调节性树突状细胞诱导CD4~+T细胞凋亡及促进记忆性CD4~+T细胞产生的实验研究
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摘要
树突状细胞(Dendritic cells,DC)是功能最强大的专职抗原提呈细胞,在免疫应答与免疫耐受中发挥重要的作用。DC的不同功能是由于不同亚群的存在或处于不同发育阶段的细胞所介导的。近年来,具有免疫调节功能的DC的鉴定、特征及其在免疫调节与免疫性疾病发病机制与治疗中的作用是免疫学领域的研究热点。调节性DC可在多种条件下产生,譬如应用抑制性细胞因子条件培养(如IL-10或TGF-β,VIP,TSLP等),或者基质细胞共培养。目前发现,调节性DC可诱导调节性T细胞产生,抑制T细胞增殖或诱导T细胞增殖阻滞。在前期研究中,我们发现脾脏基质能诱导成熟DC分化成一群新型的调节性树突状细胞亚群(diffDC),diffDC可通过分泌NO而抑制抗原特异性活化型CD4~+T细胞的增殖,但不会诱导调节性T细胞的产生。目前为止,对于diffDC发挥免疫调节功能的详细机制仍不完全清楚。
机体可精细调节T细胞的凋亡以维持免疫稳态。在抗原识别后,外周初始CD4~+T细胞增殖并分化成效应T细胞以启动免疫应答。当抗原被清除后,大部分效应T细胞发生凋亡以免破坏T细胞稳态及避免免疫功能过度活化而导致自身免疫等疾病的发生。凋亡的方式主要有:外在的细胞死亡受体介导与caspase依赖的凋亡,内在的线粒体与caspase依赖的凋亡,或者内在的线粒体依赖而caspase非依赖的凋亡。那么,diffDC是否能够通过诱导活化型CD4~+T细胞的凋亡而发挥其负向免疫调控呢?如果具备此功能的话,其具体的作用机制又是什么?
免疫记忆是适应性免疫的一大特点,它是由抗原特异性淋巴细胞在克隆增殖并分化成长期存在的记忆性淋巴细胞而产生的。记忆性淋巴细胞可在外周组织中为机体提供快速的保护力并可在二级淋巴器官内产生针对特异性抗原的再次免疫应答。记忆性淋巴细胞包括记忆性B细胞与记忆性T细胞,记忆性B细胞经再次抗原刺激后可分化成浆细胞分泌抗体来介导保护力;而记忆性T细胞可根据效应功能状态及归巢受体表达情况而分为效应型记忆性T细胞与中枢型记忆性T细胞,效应型记忆性T细胞不表达CCR7且可在外周炎症部位发挥快速的效应功能,中枢型记忆性T细胞则表达CCR7与CD62L,存在于二级淋巴器官的T细胞区,没有效应功能,但是可在抗原刺激下快速增殖并分化成效应T细胞。记忆性T细胞的产生可分为克隆增殖、克隆收缩及记忆细胞维持三个阶段,因此记忆性T细胞产生的数量与这三个阶段的调控密切相关。由于CD8~+T细胞在感染或活化后快速增殖并大量扩增且幅度远高于CD4~+T细胞,这为记忆性CD8~+T细胞的研究带来极大便利并已取得长足的进展,目前关于记忆性CD8~+T细胞的研究具有一个广泛认可的分化模式,即一部分完全分化的效应T细胞转变成长期存活的记忆性T细胞。然而,相对于记忆性CD8~+T细胞,记忆性CD4~+T细胞的数量明显较少而且随时间延长还会继续减少,这给记忆性CD4~+T细胞的研究带来了很大的困难,因此,目前对于记忆性CD4~+T细胞的产生和维持及其相关的机制仍不十分清楚。我们想知道是否diffDC也可能影响记忆性CD4~+T细胞的产生。
在本研究中,我们通过两部分实验对我们以往发现的调节性树突状细胞diffDC如何调节CD4~+T细胞的功能及其可能的机制进行了探讨。
一、调节性树突状细胞诱导活化型CD4~+T细胞凋亡及其机制研究
这部分研究的目的是明确diffDC是否可诱导抗原特异性活化型CD4~+T细胞的凋亡,如果能的话,进一步探讨具体的机制。
首先,我们将diffDC在体外加入到mDC诱导OVA_(323-339)特异性CD4~+T细胞活化的共培养体系中,观察它是否可以诱导活化型CD4~+T细胞的凋亡,我们发现,实验组diffDC/mDC/CD4T与对照组mDC/CD4T相比,diffDC/mDc/CD4T组内7AAD~+CD4~+T细胞(晚期凋亡)与Annexin V~+ 7AAD~- CD4~+T细胞(早期凋亡)的比例均明显高于mDC/CD4T组,这表明在共培养体系中,diffDC可以明显促进活化型CD4~+T细胞的凋亡。那么,如果将抗原特异性CD4~+T细胞预先用mDC与抗原进行活化后,再加入diffDC后是否也能促进其凋亡呢?进一步研究发现,diffDC也可以促进预活化的CD4~+T细胞的凋亡,其凋亡情况与共培养体系所得结果类似。可见,diffDC能够显著诱导活化型CD4~+T细胞的凋亡。
在发现了这一现象之后,我们进一步探讨了diffDC诱导活化型CD4~+T细胞凋亡的具体机制。首先,我们确定diffDC产生的可溶性因子还是膜分子,或者是diffDC的膜分子与mDC或活化CD4~+T细胞上的膜分子作用后产生的可溶性因子作用于活化型CD4~+T细胞而诱导其凋亡。采用Transwell系统将diffDC与mDC/CD4T细胞隔开培养,结果发现diffDC诱导活化型CD4~+T细胞凋亡显著下降,表明diffDC诱导活化型CD4~+T细胞凋亡依赖于diffDC与活化型CD4T细胞间的直接接触,即直接由diffDC上的膜分子或间接由diffDC上的膜分子与mDC或活化CD4~+T细胞作用后产生的可溶性因子来介导。鉴于可溶性因子可能参与diffDC的诱导凋亡作用以及我们前期研究发现NO介导了diffDC的抑制功能,我们检测发现diffDC/mDC/CD4T共培养体系中存在高水平IFN-γ与NO,IFN-γ与NO的分泌也依赖于diffDC与活化型CD4~+T细胞间的直接接触,结果提示IFN-γ与NO也可能参与diffDC对于活化型CD4~+T细胞的凋亡诱导作用。
下一步我们探索diffDC上的哪些膜分子介导了其凋亡诱导作用。我们采用流式细胞检测一些诱导凋亡的相关分子,发现diffDC高表达FasL与B7-H1,不表达TRAIL与B7-DC。因此,我们采用FasL缺陷的gld小鼠来源的diffDC或mDC,研究确定diffDC上的FasL是否参与诱导活化CD4~+T细胞的凋亡,发现gld小鼠来源的diffDC(gld diffDC)不能有效抑制mDC诱导的CD4~+T细胞的增殖,也不能诱导活化CD4~+T细胞的凋亡,这表明diffDC的FasL介导了diffDC对于活化型CD4~+T细胞的凋亡诱导作用。进一步我们采用体内实验证明,gld小鼠来源的diffDC也不能在体内抑制mDC诱导的CD4T细胞的增殖。鉴于FasL对于diffDC功能的重要意义,我们需要明确diffDC高表达FasL的具体机制,以及脾脏内的对应的diffDC群体是否也高表达FasL。我们以往的研究表明TGF-β在基质细胞诱导maDC向diffDC分化中发挥重要作用,因此我们研究了TGF-β对于diffDC表达FasL的影响,结果发现,脾脏基质细胞的TGF-β产生被阻断后所诱导的diffDC的FasL表达明显下降,同时ERK磷酸化水平也明显下降,并且diffDC在直接用ERK磷酸化抑制剂作用后FasL表达明显下降,因此,基质细胞来源的TGF-β通过促进diffDC的ERK磷酸化而促进diffDC高表达FasL。在体内diffDC对应群体分析上,我们发现CD11b~(hi)CD11c~(low)Ia~(low) cells(diffDC)的FasL表达也高于CD11c~(hi)Ia~(hi)(mDC),尤其是在免疫应答过程中,除了diffDC的比例上调外,FasL的表达也明显上调,这更进一步提示了FasL在diffDC免疫调控功能上的重要性。
由于diffDC膜分子依赖的可溶性因子也在diffDC诱导活化型CD4~+T细胞凋亡中发挥重要作用,我们拟明确是哪些因子介导的?由于近年来研究表明IFN-γ在CD4~+T细胞的凋亡及负向免疫调控中发挥重要作用,结合我们前期研究发现NO参与了diffDC的抑制功能而且diffDC/mDC/CD4T共培养体系中存在高水平IFN-γ与NO,因此我们采用IFN-γ阻断性抗体或外加IFN-γ及NO抑制剂,然后观察对于diffDC诱导活化型CD4~+T细胞凋亡的影响,结果发现,IFN-γ与NO均部分参与了diffDC对于活化型CD4~+T细胞的凋亡诱导作用。
由于diffDC的膜分子FasL介导了其诱导凋亡作用,而且膜分子依赖性的IFN-γ与NO也参与了诱导凋亡作用,那么它们之间是否有关联呢?是否是diffDC上的FasL促进了下游的IFN-γ与NO的产生呢?我们发现将FasL缺陷的diffDC加入mDC或者FasL缺陷的mDC诱导OVA_(323-339)特异性CD4~+T细胞活化的共培养体系中,培养上清中IFN-γ与NO的水平相对于diffDC/mDC/CD4T组均有一定程度的下降,而不论mDC是否存在FasL缺陷,diffDC均可明显上调mDC/CD4T共培养体系中的IFN-γ与NO水平,这表明diffDC/mDC/CD4T中IFN-γ与NO的高分泌依赖于diffDC上的膜分子FasL。进一步研究发现diffDC表达的FasL诱导活化型CD4~+T细胞高分泌IFN-γ,继而IFN-γ促进diffDC与活化型CD4~+T细胞高分泌NO,最终引起活化型CD4~+T细胞的凋亡。
既然diffDC表面的FasL在diffDC诱导活化CD4~+T细胞的凋亡中发挥了重要作用,那么diffDC上的FasL如何与活化CD4~+T上的Fas相互作用?是否会上调活化型CD4~+T细胞表面的Fas表达从而使活化型CD4~+T细胞更易于发生凋亡呢?鉴于此,我们研究了在共培养体系中,实验组diffDC/mDC/CD4T与对照组mDC/CD4T组中CD4~+T的Fas表达情况,发现diffDC/mDC/CD4T组中活化型CD4~+T细胞的Fas表达在第四天时明显高于mDC/CD4T组中的CD4~+T细胞,表明diffDC可通过上调活化型CD4~+T细胞的Fas表达,使活化型CD4~+T细胞更易于通过与diffDC表达的FasL相互作用而发生凋亡。进一步研究表明,这种上调依赖于diffDC与活化型CD4~+T细胞的直接接触以及diffDC表达的FasL,但是不依赖于IFN-γ与NO的作用。以上结果表明diffDC表达的FasL可上调活化型CD4~+T细胞表达Fas,从而使其更易于通过与diffDC表达的FasL相互作用而发生凋亡。
