摘要
目前,器官移植已经成为治疗各种脏器终末期疾病的唯一、有效治疗手段。许多患者通过移植同种异体器官获得新生。然而,外来器官进入机体后不可避免地会激发受者的免疫系统,产生针对移植器官的免疫应答以及排斥反应。多种新型免疫抑制剂的临床应用,虽然降低了移植排斥反应的发生率,但仍然存在两大问题：其一,免疫抑制剂并不能完全抑制排斥反应的发生；其二,长期应用免疫抑制剂存在诸多不良反应,包括药物的毒性、过度抑制机体免疫力造成的感染及新生肿瘤等。因此,抑制同种移植排斥反应并诱导机体对供者抗原的特异性免疫耐受,使机体保持正常的免疫力以抵抗微生物等病原体感染及肿瘤的发生,是解决器官移植排斥反应的最佳途径,也是移植领域研究的热点之一
树突状细胞(dendritic cells, DC)是一类功能强大,并且是唯一能够激活初始T细胞的抗原提呈细胞,在识别和提呈抗原、启动免疫应答、诱导移植排斥反应中起着重要的作用。然而,近年来的研究表明DC是一类异质性细胞群体,具有不同的亚群和不同的功能状态,不但在增强免疫反应上具有重要的作用,也具有抑制排斥反应、诱导特异性免疫耐受的潜力。有研究表明,未成熟DC缺乏共刺激分子CD80(B7-1)和CD86(B7-2)的表达,体外能够诱导同种抗原特异性的T细胞失能。然而,体内未成熟DC存在着自然发育成熟的内在机制,同时,器官移植术后受者体内产生的多种介质,如致炎性细胞因子、LPS等,均能促进DC分化成熟,使之拥有强大的免疫原性。因此,某些体外调控手段,如免疫抑制分子1,25-二羟维生素D共培养或白细胞介素10(interleukin-10, IL-10)、转化生长因子β(transforming growth factor-β, TGF-β)等基因修饰,可以使DC维持于稳定的不成熟状态,增强其耐受原性,是减轻移植排斥反应、诱导特异性免疫耐受的重要途径并具有潜在的临床应用价值。
细胞因子信号抑制因子1(suppressor of cytokine signalingl, SOCS1)作为负性调节细胞因子Janus激酶-信号传导和转录活化因子(Janus kinase-signal transducer and activator of transcription, JAK-STAT)信号途径的蛋白而被发现,可以抑制γ干扰素(interferon-γ, IFN-γ)、IL-6、生长激素(growth hormone, GH)等细胞内信号传导。新的研究显示SOCS1对DC的成熟和功能具有重要的调控作用,源于SOCS1-/-小鼠的脾脏DC与正常DC相比,表达高水平共刺激分子,对IL-4、IFN-γ刺激呈明显高反应,刺激B细胞的增殖作用更加强烈。负载抗原的SOCS1-/-DC和正常DC分别注射到同一只小鼠不同足垫,前者能够激发小鼠足垫区域引流淋巴结更加强烈的Thl型反应,该淋巴结分离的T细胞能够产生更高水平的IFN-γ。SOCS1基因沉默的DC负载卵白蛋白(ovalbumin, OVA)后,体内、外均能更有效地刺激OVA特异性T细胞的增殖反应及细胞毒性作用。SOCS1基因沉默DC以及SOCS1-/-DC具有更强的免疫刺激功能以及打破自身耐受的能力。鉴于SOCS1基因对DC发育及免疫功能的重要调控作用,我们设想采用基因转染技术体外增加未成熟DC内SOCS1基因的表达将会减缓或阻止DC的成熟过程,体内回输有利于减轻排斥反应,并可能诱导稳定、有效的供者特异性免疫耐受。
本文通过体外培养、扩增并富集小鼠骨髓来源的DC,并借助复制缺陷型腺病毒载体将SOCS1基因转入DC,研究SOCS1基因修饰的未成熟DC的表型及功能的变化以及体外诱导同种抗原特异性T细胞低反应性的作用和效果；通过建立小鼠同种异体异位心脏移植模型,观察SOCS1基因修饰的未成熟DC体内减轻排斥反应、诱导心脏移植免疫耐受的效果,并探讨了可能的机制。
第一部分小鼠骨髓树突状细胞体外培养及SOCS1基因转染
DC起源于骨髓,在体内分布广泛,但含量极少,不足外周血单核细胞的1%。体外培养DC可以为基础科研和临床治疗提供丰富的DC来源。我们采用重组小鼠粒巨噬细胞集落刺激因子(recombinant mouse granulocyte-macrophage cell clone stimulate factor, rmGM-CSF)和rmIL-4共同刺激新鲜分离的小鼠骨髓细胞,培养小鼠骨髓来源DC,并根据小鼠骨髓来源DC高表达相对特异性表面分子CD11c,采用针对CD11c免疫磁珠分离法进一步富集DC。结果显示,刚制备的骨髓细胞极少有成簇或有树枝状突起的细胞,培养3天后有的散在克隆形成,带树枝状突起的细胞逐渐增多,培养5天后克隆较多,细胞的突起逐渐明显,成典型的未成熟DC表现。经免疫磁珠分离富集后,可以得到足够数量的DC,其纯度可以高达93%以上。
