【作者】 王伟；

【导师】 沈传来；

【作者基本信息】 东南大学 ， 免疫学， 2016， 博士

【摘要】 系统性使用大剂量免疫抑制药物治疗移植排斥导致的众多副作用,促使了针对移植排斥的特异性免疫疗法的发展。选择性清除或抑制同种反应性T细胞是治疗移植排斥的理想策略之一。因此,近十余年来,非细胞性的、以抗原肽/主要组织相容性抗原(peptide/major histocompatibility complex, p/MHC)为靶向的,针对抗原特异性T细胞的特异性杀伤制剂被广泛研究。最新的进展之一是在非细胞性载体表面包被pMHC多聚体和Fas配体,制备杀伤性人工抗原递呈细胞(Killer artificial antigen-presenting cells, KaAPCs)。其中以磁珠或胶乳微球为载体的KaAPCs已经被报道能在体外选择性杀伤抗原特异性T细胞。然而,此类载体不易在体内生物降解,缺乏生物相容性,且在体内和器官移植模型中的研究鲜有报道。本研究是以可生物降解的聚乳酸-羟基乙酸(poly(lactic-co-glycolic acid), PLGA)微球(Microparticles, MPs)作为载体,在其表面共包被H-2Kb-Ig二聚体和anti-Fas单抗,制备成基于PLGA的KaAPCs。其表面的H-2Kb-Ig二聚体可以装载特定的抗原肽,形成p/MHC复合体,从而与抗原特异性T细胞的TCR靶向结合,同时其表面的anti-Fas单抗能够诱导抗原特异性T细胞凋亡,达到靶向杀伤抗原特异性T细胞的目的。在同种异体移植模型中,KaAPCs表面的H-2Kb-Ig二聚体即作为同种抗原,与受者鼠体内H-2Kb同种抗原反应性CD8+T细胞的TCR特异性结合,并利用anti-Fas单抗诱导活化的同种反应性T细胞凋亡,实现对移植排斥的特异性免疫治疗。研究目的：以OT-1转基因小鼠为模型研究KaAPCs能否在体外和其体内选择性清除OVA257-264抗原特异性T细胞；并以小鼠同利,异体皮肤移植为模型,分析KaAPCs特异性杀伤同种反应性T细胞的能力和治疗移植排斥的效果,进而探讨其在体内的作用机制。研究方法及结果：1、PLGA MPs的制备及表征：以PLGA聚合物为原材料,采用乳化溶剂挥发法制备直径约为5μm的PLGA MPs,并利用化学修饰法使其表面功能化(带有NH2+)；通过扫描电镜(Scanning Electronic Microscopy,SEM)、粒径分析仪和Zeta电位分析仪对PLGA MPs进行表征,并通过蛋白定量和流式技术分析其吸附蛋白的能力。结果显示：PLGA MPs在SEM下呈现良好的球形,粒径分布在1-10gmn,且80%的MPs集中在5-6μm,平均Zeta电位为65.2±6.7 mV,表明其带有足够电荷,2×107MPs对BSA的最大吸附量为80μg。以上结果表明自制的PLGA MPs具有良好的表征。2、KaAPCs的制备及其表型分析：把PLGA MPs和H-2Kb-Ig二聚体以及anti-Fas单抗共孵育后,制备成KaAPCs,并经特异性荧光单抗染色、流式细胞技术和激光共聚焦技术验证KaAPCs表型。结果显示,H-2Kb-Ig二聚体和anti-Fas单抗被成功共包被到PLGA MPs表面,表明所制的KaAPCs具有良好的表型。3、KaAPCs靶向杀伤OVA257-264抗原特异性T细胞：在体外,把装载OVA257-264的KaAPCs (Kb/OVA-KaAPC)与OT-1转基因小鼠(其CD8+T细胞TCR以H-2Kb限制的方式识别OVA257-264)的脾细胞共孵育24小时,通过流式细胞术检测T细胞群中CD8+T细胞的凋亡比例以及OVA257-264特异性CD8+T细胞的数量比例。结果显示Kb/OVA-KaAPC能够诱导大约80%的CD8+T细胞凋亡,OVA257-264特异性CD8+T细胞比例显著下降83%左右。而无关抗原肽对照组没有出现明显凋亡,OVA257-264特异性CD8+T细胞没有明显减少,表明KaAPCs能够在体外特异性地杀伤抗原特异性CD8+T细胞。其中anti-Fas介导的凋亡为主,活化诱导的凋亡(AICD)极少。在体内,Kb/OVA-KaAPC经尾静脉注入OT-1鼠后,在不同时间点通过流式细胞术分析其外周血中CD8+T细胞的凋亡比例及OVA257-264特异性CD8+T细胞的数量比例。结果与体外实验相似,CD8+T细胞凋亡比例最高达75%左右,OVA257-264特异性CD8+T细胞的比例最高下降大约84%,而对照组无显著变化。KaAPC的杀伤效率与其剂量、作用时间呈正相关。这表明KaAPC在体内也能靶向杀伤抗原特异性的CD8+T细胞。4、KaAPC抑制小鼠皮肤移植排斥的有效方案的研究：以C57BL/6鼠(H-2Kb)和BALB/c鼠(H-2Kd)为供受体,建立同种异体皮肤移植模型。KaAPCs(包被自制H-2Kb单体和商品化anti-Fas单抗)经尾静脉或局部注入受者鼠后,观察移植皮块的生存状况,进行临床评分,并通过免疫荧光染色检测移植皮块中CD8+T和CD4+T细胞的浸润量,同时多指标监测受者鼠的整体免疫功能。结果：比较多种方案的效果后,明确了靶向抗原和anti-Fas的包被剂量、KaAPCs的注射途径、剂量、次数和时间点等治疗措施。据此,KaAPCs能显著延长移植皮块的生存时间达4-6天,并明显减少移植皮块中CD8+T细胞的浸润数量,对CD4+T细胞无明显影响,且不明显抑制受者鼠的整体免疫功能。5、KaAPC抑制小鼠皮肤移植排斥的优化方案及其机制研究：以C57BL/6鼠(H-2Kb)和bml鼠(H-2Kbml)为供受体,建立同种异体皮肤移植模型。在优化方案下,经三次尾静脉注射KaAPCs(包被商品化H-2Kb-Ig二聚体和商品化anti-Fas)治疗受者鼠后,观察移植皮块的生存状况和排斥程度；流式细胞术检测受者鼠外周血和脾脏中T细胞的凋亡比例及H-2Kb同种反应性CD8+T细胞比例；免疫组化分析移植皮块中浸润的CD8+ T、CD4+T细胞和H-2Kb同种反应性T细胞的量；针对供体的同种增殖能力检测；脾脏及淋巴结中调节性T细胞的检测；KaAPCs对受者鼠脾脏中B、NK、 T细胞以及外周血中单核细胞、中性粒细胞和淋巴细胞群等的影响；通过抗肿瘤能力、T细胞库针对第三方抗原的同种增殖能力和NK细胞杀瘤能力的分析,监测KaAPCs对受者鼠整体免疫功能的影响；观察KaAPCs在体内的运行和分布。