DNAM-1-nectin-2信号途径介导人NK细胞对猪内皮细胞直接杀伤作用的研究
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摘要
第一部分猪nectin-2分子的生物信息学预测、克隆及分析鉴定
[目的]证明猪nectin-2基因的存在，分析并克隆出nectin-2基因，明确其分子结构特点及表达特性。
[方法]运用生物信息学方法从美国基因数据库(GenBank)获得与人nectin-2基因同源的猪EST序列，通过RT-PCR、3’RACE等技术在猪内皮细胞系SV-40-PED中验证该序列的准确性，并研究其组织表达分布特点，利用体细胞杂交板(somatic cell hybridpanel，SCHP)技术对其进行染色体定位。根据反向PCR原理扩增该基因5’端调控区的未知序列。参照同源分子人PVR蛋白胞膜外区的三级结构，利用生物序列分析软件及数据库构建猪nectin-2蛋白胞膜外区的三维空间结构模型。
[结果]通过克隆测序与同源性分析，发现猪存在necin-2分子，其包含α、δ2种剪接体，它们具有相同的胞膜外区序列与不同的跨膜区和胞浆区序列，将此结果提交至美国基因数据库(GenBank)，并认定为我们新发现的基因，分别获得登录号EF195266和EU069826。necin-2在猪的各种组织细胞中广泛表达，尤其在猪内皮细胞中表达丰富。SCHP结果显示猪necin-2定位于6号染色体q21区域。通过反向PCR技术从猪nectin-2α的5’端非编码区延伸出371bp的未知序列。三维空间结构显示猪nectin-2蛋白胞膜外区形成V-C-C 3个免疫球蛋白样结构域，其中V样结构域由9个β折叠构成。
[结论]生物信息学方法和分子生物学技术的联合使用，有助于猪的新基因的发现与鉴定。本研究证实了猪存在nectin-2基因的推测，明确了该基因相关的结构信息，为研究nectin-2的免疫学功能奠定了基础。
第二部分猪nectin-2胞膜外区融合蛋白的构建、表达与纯化
[目的]构建具有模拟天然nectin-2分子与受体DNAM-1相结合能力的胞膜外区融合蛋白nectin-2ED-IgGFc。
[方法]利用RT-PCR扩增猪nectin-2胞膜外区基因片段，连接至T载体，测序无误后，亚克隆至真核表达载体CD5lnegl，构建出重组质粒nectin-2ED-CD5lnegl。将重组质粒和空载体CD5lnegl分别转染至CHO细胞，用嘌呤霉素进行筛选；通过Western Blot、细胞免疫化学、免疫荧光和流式细胞术等方法检测，鉴定出能稳定表达融合蛋白nectin-2ED-IgGFc和IgGFc蛋白的细胞株。利用蛋白A+G琼脂糖珠亲和层析法分离、纯化融合蛋白。
[结果]经过双酶切和测序鉴定，重组质粒nectin-2ED-CD5lnegl构建成功。嘌呤霉素筛选出能稳定表达nectin-2ED-IgGFc融合蛋白和IgGFc蛋白的CHO细胞，从其细胞培养上清中分离、纯化出高纯度的融合蛋白。
[结论]成功构建并表达出融合蛋白nectin-2ED-IgGFc，该蛋白可作为猪nectin-2与人DNAM-1相互作用研究的重要工具。
第三部分DNAM-1-nectin-2途径介导入NK细胞杀伤猪内皮细胞作用的研究
[目的]研究猪nectin-2与人DNAM-1异种受体配体之间的结合能力，建立能客观有效地评价人NK细胞对猪内皮细胞杀伤效果的实验方法，比较不同人NK细胞系(包括NK92和YT细胞系)对猪内皮细胞的异种杀伤能力。
[方法]利用流式细胞术和免疫共沉淀技术检测融合蛋白nectin-2ED-IgGFc与YT细胞系的DNAM-1之间的亲和能力。通过Western Blot比较DNAM-1在NK92与YT细胞系中的表达水平，用流式细胞术检测融合蛋白和抗DNAM-1中和性抗体与YT细胞结合的效率。建立用CFSE／PI双染法检测NK92和YT细胞系对猪内皮细胞系(SV-40-PED)细胞毒作用的方法，比较NK92与YT细胞异种杀伤效应的差异。
[结果]融合蛋白nectin-2ED-IgGFc能与人YT细胞表面的DNAM-1发生特异性结合。NK92与YT细胞DNAM-1的表达量无明显差异。YT细胞在与终浓度为10μg／ml的抗DNAM-1中和性抗体于室温孵育1h后，抗体与YT细胞的结合率为48．53％。通过CFSE／PI双染法检测到，当效靶比为10：1，共同孵育时间为5、6和7小时后，对猪内皮细胞系(SV-40-PED)的杀伤率人NK92细胞分别为44．68％、53．99％和57．44％，人YT细胞分别为20．44％、20．60％和26．71％。
[结论]证明了猪nectin-2与人DNAM-1发生特异性结合的能力，这是DNAM-1-nectin-2途径发挥作用的重要前提。CFSE／PI双染法能够方便客观地检测NK细胞的杀伤效率。在杀伤条件完全相同时，NK92比YT细胞表现出更高的异种杀伤活性。以上结果为进一步研究DNAM-1-nectin-2信号途径介导人NK细胞对猪内皮细胞的杀伤作用奠定了良好的基础。
PartⅠ.Bioinformatics prediction,cloning and characterizationof pig nectin-2
Objective To confirm the existence of porcine nectin-2 gene,and to clone the novelgene followed by analysis for the molecule structure properties and expression pattern.