腺病毒载体具有基因组结构简单、病毒滴度高、感染效率高、宿主细胞范围广、无插入突变等优势,因此我们构建了编码SOCS1基因的复制缺陷型腺病毒载体(Ad-SOCS1), PCR、特异性酶切、测序鉴定以及Western Blotting等方法的检测结果已经证实,Ad-SOCS1载体构建成功,可以表达SOCS1蛋白。经过病毒扩增和滴度的检测,可以获得较高滴度的Ad-SOCS1载体。收获富集后的DC,按复合感染指数(MOI)1：100加入Ad-SOCS1或Ad-GFP病毒,未经病毒转染的未成熟DC作为对照。将上述三种细胞分别称为DC-SOCS1、DC-GFP、imDC(未成熟DC)。流式细胞仪分析DC-GFP组对照病毒载体转染效率；荧光实时定量PCR及Western Blotting检测各组DC内SOCS1基因的mRNA和蛋白表达。结果显示对照病毒载体Ad-GFP的转染效率可以达到70%以上；与imDC组及DC-GFP组相比,DC-SOCS1组SOCS1mRNA和蛋白表达水平明显升高(p<0.01)。上述结果表明我们构建的Ad-SOCS1载体能够有效转染DC,并明显上调DC内SOCS1基因的表达。
第二部分SOCS1基因修饰的树突状细胞的表型特征及体外免疫学功能分析
本部分实验,我们利用Ad-SOCS1腺病毒载体转染富集后的骨髓来源DC,观察了SOCS1基因修饰的DC的表型特征,并进一步研究其体外免疫学功能。
采用rmGM-CSF及rmIL-4体外培养法扩增C57BL/6小鼠骨髓来源的DC,于培养第5天采用免疫磁珠分离法富集DC。通过Ad-SOCS1腺病毒载体将SOCS1基因转染DC,同时设Ad-GFP转染的DC及同期培养的未成熟DC作为对照组。分析脂多糖(lipopolysaccharide, LPS)刺激前、后DC的表型、细胞因子分泌、抗原吞噬以及抗原提呈能力的变化；SOCS1基因修饰的DC与同种BALB/c小鼠的CD4+T细胞混合淋巴反应(mixed lymphocyte reaction, MLR)后,检测同种CD4+T细胞的增殖能力,培养上清中Thl或Th2来源的细胞因子水平以及IL-10分泌性T细胞的产生；同时观察SOCS1基因修饰的DC在二次同种MLR中,对诱导抗原特异性T细胞低反应性的影响。
分析各组DC表型发现,DC-SOCS1与imDC在LPS刺激前,CD40、CD8o及MHC分子的表达无明显的差异,但SOCS1基因修饰可明显抑制LPS刺激对DC的表面上述分子表达的上调作用；检测细胞培养液上清内细胞因子水平发现,LPS刺激前各组DC分泌的IL-12无明显差异(p>0.05),但LPS刺激后DC-SOCS1分泌IL-12较imDC及DC-GFP明显降低(p<0.01),而DC-SOCS1分泌IL-10水平在LPS刺激前或后均较imDC或DC-GFP明显升高(p<0.01)。上述结果表明通过基因转染技术增加未成熟DC内SOCS1表达,可阻断外源性LPS对DC的成熟刺激作用,使之维持在稳定的未成熟状态。
通过FITC-Dextran吞噬实验检测各组DC的抗原吞噬功能,发现LPS刺激前各组DC均具有较强的吞噬能力,LPS刺激后,imDC及DC-GFP的吞噬能力明显降低,DC-SOCS1的吞噬能力仅有轻度下降；进一步检测各组DC的抗原提呈功能,发现DC-SOCS1抗原提呈能力明显低于imDC和DC-GFP组(p<0.01)。上述结果表明SOCS1基因修饰可以使DC维持较强的抗原吞噬能力并抑制DC抗原提呈功能。
通过MLR分析各组DC的免疫刺激活性,发现SOCS1基因修饰的DC刺激同种CD4+T细胞增殖的能力明显减弱(p<0.01)；检测同种MLR反应体系上清中细胞因子水平,发现DC-SOCS1组Thl型的细胞因子IFN-γ水平明显降低(p<0.01),Th2亚群的细胞因子IL-10水平明显升高(p<0.01);进一步行细胞内细胞因子染色,结果表明DC-SOCS1刺激的同种CD4+T细胞,细胞内IL-10表达水平明显升高。上述结果表明,SOCS1基因修饰的DC,免疫刺激功能明显降低,可抑制Thl亚群的分化,促进免疫反应向Th2偏移,并促进IL-10分泌性T细胞的产生,能够诱导同种T细胞低反应。
进一步研究SOCS1基因修饰的DC体外诱导同种抗原特异性T细胞低反应性。在初次MLR中接受C57BL/6来源的DC-SOCS1刺激的BALB/c脾脏CD4+T细胞,在二次MLR中再次接触同种抗原时,表现出明显较低的反应性(p<0.01);在接触无关第三者抗原时,仍然具有与其他组类似的刺激活性(p>0.05),表明SOCS1基因修饰的DC诱导的T细胞低反应性具有同种抗原特异性。上述结果提示SOCS1基因修饰的DC免疫后有可能减轻移植排斥反应并诱导受者对同种移植器官的免疫耐受。