结果显示：三次尾静脉输注后KaAPCs能经血流进入淋巴结和脾脏,并与CD8+T细胞直接接触,有效地靶向杀伤外周血和脾脏中同种反应性CD8+T细胞,杀伤率达82%左右,从而大幅减少了同种反应性CD8+T细胞在移植皮块中的浸润,同时上调了淋巴结中调节性T细胞的水平,最终延长移植皮块的存活时间达42.5天,且对受者鼠体内的其他免疫细胞群没有明显影响,也没有显著损伤受者鼠的整体免疫功能。结论：基于PLGA微球的KaAPCs能够在体外和体内选择性杀伤抗原特异性CD8+T细胞,且能在不损伤受者鼠整体免疫功能的情况下,通过靶向杀伤同种反应性CD8+T细胞而有效抑制同种皮肤移植排斥反应,显著延长皮肤移植物的存活期。提示KaAPCs可以作为治疗移植排斥或自身免疫病的新型特异性免疫治疗策略。更多还原

【Abstract】 The global side effects, which are due to systemically administrating immunosuppressants, lead to the development of specific immunotherapies for treatment of allograft rejections. The strategy of selectively eliminating or suppressing alloreactive T cells is one of the promising immunotherapies of treating allograft rejections. Thus, the acellular agents using peptide/major histocompatibility complex (p/MHC) to target antigen-specific T cells have been investigated extensively for decades. Killer artificial antigen presenting cells (KaAPCs), which generated by coupling p/MHC multimers and Fas ligand (FasL) onto acellular scaffold, is one of the latest researches. The KaAPCs based on magnetic and latex beads have been shown the ability to selectively deplete antigen-specific T cells in vitro. However, magnetic and latex beads are absent of biocompatibility. Moreover, the research using the KaAPCs to deplete antigen-specific T cells in vivo and in allograft transplant model is rare.In this research, poly (lactic-co-glycolic acid) (PLGA) based KaAPCs are generated by covalently coupling H-2Kb-Ig dimer and anti-Fas monoclonal antibodies (mAbs) on PLGA microparticles (MPs) surfaces.The H-2Kb-Ig dimer onto KaAPCs are been able to load with peptideds and target to T cell receptor (TCR) of antigen-specific T cells, meanwhile the anti-Fas mAbs are capable to induce the apoptosis of the T cells. In allograft transplant, the H-2Kb-lg dimer are capable of selectively interacting with TCR of H-2Kb alloantigen-specific T cells, and induce apoptosis of these cells. Therefore, KaAPCs are able to specifically treat allograft rejections.Objectives:The ability of the KaAPC to selectively eliminate antigen-specific T cells in vitro and in vivo has been investigated in an OT-1 transgenic mice model. In addition, the ability of the KaAPCs to deplete alloreactive T cells and treat alloskin graft rejections in an alloskin graft transplant model has been studied.Methods and Results:1. Fabrication and characterization of PLGA MPs:PLGA MPs with diameter of 5μm were prepared using a double-emulsion solvent evaporation method. The surface of PLGA MPs was modified with chemistry method.The shape, size distribution and zeta