Methods The pig expressed sequence tags(EST) homologous to human nectin-2 wereobtained from GenBank database using bioinformatics method.By the way of RT-PCR or3'RACE techniques,the gene was cloned from porcine endothelial cell line(SV-40-PED)and checked for the accuracy of the nucleic acid sequence,then the expression anddistribution pattern of the gene was analyzed.Chromosomal localization was performed bymeans of somatic cell hybrid panel(SCHP).According to the inverse PCR principle,theunknown sequence of 5' control region of the gene was amplified.The three-dimensionstructure model of porcine nectin-2 ectodomain was built referring to the tertiary structureof human PVR ectodomain using biological sequence analysis softs and database.
Results Two different isoforms,pig nectin-2αand -2δ,with identical extracellularregion and unique transmembrane region and cytoplasmic tail region were identified by sequencing and homology analysis.The nucleotide sequences of the two splicing variantswere submitted to NCBI database and confirmed as novel genes and the GenBankaccession numbers were EF 195266 and EU069826.Nectin-2 was ubiquitously expressed invarious cells and tissues including SV-40-PED with high expression level。Pig nectin-2 wasmapping to chromosome 6q21.A 371bp long unknown sequence was definited in 5'uncoding region of nectin-2α.The three-dimension structure model showed that the pignectin-2 protein contained three Ig-like loops V-C-C in the extracellular region and the Vlike domain was folded into a secondary structure with nineβ-strands.
Conclusion Simultaneous utilization of bioinformatics method and molecular biologytechniques will conduce the prediction and probation of novel porcine genes,and thepresumption about pig nectin-2 is confirmed by the very way.Identification of the structureinformation of pig nectin-2 is essential to further investigat the immunologic function ofthe gene.
PartⅡ.Construction,expression and purification of pignectin-2ED-IgGFc fusion protein
Objective To construct extracellular region fusion protein of pig nectin-2(nectin-2ED-IgGFc) which can simulate the natural nectin-2 molecular to interact withDNAM- 1.
Methods The extracellular region of pig nectin-2 was amplified from SV-40-PEDusing RT-PCR and cloned into T vector,then subcloned into eukaryotic expression vectorCD51negl after sequencing.The recombinant plasmid nectin-2ED-CD51negl and the blankvector CD51negl were transfected into CHO cells respectively followed by puromycinselection.CHO cells which can stablely secrete nectin-2ED-IgGFc fusion protein andIgGFc were identified by the way of Western Blot,immunocytochemistry,immunofluorescence and flow cytometry .The fusion protein was purified from the supernatants of the CHO cells culture by protein A+G affinity chromatography.
Results The recombinated plasmid nectin-2ED-CD51negl was constructed andverified successfully by double enzyme digestion and sequencing.After puromycinselection,CHO cells which can stably secrete nectin-2ED-IgGFc fusion protein and IgGFcwere obtained,and highly purified ectogenic proteins were isolated from the CHO cellsculture supernatants.
Conclusion The nectin-2ED-IgGFc fusion protein was obtained successfully and canbe used to investigate the interaction between pig nectin-2 and human DNAM-1.
PartⅢ.Study about the effect of DNAM-1- nectin-2 pathway inmediating Xenogeneic cytolytic activity of human NKcells against porcine endothelial cells
Objective To identify the heterogeneic binding ability between pig nectin-2 andhuman DNAM-1.To establish a suitable empirical method to estimate the cytotoxic effectof human NK cells against porcine endothelial cells objectively,and contrast theheterogeneic kill efficiency between different human NK cell lines including NK92 andYT.
Methods The affinity ability between nectin-2ED-IgGFc fusion protein and DNAM-1was detected by flow cytometry and co-immunoprecipitation techniques.The expressionlevel of DNAM-1 of NK92 and YT cell lines was compared by semiquantitative analysisusing Western Blot.The binding efficiency of fusion protein and anti-DNAM-1 neutralizingantibody to YT cells was tested by flow cytometry.CFSE/PI double labeling method wasestablished in flow-cytometric NK cells cytotoxicity assay to compare heterogeneic killingability against SV-40-PED between NK92 and YT cell lines.
Results The fusion protein could bind specifically to human DNAM-1 expressed onYT cell surface.There was no significant difference of expression level of DNAM-1 between NK92 and YT cells.After 10μg/ml anti-DNAM-1 neutralizing antibody incubatingwith YT cells at room temperature for 1 hour,the binding rate of the antibody to YT cellswas 48.53%.Adopting CFSE/PI double labeling method,when the effctor/target ratio was10:1,coincubating time was 5h,6h and 7h,the detected cytotoxicity of NK92 againstSV-40-PED was 44.68%、53.99% and 57.44% respectively and that of YT was 20.44%、20.60% and 26.71% respectively.
Conclusion Identify the specifical conjunction between pig nectin-2 and humanDNAM-1 which is the important precondition in studying the effect ofDNAM-l-nectin-2 passway.CFSE/PI double labeling method can test the kill ability of NKcells conveniently and objectively.Under the same condition,NK92 shows higherheterogeneic cytolytic activity comparied with YT.All the data establish a favourablefoundation for further investigation of the potency of DNAM-l-nectin-2 pathway inmediating human NK cells heterogeneic cytotoxic effect against pig endothelial cells.