第三部分SOCS1基因修饰的树突状细胞治疗同种移植排斥的效果和免疫学机制
为进一步观察SOCS1基因修饰的DC体内环境下是否具有减轻移植排斥反应并诱导免疫耐受的作用,我们将C57BL/6小鼠来源的imDC、DC-GFP以及DC-SOCS1免疫BLALB/c小鼠后,采用同种小鼠颈部异位心脏移植模型,研究SOCS1基因修饰的DC体内治疗同种排斥反应、诱导免疫耐受的效果,并分析了可能的机制。
将体重在20-22克、周龄相同的BALB/c小鼠,随机分成四组,每组8只,作为受者。在心脏移植术前第7天,3组BALB/c受者小鼠分别经静脉注入2×106个C57BL/6供者来源的imDC、DC-GFP或DC-SOCS1,并设经尾静脉注入等量PBS的BALB/c受者小鼠为PBS对照。四组受体小鼠分别于同一条件下接受C57BL/6供者心脏的颈部异位移植。另设姊妹组,在心脏移植后第7天及第15天处死受者小鼠,进行病理学及相关免疫指标分析。结果发现,imDC和DC-GFP组的心脏移植物存活期分别为10.9±1.4天和9.5±1.1天,较PBS组同种异体心脏移植物存活期7.1±0.9天均有所延长(P<0.01);DC-SOCS1免疫组的移植心脏存活期为25.5±6.9天,较imDC和DC-GFP组明显延长(P<0.01)。结果表明采用未成熟DC或对照基因修饰的DC免疫受体小鼠均可延长同种移植物存活期,但效果有限；采用SOCS1基因修饰的DC免疫受体小鼠,可以明显减轻移植排斥反应,在一定程度上诱导同种异体心脏移植物的免疫耐受,显著延长移植物的存活期(P<0.01)。
组织学检查发现,PBS对照组移植心脏明显充血、肿胀,光镜下见心肌细胞明显变性、坏死,纤维断裂、间质水肿,肌束间及血管周围大量的炎性细胞浸润；imDC和DC-GFP组移植心脏仍有较明显的肿胀,光镜下见上述病变较PBS对照组有所减轻；DC-SOCS1组表现为心肌细胞无明显变性、水肿,血管周围仅有少量的淋巴细胞浸润。
分析体内免疫学机制发现,imDC和DC-GFP免疫组受者小鼠的脾脏淋巴细胞对供者抗原刺激反应无明显区别(P>0.05),均较PBS同种移植组降低(P<0.01);而DC-SOCS1组与imDC和DC-GFP两组相比,脾脏淋巴细胞对供者抗原的刺激反应明显降低(P<0.01)。同时,DC-SOCS1免疫组受者小鼠的脾脏效应细胞在CTL反应中对靶细胞的杀伤活性明显减弱(P<0.01)。上述结果表明SOCS1基因修饰的DC免疫受体小鼠,其脾脏淋巴细胞对同种抗原的刺激呈低反应性,同时对同种抗原的杀伤活性亦明显降低。
进一步分析各组受者小鼠脾脏及颈部淋巴结内CD4+CD25+Treg的产生,发现与PBS对照组相比,imDC和DC-GFP组受体脾脏及颈部淋巴结内CD4+CD25+Treg比例轻度增加(P<0.05); DC-SoCS1组与imDC和DC-GFP组相比,受体脾脏及颈部淋巴结内CD4+CD25+Treg比例明显增加(P<0.01)。荧光实时定量PCR检测的结果显示,DC-SOCS1组受体脾脏及颈部淋巴结内CD4+T细胞内转录因子Foxp3的表达水平明显升高(P<0.01)。结果表明SOCS1基因修饰的DC免疫受体小鼠,可明显诱导体内CD4+CD25+Treg的产生并促进CD4+T细胞内Foxp3的表达。
结论：重组复制缺欠型腺病毒载体介导的SOCS1基因转染可以明显增加DC内SOCS1表达,且病毒载体本身在转染过程中对DC的影响较小。即使存在外源性刺激物时,SOCS1基因修饰的未成熟DC仍然能够稳定于未成熟状态,表达低水平的MHC分子和共刺激分子,分泌高水平的免疫抑制性细胞因子IL-10,拥有较强抗原吞噬功能,较弱的抗原刺激能力,体外能够诱导同种T细胞抗原特异性低反应性。体内免疫SOCS1基因修饰的未成熟DC,能够抑制同种排斥反应,延长同种移植物存活期,抑制受体淋巴细胞的抗原反应性及CTL杀伤活性,诱导CD4+CD25+Treg的产生并促进Foxp3的表达,在一定程度上诱导受者形成免疫耐受。因此,进一步研究基于SOCS1基因修饰的DC的免疫治疗策略对于诱导移植免疫耐受有重要意义,具有潜在的临床应用价值