以上结果表明在初次免疫应答过程中,调节性树突状细胞(diffDC)可通过FasL/Fas作用途径激发下游多种机制来诱导活化型CD4~+T细胞的凋亡继而抑制其增殖,从而在初次免疫应答中发挥重要的负向调控功能。
二、调节性树突状细胞对记忆性CD4~+T细胞产生的促进作用
在免疫应答过程中,成熟DC进入二级淋巴器官执行抗原提呈功能,之后走向凋亡以避免免疫过度活化。我们以往的研究发现成熟DC可在脾脏基质微环境的作用下继续分化为一群具有新型的具有调节功能的树突状细胞亚群(diffDC)。已知初始CD4~+T细胞在活化、增殖并分化成效应T细胞后,其中大部分将发生凋亡,只有一小部分细胞最终形成记忆性细胞,而diffDC作为初次免疫应答中产生的新的细胞,除了负向调控初次免疫应答外,是否对记忆性T细胞尤其是记忆性CD4~+T细胞的产生也具有重要的调控功能昵?
为了研究diffDC对记忆性CD4~+T细胞产生的调控作用,我们首先研究diffDC是否可在体外长期培养并维持其特征?我们研究发现diffDC可在脾脏基质细胞上清存在的条件下贴壁生长且存活,至少存活30天以上,而且保持diffDC原有的特征,即抗原提呈功能弱但可显著抑制活化型CD4~+T细胞的增殖。结果表明diffDC可在基质细胞上清存在的条件下在体外长期培养并维持其原有的功能特征,这可部分模拟diffDC在体内的生存条件。而且,diffDC脾脏内对应亚群在正常小鼠内始终存在,并在免疫应答时明显增加。以上结果提示diffDC可能参与调控记忆性T细胞的产生存在相应的时间和空间证据。
由于记忆性T细胞研究尤其是记忆性CD4~+T细胞研究的难度大,时间跨度长,目标细胞少,因此关于记忆性CD4~+T细胞的产生和维持及其相应的机制等有待于深入研究。近年来,很多研究表明效应CD4~+T细胞在体外或体内静息后可快速转变成具有记忆细胞特征的CD4~+T细胞,因此我们采用类似模型,研究了diffDC是否及如何调控静息效应CD4~+T细胞转变成记忆性CD4~+T细胞。体外研究表明,效应CD4~+T细胞在静息时大部分细胞将发生凋亡,而我们发现,与diffDC共培养后CD4~+T细胞的生长更好,细胞凋亡明显减少,存活的细胞数量明显多于单独培养的CD4~+T细胞,至少可在体外培养21天以上。进一步采用CFSE及细胞周期分析与diffDC共培养的效应CD4~+T细胞的分裂情况,发现与单独培养相比并没有明显的细胞分裂,只是凋亡细胞明显少于单独培养的CD4~+T细胞。深入分析与diffDC长期共培养的静息效应CD4~+T细胞的细胞因子分泌、表型等特征,发现与diffDC长期共培养的静息效应CD4~+T细胞,与初始CD4~+T细胞或效应CD4~+T细胞相比,可更快更强地产生细胞因子IFN-γ与IL-2,这是记忆性CD4~+T的重要特征之一;在表型上,与单独静息3天的效应CD4T细胞相比,与diffDC长期共培养的静息效应CD4T细胞的大部分表型类似,即FSC~(low)SSC~(low) CD25~(-/low)CD69~-CD45RA~-CD122~-FasL~-CD44~(hi)CD127~+,所不同的是Fas表达明显降低,CD62~(hi)的比例较高,CD45RB~(hi)的比例也较高,这些结果提示diffDC可以促进静息效应CD4~+T细胞的存活并使其转变成记忆样CD4~+T细胞,同时所产生的记忆样CD4~+T细胞有一些独特的特征。那么diffDC是通过什么来调控效应CD4~+T细胞转变成记忆性CD4~+T细胞呢?体外实验初步发现diffDC促进静息效应CD4~+T细胞的存活依赖于diffDC与效应CD4~+T细胞间的直接接触,但是具体的机制目前尚不清楚,有待于进一步探索。重要的是,将效应CD4~+T细胞过继回输到受体鼠后12天检测存活的记忆性CD4~+T细胞的数量,我们发现在过继回输效应CD4~+T细胞同时过继diffDC后12天,在脾脏、淋巴结及外周血中供体细胞数明显高于单独过继回输效应CD4~+T细胞组,这表明diffDC可在体内促进记忆性CD4~+T细胞的存活。
以上结果表明,diffDC可以促进静息效应CD4~+T细胞的存活并促进其转变成具有一些独特特征的记忆样CD4~+T细胞。
综上所述,diffDC可诱导活化型CD4~+T细胞的凋亡从而参与抑制CD4~+T细胞的增殖,发挥负向免疫调控功能,而在免疫记忆的形成中,diffDC对于记忆性CD4~+T细胞的产生具有促进作用,其具体机制有待于进一步系统的研究。本研究结果将有助于更好地理解调节性DC参与免疫调控的机制,为临床疾病的发病机制和免疫治疗的设计提供了理论基础。
Dendritic cells(DCs) are the most potent professional antigen presenting cells that play pivotal roles in the initiation of primary immune responses and induction of immunological tolerances.Different functions of DCs may be explained by existence of different subsets of DCs or DCs at different developmental stages.In recent years, identification and characterization of phenotype and cytokine profile of DCs with regulatory properties(so-called regulatory DCs),and,inveatigation of their roles in the immune regulation and the pathogenesis of immune disorders attract much attention. Now,regulatory DCs can be generated in vitro or in vivo by coculture with stromal cells or inhibitory cytokines including IL-10 or TGF-β,or other substances such as Vitamin D receptor ligands,vasoactive intestinal peptide(VIP),thymic stromal lymphopoietin (TSLP).Regulatory DCs can induce regulatory T cells,inhibit T cell proliferation or induce T cell anergy.In our previous study,we found that splenic stroma could drive mature dendritic cells(mDCs) to differentiate into a novel subset of regulatory dendritic cells(diffDCs) which strongly inhibit mDCs-induced CD4~+T cells proliferation via nitric oxide(NO),however,diffDCs could not induce generation of regulatory T cells.Up to now,the detailed mechanisms for the inhibitory functions of diffDCs remain to be further investigated.
Apoptosis of T cells is well regulated to maintain immune homeostasis.After antigen recognition,peripheral naive CD4~+ T cells proliferate and differentiate into effector T cells to initiate immune responses.When the pathogenic antigen is deleted, effector T cells can go to apoptosis to prevent from disturbing T cell homeostasis and immune diseases such as autoimmunity and leukemogenesis.T cell apoptosis is regulated by several mechanisms:extrinsic cell-death-receptor-mediated and caspase dependent apoptosis,intrinsic mitochondria- and caspase-dependent apoptosis,or caspase-independent cell death.Considering that diffDCs can inhibit T cell proliferation, we wonder whether diffDCs could induce apoptosis of the activated CD4~+T cells to exert their inhibitory effects.If so,what are the underlying mechanisms?