potential of PLGA MPs were characterized through scanning electron microscopy (SEM), dynamic light scattering technique and PALS zeta instrument, respectively. The flow cytometry was used to detect the ability of PLGA MPs to couple protein.The results showed that the MPs displayed a spherical shape under the SEM, the diameter was range from 1 to 10 um and 80% of these MPs were 5-6μm in diameter, the mean zeta potential was 65.2±6.7 mV and a strong capacity to couple protein (2×107 PLGA MPs can be loaded with about 80μg of protein).2. Generation and Phenotypic analysis of KaAPCs:The KaAPCs were generated by coupling H-2Kb-Ig and anti-Fas mAbs onto PLGA MPs. Flow cytometry and immunofluorescence staining were used to detect phenotype of the KaAPCs. The results revealed that the KaAPCs displayed both H-2Kb-Ig and anti-Fas mAbs immobilized to the surface of PLGA MPs.3. KaAPCs selectively deplete antigen-specific T cells:in vitro, KaAPCs loaded with OVA257-264 peptides (Kb/OVA-KaAPC) were cocultured with splenocytes from OT-1 transgenic mice (whose CD8+T cells recognize OVA257-264 peptides in the context of H-2Kb) for 24 hours. The apoptosis and frequency of OVA257-264-specific CD8+T cells were detected by flow cytometry. Coculture of Kb/OVA-KaAPC resulted in a apoptosis of about 80% CD8+T cells and remarkable reduce of OVA257-264-specific CD8+ T cells by about 83% in vitro. In contrast, control cocultures did not lead to significant increase of CD8+T cells apoptosis and decrease frequency of OVA257-264-specific CD8+T cells. The killing effect of KaAPCs is mainly dependent on anti-Fas mAbs, not the activation induction cell death (AICD). In vivo, Kb/OVA-KaAPC were injected (via tail vein) into OT-1 transgenic mice, and the apoptosis and proportion of OVA257-264-specific CD8+ T cells in peripheral blood were analyzed by flow cytometryat different time points. Similarly, injections of Kb/OVA-KaAPC caused a strong apoptosis (about 83% of CD8+ T cells) and significant decrease of OVA257-264-specific CD8+ T cells by 84% in vivo.These results demonstrated that the KaAPCs were able to selectively deplete antigen-specific T cells in vitro and in vivo.4. The study of effective regimen for suppressing allograft rejections by KaAPCs:using C57BL/6 mice (H-2Kb) and BALB/c mice (H-2Kd) as donor and recipient, respectively, to establish the alloskin transplant model. After injection (via tail vein or local of alloskin graft) of KaAPC (generated by coupling in-house H-2Kb monomer and commercial anti-Fas mAbs) into recipients, alloskin graft survival was assessed, CD4+ and CD8+ T cells infiltrated into alloskin graft were detected