【作者】 肖亮；

【导师】 丁国善；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 CD4^＋T辅助细胞的活化、增殖和分化是抗原特异性免疫反应产生的关键环节。该反应的产生除了需要抗原递呈细胞（Antigen presenting cells, APCs）提供抗原肽MHC复合物与T细胞受体特异性结合外，还必须有APCs提供协同刺激分子如免疫球蛋白家族成员（CD80、ICOSL等）、TNF家族成员（CD40、OX40、4-1BB、CD27、LIGHT等）和多种细胞因子的参与。随着研究的深入，不断有新的分子被发现参与、调控了T细胞的活化过程。因此，T细胞的活化与调节仍然是基础免疫学研究的重点，也是移植免疫学关注的热点。T细胞免疫球蛋白粘蛋白（T cell immunoglobulin mucin，Tim）是2001年发现的表达在T细胞和DCs表面与细胞活化相关的新分子。小鼠TIM基因家族位于11B1.1，目前共发现8个基因，编码蛋白Tim-l～4和4个假想蛋白Tim-5～8。人类TIM基因家族只有3个成员，定位于染色体5q33.2，分别编码蛋白Tim-l, 3, 4（没有Tim-2）。Tim是一类具有共同基序的跨膜糖蛋白，其结构包括免疫球蛋白（Ig）样区、粘蛋白样区、跨膜区和胞内区。除Tim-4外，Tim-1，2，3的胞内区均含有酪氨酸激酶磷酸化位点，直接参与信号转导。早期研究发现，Tim-1表达于所有活化的CD4⁺ T细胞，并在其分化后仍高表达于Th2细胞，而低表达于Th1，Th17，CD11c⁺骨髓源性树突状细胞（Dendritic cells,DCs）和CD19⁺ B细胞。Tim-1的配体为Tim-4，与其它Tim蛋白不同，小鼠Tim-4只表达于APCs。它作为Tim-1的配体参与T细胞的活化和增殖，并能减少其凋亡。Tim-主要表达于Th1细胞和DCs，它与DCs上的Galectin-9结合后能导致Th1细胞的凋亡。然而，随着研究的深入，人们发现Tim-1和Tim-4在免疫调节中的作用比预想的更为复杂。最近的研究显示，小鼠Tim-4与人IgG₁ Fc段融合蛋白（Tim-4-Ig）在不同的终浓度下能刺激或抑制培养体系中T细胞的活化。另外, Tim-4-Ig可以通过与小鼠初始CD4⁺ T细胞上的某种配体结合抑制其活化，经证实，这种配体并非Tim-1。然而，Tim-4-Ig的这种作用只局限于不表达Tim-1的小鼠初始T细胞，它不能抑制预先激活的T细胞增殖。上述研究提示Tim-4-Ig可以与T细胞上的不同配体结合，介导不同的免疫效应。此外，Tim-1的不同单克隆抗体（3B3和RMT1-10）也可以对T细胞活化产生相反的作用，这可能取决于它们与Tim-1结合的亲和力不同或对细胞骨架结构的影响不同。因此，进一步阐明Tim-1调节T细胞反应的机制，对于开发治疗自身免疫性疾病和移植物排斥反应的新药物，具有重要的意义。基于上述研究，本课题拟进一步：（1）合成人源化Tim-1胞外段和IgG₁ Fc段融合蛋白（Tim-1-Fc），检测其与小鼠不同种类细胞的结合情况，验证其是否具有交叉活性，并判定这种可能的结合是否是Tim-4依赖性的；（2）体外研究Tim-1-Fc对anti-CD3, anti-CD28 mAbs介导的小鼠CD4⁺ T细胞活化，增殖，凋亡，细胞周期和对同种DCs介导的混合淋巴细胞反应（Allogeneic mixed lymphocyte response, allo-MLR）的影响，探讨其胞内信号转导途径；观察其对调节性T细胞（Regulatory T cells, Tregs）增殖和叉头状转录因子p3（Forkhead transcription factor p3, Foxp3）表达的影响；（3）通过腹腔注射Tim-1-Fc（对照组注射PBS和人IgG₁），阻断Tim-4-Tim-1介导的T细胞活化，观察其对小鼠颈部异位心脏移植物生存期的影响，初步揭示其抑制移植排斥反应的作用和体内机制；通过本课题研究，有望进一步认识Tim-1-Fc分子在T细胞介导的免疫反应和抑制同种移植物排斥反应中的作用，为寻找临床免疫抑制治疗的新靶点提供实验依据。目的：合成人源化Tim-1胞外段和IgG₁ Fc段融合蛋白（Tim-1-Fc），检测其与小鼠不同细胞结合情况，验证其是否具有交叉活性，并判定这种可能的结合是否是Tim-4依赖性的。方法：从基因库中搜索人Tim-1胞外段和IgG₁ Fc段的cDNA序列，克隆到哺乳动物细胞表达载体pcDNA3.1上，利用转染试剂将重组质粒转入中国仓鼠卵巢细胞（Chinese hamster ovary, CHO）中，通过Western-blot检测Tim-1-Fc表达情况，经亲和、离子交换等多种层析方法纯化蛋白。采用流式细胞仪（Flow cytometry, FCM）检测Tim-1-Fc与未接受刺激的或刺激3天后的小鼠DCs，CD4⁺CD25^- T细胞，CD4⁺CD25⁺ T细胞（Natural regulatory T cells, nTregs）结合情况，加入Tim-4单抗，阻断可能的Tim-4-Tim-1-Fc结合。结果：经Western-blot检测，合成的Tim-1-Fc分子量约62KD，纯度>92%。Tim-1-Fc不结合DCs，nTregs和未活化的CD4⁺CD25^- T细胞，但能选择性结合活化后的CD4⁺CD25^- T细胞（Effector T cells, Teffs），且该作用不被anti-Tim-4 mAb阻断。结论：Tim-1-Fc构建成功。小鼠Teffs上存在人Tim-1的新型配体。目的：观察Tim-1-Fc在体外对CD4⁺ T细胞活化，增殖，凋亡和细胞周期，胞内信号转导，allo-MLR以及nTregs增殖、Fxop3表达的影响。方法：在CD4⁺ T细胞培养体系中加入不同终浓度的Tim-1-Fc（1，5，10μg/ml），按不同比例（1:40，1:20，1:10）将DC与CD4⁺ T细胞混合培养，采用3H掺入法检测细胞增殖，CSFE检测细胞分裂，ELISA检测培养上清液中Th1/Th2细胞因子分泌情况，PI/Annexin V双染色法检测各组CD4⁺ T细胞凋亡的比例，RT-PCR法检测Tim-1-Fc对nTregs内Foxp3基因转录的影响，Western-blot检测Tim-1-Fc对细胞周期蛋白Cdk2，Cdk4，p27^kip表达及anti-CD3（2μg/ml）, anti-CD28（1μg/ml）mAbs刺激后不同时间点（5, 15, 30min）CD4⁺ T细胞内信号分子AKT，ERK1/2磷酸化情况的影响。