和B细胞上的CD40直接接触也参与介导了diffDC诱导调节B细胞高分泌IL-10。为了明确我们在体外用diffDC诱导的CD19^hiFcγRIIb^hi调节性B细胞是否在小鼠体内存在相应的亚群,我们利用磁珠分选小鼠的脾脏和淋巴结CD19+ B细胞,利用流式检测证实了小鼠体内存在着这类CD19^hiFcγRIIb^hi B细胞。进一步通过从体内分选出CD19^hiFcγRIIb^hi B细胞并对其功能进行了分析,研究表明,无论是小鼠脾脏还是淋巴结中的CD19^hiFcγRIIb^hi B细胞,都与体外diffDC所诱导产生的新型CD19^hiFcγRIIb^hi调节性B细胞亚群的功能类似,可以高分泌IL-10,对IC的吞噬能力增强以及抑制抗原特异性CD4+ T细胞的增殖,从而证明小鼠体内存在着这类我们发现的新型CD19^hiFcγRIIb^hi B细胞。综上所述,在本部分实验中,我们发现diffDC可通过产生IFN-β和NO以及表达的CD40L作用于B细胞,诱导其分化为高分泌IL-10并具有CD19^hiFc?RIIb^hi表型特征的调节性B细胞。diffDC所诱导的CD19^hiFc?RIIb^hi调节性B细胞对IC的吞噬能力增强,并且可以抑制抗原特异性T细胞的增殖反应。最后,我们在小鼠体内证实了这群调节性B细胞的存在。总之,我们的实验结果表明,diffDC可通过分泌IFN-β和NO以及CD40L/CD40相互作用而诱导脾脏B细胞分化为高分泌IL-10的具有独特表型(CD19^hiFc?RIIb^hi)的新型调节性B细胞亚群,从而进一步扩大了其独特的免疫负向调控功能。本实验结果丰富了对免疫微环境负向调控整个免疫应答多细胞网络的认识,为今后进一步研究新型调节性DC（diffDC）参与免疫负向调控的机制提供了新的方向和一定的实验基础,为探索肿瘤、自身耐受和自身免疫性疾病等的发生机制及免疫治疗的应用提供了新的研究途径。更多还原

【Abstract】 Dendritic cells （DCs）, the most potent professional antigen-presenting cells （APCs）, have the unique capacity to integrate a wide array of incoming signals and convey them to lymphocytes, directing the appropriate immune responses. It is well known that immature DCs （imDCs） undergo rapid maturation following microbial infection or transplantation. Mature DCs lose endocytic activity, upregulate the surface expression of adhesion and costimulatory molecules, secrete proinflammatory cytokines and initiate primary T cell-mediated immune responses. In recent years, more and more studies have shown that DCs are heterogeneous cell population, with many kinds of DC subsets exhibiting different phenotype and functions. Among the DCs subsets, one population of DCs with negative immune regulation, so-called regulatory DCs, attracts much attention, and more evidences show that the regulatory DCs negatively regulate immune response by promoting naive CD4⁺ and CD8⁺ T cells to differentiate into IL-10-producing T regulatory/suppressor cells or inducing a preferential Th2 response. However, the machanisms by which regulatory DCs negatively regulate immune response remain to be fully understood to date.Our previous studies have shown that splenic stromal cells, mimicking the secondary lymph organ microenvironment, can drive mature DCs to proliferate and differentiate into a novel subtype of regulatory DCs （diffDCs）, which express a higher level of IL-10 but minimal IL-12p70 and inhibit antigen-specific T-cell proliferation. Up-regulation of extracellular signal-regulated kinase （ERK1/2） activation was shown to be responsible for IL-10 preferential production, and suppression of p38 activation was for impaired IL-12p70 production in diffDCs. diffDCs can interact with other immune cells in physiology and pathology conditions. For example, diffDCs can active natural killer （NK） cells through diffDC-derived IL-10 and selectively recruit more Th1 cells through diffDC-derived IP-10 and inhibit Th1 proliferation. We have identified an in vivo counterpart of diffDCs in the spleen with similar phenotype and functions.B cells positively regulate immune responses through antibody production and optimal CD4⁺ T-cell activation. However, B cells possess additional immune functions, including the production of cytokines and the ability to function as APC. B cell development encompasses a continuum of stages that begin in primary lymphoid tissue; with subsequent functional maturation in secondary lymphoid tissue. Also, regulatory B cells have been recently identified and play important roles in the immune hematostasis. These findings suggest that secondary lymphoid tissue may be potential locations for diffDC-B cell interaction. However, whether diffDCs and B cells interact in these locations and what the consequence of these interactions may be have not been elucidated.Up to now, little is known about how regulatory diffDCs modulate the B cells function in the late period of the immune response. Therefore, in this study, we have two parts to investigate the effects of diffDCs on the function of B cells and then we further investigated the underlying mechanisms for the regulation of B cells by diffDCs.1. Regulatory dendritic cells （diffDCs） induce freshly isolated splenic B