Immunological memory is the hallmark of aquired immune system.It results from clonal expansion and differentiation of antigen-specific lymphocytes that ultimately persist for a lifetime.Memory lymphocytes confer immediate protection in peripheral tissues and mount recall responses to antigens in secondary lymphoid organs.Memory lymphocytes include memory B cells and memory T cells.Memory B cells can proliferate and differentiate to plasma cells that secrete antibody in response to antigenic stimulation. Memory T cells can be divided into effector memory T cells and central memory T cells according to immediate effector function and homing receptor expression.Effector memory T cells migrate to inflamed peripheral tissues and display immediate effector function,whereas central memory T cells expressing CCR7 and CD62L home to T cell areas of secondary lymphoid organs,have little or no effector function,but readily proliferate and differentiate to effector T cells in response to antigenic stimulation.The generation of memory T cells can be divided into three phases including clonal expansion, clonal contraction and memory cell maintainance.The quantity of memory T cells can be regulated during these three phase.The large clonal expansion of CD8~+ T cells in response to some infections has made the development of CD8~+T cell memory more amenable to study,giving rise to a model of memory cell differentiation in which a fraction of fully competent effector T cells transition into longlived memory T cells.However,due to the relatively low frequencies of memory CD4~+ T cells,the studies about memory CD4~+ T cells are difficult,how memory CD4~+T cells are generated and then modulated remain to be fully understood.So,we wonder whether diffDCs may affect the generation of memory CD4~+ T cells.
Therefore,we have two parts for our investigation of the effects of regulatory DCs (diffDCs) on the CD4~+T cells as follows.
1.Induction of CD4~+T cell apoptosis by regulatory dendritic cells and the underlying mechanisms
The primaty aim of this part of our study is to investigate whether diffDCs can induce apoptosis of antigen-specific CD4~+T cells,and if so,what are the underlying mechanisms.
Firstly,we added diffDC to of mDC and OVA323-339 specific CD4~+ T cells coculture system and observed whether diffDC could induce apoptosis of activtated CD4~+ T cells. We found that the percentage of late and early apoptotic activated CD4~+ T cells increased markedly in the diffDC/mDC/CD4T coculture system,indicating diffDCs could significantly induce apoptosis of the activated CD4~+ T cells.Then we wondered whether diffDCs could induce apoptosis of T cells that had been already primed and activated by antigen-pulsed mDCs.To determine this,naive antigen-specific CD4~+ T cells were stimulated by mDCs and antigen peptide for 24 hours,and then diffDCs or mDC were added into the culture system.It was found that diffDCs could increase the percentage of late and early apoptosis of the activated CD4~+ T cells,whereas mDCs failed.The data indicated that diffDC could directly induce apoptosis of the activated CD4~+ T cells.
Next,we went further to investigate the detailed mechanisms.We first determined whether soluble factors from diffDCs or cell-cell contact between diffDCs and activated CD4~+ T cells is required for the apoptosis induction of the activated CD4~+ T cells by diffDCs.So we performed Transwell assays.It was demonstrated that cell-cell contact between diffDCs and activated CD4~+ T cells or cell-ceU contact dependent soluble factors was required for diffDCs-induced apoptosis of activated CD4~+ T cells.Meanwhile, we found that higher levels of IFN-γand NO existed in diffDC/mDC/CD4T supernatants as compared to that in mDC/CD4T supematants,and their production was dependent on cell-cell contact between diffDCs and activated CD4~+ T cells.The data indicated IFN-γand NO might be involved in diffDCs-induced apoptosis of the activated CD4~+ T cells.