Organ transplantation has become the only effective treatment for end-stage organ disease at present and many patients have gained new life through the transplantation of allogeneic organs. However, the immune system will be inevitably stimulated by the allograft and a series of immune response and rejection will be generated against the transplanted organ. The incidence of organ transplant rejection is lowered after the application of a variety of new clinical immunosuppressants, but two major problems are still remained:first, new immunosuppressants can not completely suppress the occurrence of rejection; second, many adverse reactions will occur after long-term application of immunosuppressive agents, such as drug toxicity, infection and neonatal immunity induced tumors. Therefore, inducing specific immune tolerance for allo-antigen is the best way to solve the transplant rejection, with witch the recipients maintain normal immunity to resist pathogens and other microbial induced infections and tumors, and has become the hot spots of transplantation research.
Dendritic cells (DC), which are potent professional antigen presenting cells (APC), have appeared to be central for immune systems because of their abilities to prime naive T cells and initiate a primary immune response. Recent studies have outlined that DC may function as immune-stimulating cells as well as tolerance-inducing cells. The immunophenotype and function of DC will change during maturation. Immature DC (imDC) display tolerance-inducing activities, though they turn to exhibit their immunogenicity after maturation. imDC lacking CD80 (B7-1) and CD86 (B7-2) could induce antigen-specific anergy in vitro. However, there is a natural mechanism of immature for DC in vivo, and at the same time, a variety of inflammatory mediators produced by recipients after organ transplant including inflammatory cytokines, LPS, etc promote DC differentiation and maturation, which make DC possessing strong immunogenicity. Gene modifications using immunosuppressive molecules, such as 1,25-dihydroxyvitamin D, IL-10, TGF-β, will confer DCs with greater tolerance-inducing effect, making DCs an attractive candidate for tolerance induction and rejection prevention.