结果：Tim-1-Fc不但能抑制CD4⁺ T细胞表面活化标志CD25，CD69表达、抑制细胞分裂增殖，该作用不依赖于Tim-4（RT-PCR和FCM检测也未发现小鼠T细胞表达Tim-4）；还能显著下调Cdk2，Cdk4而增加p27^kip表达，使细胞停滞在G1期；抑制CD4⁺ T细胞内AKT，ERK1/2磷酸化；抑制allo-MLR，诱导T细胞对同种抗原的低反应性，减少IL-2，IFN-γ而促进IL-10的分泌。但Tim-1-Fc对CD4⁺ T细胞凋亡，nTregs增殖和Foxp3表达无明显影响。结论：Tim-1-Fc在体外能通过与T细胞表面新型受体结合抑制CD4⁺ T细胞活化、增殖和Th1源性细胞因子表达，使细胞停滞在G₀/G₁期，并下调T细胞内信号分子AKT，ERK1/2磷酸化水平。还能抑制小鼠allo-MLR，诱导T细胞对同种抗原的低反应性；但它不影响CD4⁺ T细胞凋亡，nTregs增殖和Foxp3表达。目的：观察Tim-1-Fc对小鼠心脏移植物存活期的影响并探讨其抑制排斥反应的体内机制。方法：采用BALB/c→C57BL/6小鼠颈部异位心脏移植模型，将受体分为3组（每组n=10）：组1，心脏移植+PBS治疗组；组2，心脏移植+ hIgG₁（10μg/g/天术后0⁶天）治疗组；组3，心脏移植+Tim-1-Fc（10μg/g/天术后0⁶天）腹腔注射治疗组。专人记录心脏移植物存活时间。于术后第7天处死小鼠，收集血液、心脏、脾脏标本。Real-time PCR检测心脏移植物内淋巴细胞表面分子CD3, CD11b和炎性细胞因子IL-2, IFN-γ, IL-4, IL-10和转录因子Foxp3表达情况，病理检查心脏移植物内炎性细胞浸润和组织破坏情况。磁珠分选受体小鼠脾脏CD4⁺ T细胞，用BALB/c小鼠DCs刺激，³H掺入法检测其增殖情况，ELISA检测Th1，Th2细胞因子水平。FCM检测受体小鼠脾脏中CD4⁺Foxp3⁺调节性T细胞占总CD4⁺T细胞比例，以及CD4⁺CD25^- T细胞培养体系中加入Tim-1-Fc后Foxp3的表达变化。结果：三组小鼠心脏移植物存活时间分别为6.5±0.5，6.7±0.4和17.1±1.1天。Tim-1-Fc治疗组移植物内IL-2、IFN-γ、CD11b、CD3 mRNA表达水平显著较低，病理损害亦明显减轻，但IL-10水平升高（P<0.05）；Tim-1-Fc组受体脾脏来源CD4⁺ T细胞接受同种异基因DCs刺激后增殖水平显著较低（P<0.05）；受体小鼠脾脏中CD4⁺Foxp3⁺调节性T细胞占总CD4⁺T细胞比例显著升高（P<0.05），但Tim-1-Fc并不能直接诱导CD4⁺CD25^- T细胞表达Foxp3。结论：Tim-1-Fc能减轻小鼠心脏移植物Th1细胞因子表达和炎性细胞浸润，促进Th2细胞因子表达，降低受体脾脏内供体反应性T细胞对同种抗原的反应能力，显著延长移植物存活期；Tim-1-Fc虽不能直接诱导Tregs，但它通过调节Th1/Th2平衡向Th2发展，提高脾脏内CD4⁺Foxp3⁺ T细胞比例，调节Teffs/Tregs平衡向Tregs偏移，成功抑制了小鼠心脏移植物排斥反应，使其有望成为抗排斥治疗的新药物。更多还原

【Abstract】 Activation, proliferation and differentiation of CD4⁺ T helper cell are crucial elementsof antigen-specific immune response. This process needs not only the specific binding of Tcellreceptors with antigenic peptide/MHC complexes provided by antigen-presenting cells（APCs）, but also needs co-stimulatory molecules such as immunoglobulin family members（CD80, ICOSL, etc.）, TNF family members （CD40, OX40, 4-1BB, CD27, LIGHT, etc.）and a variety of inflammatory cytokines. With the in-depth research, new co-stimulatorymolecules have being discovered to be involved in T cells activation. Thus, the activationand regulation of T cells remain major scientific issues to basic immunology research andhot spots to transplantation immunology research.T cell immunoglobulin mucin （Tim） is a newly discovered molecular expressed on thesurface of T cells and APCs and is associated to T-cell activation. Mouse TIM genes arelocated on chromosome 11B1.1, encoding protein Tim-1⁴, and four hypothetical proteinTim-5⁸. Human TIM genes are located on chromosome 5q33.2, encoding protein Tim-l, 3,4, but not Tim-2. Tim represents a group of transmembrane glycoproteins with commonmotifs: immunoglobulin （Ig）-like domain, mucin-like domain, transmembrane region andintracellular domain. In addition to Tim-4, Tim-1, 2 and 3 all contain an intracellulartyrosine kinase phosphorylation site, which is directly involved in the intracellular signaltransduction.Tim-1 is expressed on all activated CD4⁺ T cells, and highly