As cell-cell contact between diffDCs and the activated CD4~+ T cells was required for diffDCs-induced apoptosis of the activated CD4~+ T cells,we wondered what membrane molecule(s) on diffDCs triggered apoptosis of the activated CD4~+ T cells.It is well known that various death ligands such as Fas ligand,TRAIL,B7-Hland B7-DC can trigger apoptosis of activated CD4~+ T cells.Therefore we first detected whether diffDCs expressed these molecules.We found that diffDCs expressed high level of FasL and B7-H1,but did not express TRAIL and B7-DC.To prove whether FasL on diffDC plays vital role in the apoptosis induction of the activated CD4~+ T cells,we prepared diffDCs or mDCs from wild-type C57BL/6J mice or FasL-mutant gld mice,and then used them in DCs-CD4~+ T cells coculture system.It was shown that diffDCs from gld mice (gld-diffDC) failed to induce apoptosis of activated CD4~+ T cells and lost their ability to inhibit CD4~+ T cell proliferation almost completely.However,gld-mDCs could prime CD4~+ T cells proliferation normally.These results demonstrated that FasL on diffDCs played vital role in diffDCs-induced apoptosis of activated CD4~+ T cells and could trigger apoptosis of activated CD4~+ T cells effectively.Further in vivo results demonstrated that gld-diffDCs was not able to inhibit mDC-induced CD4~+T cell proliferation in vivo.Due to the importance of FasL for diffDC's function,we want to know the mechanism of high expression of FasL on diffDCs and whether the in vivo counterparts of diffDCs in the spleen also highly express FasL.As stromal cell-derived TGF-βplays a vital role in the differentiation of diffDCs,we observed the effect of TGF-βon FasL expression of diffDC,and found that stromal cell-derived TGF-β contributed to the high expression of FasL on diffDCs through activation of ERK in diffDCs.Meanwhile,we found that the in vivo counterpart of diffDC in spleen (CD11b~(hi)CD11c~(low)Ia~(low) cells) expressed FasL higher than CD11c~(hi)Ia~(hi) cells(mDC). Furthermore,during immune response,in addition to the increased percentage of diffDCs, FasL expression on diffDCs was up-regulated significantly,further indicating the importance of FasL for diffDC's function.
As soluble factor(s) produced by cell-cell contact between diffDC and mDC/CD4T was also involved in diffDCs-induced apoptosis of CD4~+ T cells,we want to know what these soluble factors were.Recent studies indicate that IFN-γplays important roles in inducing apoptosis or loss of CD4~+ T cells during infectious diseases and antitumor responses.In our previous study,we found that the inhibiting function of diffDCs was mediated by nitric oxide(NO).Furthermore,our previous results showed that the level of IFN-γand NO in the supernatants in diffDC/mDC/CD4T coculture system was higher than that in mDC/CD4T coculture system which depended on cell-cell contact between diffDCs and activated CD4~+ T cells.So we wondered whether IFN-γor nitric oxide played a role in diffDCs-induced apoptosis of CD4~+ T cells.Using anti-IFN-γblocking antibody and the inhibitor of NO production,we found that both IFN-γand nitric oxide were partially involved in diffDCs-induced apoptosis of activated CD4~+ T cells.As FasL on diffDCs triggered apoptosis of CD4~+ T cells and both IFN-γ,and NO were involved in diffDCs-induced apoptosis of CD4~+ T cells,we wondered whether high level of IFN-γand NO in diffDC/mDC/CD4T coculture system was related to FasL on diffDCs.Our results indicated that high level secretion of IFN-γ,and NO in diffDC/mDC/CD4T supernatants was dependent on FasL on diffDCs because the production of IFN-γand NO decreased in the coculture system of FasL-deficient diffDCs and mDC/CD4 T cells. Further results demonstrated that FasL-dependent high level of IFN-γ,derived from the activated CD4~+ T cells was responsible for high level of NO produced by diffDCs and activated CD4~+ T cells.
In addition,we found that diffDCs could upregulate Fas expression of activated CD4~+ T cells,the process was dependent on cell-cell contact and FasL on diffDCs but independent on IFN-γand NO.The data suggest that diffDCs may sensitize the activated CD4 T cells to FasL-induced apoptosis.
All together,the results demonstrate that regulatory dendritic cells(diffDCs) can induce apoptosis of the activated CD4~+ T cells via IFN-γ-induced NO in a FasL-dependent manner,thus inhibiting the proliferation of the activated CD4~+ T cells and exerting negative immune regulation.
2.Promotion of memory CD4~+T cell generation by regulatory dendritic cells
The primaty aim of this part of our study is to investigate whether diffDCs can affect the generation of memory CD4 T cells.
First,we observed whether diffDCs could survive and keep their function after long-term culture in vitro.We found that diffDCs could be cultured in the presence of ESSC supernatant for a long period,survived at least 30 days in vitro and still kept the the function of inhibiting mDC-induced activated CD4~+ T cell proliferation.Meanwhile, the in vivo counterpart in the spleen of diffDCs exists in na(?)ve mice and the number of diffDCs increases during immune response.These data provide the time and space evidence for the possibility that diffDCs may modulate the generation of memory T cells.
As the studies about memory T cells especially memory CD4~+ T cells are difficult for long time-span and the relatively low frequencies of memory CD4~+ T cells,how memory CD4~+ T cells are generated and then modulated remain to be fully understood. Some recent studies showed that after antigen clearance CD4~+ T effector cells rested and transited to functional memory cells in vitro and in vivo.So we used similar experimental system to study how diffDCs modulate the transition of CD4~+ T cell effectors to memory cells.In vitro results showed that,after resting,most cells of CD4~+ T cell effectors would die via apoptosis,however,while being cocultured with diffDCs,the rested CD4~+ T cell effectors(diffDC-rested effector CD4~+T cells) grew better,died less and survived longer than the rested CD4~+ T cell effectors alone beyond at least 21 days in Vitro.Using adoptive transfer model,we found that adoptive transfer of diffDCs also prolonged the survival of rested effector CD4~+T cells in vivo.Furthermore,by detecting CFSE division and cell cycle,we found that both diffDC-rested effector CD4~+T cells and the rested effector CD4~+T cells alone underwent no active cell division,indicating diffDC prolonged the survival of the rested effector CD4~+T cells without further dvision.
Next,we analyzed the characteristics of the surviving diffDC-rested effector CD4~+T cells.As compared with cytokine production by naive CD4~+T and effector CD4~+T cells, diffDC-rested effector CD4~+T cells produced IL-2 and IFN-γmore rapidly and effectively after TCR stimulation,which is one of the major features of memory CD4~+T cells,diffDC-rested effector CD4~+T cells showed similar phenotypcs of the effector CD4~+T cells rested for 3 days in that FSC~(low) SSC~(low) CD25~-/low CD69~- CD45RA~- CD122~-FasL~- CD44~(hi) CD127~+.However,diffDC-rested effector CD4~+T cells expressed lower Fas and had more CD62L~(hi) and CD45RB~(hi) cells.These data indicate that diffDCs prolong the survival of rested effector CD4~+T cells and promote their transition to memory-like CD4~+T cells in vitro.In vitro results showed that the enhanced survival of rested effector CD4~+T cells by diffDCs required cell-cell contact between diffDCs and effector CD4~+T cells.As to what are the underlying mechanisms by which diffDCs promote the transition of effector CD4~+T cells to memory-like CD4~+T cells remain to be further investigated.