cells to differentiate into a novel subtype of regulatory B cell characterized by the ability to preferentially produce IL-10.The primary aim of this part of our study is to investigate whether diffDCs can interact with B cells and what the consequence of these interactions.Firstly, we analyzed the proliferation, apoptosis, antibody secretion and cytokine production by B cells after interaction with imDCs, maDCs or diffDCs. The proliferation and apoptosis of B cells were not significantly affected after interaction with imDCs, maDCs or diffDCs. Moreover, no difference in IgM and IgG2b production was detected between B cells after interaction with diffDCs and B cells after interaction with imDCs or maDCs. On the other hand, by screening cytokine production in B cells cocultured with diffDCs, we found there was no significant difference in production of IL-12, IFN-γ, IL-6, IP-10, PGE2 and TGF-βin B cells cocultured with diffDCs and cultured alone. Interestingly; B cells cocultured with diffDCs compared with B cells cocultured with imDCs or maDCs produced higher levels of IL-10 at varying ratios of DC: B. The ratio of 1:10 DC to B was closer to the ratio of DC to B in vivo. Therefore, cells were cocultured at the ratio of 1:10 DC to B in the following experiments. Furthermore, IL-10 production by B cells cocultured with diffDCs was increased gradually and reached a maximum after 48 hours of coculture and the increased IL-10 secretion was mainly from B cells as demonstrated by intracellular staining. Taken together, these results suggested that regulatory diffDCs can induce splenic B cells to differentiate into regulatory B cells with preferential IL-10 secretion.To investigate whether diffDCs could affect the expression of membrane molecules on B cells, experiments were performed in which the B-cell phenotype was analyzed by flow cytometry both in freshly isolated B-cell populations and in B cells that had been cultured for 48 hours with diffDCs. After culture with diffDCs, B cells displayed increased expression of CD19, CD62L and CD16/CD32. Available anti-CD16/CD32 monoclonal antibody recognizes activating receptors FcγRIII （CD16）, FcγRIIa （CD32a） and inhibitory receptor FcγRIIb （CD32b）. To investigate which receptor is overexpressed on CD19^hiCD16/CD32hi B cells, FcγRIIb expression in splenic B cells and sorted CD19^hiCD16/CD32hi B cells at mRNA level was analyzed. The FcγRIIb mRNA expression in CD19^hi B cells was up-regulated more significantly than that in splenic B cells .These data suggested that it is FcγRIIb that is overexpressed on CD19^hi B cells.We then wondered whether the CD19^hiFcγRIIb^hi B cells produce preferentially IL-10 than B cells. After cocultured with diffDCs, B cells could be divided into two subpopulations: CD19loFcγRIIblo and CD19^hiFcγRIIb^hi. We sorted CD19loFcγRIIblo and CD19^hiFcγRIIb^hi populations and stimulated them with polyI:C、LPS and CpG ODN for 24 hours respectively, then assayed IL-10 production in the supernatants. The results showed that sorted CD19^hiFcγRIIb^hi populations secreted significantly higher IL-10 production than CD19loFcγRIIblo populations or splenic B cells. Furthermore, LPS could stimulate CD19^hiFcγRIIb^hi populations to produce more IL-10 protein than for CD19loFcγRIIblo populations or splenic B cells. However, IL-10 production in CD19^hiFcγRIIb^hi populations could not be enhanced by stimulation with CpG ODN or polyI:C. Together, these results demonstrated that diffDCs can induce an IL-10-secreting B cell population with CD19^hiFcγRIIb^hi phenotype.In summary of this part of our study, we demonstrate that diffDCs induce splenic B cells to differentiate into a novel subtype of regulatory B cells （Bregs） with high CD19, FcγIIb and CD62L expression and high IL-10 secretion.2. The functions of CD19^hiFcγIIb^hi B cells and mechanisms underlying differentiation of CD19^hiFcγIIb^hi B cells driven by regulatory diffDCs. The primary aim of this part of our study is to investigate the functions of CD19 hiFcγIIb^hi Bregs and the detailed mechanisms of CD19^hiFcγRIIb^hi Bregs differentiation and identification of an in vivo