Suppressor of cytokine signaling 1 (SOCS1), which was discovered as a negative regulator of cytokine signaling proteins, plays an important role on the maturation of DC in resent research. By highly expressing costimulatory molecules, splenic DC of SOCS1-/- mice responded significantly higher on IL-4, IFN-γand intensely stimulated the proliferation of B cell. Antigen loaded SOCS1-/-DC and normal DC were injected into the different foot pad of one mice, the former could cause stronger Thl-type response in foot pad draining lymph nodes, proved by that the isolated lymph node T cells could produce higher levels of IFN-y. SOCS gene silenced DC were also more effective in triggering the proliferation and cytotoxic function of OVA-specific cytotoxic T cells than normal DC, both in vitro and in vivo. SOCS1 gene silencing DC, as well as SOCS1-/- DC have a stronger immune-stimulating functions to break self-tolerance.Therefore, that using genetic engineering techniques exogenously increase the expression of SOCS1 within DC to inhibit DC maturation and immunostimulatory function is likely to be an effective approach to prevent rejection and induce immune tolerance.
In the present study, bone marrow-derived DC was cultivated, expanded and enriched in vitro. After SOCS1 gene transfer by adenovirus vector, the phenotype and function of SOCS1 gene modificated DC was investigated and the effect on inducing allogeneic T cells hyporesponsiveness in vitro was studied. By means of cervical heterotopic heart transaplantation model in mice, we also analyzed effects of SOCS 1 gene modificated immature DC in inducting alloantigen-specific immune tolerance in vivo and the related mechanism.
PARTⅠCulturing mouse bone marrow dendritic cells in vitro and SOCS1 gene transfer
DC derived from bone marrow, are widely distributed in the body with the extremely low percentage less than 1% in peripheral blood mononuclear cells. In vitro, DC culture technology can provide a rich DC source for basic research and clinical treatment. We cultured freshly isolated mouse bone marrow-derived DC with rmGM-CSF and rmIL-4. Given that mouse bone marrow-derived DC relatively highly expressed specific surface molecules CD11c, we used immunomagnetic bead separating kits to enrich CD11+ DC. The results showed that newly prepared bone marrow cells were very rare in clusters or dendritic processes of cells, cultured for 3 days scattered colony formation can be clearly seen, and dendritic processes gradually increased, for 5 days the a larger cloning and partially connected pieces, cell processes became clear, performance of a typical immature DC. After immunomagnetic bead separation and enrichment, a sufficient number of DC with more than 93% purity could be obtained.
Adenovirus vector possessing simple structure of the genome, high viral titers, high infection efficiency, wide range of host cells, non-insertion mutation and other advantages, so AdEasyTM Adenoviral vector system was selected to constructure of adenovirus vector for encoding SOCS1 genes (Ad-SOCSl).The resuts from PCR, specific enzyme digestion, sequencing, and Western Blotting have confirmed that Ad-SOCS1 vector was successfully constructed and could express SOCS1 protein. After amplification, a higher titer of Ad-SOCS1 vector was obtained. The enriched imDC was transfected with Ad-SOCS1 or Ad-GFP at 1:100 MOI, and imDC without virus transfection served as control.Accordingly, the resulting three groups of DC were known as DC-SOCS1, DC-GFP and imDC. Then, we detected the transfection efficiency of control viral vector in DC-GFP group by Flow cytometry, and examined the mRNA and protein expression of SOCS1 with real-time quantitative PCR and Western Blotting. The results showed the transfection efficiency of control Ad-GFP vector could reach more than 70%. The mRNA and protein expression of SOCS1 in DC-SOCS1 group was significantly increased as compared with that in imDC group and DC-GFP group (P<0.01). The above results demonstrated that our constructed Ad-SOCS1 vector could effectively transfect DC, and significantly up-regulated SOCS1 gene expression in DC.