expressed on Th2 cellsafter differentiation, while lowly expressed on Th1, Th17, CD11c⁺ bone marrow-deriveddendritic cells （DCs） and CD19⁺ B cells. Tim-1 is the natural ligand for Tim-4, which isonly expressed on APCs and is involved in T cell activation, proliferation, and apoptosisinhibition. Tim-3 is mainly expressed on Th1 cells and DCs, and after cross-linking withGalectin-9, it can lead to the apoptosis of Th1 cells.However, the mechanisms for immune regulation by Tim-1 and Tim-4 are morecomplex than initially expected. Recent studies showed that Tim-4-Ig may either stimulateor inhibit T cell proliferation depending on its concentration. Moreover, Tim-4-Ig wasdemonstrated to inhibit naive mouse CD4⁺ T cell activation through a novel ligand otherthan Tim-1, as such an inhibitory effect of Tim-4-Ig was specific to naive T cells that do notexpress Tim-1, and the effect disappeared in pre-activated T cells. This suggests thepossibility that the opposite effect of Tim-4-Ig on T cell activation observed in the previous studies could be resulted from its engagement with different receptors expressed on T cells.On the other hand, anti-Tim-1 mAbs （3B3 and IRM-10） were also found to mediate either astimulatory or an inhibitory effect on T cell activation depending on their binding affinity toTim-1 or their influence on the cytoskeleton. Thus, further elucidation of the role of Tim-1in regulating T cell responses is highly important for developing novel therapeuticstrategies targeting Tim-1 for the treatment of autoimmune diseases and allograft rejection.Based on the above studies, further efforts of this study include: （1） Synthesization ofhuman Tim-1 excellular domain and IgG₁ Fc domain fusion protein （Tim-1-Fc）. To analyzethe biding activity of Tim-1-Fc with different kinds of mouse cell types such asunstimulated or stimulated dendritic cells （DCs）, CD4⁺CD25^- T cells and CD4⁺Foxp3+regulatory T cells （nTregs） to identify the cross-reactive, and to test whether this bindingwas Tim-4 dependent; （2） Exploration of the influence of Tim-1-Fc on allogeneic mixedlymphocyte response, activation and proliferation of CD4⁺ T cells, cells cycle and apoptosisevoked by anti-CD3 and anti-CD28 mAbs. Investigation of the intracellular signalingpathways in CD4⁺ T cells after ligation with Tim-1-Fc. Observation of its effect on theproliferation and Foxp3 expression of nTregs. （3） Intraperitoneal injection of Tim-1-Fc tomice that receive heterotopic cardiac transplantation, while PBS and hIgG₁ treatment wereused as controls. The mechanisms of Tim-1-Fc in suppressing allograft acute rejection areto be explored. Through this research, we could get a better understanding of human Tim-1in mouse T cell-mediated immune responses and allograft rejection suppression, which mayprovide a new target for clinical immunosuppressive