counterpart of these Bregs.As a very important immune regulatory molecule, FcγRIIb is overexpressed on the IL-10-producing Bregs identified in our system. It has been shown that Fc receptor mediates effective internalization of Ag-IgG complexes. To investigate whether FcγRIIb is involved in the phagocytosis of immune complex （IC）, B cells and Bregs were incubated with FITC-OVA-IC, we found that Bregs had enhanced phagocytic capacity compared with that of B cells by FACS and immunofluorescence microscopy. To further confirm FcγRIIb mediates phagocytosis of FITC-OVA-IC by Bregs, FcγRIIb-/- Bregs were generated and then used to incubate with FITC-OVA-IC. The results showed that the ability of FcγRIIb-/- Bregs for the uptake of FITC-OVA-IC as determined by FACS significantly decreased as compared with that of CD19^hiFcγRIIb^hi Bregs. In addition, CD19^hiFcγRIIb^hi Bregs significantly inhibited maDC-induced CD4+ T cell proliferation compared with B cells, without depressing IL-2 or IFN-γsecretion. Our results indicated that CD19^hiFcγRIIb^hi Bregs can regulate immune response by inhibiting CD4+ T cell proliferation with the enhanced phagocytic capacity.To determine the differentiation process of CD19^hiFcγRIIb^hi Bregs depends on soluble mediators released by diffDCs or direct contact with diffDCs, we performed the experiments both in cell-cell contact and in transwell systems. Our data demonstrated that IL-10 production in CD19^hiFcγRIIb^hi Bregs depended on soluble mediators and also cell-cell contact. As reported previously by us, diffDCs secrete considerable amounts of IL-10, IL-6, IP-10, NO and IFN-β. We generated IL-10-/- diffDCs or used specific neutralizing mAbs and the selective NO synthase inhibitor dihydrobromide （1,4 PBIT） to test the role of these soluble mediators secreted by diffDCs in inducing IL-10-producing B cells, we found that antibody blockade of IFN-βand the NO synthase inhibitor PBIT partially inhibited IL-10 production by B cells induced by diffDCs. As reported that CD40L-CD40 pathway is an important pathway in DCs to B cells contact. CD40-/- B cells were generated to coculture with diffDCs. Surprisingly, absence of CD40 on B cells impaired their ability to produce IL-10, indicating that CD40 is also involved in the differentiation of CD19^hiFcγRIIb^hi Bregs. In contrast, B7H1 blockade had little effect on B-cell IL-10 production induced by diffDCs. Together, these data suggest that both diffDC-B direct contact in a CD40L-CD40 dependent way and diffDC-derived IFN-βand NO play critical roles in CD19^hiFcγRIIb^hi Bregs differentiation. Considering the above data are just based on the differentiated CD19^hiFcγRIIb^hi Bregs in vitro, we next investigated whether the natural counterpart of CD19^hiFcγRIIb^hi Bregs exist in vivo. We analyzed CD19⁺ B cells in spleen and lymph node of mice on the basis of the phenotype of CD19^hiFcγRIIb^hi, and only about 4.30% of CD19+ B cells were CD19^hiFcγRIIb^hi B cells, which were similar to CD19^hiFcγRIIb^hi B cells induced by diffDCs in vitro with respect to cytokine profile, phagocytic capacity and inhibition of CD4+ T cell proliferation. These results indicate that the natural counterpart of CD19^hiFcγRIIb^hi Bregs do exist in vivo.In conclusion, we show that diffDCs can induce freshly isolated splenic B cells to differentiate into a distinct regulatory B subset, CD19^hiFcγIIb^hi Bregs, which preferentially secret IL-10 and have regulatory functions in vitro and in vivo. These Bregs inhibit maDCs-initiated the proliferative response of antigen-specific CD4+ T cells. Also, CD19^hiFcγIIb^hi Bregs have enhanced phagocytic capacity as compared with that of B cells, and FcγRIIb mediates the uptake of IC by CD19^hiFcγIIb^hi Bregs. diffDC-derived IFN-β, NO and CD40L/CD40 interaction between diffDC/B cells play critical roles in the differentiation of CD19^hiFcγIIb^hi Bregs. Our results suggest that the regulatory DCs provide a new manner for negative feedback control of T cell immune response and maintenance of immune homeostasis by, at least partially, inducing differentiation of B cells into regulatory B cells.更多还原