In summary,diffDC can induce apoptosis of the activated CD4~+T cells through IFN-γ-induced NO in a FasL-dependent manner,providing another manner for the negative regulation of T cell response by regulatory DC.Also,regulatory DC can prolong the survival of the rested effector CD4~+T cells and promote the transition of effector CD4~+T cells to memory-like CD4~+T cells in cell-to-cell contact manner.Our results will contribute to the beeter understanding of the immune regulation and the pathogenesis of the immunological disorders,and also may be useful to the design of immunotherapy for the immunological diseases.
机体可精细调节T细胞的凋亡以维持免疫稳态。在抗原识别后,外周初始CD4~+T细胞增殖并分化成效应T细胞以启动免疫应答。当抗原被清除后,大部分效应T细胞发生凋亡以免破坏T细胞稳态及避免免疫功能过度活化而导致自身免疫等疾病的发生。凋亡的方式主要有:外在的细胞死亡受体介导与caspase依赖的凋亡,内在的线粒体与caspase依赖的凋亡,或者内在的线粒体依赖而caspase非依赖的凋亡。那么,diffDC是否能够通过诱导活化型CD4~+T细胞的凋亡而发挥其负向免疫调控呢?如果具备此功能的话,其具体的作用机制又是什么?
免疫记忆是适应性免疫的一大特点,它是由抗原特异性淋巴细胞在克隆增殖并分化成长期存在的记忆性淋巴细胞而产生的。记忆性淋巴细胞可在外周组织中为机体提供快速的保护力并可在二级淋巴器官内产生针对特异性抗原的再次免疫应答。记忆性淋巴细胞包括记忆性B细胞与记忆性T细胞,记忆性B细胞经再次抗原刺激后可分化成浆细胞分泌抗体来介导保护力;而记忆性T细胞可根据效应功能状态及归巢受体表达情况而分为效应型记忆性T细胞与中枢型记忆性T细胞,效应型记忆性T细胞不表达CCR7且可在外周炎症部位发挥快速的效应功能,中枢型记忆性T细胞则表达CCR7与CD62L,存在于二级淋巴器官的T细胞区,没有效应功能,但是可在抗原刺激下快速增殖并分化成效应T细胞。记忆性T细胞的产生可分为克隆增殖、克隆收缩及记忆细胞维持三个阶段,因此记忆性T细胞产生的数量与这三个阶段的调控密切相关。由于CD8~+T细胞在感染或活化后快速增殖并大量扩增且幅度远高于CD4~+T细胞,这为记忆性CD8~+T细胞的研究带来极大便利并已取得长足的进展,目前关于记忆性CD8~+T细胞的研究具有一个广泛认可的分化模式,即一部分完全分化的效应T细胞转变成长期存活的记忆性T细胞。然而,相对于记忆性CD8~+T细胞,记忆性CD4~+T细胞的数量明显较少而且随时间延长还会继续减少,这给记忆性CD4~+T细胞的研究带来了很大的困难,因此,目前对于记忆性CD4~+T细胞的产生和维持及其相关的机制仍不十分清楚。我们想知道是否diffDC也可能影响记忆性CD4~+T细胞的产生。
在本研究中,我们通过两部分实验对我们以往发现的调节性树突状细胞diffDC如何调节CD4~+T细胞的功能及其可能的机制进行了探讨。
一、调节性树突状细胞诱导活化型CD4~+T细胞凋亡及其机制研究
这部分研究的目的是明确diffDC是否可诱导抗原特异性活化型CD4~+T细胞的凋亡,如果能的话,进一步探讨具体的机制。
首先,我们将diffDC在体外加入到mDC诱导OVA_(323-339)特异性CD4~+T细胞活化的共培养体系中,观察它是否可以诱导活化型CD4~+T细胞的凋亡,我们发现,实验组diffDC/mDC/CD4T与对照组mDC/CD4T相比,diffDC/mDc/CD4T组内7AAD~+CD4~+T细胞(晚期凋亡)与Annexin V~+ 7AAD~- CD4~+T细胞(早期凋亡)的比例均明显高于mDC/CD4T组,这表明在共培养体系中,diffDC可以明显促进活化型CD4~+T细胞的凋亡。那么,如果将抗原特异性CD4~+T细胞预先用mDC与抗原进行活化后,再加入diffDC后是否也能促进其凋亡呢?进一步研究发现,diffDC也可以促进预活化的CD4~+T细胞的凋亡,其凋亡情况与共培养体系所得结果类似。可见,diffDC能够显著诱导活化型CD4~+T细胞的凋亡。
在发现了这一现象之后,我们进一步探讨了diffDC诱导活化型CD4~+T细胞凋亡的具体机制。首先,我们确定diffDC产生的可溶性因子还是膜分子,或者是diffDC的膜分子与mDC或活化CD4~+T细胞上的膜分子作用后产生的可溶性因子作用于活化型CD4~+T细胞而诱导其凋亡。采用Transwell系统将diffDC与mDC/CD4T细胞隔开培养,结果发现diffDC诱导活化型CD4~+T细胞凋亡显著下降,表明diffDC诱导活化型CD4~+T细胞凋亡依赖于diffDC与活化型CD4T细胞间的直接接触,即直接由diffDC上的膜分子或间接由diffDC上的膜分子与mDC或活化CD4~+T细胞作用后产生的可溶性因子来介导。鉴于可溶性因子可能参与diffDC的诱导凋亡作用以及我们前期研究发现NO介导了diffDC的抑制功能,我们检测发现diffDC/mDC/CD4T共培养体系中存在高水平IFN-γ与NO,IFN-γ与NO的分泌也依赖于diffDC与活化型CD4~+T细胞间的直接接触,结果提示IFN-γ与NO也可能参与diffDC对于活化型CD4~+T细胞的凋亡诱导作用。
下一步我们探索diffDC上的哪些膜分子介导了其凋亡诱导作用。我们采用流式细胞检测一些诱导凋亡的相关分子,发现diffDC高表达FasL与B7-H1,不表达TRAIL与B7-DC。因此,我们采用FasL缺陷的gld小鼠来源的diffDC或mDC,研究确定diffDC上的FasL是否参与诱导活化CD4~+T细胞的凋亡,发现gld小鼠来源的diffDC(gld diffDC)不能有效抑制mDC诱导的CD4~+T细胞的增殖,也不能诱导活化CD4~+T细胞的凋亡,这表明diffDC的FasL介导了diffDC对于活化型CD4~+T细胞的凋亡诱导作用。进一步我们采用体内实验证明,gld小鼠来源的diffDC也不能在体内抑制mDC诱导的CD4T细胞的增殖。鉴于FasL对于diffDC功能的重要意义,我们需要明确diffDC高表达FasL的具体机制,以及脾脏内的对应的diffDC群体是否也高表达FasL。我们以往的研究表明TGF-β在基质细胞诱导maDC向diffDC分化中发挥重要作用,因此我们研究了TGF-β对于diffDC表达FasL的影响,结果发现,脾脏基质细胞的TGF-β产生被阻断后所诱导的diffDC的FasL表达明显下降,同时ERK磷酸化水平也明显下降,并且diffDC在直接用ERK磷酸化抑制剂作用后FasL表达明显下降,因此,基质细胞来源的TGF-β通过促进diffDC的ERK磷酸化而促进diffDC高表达FasL。在体内diffDC对应群体分析上,我们发现CD11b~(hi)CD11c~(low)Ia~(low) cells(diffDC)的FasL表达也高于CD11c~(hi)Ia~(hi)(mDC),尤其是在免疫应答过程中,除了diffDC的比例上调外,FasL的表达也明显上调,这更进一步提示了FasL在diffDC免疫调控功能上的重要性。
由于diffDC膜分子依赖的可溶性因子也在diffDC诱导活化型CD4~+T细胞凋亡中发挥重要作用,我们拟明确是哪些因子介导的?由于近年来研究表明IFN-γ在CD4~+T细胞的凋亡及负向免疫调控中发挥重要作用,结合我们前期研究发现NO参与了diffDC的抑制功能而且diffDC/mDC/CD4T共培养体系中存在高水平IFN-γ与NO,因此我们采用IFN-γ阻断性抗体或外加IFN-γ及NO抑制剂,然后观察对于diffDC诱导活化型CD4~+T细胞凋亡的影响,结果发现,IFN-γ与NO均部分参与了diffDC对于活化型CD4~+T细胞的凋亡诱导作用。
由于diffDC的膜分子FasL介导了其诱导凋亡作用,而且膜分子依赖性的IFN-γ与NO也参与了诱导凋亡作用,那么它们之间是否有关联呢?是否是diffDC上的FasL促进了下游的IFN-γ与NO的产生呢?我们发现将FasL缺陷的diffDC加入mDC或者FasL缺陷的mDC诱导OVA_(323-339)特异性CD4~+T细胞活化的共培养体系中,培养上清中IFN-γ与NO的水平相对于diffDC/mDC/CD4T组均有一定程度的下降,而不论mDC是否存在FasL缺陷,diffDC均可明显上调mDC/CD4T共培养体系中的IFN-γ与NO水平,这表明diffDC/mDC/CD4T中IFN-γ与NO的高分泌依赖于diffDC上的膜分子FasL。进一步研究发现diffDC表达的FasL诱导活化型CD4~+T细胞高分泌IFN-γ,继而IFN-γ促进diffDC与活化型CD4~+T细胞高分泌NO,最终引起活化型CD4~+T细胞的凋亡。
