CTLA4Ig融合蛋白体外免疫抑制作用机制探讨

本文在建立EAEWistar大鼠模型的基础上 ,借助MBP特异反应的T细胞 ,体外探讨CTLA4Ig免疫抑制作用。实验结果发现 ,CTLA4Ig (10 μg/ml)可完全抑制MBP反应的大鼠T细胞增殖 ,而外源性IL 2的同时加入可恢复T细胞针对MBP的增殖反应。FACS检测经CTLA4Ig处理的T细胞表面仍有少量IL 2R的表达 ,且CTLA4Ig能诱导T细胞凋亡。RT PCR结果显示CTLA4Ig能明显抑制IL 2mRNA的表达 ,轻度抑制IFN γmRNA和TNF αmRNA的表达 ,而对IL 10mRNA的表达无明显影响。本研究旨在为CTLA4Ig进一步实验室及临床应用提供依据     

CTLA4-Ig基因修饰的树突状细胞体外对Th1/Th2平衡的影响

目的:探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白(CTLA4 Ig)基因修饰的树突状细胞(CTLA4 Ig-DCs)体外对Th1/Th2平衡的影响。方法:通过腺病毒载体将目的基因(CTLA4 Ig)转染至小鼠骨髓来源的树突状细胞(DC)。采用流式细胞术(FCM)检测DC表面分子和胞内CTLA4 Ig的表达;采用混合淋巴细胞反应检测DC刺激同种异体T细胞的能力,ELISA法检测DC抗原提呈反应中Th1和Th2类细胞因子IFN-γ和IL-4分泌水平。结果:CTLA4 Ig基因成功转染至DC,转染率约为80%,制备的CTLA4 Ig-DCs稳定表达CTLA4Ig,表面分子CD86呈现低表达;CTLA4 Ig-DCs可有效抑制T细胞增殖,降低抗原提呈反应上清中IFN-γ和IL-4的分泌,并增加IFN-γ/IL-4比值。结论:通过腺病毒将CTLA4 Ig转染DC并且高效表达,可有效降低DC表面CD86分子,抑制同种异体T细胞反应,并能影响体外Th1/Th2水平。     

负载CTLA4Ig重组腺病毒的未成熟树突状细胞对大鼠Th细胞增殖的影响

目的探讨负载CTLA4Ig重组腺病毒的未成熟树突状细胞对大鼠Th细胞增殖的影响。方法将CTLA4Ig重组腺病毒与Wistar大鼠未成熟树突状细胞于37℃共孵育6h后,经尾静脉注射该大鼠作实验组,另外分别设立Wistar大鼠未成熟树突状细胞、CTLA4Ig重组腺病毒、生理盐水经尾静脉注射为对照。1周后,用0.3%戊巴比妥麻醉各组大鼠后抽血检测CTLA4Ig。取脾脏,经流式细胞术分选出Th1、Th2细胞及CD4 T细胞,行混合淋巴细胞培养检测Th细胞的增殖。用免疫组织化学法检测Th1、Th2细胞的比例。结果实验组血清CTLA4Ig水平(0.654±0.13)显著高于CTLA4Ig重组腺病毒组(0.392±0.10,P<0.01),树突状细胞组及生理盐水组未检出。实验组Th1细胞的增殖指数(742±161)、Th1/Th2(0.16±0.05)均显著低于各对照组(分别与未成熟树突状细胞组、CTLA4Ig重组腺病毒组、生理盐水组相比,P均<0.01);而Th2细胞的增殖指数(9162±598)显著高于各对照组(P均<0.01)。结论负载CTLA4Ig重组腺病毒的未成熟树突状细胞可显著抑制大鼠Th1细胞增殖,促进Th2细胞增殖,使Th细胞由Th1向Th2显著偏移,诱导有效的免疫耐受。     