[目的]证明猪nectin-2基因的存在，分析并克隆出nectin-2基因，明确其分子结构特点及表达特性。
[方法]运用生物信息学方法从美国基因数据库(GenBank)获得与人nectin-2基因同源的猪EST序列，通过RT-PCR、3’RACE等技术在猪内皮细胞系SV-40-PED中验证该序列的准确性，并研究其组织表达分布特点，利用体细胞杂交板(somatic cell hybridpanel，SCHP)技术对其进行染色体定位。根据反向PCR原理扩增该基因5’端调控区的未知序列。参照同源分子人PVR蛋白胞膜外区的三级结构，利用生物序列分析软件及数据库构建猪nectin-2蛋白胞膜外区的三维空间结构模型。
[结果]通过克隆测序与同源性分析，发现猪存在necin-2分子，其包含α、δ2种剪接体，它们具有相同的胞膜外区序列与不同的跨膜区和胞浆区序列，将此结果提交至美国基因数据库(GenBank)，并认定为我们新发现的基因，分别获得登录号EF195266和EU069826。necin-2在猪的各种组织细胞中广泛表达，尤其在猪内皮细胞中表达丰富。SCHP结果显示猪necin-2定位于6号染色体q21区域。通过反向PCR技术从猪nectin-2α的5’端非编码区延伸出371bp的未知序列。三维空间结构显示猪nectin-2蛋白胞膜外区形成V-C-C 3个免疫球蛋白样结构域，其中V样结构域由9个β折叠构成。
[结论]生物信息学方法和分子生物学技术的联合使用，有助于猪的新基因的发现与鉴定。本研究证实了猪存在nectin-2基因的推测，明确了该基因相关的结构信息，为研究nectin-2的免疫学功能奠定了基础。
第二部分猪nectin-2胞膜外区融合蛋白的构建、表达与纯化
[目的]构建具有模拟天然nectin-2分子与受体DNAM-1相结合能力的胞膜外区融合蛋白nectin-2ED-IgGFc。
[方法]利用RT-PCR扩增猪nectin-2胞膜外区基因片段，连接至T载体，测序无误后，亚克隆至真核表达载体CD5lnegl，构建出重组质粒nectin-2ED-CD5lnegl。将重组质粒和空载体CD5lnegl分别转染至CHO细胞，用嘌呤霉素进行筛选；通过Western Blot、细胞免疫化学、免疫荧光和流式细胞术等方法检测，鉴定出能稳定表达融合蛋白nectin-2ED-IgGFc和IgGFc蛋白的细胞株。利用蛋白A+G琼脂糖珠亲和层析法分离、纯化融合蛋白。
[结果]经过双酶切和测序鉴定，重组质粒nectin-2ED-CD5lnegl构建成功。嘌呤霉素筛选出能稳定表达nectin-2ED-IgGFc融合蛋白和IgGFc蛋白的CHO细胞，从其细胞培养上清中分离、纯化出高纯度的融合蛋白。
[结论]成功构建并表达出融合蛋白nectin-2ED-IgGFc，该蛋白可作为猪nectin-2与人DNAM-1相互作用研究的重要工具。
第三部分DNAM-1-nectin-2途径介导入NK细胞杀伤猪内皮细胞作用的研究
[目的]研究猪nectin-2与人DNAM-1异种受体配体之间的结合能力，建立能客观有效地评价人NK细胞对猪内皮细胞杀伤效果的实验方法，比较不同人NK细胞系(包括NK92和YT细胞系)对猪内皮细胞的异种杀伤能力。
[方法]利用流式细胞术和免疫共沉淀技术检测融合蛋白nectin-2ED-IgGFc与YT细胞系的DNAM-1之间的亲和能力。通过Western Blot比较DNAM-1在NK92与YT细胞系中的表达水平，用流式细胞术检测融合蛋白和抗DNAM-1中和性抗体与YT细胞结合的效率。建立用CFSE／PI双染法检测NK92和YT细胞系对猪内皮细胞系(SV-40-PED)细胞毒作用的方法，比较NK92与YT细胞异种杀伤效应的差异。
[结果]融合蛋白nectin-2ED-IgGFc能与人YT细胞表面的DNAM-1发生特异性结合。NK92与YT细胞DNAM-1的表达量无明显差异。YT细胞在与终浓度为10μg／ml的抗DNAM-1中和性抗体于室温孵育1h后，抗体与YT细胞的结合率为48．53％。通过CFSE／PI双染法检测到，当效靶比为10：1，共同孵育时间为5、6和7小时后，对猪内皮细胞系(SV-40-PED)的杀伤率人NK92细胞分别为44．68％、53．99％和57．44％，人YT细胞分别为20．44％、20．60％和26．71％。
[结论]证明了猪nectin-2与人DNAM-1发生特异性结合的能力，这是DNAM-1-nectin-2途径发挥作用的重要前提。CFSE／PI双染法能够方便客观地检测NK细胞的杀伤效率。在杀伤条件完全相同时，NK92比YT细胞表现出更高的异种杀伤活性。以上结果为进一步研究DNAM-1-nectin-2信号途径介导人NK细胞对猪内皮细胞的杀伤作用奠定了良好的基础。
PartⅠ.Bioinformatics prediction,cloning and characterizationof pig nectin-2
Objective To confirm the existence of porcine nectin-2 gene,and to clone the novelgene followed by analysis for the molecule structure properties and expression pattern.