by immunofluorescence, and general immune functions of recipients was evaluated. The effective regimen for suppressing allograft rejections were determined by comparing some treatment regimen including the dosage, route and times of injection. In compare to control groups, treatment of KaAPCs was able to prolong the allograft survival for 4-6 days, significantly reduce the CD8+ T cells infiltrated into alloskin graft but not CD4+ T cells, and did not impair the general immune functions of recipients.5. The study of optimizing regimen and mechanism of allograft rejections suppression by KaAPCs:using C57BL/6 mice (H-2Kb) and bml mice (H-2Kbml) as donor and recipient, respectively, to establish the alloskin transplant model. After injection (via tail vein) of KaAPC (generated by coupling commercial H-2Kb-Ig dimer and anti-Fas mAbs) into recipients, alloskin graft survival was assessed, the percentage of H-2Kb alloreactive CD8+ T cells in peripheral blood and spleen of recipients was detected by flow cytometry, the H-2Kb alloreactive CD8+T, CD4+T and CD8+T cells infiltrated into alloskin graft was analyzed with immunohistochemical (IHC), the apoptosis of T cells was detected by flow cytometry, the proliferation of host CD3+ T cells was evaluated in a host-versus-donor mixed lymphocytes reaction (MLR), the B, NK and regulatory T cells was detected by flow cytometry, the lymphocytes, monocytes and neutrophils in peripheral blood were detected by automated hematology analyzer, the host antitumor effects and proliferative response in a third-party MLR were evaluated. The results exhibited that KaAPCs were able to enter into spleen and lymphoid node (LN) and interact with CD8+ T cells after three intravenous injections of KaAPCs. Treatment of KaAPCs resulted in a dramatic depletion of alloreactive CD8+T cells in the spleen, peripheral blood and infiltrating into the alloskin graft, and prolonged alloskin graft survival for 42.5 days. Furthermore, KaAPCs were able to contract the proliferation of host CD3+ T cells in a host-versus-donor MLR. A significant increase of CD4+CD25+Foxp3+ regulatory T cells were induced in LN, but not spleen, by KaAPCs. Additionally, KaAPCs did not markedly suppress the immune cells and general immune function of host.In conclusion, the PLGA based KaAPCs are capable of depleting antigen-specific CD8+ T cells in vitro and in vivo, and suppressing the allograft rejections by targeting alloreactive T cells without impairment of general immune functions of host. Thus, the strategy of KaAPCs represents a potential specific immunotherapy for treating allograft rejections and immune disorders.更多还原