既然diffDC表面的FasL在diffDC诱导活化CD4~+T细胞的凋亡中发挥了重要作用,那么diffDC上的FasL如何与活化CD4~+T上的Fas相互作用?是否会上调活化型CD4~+T细胞表面的Fas表达从而使活化型CD4~+T细胞更易于发生凋亡呢?鉴于此,我们研究了在共培养体系中,实验组diffDC/mDC/CD4T与对照组mDC/CD4T组中CD4~+T的Fas表达情况,发现diffDC/mDC/CD4T组中活化型CD4~+T细胞的Fas表达在第四天时明显高于mDC/CD4T组中的CD4~+T细胞,表明diffDC可通过上调活化型CD4~+T细胞的Fas表达,使活化型CD4~+T细胞更易于通过与diffDC表达的FasL相互作用而发生凋亡。进一步研究表明,这种上调依赖于diffDC与活化型CD4~+T细胞的直接接触以及diffDC表达的FasL,但是不依赖于IFN-γ与NO的作用。以上结果表明diffDC表达的FasL可上调活化型CD4~+T细胞表达Fas,从而使其更易于通过与diffDC表达的FasL相互作用而发生凋亡。
以上结果表明在初次免疫应答过程中,调节性树突状细胞(diffDC)可通过FasL/Fas作用途径激发下游多种机制来诱导活化型CD4~+T细胞的凋亡继而抑制其增殖,从而在初次免疫应答中发挥重要的负向调控功能。
二、调节性树突状细胞对记忆性CD4~+T细胞产生的促进作用
在免疫应答过程中,成熟DC进入二级淋巴器官执行抗原提呈功能,之后走向凋亡以避免免疫过度活化。我们以往的研究发现成熟DC可在脾脏基质微环境的作用下继续分化为一群具有新型的具有调节功能的树突状细胞亚群(diffDC)。已知初始CD4~+T细胞在活化、增殖并分化成效应T细胞后,其中大部分将发生凋亡,只有一小部分细胞最终形成记忆性细胞,而diffDC作为初次免疫应答中产生的新的细胞,除了负向调控初次免疫应答外,是否对记忆性T细胞尤其是记忆性CD4~+T细胞的产生也具有重要的调控功能昵?
为了研究diffDC对记忆性CD4~+T细胞产生的调控作用,我们首先研究diffDC是否可在体外长期培养并维持其特征?我们研究发现diffDC可在脾脏基质细胞上清存在的条件下贴壁生长且存活,至少存活30天以上,而且保持diffDC原有的特征,即抗原提呈功能弱但可显著抑制活化型CD4~+T细胞的增殖。结果表明diffDC可在基质细胞上清存在的条件下在体外长期培养并维持其原有的功能特征,这可部分模拟diffDC在体内的生存条件。而且,diffDC脾脏内对应亚群在正常小鼠内始终存在,并在免疫应答时明显增加。以上结果提示diffDC可能参与调控记忆性T细胞的产生存在相应的时间和空间证据。
由于记忆性T细胞研究尤其是记忆性CD4~+T细胞研究的难度大,时间跨度长,目标细胞少,因此关于记忆性CD4~+T细胞的产生和维持及其相应的机制等有待于深入研究。近年来,很多研究表明效应CD4~+T细胞在体外或体内静息后可快速转变成具有记忆细胞特征的CD4~+T细胞,因此我们采用类似模型,研究了diffDC是否及如何调控静息效应CD4~+T细胞转变成记忆性CD4~+T细胞。体外研究表明,效应CD4~+T细胞在静息时大部分细胞将发生凋亡,而我们发现,与diffDC共培养后CD4~+T细胞的生长更好,细胞凋亡明显减少,存活的细胞数量明显多于单独培养的CD4~+T细胞,至少可在体外培养21天以上。进一步采用CFSE及细胞周期分析与diffDC共培养的效应CD4~+T细胞的分裂情况,发现与单独培养相比并没有明显的细胞分裂,只是凋亡细胞明显少于单独培养的CD4~+T细胞。深入分析与diffDC长期共培养的静息效应CD4~+T细胞的细胞因子分泌、表型等特征,发现与diffDC长期共培养的静息效应CD4~+T细胞,与初始CD4~+T细胞或效应CD4~+T细胞相比,可更快更强地产生细胞因子IFN-γ与IL-2,这是记忆性CD4~+T的重要特征之一;在表型上,与单独静息3天的效应CD4T细胞相比,与diffDC长期共培养的静息效应CD4T细胞的大部分表型类似,即FSC~(low)SSC~(low) CD25~(-/low)CD69~-CD45RA~-CD122~-FasL~-CD44~(hi)CD127~+,所不同的是Fas表达明显降低,CD62~(hi)的比例较高,CD45RB~(hi)的比例也较高,这些结果提示diffDC可以促进静息效应CD4~+T细胞的存活并使其转变成记忆样CD4~+T细胞,同时所产生的记忆样CD4~+T细胞有一些独特的特征。那么diffDC是通过什么来调控效应CD4~+T细胞转变成记忆性CD4~+T细胞呢?体外实验初步发现diffDC促进静息效应CD4~+T细胞的存活依赖于diffDC与效应CD4~+T细胞间的直接接触,但是具体的机制目前尚不清楚,有待于进一步探索。重要的是,将效应CD4~+T细胞过继回输到受体鼠后12天检测存活的记忆性CD4~+T细胞的数量,我们发现在过继回输效应CD4~+T细胞同时过继diffDC后12天,在脾脏、淋巴结及外周血中供体细胞数明显高于单独过继回输效应CD4~+T细胞组,这表明diffDC可在体内促进记忆性CD4~+T细胞的存活。
以上结果表明,diffDC可以促进静息效应CD4~+T细胞的存活并促进其转变成具有一些独特特征的记忆样CD4~+T细胞。
综上所述,diffDC可诱导活化型CD4~+T细胞的凋亡从而参与抑制CD4~+T细胞的增殖,发挥负向免疫调控功能,而在免疫记忆的形成中,diffDC对于记忆性CD4~+T细胞的产生具有促进作用,其具体机制有待于进一步系统的研究。本研究结果将有助于更好地理解调节性DC参与免疫调控的机制,为临床疾病的发病机制和免疫治疗的设计提供了理论基础。
Dendritic cells(DCs) are the most potent professional antigen presenting cells that play pivotal roles in the initiation of primary immune responses and induction of immunological tolerances.Different functions of DCs may be explained by existence of different subsets of DCs or DCs at different developmental stages.In recent years, identification and characterization of phenotype and cytokine profile of DCs with regulatory properties(so-called regulatory DCs),and,inveatigation of their roles in the immune regulation and the pathogenesis of immune disorders attract much attention. Now,regulatory DCs can be generated in vitro or in vivo by coculture with stromal cells or inhibitory cytokines including IL-10 or TGF-β,or other substances such as Vitamin D receptor ligands,vasoactive intestinal peptide(VIP),thymic stromal lymphopoietin (TSLP).Regulatory DCs can induce regulatory T cells,inhibit T cell proliferation or induce T cell anergy.In our previous study,we found that splenic stroma could drive mature dendritic cells(mDCs) to differentiate into a novel subset of regulatory dendritic cells(diffDCs) which strongly inhibit mDCs-induced CD4~+T cells proliferation via nitric oxide(NO),however,diffDCs could not induce generation of regulatory T cells.Up to now,the detailed mechanisms for the inhibitory functions of diffDCs remain to be further investigated.
Apoptosis of T cells is well regulated to maintain immune homeostasis.After antigen recognition,peripheral naive CD4~+ T cells proliferate and differentiate into effector T cells to initiate immune responses.When the pathogenic antigen is deleted, effector T cells can go to apoptosis to prevent from disturbing T cell homeostasis and immune diseases such as autoimmunity and leukemogenesis.T cell apoptosis is regulated by several mechanisms:extrinsic cell-death-receptor-mediated and caspase dependent apoptosis,intrinsic mitochondria- and caspase-dependent apoptosis,or caspase-independent cell death.Considering that diffDCs can inhibit T cell proliferation, we wonder whether diffDCs could induce apoptosis of the activated CD4~+T cells to exert their inhibitory effects.If so,what are the underlying mechanisms?