负载CTLA4Ig重组腺病毒的未成熟树突状细胞对大鼠Th细胞增殖的影响

目的 探讨负载CTLA4Ig重组腺病毒的未成熟树突状细胞对大鼠Th细胞增殖的影响.方法 将CTLA4Ig重组腺病毒与Wistar大鼠未成熟树突状细胞于37C共孵育6h后,经尾静脉注射该大鼠作实验组,另外分别设立Wistar大鼠未成熟树突状细胞、CTLA4Ig重组腺病毒、生理盐水经尾静脉注射为对照.1周后,用0.3%戊巴比妥麻醉各组大鼠后抽血检测CTLA4Ig.取脾脏,经流式细胞术分选出Th1、Th2细胞及CD4+T细胞,行混合淋巴细胞培养检测Th细胞的增殖.用免疫组织化学法检测Th1、Th2细胞的比例.结果 实验组血清CTLA4Ig水平(0.654±0.13)显著高于CTLA4Ig重组腺病毒组(0.392±0.10,P＜0.01),树突状细胞组及生理盐水组未检出.实验组Th1细胞的增殖指数(742±161)、Th1/Th2(0.16±0.05)均显著低于各对照组(分别与未成熟树突状细胞组、CTLA4Ig重组腺病毒组、生理盐水组相比,P均＜0.01);而Th2细胞的增殖指数(9162 ±598)显著高于各对照组(P均＜0.01).结论 负载CTLA4Ig重组腺病毒的未成熟树突状细胞可显著抑制大鼠Th1细胞增殖,促进Th2细胞增殖,使Th细胞由Th1向Th2显著偏移,诱导有效的免疫耐受.     

CTLA-4 Ig腺病毒基因治疗联合供体细胞输注诱导混合嵌合体和大鼠心脏移植耐受

目的　在心脏移植手术中实施CTLA 4Ig腺病毒基因治疗并联合术后输注供体骨髓细胞 ,诱导异基因大鼠心脏移植耐受 ,并对相关机制进行研究。方法　将异基因DA大鼠的心脏移植给受体LEW大鼠 ,同时经门静脉输注供体DA的脾细胞 (SC ,3× 10 8)、CTLA 4Ig腺病毒 [( 1～ 5 )× 10 9PFU ml],第 4天由舌静脉输注DA的骨髓细胞 (BMC ,3× 10 8)。观察、记录心脏移植物的存活时间。并对皮肤移植的受体作同样处理 ,观察皮肤移植存活情况。通过MLR、IL 2逆转实验及嵌合体的测定 ,探讨耐受机理 ,并检测了CTLA 4Ig的体内表达、TH1 TH2型细胞因子的表达。结果　单用CTLA 4Ig腺病毒基因治疗 ,或CTLA 4Ig腺病毒基因治疗联合单独的供体脾细胞或骨髓细胞能不同程度地延长异基因心脏移植物的存活 ,但不能延长皮肤移植物的存活。脾细胞、CTLA 4Ig腺病毒和骨髓细胞(SC Ad BMC)处理组的心脏移植物存活时间明显超过其它各耐受诱导组 ,并且能够诱导皮肤耐受。RT PCR实验证明 ,在受体内不同的组织CTLA 4Ig基因的表达量有所不同 ,并且随着时间的推移表达下降。TH1和TH2型细胞因子的检测显示 ,耐受大鼠体内未发现这两类细胞因子的偏移现象。MLR证明耐受大鼠的免疫应答表现为供体特异性降低 ,IL 2逆转实验、嵌合体检测表明 ,该耐受可能与     

CTLA-4在超抗原诱导T细胞无能中的作用研究

目的 探讨CTLA—4对T细胞无能的诱导作用。方法 通过使用超抗原SEA作为一种无能诱导剂，建立体外无能模型，检测了无能T细胞在受到SEA的再次刺激时其膜分子CD28和CTLA—4的表达。结果 与活化T细胞相比，无能T细胞在免疫应答的后期阶段表面表达高水平的CTLA—4分子，而CD28的表达则只较活化组T细胞稍高一些。结论 CTLA—4表面表达水平的升高很可能与T细胞无能状态的诱导有关。     