Methods The pig expressed sequence tags(EST) homologous to human nectin-2 wereobtained from GenBank database using bioinformatics method.By the way of RT-PCR or3'RACE techniques,the gene was cloned from porcine endothelial cell line(SV-40-PED)and checked for the accuracy of the nucleic acid sequence,then the expression anddistribution pattern of the gene was analyzed.Chromosomal localization was performed bymeans of somatic cell hybrid panel(SCHP).According to the inverse PCR principle,theunknown sequence of 5' control region of the gene was amplified.The three-dimensionstructure model of porcine nectin-2 ectodomain was built referring to the tertiary structureof human PVR ectodomain using biological sequence analysis softs and database.
Results Two different isoforms,pig nectin-2αand -2δ,with identical extracellularregion and unique transmembrane region and cytoplasmic tail region were identified by sequencing and homology analysis.The nucleotide sequences of the two splicing variantswere submitted to NCBI database and confirmed as novel genes and the GenBankaccession numbers were EF 195266 and EU069826.Nectin-2 was ubiquitously expressed invarious cells and tissues including SV-40-PED with high expression level。Pig nectin-2 wasmapping to chromosome 6q21.A 371bp long unknown sequence was definited in 5'uncoding region of nectin-2α.The three-dimension structure model showed that the pignectin-2 protein contained three Ig-like loops V-C-C in the extracellular region and the Vlike domain was folded into a secondary structure with nineβ-strands.
Conclusion Simultaneous utilization of bioinformatics method and molecular biologytechniques will conduce the prediction and probation of novel porcine genes,and thepresumption about pig nectin-2 is confirmed by the very way.Identification of the structureinformation of pig nectin-2 is essential to further investigat the immunologic function ofthe gene.
PartⅡ.Construction,expression and purification of pignectin-2ED-IgGFc fusion protein
Objective To construct extracellular region fusion protein of pig nectin-2(nectin-2ED-IgGFc) which can simulate the natural nectin-2 molecular to interact withDNAM- 1.
Methods The extracellular region of pig nectin-2 was amplified from SV-40-PEDusing RT-PCR and cloned into T vector,then subcloned into eukaryotic expression vectorCD51negl after sequencing.The recombinant plasmid nectin-2ED-CD51negl and the blankvector CD51negl were transfected into CHO cells respectively followed by puromycinselection.CHO cells which can stablely secrete nectin-2ED-IgGFc fusion protein andIgGFc were identified by the way of Western Blot,immunocytochemistry,immunofluorescence and flow cytometry .The fusion protein was purified from the supernatants of the CHO cells culture by protein A+G affinity chromatography.
Results The recombinated plasmid nectin-2ED-CD51negl was constructed andverified successfully by double enzyme digestion and sequencing.After puromycinselection,CHO cells which can stably secrete nectin-2ED-IgGFc fusion protein and IgGFcwere obtained,and highly purified ectogenic proteins were isolated from the CHO cellsculture supernatants.
Conclusion The nectin-2ED-IgGFc fusion protein was obtained successfully and canbe used to investigate the interaction between pig nectin-2 and human DNAM-1.
PartⅢ.Study about the effect of DNAM-1- nectin-2 pathway inmediating Xenogeneic cytolytic activity of human NKcells against porcine endothelial cells
Objective To identify the heterogeneic binding ability between pig nectin-2 andhuman DNAM-1.To establish a suitable empirical method to estimate the cytotoxic effectof human NK cells against porcine endothelial cells objectively,and contrast theheterogeneic kill efficiency between different human NK cell lines including NK92 andYT.
Methods The affinity ability between nectin-2ED-IgGFc fusion protein and DNAM-1was detected by flow cytometry and co-immunoprecipitation techniques.The expressionlevel of DNAM-1 of NK92 and YT cell lines was compared by semiquantitative analysisusing Western Blot.The binding efficiency of fusion protein and anti-DNAM-1 neutralizingantibody to YT cells was tested by flow cytometry.CFSE/PI double labeling method wasestablished in flow-cytometric NK cells cytotoxicity assay to compare heterogeneic killingability against SV-40-PED between NK92 and YT cell lines.
Results The fusion protein could bind specifically to human DNAM-1 expressed onYT cell surface.There was no significant difference of expression level of DNAM-1 between NK92 and YT cells.After 10μg/ml anti-DNAM-1 neutralizing antibody incubatingwith YT cells at room temperature for 1 hour,the binding rate of the antibody to YT cellswas 48.53%.Adopting CFSE/PI double labeling method,when the effctor/target ratio was10:1,coincubating time was 5h,6h and 7h,the detected cytotoxicity of NK92 againstSV-40-PED was 44.68%、53.99% and 57.44% respectively and that of YT was 20.44%、20.60% and 26.71% respectively.
Conclusion Identify the specifical conjunction between pig nectin-2 and humanDNAM-1 which is the important precondition in studying the effect ofDNAM-l-nectin-2 passway.CFSE/PI double labeling method can test the kill ability of NKcells conveniently and objectively.Under the same condition,NK92 shows higherheterogeneic cytolytic activity comparied with YT.All the data establish a favourablefoundation for further investigation of the potency of DNAM-l-nectin-2 pathway inmediating human NK cells heterogeneic cytotoxic effect against pig endothelial cells.