PART II The phenotype and immunologic function of SOCS1 gene modified DC in vitro
Studies have shown that SOCS1 knock-out mice (SOCS1-/-) DC, expressing high levels of MHC molecules and costimulatory molecules, possessed significantly enhanced immunostimulation capability than normal DC. SOCS1-/- DC could stimulate immune response against tumors after loading tumor antigens, and was assumed as an effective adjuvant treatment of cancer. In view of the important role of SOCS1 gene in immune function, we assumed that increasing the expression of SOCS1 within DC in vitro by biotechnology could prevent the DC maturation process and contribute to induce an efficient and long-time donor-specific immunologic tolerance. In this section, we used Ad-SOCS1 to transfect the enriched imDC, observed the phenotypic features and functional characteristics of SOCS1 gene modified DC, and further investigated the effect of inducing immune tolerance and its mechanism.
RmGM-CSF and rmIL-4 were applied to culture bone marrow-derived DC (C57BL/6) in vitro, and imDC were enriched with immunomagnetic beads separating kits.Then, SOCS1 gene was transferred into the imDC by Ad-SOCS1, and at the same time, Ad-GFP modified imDC and same period cultured imDC were set up as the control group. We analyzed the phenotype, cytokine secretion, antigen phagocytic capacity and antigen-presenting ability of DC-SOCS1, DC-GFP and imDC in the absence or the presence of LPS stimulation.The allostimulation of DC-SOCS1, DC-GFP and imDC was examined with MLR, and the cytokines in supernatant of MLR was detected with ELISA.The generation of IL-10 producing T cells and antigen-specific hyporesponseness was also studied.
The results from analyzing DC phenotype showed that before LPS stimulation, DC-SOCS1 and imDC expressed low levels of CD40, CD80 and MHC molecules with no significant difference. Hower, SOCS1 gene modification could significantly inhibit the up-regulating effect of these molecules by LPS stimulation. For cytokines secretion, there had no significant difference of IL-12 before LPS stimulation in the groups (p>0.05), but DC-SOCS1 secreted lower levels of IL-12 in comparison with imDC and DC-GFP after LPS stimulation (p<0.01). High levels of IL-10 were found in the supernatant of DC-SOCSl group as compared with DC-GFP and imDC group in spite of LPS stimulation (p<0.01). These results suggested that increasing SOCS1 expression could block DC from maturation to exogenous LPS stimulation and maintained a stable immature condition.
FITC-Dextran endocytosis examination showed that DC in each group all had strong phagocytic capacity before LPS stimulation. After that, imDC and DC-GFP significantly reduced their phagocytic capacity, hower, there was a slight decline in phagocytic capacity of DC-SOCS1. Testing antigen-presenting function of DC in each group, we found that antigen presenting ability of DC-SOCS1 was significantly lower than imDC and DC-GFP group (p<0.01). The results showed that SOCS1 gene modification could maintain the DC of a strong antigen phagocytosis and inhibit DC antigen-presenting function.
In MLR, we found that allogeneic CD4+ T cell proliferation stimulated by SOCS1 modified DC were markedly reduced (p<0.01). After detecting cytokine levels in supernatants, we found that Thl subsets cytokines IFN-γlevels were significantly lower in DC-SOCS1 group (P＜0.01), while Th2 subsets cytokines IL-10 levels were significantly higher (P＜0.01). Results from intracellular cytokine staining showed that IL-10 producing CD4+ T in DC-SOCS1 group was significantly increased as compared with that in imDC and DC-GFP group. These results indicated that SOCS1 gene modified DC had lower levels of immune stimulating function, and could inhibit Thl differentiation, divert thl to Th2 bias, and promote IL-10 producing T cells generation in MLR.
Further studied the alloantigenic T cells hyporesponsiveness, we found that CD4+ T stimulated with DC-SOCS1 in primary MLR showed low reactivity when re-stimulation with allogeneic antigen in secondary MLR, which was in contrast to CD4+ T stimulated with imDC and DC-GFP (P＜0.01). Hower, CD4+ T stimulated with DC-SOCS1, imDC or DC-GFP in primary MLR showed similar responsiveness to thirt part antigen (p>0.05). The results suggested that SOCS1 modified DC could induce alloantigenic specific hyporesponsiveness, and possessed the potential to induce allograft immune tolerance.