Immunological memory is the hallmark of aquired immune system.It results from clonal expansion and differentiation of antigen-specific lymphocytes that ultimately persist for a lifetime.Memory lymphocytes confer immediate protection in peripheral tissues and mount recall responses to antigens in secondary lymphoid organs.Memory lymphocytes include memory B cells and memory T cells.Memory B cells can proliferate and differentiate to plasma cells that secrete antibody in response to antigenic stimulation. Memory T cells can be divided into effector memory T cells and central memory T cells according to immediate effector function and homing receptor expression.Effector memory T cells migrate to inflamed peripheral tissues and display immediate effector function,whereas central memory T cells expressing CCR7 and CD62L home to T cell areas of secondary lymphoid organs,have little or no effector function,but readily proliferate and differentiate to effector T cells in response to antigenic stimulation.The generation of memory T cells can be divided into three phases including clonal expansion, clonal contraction and memory cell maintainance.The quantity of memory T cells can be regulated during these three phase.The large clonal expansion of CD8~+ T cells in response to some infections has made the development of CD8~+T cell memory more amenable to study,giving rise to a model of memory cell differentiation in which a fraction of fully competent effector T cells transition into longlived memory T cells.However,due to the relatively low frequencies of memory CD4~+ T cells,the studies about memory CD4~+ T cells are difficult,how memory CD4~+T cells are generated and then modulated remain to be fully understood.So,we wonder whether diffDCs may affect the generation of memory CD4~+ T cells.
Therefore,we have two parts for our investigation of the effects of regulatory DCs (diffDCs) on the CD4~+T cells as follows.
1.Induction of CD4~+T cell apoptosis by regulatory dendritic cells and the underlying mechanisms
The primaty aim of this part of our study is to investigate whether diffDCs can induce apoptosis of antigen-specific CD4~+T cells,and if so,what are the underlying mechanisms.
Firstly,we added diffDC to of mDC and OVA323-339 specific CD4~+ T cells coculture system and observed whether diffDC could induce apoptosis of activtated CD4~+ T cells. We found that the percentage of late and early apoptotic activated CD4~+ T cells increased markedly in the diffDC/mDC/CD4T coculture system,indicating diffDCs could significantly induce apoptosis of the activated CD4~+ T cells.Then we wondered whether diffDCs could induce apoptosis of T cells that had been already primed and activated by antigen-pulsed mDCs.To determine this,naive antigen-specific CD4~+ T cells were stimulated by mDCs and antigen peptide for 24 hours,and then diffDCs or mDC were added into the culture system.It was found that diffDCs could increase the percentage of late and early apoptosis of the activated CD4~+ T cells,whereas mDCs failed.The data indicated that diffDC could directly induce apoptosis of the activated CD4~+ T cells.
Next,we went further to investigate the detailed mechanisms.We first determined whether soluble factors from diffDCs or cell-cell contact between diffDCs and activated CD4~+ T cells is required for the apoptosis induction of the activated CD4~+ T cells by diffDCs.So we performed Transwell assays.It was demonstrated that cell-cell contact between diffDCs and activated CD4~+ T cells or cell-ceU contact dependent soluble factors was required for diffDCs-induced apoptosis of activated CD4~+ T cells.Meanwhile, we found that higher levels of IFN-γand NO existed in diffDC/mDC/CD4T supernatants as compared to that in mDC/CD4T supematants,and their production was dependent on cell-cell contact between diffDCs and activated CD4~+ T cells.The data indicated IFN-γand NO might be involved in diffDCs-induced apoptosis of the activated CD4~+ T cells.