引文
1. Panepinto LM, Phillips RW. The Yucatan miniature pig : characterization and utilization in biomedical research. Lab Anim Sci, 1986, 36: 344-3471
2. Sachs DH. The pig as a potential xenograft donor . Vet Immunol Immunopathol, 1994,43:185-1911
3. Shibuya A, Campbell D, Hannum C, et al.DNAM-l,a novel adhesion molecule involved in the cytolytic function of T lymphochtes. Immunity. 1996, 4: 573-581
4. Delia Chiesa M, Romagnani C, Thiel A, et al. Multidirectional interactions are bridging human NK cells with plasmacytoid and monocyte-derived dendritic cells during innate immune responses. Blood, 2006, 108: 3851-3858
5. Pende D, Castriconi R, Romagnani P,et al. Expression of the DNAM-1 ligands, Nectin-2(CD112) and poliovirus receptor(CD155), on dendritic cells:relevance for natural killer-dendritic cell interaction. Blood, 2006, 107: 2030-2036
6. Spaggiari GM,Capobianco A, Becchetti S et al. Mesenchymal stem cell-natural killer cell interactions:evidence that activated NK cells are capable of killing MSCs,whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood, 2006, 107: 1484-1490
7. Bottino C, Castriconi R, Pende D, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule.J Exp Med, 2003, 198:557-67
8. Lopez M, Aoubala M, Jordier F, et al. The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule. Blood, 1998, 92:4602-4611
9. Kozak M. A consideration of alternative models for the initiation of translation in eukaryotes. Crit Rev Biochem Mol Biol, 1992, 27: 385-402
10. Eberle F, Dubreuil P, Mattei MG, et al. The human PRR2 gene, related to the human poliovirus receptor gene(PVR), is the true homolog of the murine MPH gene. Gene,1995. 159:267-272
11. Takahashi K, Nakanishi H, Miyahara M,et al. Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to Cadherin-Based adherens junctions through interaction with afadin,a PDZ domain-containing protein. J Cell Biol, 1999, 145:539-549
12. Irie K. Shimizu K, Sakisaka T, et al. Roles and modes of action of nectins in cell-cell adhesion. Semin Cell Dev Biol, 2004, 15: 643-656
13. Takai Y. Nakanishi H. Nectin and afadin: novel organizers of intercellular junctions. J Cell Sci. 2003, 116:17-27
14. Takai Y, Irie K, Shimizu K, et al. Nectins and nectinlike molecules: roles in cell adhesion, migration, and polarization. Cancer Sci, 2003, 94: 655-667
15．Campadelli-Fiume G，Cocchi F．Menotti L，et al．The novel receptors that mediate the entry of herpes simplex viruses and animal alphaherpesviruses into cells．Rev Med Virol，2000，10：305-319
16．Davoli R，Bigi D，Fontanesi L，et al．Mapping of 14 expressed sequence tags(ESTs)from porcine skeletal muscle by somatic cell hybrid analysis．Animal Genetics，2000，31：400-403
17．Chevalet C，Gouzy J，SanCristobal-Gaudy M．Regional assignment of genetic markers using a somatic cell hybrid panel：a WWW interactive program available for the pig genome．Comput Appl Biosci，1997，13：69-73
18．Hawken RJ，Murtaugh J，Flickinger GH，et al．A first-generation porcine whole-genome radiation hybrid map．Mamm Genome，1999，10：824-830
19．朱正茂．应用cDNA宏阵列技术发现猪某些产肉性状的候选基因．[博士学位论文]．华中农业大学图书馆，2003
20．余梅．猪12号染色体上四个新基因的分离、鉴定和物理定位．[博士学位论文]．华中农业大学图书馆，2002
21．Goureau A，Yerle M，Schmitz A，et al．Human and porcine correspondence of chromosome segments using bidirectional chromosome painting．Genomics，1996，36：252-262
22．Reymond N，Imbert AM，Devilard E，et al．DNAM-1 and PVR Regulate Monocyte Migration through Endothelial Junctions．J Exp Med，2004，199：1331-1341
23．Miller DA，Miller OJ，Dev VG，et al．Human chromosome 19 carries a poliovirus receptor gene．Cell，1974，1：167-173
24．邹文雄，黄明经．蛋白质结构之电脑预测．Chemistry，1997，55：101-109
25．Mart(?)-Renom M A，Stuart A C，Fiser A，et al．Comparative protein structure modeling of genes and genomes．Annu Rev Biophys Biomol Struct，2000，29：291-325
26．He Y，Bowman VD，Mueller S，et al．Interaction of the poliovirus receptor with poliovirus．Proc Natl Acad Sci，2000，97：79-84
27．Du Pasquier L，Zucchetti I，De Santis R．Immunoglobulin superfamily receptors in protochordates：before RAG time．Immunol Rev，2004，198：233-248
28．Fabre S，Reymond N，Cocchi F，et al．Prominent role of the Ig-like V domain in trans-interactions of nectins．Nectin3 and nectin 4 bind to the predicted C-C′-C″-D beta-strands of the nectinl V domain．J Biol Chem，2002，277：27006-27013
1．Yasumi M，Shimizu K，Honda T，et al．Role of each immunoglobulin-like loop of nectin for its cell-cell adhesion activity．Biochem Biophys Res Commun，2003，302：61-66