therapy.Objective: To synthesize humanized excellular domain of Tim-1 and IgG₁ Fc domainfusion protein （Tim-1-Fc）. To analyze the biding activity of Tim-1-Fc with different kindsof mouse cell types such as DCs, CD4⁺CD25^- T cells and nTregs, and to test whether thisbinding activity was Tim-4 dependent. Methods: cDNA sequences of human Tim-1extracellular domain and IgG₁ Fc fragment were got from the gene bank and cloned intothe mammalian cell expression vector pcDNA3.1, the recombinant plasmids weretransformed into CHO cells using transfection reagent. Tim-1-Fc expression was detected by SDS-PAGE and Western-blot, and purified by affinity column. The binding activity ofTim-1-Fc （5μg/ml） with fresh isolated or activated DCs, CD4⁺CD25^- T cells and nTregswas labeled using mouse anti-human IgG₁（Fc）-FITC and measured by FCM. Anti-Tim-4mAb （10μg/ml） was added to block the possible Tim-4-Tim-1-Fc engagement. ResultsResults:Western-blot test revealed that Tim-1-Fc molecular weight was about 62KD, purity was>92%. Tim-1-Fc could not bind to unstimulated or stimulated DCs or nTregs, but couldselectively bind to activated CD4⁺CD25^- T cells （Teffs）, and this effect could not bereversed by anti-Tim-4 mAb. Conclusion: Tim-1-Fc was constructed successfully. Theremust be a novel ligand for human Tim-1 expressed on mouse Teffs other than Tim-4.Objective: To test the influence of Tim-1-Fc on allo-MLR and on the activation,proliferation, cell cycles, apoptosis and Fxop3 expression of CD4⁺ T cells; to explore theTim-1-Fc-triggered intracellular signaling pathways in CD4⁺ T cells. Methods: Differentconcentrations of Tim-1-Fc （1, 5, 10μg/ml） and different proportions （1:40, 1:20, 1:10） ofDC:T-cell were added to test the influence of Tim-1-Fc on allo-MLR. 3H-TdR, CFSE andELISA were used to assay the proliferation and cytokines production of CD4⁺ T cells coculturedwith Tim-1-Fc. FCM was used to test the influence of Tim-1-Fc on the apoptosis（PI/Annexin V） and Foxp3 expression of CD4⁺ T cells. Western blot was used to explore thephosphorylation of intracellular signal moleculars. Results: Tim-1-Fc significantlyinhibited CD4⁺ T cells activation, proliferation and allo-MLR but not the apoptosis. Theseeffects were Tim-4-independent. Also, Tim-1-Fc reduced IL-2，IFN-γwhile promoted IL-10production, and it negatively regulated Cdk2 and Cdk4 expression while increased p27^kipexpression. Moreover, the phosphorylation of AKT and ERK1/2 in CD4⁺ T cells was alsoinhibited, but the proliferation of nTregs or Foxp3 expression were not inhibited by Tim-1-Fc. Conclusion: Tim-1-Fc was able to inhibit the activation, proliferation, differentiation,Th1 cytokines production and allo-MLR of CD4⁺ T cells, and to suppress thephosphorylation of AKT, ERK1/2 in CD4⁺ T cells, but has not influence on the proliferation and Foxp3 expression of nTregs in