PARTⅢThe therapeutic effect on allograft rejection of DC-SOCS1 and its related immunological mechanisms in vivo
To further study whether SOCS1 gene modified DC could induce immune tolerance in vivo, we immunized BALB/c mice with imDC, DC-GFP or DC-SOCS1 dirived from C57BL/6 mice, observed the allograft survival time utilizing cervical heterotopic heart transplantation mode and investigated the related mechanism.
Same week-old BALB/c mice (20-22g) were divided into four groups randomly, and three groups were intravenously injected 2×106 imDC, DC-GFP or DC-SOCS1 seven days before heart transplant, respectively. Recipient received PBS injection served as blank group. Four groups recipient received the cervical heterotopic heart transplantation under same conditions, and were palpated everyday to record allograft survival. A sister group recipient were sacrificed at seven days and fifteen days to analyze pathology and immune parameters. The results showed that allograft survival in imDC and DC-GFP group was 10.9±1.4 days and 9.5±1.1 days, respectively, which was moderately extended as compared with PBS group with 7.1±0.9 days survival (P<0.01). However, allograft survival in DC-SOCS1 group with 25.5±6.9 days was significantly longer as compared with imDC and DC-GFP group (P <0.01). The results demonstrated that imDC and DC-GFP immunization could prolong allograft survival with limited effect. However, DC-SOCS1 immunization could induce allograft immune tolerance and significantly prolong allograft survival.
Histological examination revealed heart graft of PBS control group were significantly congestive and swelling, and myocardial cells degeneration, necrosis, fiber fracture, interstitial edema and inflammatory cell infiltration in the muscle bundle and perivascular in light microscope. imDC and DC-GFP group showed the lesion was slightly reduced compared with PBS control group. In contrast, Allograft in DC-SOCS1 group showed no significant degeneration of myocardial cells, edema, and slight perivascular lymphocytic infiltration.
Analysis of immunological mechanisms revealed that there was no difference in antigenic stimulus response of spleen lymphocytes in imDC and DC-GFP recipient (P>0.05), which was lower that in in PBS recipient (P＜0.01). However, spleen lymphocytes in DC-SOCS1 group showed significantly reduced alloresponsiveness as compared with that in in imDC and DC-GFP recipient (P＜0.01). At the same time, spleen lymphocytes in DC-SOCS1 group exhibited low cytotoxicity in CTL response (P＜0.01). The results showed that immunization with SOCS1 gene modified DC could reduce alloantigenic stimulation and alloantigenic killing activity of recipient lymphocytes.
Detecting the generation of CD4+CD25+Treg in the spleen and cervical lymph node in each group, we found that the proportion of CD4+CD25+Treg in imDC and DC-GFP was moderately increased than that in PBS group (P＜0.05), however, the proportion of CD4+CD25+Treg in DC-SOCS1 group was significantly increased than that in imDC and DC-GFP group (P<0.01). Real-time quantitative PCR was used to detect the Foxp3 mRNA in CD4+ T in each group, and the results showed that Foxp3 mRNA levels in spleen and cervical lymph node CD4+ T in DC-SOCS1 group was significantly higher than that in other groups (P<0.01). The results demonstrated that immunization with SOCS1 gene modified DC could induce the generation of CD4+CD25+ Treg in vivo and promote the expression of Foxp3 within CD4+ T cells.
Conclusion
Replication defective adenovirus vector Ad-SOCS1 can effectively transfect DC, and increase the expression of SOCS1 mRNA and protein with harmlessness to DC. In spite of exogenous stimulus, SOCS1 gene modified immature DC can still stabilize in the immature state, expressing low levels of MHC molecules and costimulatory molecules, secreting high levels of immune suppressive cytokines IL-10, and has a stronger antigen phagocytosis, a weaker antigen stimulation activity, which can induce allogeneic T-cell antigenic specific hyporesponsiveness in vitro. Immunization with SOCS1 gene modified immature DC in vivo could prolong the allograft survival time, inhibit the allostimulatory and cytotoxicity activity of recipient lymphocyte, and induce CD4+CD25+Treg generation and promote the expression of Foxp3, exhibiting potential to induce antigen-specific immune tolerance in organ transplantation.
引文
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