2．Momose Y，Honda T，Inagaki M，et al．Role of the second immunoglobulin-like loop of nectin in cell-cell adhesion．Biochem Biophys Res Commun，2002，293：45-49
3．John FM，Paul B．Cleavage of Simian Virus 40 DNA at a Unique Site by a Bacterial Restriction Enzyme．Proc Natl Acad Sci，1972，69：3365-3369
4．冯作化．医学分子生物学．人民卫生出版社．2001年9月第1版，139
5．Lin SJ，Wang JY，Klickstein LB，et al．Lack of age-associated LFA-1 up-regulation and impaired ICAM-1 binding in lymphocytes from patients with Down syndrome．Clin Exp Immunol，2001，126：54-63
6．Shreffler WG，Castro RR，Kucuk ZY，et al．The major glycoprotein allergen from Arachis hypogaea．Ara h 1，is a ligand of dendritic cell-specific ICAM-grabbing nonintegrin and acts as a Th2 adjuvant in vitro．J Immunol，2006，177：3677-3685
7．Smyth N，Odenthal U，Merkl B，et al．Eukaryotic expression and purification of recombinant extracellular matrix proteins carrying the strep Ⅱ tag．Methods Mol Biol，2009，522：63-72
8．Taatjes DJ，Chen TH，Ackerstr(o|¨)m B，et al．Streptococcal protein G-gold complex：comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling．Eur J Cell Biol，1987，45：151-159
1．Shibuya A，Campbell D，Hannum C，et al．DNAM-1，a novel adhesion molecule involved in the cytolytic function of T lymphocytes．Immunity，1996，4：573．
2．Tahara-Hanaoka S，Shibuya K，Kai H，et al．Tumor rejection by the poliovirus receptor family ligands of the DNAM-1(CD226)receptor．Blood，2006，107：1491-1496
3．Della Chiesa M，Romagnani C，Thiel A，et al．Multidirectional interactions are bridging human NK cells with plasmacytoid and monocyte-derived dendritic cells during innate immune responses．Blood，2006，108：3851-3858
4．Pende D，Castriconi R，Romagnani P，et al．Expression of the DNAM-1 ligands，Nectin-2(CD112)and poliovirus receptor(CD155)，on dendritic cells：relevance for natural killer-dendritic cell interaction．Blood，2006，107：2030-2036
5．Spaggiari GM，Capobianco A，Becchetti S et al．Mesenchymal stem cell-natural killer cell interactions：evidence that activated NK cells are capable of killing MSCs，whereas MSCs can inhibit IL-2-induced NK-cell proliferation．Blood，2006，107：1484-1490
6. Bottino C. Castriconi R, Pende D, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule.J Exp Med. 2003, 198: 557-567
7. Pende D. Bottino C, Castriconi R, et al. PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in tumor cell lysis. Mol Immunol, 2005, 42: 463-469
8. Lopez M. Aoubala M, Jordier F, et al. The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule. Blood, 1998. 92: 4602-4611
9. Shibuya A, Campbell D. Hannum C, et al. DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes. Immunity, 1996, 4: 573-581
10. Irie K, Shimizu K, Sakisaka T, et al. Roles and modes of action of nectins in cell-cell adhesion. Semin Cell Dev Biol, 2004, 15: 643-656
11. Shibuya A D, Campbell C, Hannum H, et al. DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes. Immunity, 1996, 4: 573-581
12. Ralston KJ, Hird SL, Zhang X, et al.The LFA-1-associated molecule PTA-1(CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large. J Biol Chem, 2004, 279: 33816-33828
13. Shibuya K, Lanier LL, Phillips JH et al. Physical and functional association of LFA-1 with DNAM-1 adhesion molecule. Immunity, 1999, 11: 615-623
14. Chen L, Xin X, Zhang XH. et al. The expression,regulation and adhesion function of a novel CD molecule,CD226,on human endothelial cells. Life Science, 2003.73:2373-2382
15. Jia W, Xu X, Jin B, et al. CD226 is expressed on the megakaryocytic lineage from hematopoietic stem cells/progenitor cells and involved in its polyploidyization. Eur J Haematol, 2005, 74: 228-240
16. Dunn SM, Jin B, Hillam AJ, et al. Characterization of a novel membrane glycoprotein involved in platelet activation. J Bio Chem, 1989, 264: 13475-13482
17. Zuzel M, Walton S, Burns GF, et al. A monoclonal antibody to a 67kD cell membrane glycoprotein directly induces persistent platelet aggregation independently of granule secretion.Brit J Haematol, 1991, 79: 466-473
18. Shirakawa J, Wang Y, Tahara-Hanaoka S, et al. LFA-1-dependent lipid raft recruitment of DNAM-1 (CD226) in CD4+ T cell. Int Immunol, 2006, 18: 951-957
19. Shirakawa J, Shibuya K, Shibuya A. Requirement of the serine at residue 329 for lipid raft recruitment of DNAM-1 (CD226). Int Immunol, 2005, 17: 217-223
20. Tahara-Hanaoka S, Shibuya K, Onoda Y, et al. Functional characterization of DNAM-1 (CD226) interaction with its ligands PVR (CD 155) and nectin-2 (PRR-2/CD112). Int Immunol, 2004, 16: 533-538.