vitro.Objective: To observe the influence of Tim-1-Fc on cardiac allograft survival andallograft acute rejection and to explore the in vivo mechanisms. Methods:BALB/c→C57BL/6 mouse model of cervical heterotopic heart transplantation wasperformed, the recipients were divided into 3 groups according to the different treatments（for each group, n=10）: Group 1, transplantation + PBS treatment group; Group 2,transplantation + human IgG₁ treatment group （10μg/g/day, intraperitoneal injection, 0-6days post-operatively）; Group 3, transplantation+Tim-1-Fc treatment group （10μg/g/day,intraperitoneal injection, 0-6 days post-operatively）. The survival time of cardiac allograftwas recorded. At the 7th day after transplant, the mice were killed, the bloods, hearts andspleens were collected. ELISA test for blood cytokines （IL-2, IFN-γ, IL-4 and IL-10） levelsand intragraft RT-PCR detection for inflammatory cytokines production, lymphocytessurface molecules （CD3 and CD11b） and Foxp3 expression were conducted. Recipients’splenic CD4⁺ T cells were isolated by magnetic activated cell sorting and stimulated withallogenetic DCs, the proliferation was detected by using ³H thymidine incorporation using aliquid scintillation counter. The ratio of Tregs in recipient mice spleens was also assessed.CD4⁺CD25^- T cells were isolated and cultured with Tim-1-Fc and/or recombinant humanTGF-β1，then the expression of Foxp3 were determined by FCM. Results: Recipientstreated with Tim-1-Fc showed significantly prolonged allograft survival （17.1±1.1 days）compared to those treated with PBS （6.5±0.5 days; P<0.01） or control hIgG₁ （6.7±0.4 days;P<0.01）. Histological analysis of heart grafts at POD 7 revealed severe mononuclear cellsinfiltration and myocardial necrosis in the control groups, whereas almost normal tissuestructure with minimal mononuclear cell infiltration was seen in Tim-1-Fc-treated mice.Tim-1-Fc significantly reduced the intragraft transcription for IL-2, IFN-γ, CD3 and CD11b. The proliferation of CD4⁺ T cells in response to allogenetic DCs was significantlyinhibited in Tim-1-Fc treated group. Moreover, the proportion of CD4⁺Foxp3⁺ Tregs insplenic CD4⁺ T cells was significantly increased, although Tim-1-Fc did not induce Foxp3expression in CD4⁺CD25^- T cells. ConclusionConclusion: Tim-1-Fc was able to prolong mouse heartallograft survival though reducing the inflammatory cells infiltration and Th1 cytokinesproduction in the graft and decreasing the proliferative capability of donor reactive T cellsto allogenetic antigens, while increasing the proportion of Tregs in the spleen, thus shiftingthe balance of Th1/Th2 towards Th2 and the balance of Tregs/Teffs towards regulators,although it did not induce Foxp3 expression in CD4⁺CD25^- T cells directly. These madeTim-1-Fc become a promising drug for anti-rejection therapy.更多还原