CTLA4Ig基因修饰的BMSCs支持CD34+细胞扩增的实验研究

目的：通过腺病毒介导CTLA4Ig在骨髓基质细胞（BMSCs)中的表达，探讨CTLA4Ig基因修饰的BMSCs支持CD34^ 细胞扩增的功能变化，为该基因修饰的BMSCs联合造血干细胞移植（HSCT），达到预防移植物抗宿主病（GVHD）和纠正预处理损伤的造血微环境（HIM）积累实验依据。方法：以CTLA4Ig-重组腺病毒按感染复数（Multiplicity of infection,MOI)50转染BMSCs，以RT－PCR检测目的基因转录；免疫磁珠（MACS）阳性选择分选骨髓CD34^ 细胞，流式细胞仪检测其纯度；通过骨髓基质细胞支持CD34^ 扩增的细胞总数及集落形成细胞数（CFC）的变化，比较转染和未转染BMSCs在支持CD34^ 细胞扩增方面的差异。结果：RT－PCR在转染组及转染后传两代BMSCs中检测到CTLA4Ig基因的转录；通过观察扩增后细胞总数及CFC数的变化，发现CTLA4Ig基因转染组与未转染组BMSCs支持CD34^ 细胞扩增的能力也无显著差异（P＞0．05）。结论：CTLA4Ig－重组腺病毒有效介导目的基因对BMSCs的转染，在MOI为50时对其支持造血的功能无显著影响。     

CTLA-4与超抗原对SEA诱导T细胞无能的关联研究

在建立超抗原SEA诱导T细胞无能的体外模型基础上 ,观察了无能T细胞受SEA刺激时共刺激分子CD2 8和CTLA 4的表达。结果发现 ,与活化组相比 ,在SEA加入后的不同时相点无能T细胞上CD2 8的表达都是正常的 ,而CTLA 4分子在SEA加入的第 60小时细胞表面有高水平的表达。这些结果表明 ,超抗原SEA诱导的这种无能状态与CTLA 4所介导的抑制作用增强有关     

CTLA4Ig诱导T细胞对氧化修饰低密度脂蛋白无能的体外研究

目的 在体外诱导T细胞对氧化修饰低密度脂蛋白(ox-LDL)的免疫无能,以期预防免疫损伤在动脉粥样硬化(AS)发病中的作用,为防治AS提供新的思路.方法 分离外周血单个核细胞诱导树突状细胞(DC).分别加入LPS、LDL、ox-LDL等刺激48 h,与同种异体淋巴细胞行混合淋巴细胞反应(MLR).ox-LDL组的MLR中,分别加入不同浓度的CTLA4Ig,以MTY法检测T细胞的增殖.流式细胞仪检测MLR中T细胞活化和T细胞凋亡.ELISPOT检测MLR中T细胞分泌IL-2、IFN-γ和IL-4的情况.结果 ox-LDL组MTT中的刺激指数(SI)明显高于LDL组(DC:T-1:5,1.6717±0.3152 vs 1.4250±0.2874,P<0.05;DC:T=1:10,1.5458±0.2748 vs 1.3352士0.2991,P<0.05);应用CTLA4Ig后,SI较未应用时明显降低(CTLA4Ig 1.25 Ixg/ml,0.96±0.30 vs 1.64±0.33,P<0.01;CTLA4Ig0.62μg/ml,1.12±0.33 vs 1.64±0.33,P<0.05;CTLA4Ig 0.31μg/ml,1.29±0.28vs 1.64±0.33,P<0.05);CTLA4Ig可明显减少T细胞CD25的表达(CTLA4Ig 1.25μg/ml,11.26士0.58 vs 14.25±1.02,P<0.05;CTLA4Ig 10μg/ml,8.42±0.45,P<0.01),增加T细胞的凋亡(CTLA4Ig 1.25μg/ml,12.54±3.69 vs 6.09±2.24,P<0.05;CTLA4Ig 10μg/ml,26.87±5.06 vs 6.09±2.24,P<0.01).ELISPOT表明,CTLA4Ig可减少IL-2(CTLA4Ig 1.25μg/ml,386±42 vs 534±54,P<0.05;CTLA4Ig 10μg/ml,230±27 vs 534±54,P<0.01)和IFN-γ(CTLA4Ig 1.25μg/ml,445±48 v8672±46,P<0.05;CTLA4Ig 10μg/ml,193±39 vs 672±46,P<0.01)的ELISPOT计数,增加IL-4的ELISPOT计数(CTLA4Ig 1.25μg/ml,401±32 vs 332±41,P<0.05;CTLA4Ig 10μg/ml,453±57 vs332±41,P<0.05).结论 CTLA4Ig可在体外诱导T细胞对ox-LDL的免疫无能;CTLA4Ig通过抑制T细胞活化、诱导T细胞凋亡和促进TH1/TH2免疫偏移等机制,诱导T细胞免疫无能.     