21．Moretta A C，Bottino M，Vitale D，et al．Activating receptors and co-receptors involved in human natural killer cell-mediated cytolysis．Annu Rev Immunol，2001，19：197-223
22．Marcusson-Stahl M，Cederbrant K．A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies．Toxicology，2003，193：269-279
23. 王晓祺，谭岩，段秀梅，等．流式细胞术检测细胞毒方法的建立．中国免疫学杂 志．2004，20：704-707
24．Munn DH，Sharma MD，Mellor AL．Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2，3-dioxygenase activity in dendritic cells．J Immunol，2004，172：4100-4110
25．Current Opinion in Organ Transplantation．Current world literature．Curr Opin Organ Transplant，2008，13：664-682
26．Rodig N，Ryan T，Allen JA，et al．Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis．Eur J Immunol，2003，33：3117-3126
27．Grabie N，Gotsman I，DaCosta R，et al．Endothelial programmed death-1 ligand 1(PD-L1)regulates CD8+ T-cell mediated injury in the heart．Circulation，2007，116：2062-2071
1．Dieckmann D，Bruett CH，Ploettner H，et al．Human CD4(+)CD25(+)regulatory．contact-dependent T cells induce interleukin 10-producing，contact-independent type 1-like regulatory T cells．J Exp Med，2002，196：247-253．
2．Liu Z，Tugulea S，Cortesini R，et al．Specific suppression of T helper alloreactivity by allo-MHC class I-restricted CD8+CD28 T cells．Int Immunol，1998，10：775-783．
3．Zhang Z，Yang L，Young K，et al．Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression．Nat Med，2000．6：782-789．
4．Seino KI，Fukao K，Muramoto K，et al．Requirement for natural killer T(NKT)cells in the induction of allograft tolerance．Proc Natl Acad Sci，2001，98：2577-2581．
5. Jordan MS, Boesteanu A, Reed AJ, et al. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat Immunol, 2001, 2:301-306.
6. Bensinger SJ, Bandeira A, Jordan MS, et al. Major histocompatibility complex class ######-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells. J Exp Med, 2001, 194:427-438.
7. Jonuleit H, Schmitt E, Kakirman H, et al. Infectious tolerance: human CD25(+) regulatory T cells convey suppressor activity to conventional CD4(+) T helper cells. J Exp Med, 2002, 196:255-260.
8. Annunziato F, Cosmi L, Liotta F, et al. Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+) human thymocytes, J Exp Med, 2002, 196:379-387.
9. Dieckmann D, Plottner H, Berchtold S, et al. Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood, J Exp Med,2001. 193:1303-1310.
10. Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med, 1998, 188:287-296.
11. Malek TR, Bayer AL. Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol, 2004, 4:665-674.
12. Willerford DM, Chen J, Ferry JA, et al. Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity, 1995, 3:521-530.
13. Lin JX, Leonard WJ. Signaling from the IL-2 receptors to the nucleus. Cytokine Growth Factor Rev, 1997, 8:313-332.
14. Suto A, Nakajima H, Ikeda K, et al. CD4(+)CD25(+) T-cell development is regulated by at least 2 distinct mechanisms. Blood, 2002, 99:555-560.
15. Horak I, Lohler J, Ma A, et al. Interleukin-2 deficient mice: a new model to study autoimmunity and self-tolerance. Immunol Rev, 1995, 148:35-44.
16. de la Rosa M, Rutz S, Dorninger H, et al. Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur J Immunol, 2004, 34:2480-2488.
17. Klein L, Khazaie K,von Boehmer H. In vivo dynamics of antigenspecific regulatory T cells not predicted from behavior in vitro. Proc Natl Acad Sci. 2003, 100: 8886-8891.
18. Furtado GC, Curotto de Lafaille MA, Kutchukhidze N, et al. Interleukin 2 signaling is required for CD4(+) regulatory T cell function. J Exp Med, 2002, 196:851-857.
19. Thornton AM, Donovan EE, Piccirillo CA,et al. Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function. J Immunol, 2004, 172:6519-6523.
20. Barthlott T, Moncrieffe H, Veldhoen M, et al. CD25+CD4+ T cells compete with naive CD4+ Tcells for IL-2 and exploit it for the induction of IL-10 production. Int Immunol, 2005. 17:279-288.
21. Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers. Nat Rev Immunol, 2002, 2: 389-400.
22. Brunkow ME, Jeffery EW, Hjerrild K.A, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet, 2001, 27:68-73.
23. Chatila TA, Blaeser F, Ho N, et al. JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunity-allergic disregulation syndrome. J Clin Invest, 2000,106:R75-81.
24. Bennett CL, Christie J, Ramsdell F, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet, 2001, 27:20-21.
25. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science, 2003, 299:1057-1061.
26. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol, 2003, 4:330-336.
27. Yagi H. Nomura T, Nakamura K, et al. Crucial role of FOXP3 in the development and function of human CD25~+CD4~+ regulatory T cells. Int Immunol, 2004, 16:1643-1656.
28. Khattri R, Cox T, Yasayko SA, et al. An essential role for Scurfin in CD4~+CD25~+ T regulatory cells. Nat Immunol, 2003, 4:337-342.
29. Walker MR, Kasprowicz DJ, Gersuk VH, et al. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4~+CD25~- T cells. J Clin Invest, 2003,112: 1437-1443.
30. Salomon B, Lenschow DJ, Rhee L,et al. B7/CD28 costimulation is essential for the homeostasis of the CD4~+CD25~+ immunoregulatory T cells that control autoimmune diabetes. Immunity, 2000,12:431-440.
31. Read S. Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med, 2000, 192:295-302.
32. Takahashi T, Tagami T, Yamazaki S, et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med, 2000, 192:303-310.
33. Manzotti CN, Tipping H, Perry LC,et al. Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25+ regulatory T cells. Eur J Immunol, 2002,32:2888-2896.