As cell-cell contact between diffDCs and the activated CD4~+ T cells was required for diffDCs-induced apoptosis of the activated CD4~+ T cells,we wondered what membrane molecule(s) on diffDCs triggered apoptosis of the activated CD4~+ T cells.It is well known that various death ligands such as Fas ligand,TRAIL,B7-Hland B7-DC can trigger apoptosis of activated CD4~+ T cells.Therefore we first detected whether diffDCs expressed these molecules.We found that diffDCs expressed high level of FasL and B7-H1,but did not express TRAIL and B7-DC.To prove whether FasL on diffDC plays vital role in the apoptosis induction of the activated CD4~+ T cells,we prepared diffDCs or mDCs from wild-type C57BL/6J mice or FasL-mutant gld mice,and then used them in DCs-CD4~+ T cells coculture system.It was shown that diffDCs from gld mice (gld-diffDC) failed to induce apoptosis of activated CD4~+ T cells and lost their ability to inhibit CD4~+ T cell proliferation almost completely.However,gld-mDCs could prime CD4~+ T cells proliferation normally.These results demonstrated that FasL on diffDCs played vital role in diffDCs-induced apoptosis of activated CD4~+ T cells and could trigger apoptosis of activated CD4~+ T cells effectively.Further in vivo results demonstrated that gld-diffDCs was not able to inhibit mDC-induced CD4~+T cell proliferation in vivo.Due to the importance of FasL for diffDC's function,we want to know the mechanism of high expression of FasL on diffDCs and whether the in vivo counterparts of diffDCs in the spleen also highly express FasL.As stromal cell-derived TGF-βplays a vital role in the differentiation of diffDCs,we observed the effect of TGF-βon FasL expression of diffDC,and found that stromal cell-derived TGF-β contributed to the high expression of FasL on diffDCs through activation of ERK in diffDCs.Meanwhile,we found that the in vivo counterpart of diffDC in spleen (CD11b~(hi)CD11c~(low)Ia~(low) cells) expressed FasL higher than CD11c~(hi)Ia~(hi) cells(mDC). Furthermore,during immune response,in addition to the increased percentage of diffDCs, FasL expression on diffDCs was up-regulated significantly,further indicating the importance of FasL for diffDC's function.
As soluble factor(s) produced by cell-cell contact between diffDC and mDC/CD4T was also involved in diffDCs-induced apoptosis of CD4~+ T cells,we want to know what these soluble factors were.Recent studies indicate that IFN-γplays important roles in inducing apoptosis or loss of CD4~+ T cells during infectious diseases and antitumor responses.In our previous study,we found that the inhibiting function of diffDCs was mediated by nitric oxide(NO).Furthermore,our previous results showed that the level of IFN-γand NO in the supernatants in diffDC/mDC/CD4T coculture system was higher than that in mDC/CD4T coculture system which depended on cell-cell contact between diffDCs and activated CD4~+ T cells.So we wondered whether IFN-γor nitric oxide played a role in diffDCs-induced apoptosis of CD4~+ T cells.Using anti-IFN-γblocking antibody and the inhibitor of NO production,we found that both IFN-γand nitric oxide were partially involved in diffDCs-induced apoptosis of activated CD4~+ T cells.As FasL on diffDCs triggered apoptosis of CD4~+ T cells and both IFN-γ,and NO were involved in diffDCs-induced apoptosis of CD4~+ T cells,we wondered whether high level of IFN-γand NO in diffDC/mDC/CD4T coculture system was related to FasL on diffDCs.Our results indicated that high level secretion of IFN-γ,and NO in diffDC/mDC/CD4T supernatants was dependent on FasL on diffDCs because the production of IFN-γand NO decreased in the coculture system of FasL-deficient diffDCs and mDC/CD4 T cells. Further results demonstrated that FasL-dependent high level of IFN-γ,derived from the activated CD4~+ T cells was responsible for high level of NO produced by diffDCs and activated CD4~+ T cells.
In addition,we found that diffDCs could upregulate Fas expression of activated CD4~+ T cells,the process was dependent on cell-cell contact and FasL on diffDCs but independent on IFN-γand NO.The data suggest that diffDCs may sensitize the activated CD4 T cells to FasL-induced apoptosis.
All together,the results demonstrate that regulatory dendritic cells(diffDCs) can induce apoptosis of the activated CD4~+ T cells via IFN-γ-induced NO in a FasL-dependent manner,thus inhibiting the proliferation of the activated CD4~+ T cells and exerting negative immune regulation.
2.Promotion of memory CD4~+T cell generation by regulatory dendritic cells
The primaty aim of this part of our study is to investigate whether diffDCs can affect the generation of memory CD4 T cells.
First,we observed whether diffDCs could survive and keep their function after long-term culture in vitro.We found that diffDCs could be cultured in the presence of ESSC supernatant for a long period,survived at least 30 days in vitro and still kept the the function of inhibiting mDC-induced activated CD4~+ T cell proliferation.Meanwhile, the in vivo counterpart in the spleen of diffDCs exists in na(?)ve mice and the number of diffDCs increases during immune response.These data provide the time and space evidence for the possibility that diffDCs may modulate the generation of memory T cells.
As the studies about memory T cells especially memory CD4~+ T cells are difficult for long time-span and the relatively low frequencies of memory CD4~+ T cells,how memory CD4~+ T cells are generated and then modulated remain to be fully understood. Some recent studies showed that after antigen clearance CD4~+ T effector cells rested and transited to functional memory cells in vitro and in vivo.So we used similar experimental system to study how diffDCs modulate the transition of CD4~+ T cell effectors to memory cells.In vitro results showed that,after resting,most cells of CD4~+ T cell effectors would die via apoptosis,however,while being cocultured with diffDCs,the rested CD4~+ T cell effectors(diffDC-rested effector CD4~+T cells) grew better,died less and survived longer than the rested CD4~+ T cell effectors alone beyond at least 21 days in Vitro.Using adoptive transfer model,we found that adoptive transfer of diffDCs also prolonged the survival of rested effector CD4~+T cells in vivo.Furthermore,by detecting CFSE division and cell cycle,we found that both diffDC-rested effector CD4~+T cells and the rested effector CD4~+T cells alone underwent no active cell division,indicating diffDC prolonged the survival of the rested effector CD4~+T cells without further dvision.
Next,we analyzed the characteristics of the surviving diffDC-rested effector CD4~+T cells.As compared with cytokine production by naive CD4~+T and effector CD4~+T cells, diffDC-rested effector CD4~+T cells produced IL-2 and IFN-γmore rapidly and effectively after TCR stimulation,which is one of the major features of memory CD4~+T cells,diffDC-rested effector CD4~+T cells showed similar phenotypcs of the effector CD4~+T cells rested for 3 days in that FSC~(low) SSC~(low) CD25~-/low CD69~- CD45RA~- CD122~-FasL~- CD44~(hi) CD127~+.However,diffDC-rested effector CD4~+T cells expressed lower Fas and had more CD62L~(hi) and CD45RB~(hi) cells.These data indicate that diffDCs prolong the survival of rested effector CD4~+T cells and promote their transition to memory-like CD4~+T cells in vitro.In vitro results showed that the enhanced survival of rested effector CD4~+T cells by diffDCs required cell-cell contact between diffDCs and effector CD4~+T cells.As to what are the underlying mechanisms by which diffDCs promote the transition of effector CD4~+T cells to memory-like CD4~+T cells remain to be further investigated.
In summary,diffDC can induce apoptosis of the activated CD4~+T cells through IFN-γ-induced NO in a FasL-dependent manner,providing another manner for the negative regulation of T cell response by regulatory DC.Also,regulatory DC can prolong the survival of the rested effector CD4~+T cells and promote the transition of effector CD4~+T cells to memory-like CD4~+T cells in cell-to-cell contact manner.Our results will contribute to the beeter understanding of the immune regulation and the pathogenesis of the immunological disorders,and also may be useful to the design of immunotherapy for the immunological diseases.
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