CTLA4Ig诱导对氧化修饰低密度脂蛋白免疫耐受的机制研究

目的： 动脉粥样硬化（AS）是一种炎症过程，获得性免疫应答参与AS发生和发展。氧化修饰的低密度脂蛋白（ox-LDL）是目前认为最重要的AS相关自身抗原。本研究拟应用融合蛋白CTLA4Ig，在体外建立对ox-LDL的免疫耐受模型，从而有可能预防免疫应答导致的炎症损伤在AS发病中的作用，为防治AS提供新的策略。方法: 分离人外周血单个核细胞诱导树突状细胞（DC）。分别加入LPS、LDL、 ox-LDL刺激48 h，与同种异体淋巴细胞行混合淋巴细胞反应（MLR）。ox-LDL组的MLR中，分别加入不同浓度的CTLA4Ig。以MTT法检测T细胞的增殖。流式细胞仪检测MLR中T细胞活化和T细胞凋亡。ELISpot检测MLR中T细胞分泌IL-2、IFN-γ和IL-4的情况。结果: ox-LDL组MTT中的刺激指数（SI）明显高于LDL组（P<0.05）；应用CTLA4Ig后，SI较未应用时明显降低（P<0.05，P<0.01）；CTLA4Ig可明显减少T细胞CD25的表达（P<0.05，P<0.01），增加T细胞的凋亡（P<0.05，P<0.01）。CTLA4Ig可减少T细胞分泌IL-2和IFN-γ的ELISpot计数(P<0.01），增加IL-4的ELISpot计数（P<0.05）。结论: CTLA4Ig可在体外诱导对ox-LDL的免疫耐受；CTLA4Ig通过抑制T细胞活化、诱导T细胞凋亡和促进Th1/Th2免疫偏移等机制，诱导免疫耐受。     

NKG2D blockade attenuated cardiac allograft vasculopathy in a mouse model of cardiac transplantation

A previous paper has reported that blockade of NKG2D was effective in protecting allograft in murine models of cardiac transplantation, but the mechanism of NKG2D blockade on attenuated cardiac allograft vasculopathy (CAV) was still unknown. In our current study, we found that wild‐type recipients treated with anti‐NKG2D monoclonal antibody (mAb) plus cytotoxic T lymphocyte antigen (CTLA)‐4‐immunoglobulin (I)g showed prolonged allograft survivals (>90 days, P < 0·001) significantly and attenuated CAV. These in‐vivo results correlated with reduced alloantibody production, low expression of interleukin (IL)‐17 and IL‐6, while infiltration of regulatory T cells increased. IL‐6 administration induced shorter allograft survival and higher CAV grade in CTLA‐4–Ig plus anti‐NKG2D mAb‐treated recipients, whereas IL‐17 had no significant effect on allograft survival and CAV grade in CTLA‐4–Ig plus anti‐NKG2D mAb‐treated recipients. Furthermore, the prolonged allograft survival induced by NKG2D blockade was abrogated partially with depletion of regulatory T cells. In conclusion, blockade of NKG2D combined with CTLA‐4–Ig attenuated CAV and this effect was associated with lower alloantibody production, inhibited IL‐6 expression and enhanced expansion of regulatory T cells.     

Immature dendritic cells convert anergic nonregulatory T cells into Foxp3−IL‐10+ regulatory T cells by engaging CD28 and CTLA‐4

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide‐specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL‐10^?Foxp3^?CTLA‐4⁺CD25^lowEgr2⁺ T cells could be converted into suppressive IL‐10⁺Foxp3^?CTLA‐4⁺CD25^highEgr2⁺ cells resembling type‐1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF‐β during anergy induction favored Foxp3⁺ Treg‐cell induction, while TGF‐β had little effect when added to the second stimulation. Expression of both CD28 and CTLA‐4 molecules on anergic T cells was required to allow their conversion into Tr1‐like cells. Suppressor activity was enabled via CD28‐mediated CD25 upregulation, acting as an IL‐2 sink, together with a CTLA‐4‐mediated inhibition of NFATc1/α activation to shut down IL‐2‐mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1‐like cells.     

Upregulation of CD4+CD25+ T lymphocyte by adenovirus-mediated gene transfer of CTLA4Ig fusion protein in experimental autoimmune myocarditis

OBJECTIVE: To explore the effects of adenovirus vector-mediated gene transfer of CTLA4Ig fusion protein on CD4+CD25+ T cells in experimental autoimmune myocarditis (EAM). METHODS: EAM was induced by porcine cardiac myosin as previously described. Adenovirus vector-mediated CTLA4Ig gene was administrated intravenously in EAM rats on days 1, 4 and 7, with EGFP as control. On day 21, myocardium histopathology was examined and CD4+CD25+ T cells were isolated. Proliferation and suppression assays were used to evaluate the suppressive capacity of CD4+CD25+ T cells in vitro. Relative mRNA level of Foxp3 and TGF-beta was determined by quantitative real-time RT-PCR; expression of CTLA-4, B7-1 and B7-2 protein was compared with Western blot in CD4+CD25+ Tregs. RESULTS: Severe inflammatory lesions were observed in the hearts of EGFP-treated EAM rats and the untreated ones, while Ad-CMV-CTLA4Ig alleviated the myocarditis histologically. Adenovirus vector-mediated CTLA4Ig gene transfer up-regulated the proportion of CD4+CD25+ Tregs significantly. T cell proliferation was greatly inhibited in the CTLA4Ig group compared with the untreated and EGFP-treated groups in vitro. CTLA-4 and B7-2 proteins were down-regulated in the CTLA4Ig group, Foxp3 and TGF-beta mRNA was up-regulated significantly by CTLA4Ig treatment. CONCLUSIONS: Adenovirus vector-mediated CTLA4Ig gene transfer alleviated inflammation in EAM, one of the potential mechanisms is up-regulation of CD4+CD25+ Tregs.     

Cytotoxic T lymphocyte antigen 4‐immunoglobulin G is a potent adjuvant for experimental allergen immunotherapy

Allergen‐specific immunotherapy (SIT) is the only treatment for allergic diseases that targets allergen‐specific T helper type 2 (Th2) cells, which are the cause of the disease. There is an unmet requirement for adjuvants that increase the clinical efficacy of SIT allowing application of lower doses of the allergen, thereby reducing the risk of anaphylactic reactions. Cytotoxic T lymphocyte antigen 4–immunoglobulin (CTLA‐4–Ig) has been shown to induce immunological tolerance in autoimmunity and allograft transplantation by blocking T cell co‐stimulation and induction of the immunoregulatory enzyme indoleamine 2,3 dioxygenase (IDO). Previously, we showed that CTLA‐4–Ig treatment at the time of allergen inhalation induced tolerance to subsequent allergen exposure in a mouse model of asthma. In this study, we test the hypothesis that CTLA‐4–Ig acts as an adjuvant for experimental SIT. We evaluated the adjuvant effects of CTLA‐4–Ig on SIT in a mouse model of ovalbumin‐driven asthma. We used both wild‐type and IDO‐deficient mice to assess the role of IDO in the adjuvant effects of CTLA‐4–Ig. Co‐administration of CTLA‐4–Ig strongly increased SIT‐induced suppression of airway hyperreactivity (AHR), specific IgE in serum, airway eosinophilia and Th2 cytokine levels. Moreover, we found that CTLA‐4–Ig, as an adjuvant for SIT, is equally effective in IDO‐deficient and wild‐type mice, demonstrating that the effect of CTLA‐4–Ig is independent of IDO expression. We show that CTLA‐4–Ig acts as a potent adjuvant to augment the therapeutic effects of SIT. As the adjuvant activity of CTLA‐4–Ig is independent of IDO, we conclude that it acts by blocking CD28‐mediated T cell co‐stimulation.     

超抗原SEA诱导T细胞无能的作用机制探讨

目的 探讨超抗原诱导T细胞无能的分子机制。方法 采用ELISA的FACS，分别检测超抗原对诱导T细胞无能的过程中，IL－2，IL－10的产生和IL－2Rα链（CD25）的表达，并以^3H-TdR掺入法，测定无能T细胞对rhIL-2,PMA及ionomycin的应答能力，而L－10的产生则逐渐升高；CD25的表达与活化组相比较无显著差异。rhIL-2的加入可恢复T细胞增殖。PMA单独作用能诱导无能T细胞的部分增殖能力，但PMA＋ionomycin则能更大程度地恢复T细胞的增殖。结论 超抗原SEA对T细胞无能的诱导，可能与降低IL－2的水平和升高IL－10的水平有关。超抗原SEA的反复刺激对T细胞无能的诱导，可能是干扰了TCR信号途径的近端事件，导致Ras/MAPK途径和Ca/calcineurin途径受阻，而使IL－2基因不能转录所致。