34. Fallarino F, Grohmann U, Hwang KW, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol. 2003, 4:1206-1212.
35. Munn DH, Sharma MD, Mellor AL. Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J Immunol, 2004, 172:4100-4110.
36. Paust S, Lu L, McCarty N, et al. Engagement of B7 on effector T cells by regulatory T cells prevents autoimmune disease. Proc Natl Acad Sci, 2004, 101:10398-10403.
37. Tang Q, Boden EK, Henriksen KJ, et al. Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function. Eur J Immunol, 2004, 34:2996-3005.
38. Salomon B, Bluestone JA. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu Rev Immunol, 2001, 19:225-252.
39. Tang Q, Henriksen KJ, Boden EK. et al. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J Immunol, 2003, 171:3348-3352.
40. Setoguchi R, Hori S, Takahashi T,et al. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med, 2005, 201:723-735.
41. Takahashi T, Kuniyasu Y, Toda M, et al. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol, 1998, 10:1969-1980.
42. Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol, 2002, 3: 135-142.
43. McHugh RS, Whitters MJ, Piccirillo CA, et al. CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity, 2002, 16:311-323.
44. Ji HB, Liao G, Faubion WA, Cutting edge: the natural ligand for glucocorticoid-induced TNF receptor-related protein abrogates regulatory T cell suppression. J Immunol, 2004, 172:5823-5827.
45. Ronchetti S, Zollo O, Bruscoli S,et al. GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur J Immunol, 2004,34:613-622.
46. Esparza EM, Arch RH. Signaling triggered by glucocorticoid-induced tumor necrosis factor receptor family-related gene: regulation at the interface between regulatory T cells and immune effector cells. Front Biosci, 2006, 11:1448-1465.
47. Stephens GL, McHugh RS, Whitters MJ, et al. Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J Immunol, 2004, 173:5008-5020.
48. Tone M, Tone Y, Adams E, et al. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc Natl Acad Sci, 2003,100:15059-15064.
49. Iellem A, Mariani M, Lang R, et al. Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells. J Exp Med, 2001. 194:847-853.
50. Iwasaki ARM. Toll-like receptor control of the adaptive immune responses. Nat. Immunol, 2004, 5: 987
51. Caramalho I, Lopes-Carvalho T, Ostler D, et al. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med. 2003,197:403-411.
52. Pasare C, Medzhitov R. Toll Pathway-Dependent Blockade of CD4_CD25_ T Cell-Mediated Suppression by Dendritic Cells. Science, 2003, 299: 1033.
53. Pasare C, Medzhitov R. Toll-Dependent Control Mechanisms of CD4 T Cell Activation. Immunity, 2004, 21: 733.
54. Peng G, Guo Z, Kiniwa Y. Toll-Like Receptor 8-Mediated Reversal of CD4~+Regulatory T Cell Function. Science, 2005, 309:1380-1384.
55. Obhrai J, Goldstein DR. The role of toll-like receptors in solid organ transplantation. Transplantation, 2006, 81:497-502.
56. Kumanogoh A, Wang X, Lee I, et al. Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development. J Immunol, 2001, 166: 353-360.
57. Serra P, Amrani A, Yamanouchi J, et al. CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells. Immunity, 2003, 19:877-889.
58. Grossman WJ, Verbsky JW, Barchet W, et al.Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity, 2004, 21:589-601.
59. Marski M, Kandula S, Turner JR, et al. CD 18 is required for optimal development and function of CD4+CD25+ T regulatory cells. J Immunol, 2005, 175:7889-7897.
60. Bacchetta R, Bigler M, Touraine JL, et al. High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells. J Exp Med, 1994, 179:493-502.
61. Hara M, Kingsley CI, Niimi M, et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol, 2001, 166: 3789-3796.
62. VanBuskirk A, Burlingham W, Jankowska-Gan E, et al. Human allograft acceptance is associated with immune regulation. J Clin Invest, 2000, 106:145.
63. Suri-Payer E, Cantor H. Differential cytokine requirements for regulation of autoimmune gastritis and colitis by CD4(+)CD25(+) T cells. J Autoimmun, 2001,16:115-23.
64. Jiang S, Lechler RI. Regulatory T cells in the control of transplantation tolerance and autoimmunity. Am J Transplant, 2003, 3:516-524.
65. Alpan O, Bachelder E, Isil E, et al. 'Educated' dendritic cells act as messengers from memory to naive T helper cells. Nat Immunol, 2004,5:615-622.
66. Horwitz DA, Zheng SG, Gray JD. The role of the combination of IL-2 and TGF-beta or IL-10 in the generation and function of CD4+ CD25+ and CD8+ regulatory T cell subsets. J Leukoc Biol, 2003, 74: 471-478.
67. Yamagiwa S, Gray JD, Hashimoto S, et al. A role for TGF-beta in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J Immunol,2001, 166:7282-7289.
68. Zheng SG, Gray JD, Ohtsuka K, et al. Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors. J Immunol, 2002, 169:4183-4189.
69. Madisen L, Webb NR, Rose TM et al. Transforming growth factor-beta 2: cDNA cloning and sequence analysis. DNA, 1988, 7:1-8.
70. Marie JC, Letterio JJ, Gavin M,et al. TGF-betal maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. J Exp Med, 2005,201:1061-1067.
71. Coombes JL. Robinson NJ, Maloy KJ, et al. Regulatory T cells and intestinal homeostasis. Immunol Rev, 2005, 204:184-194.
72. Fu S, Zhang N, Yopp AC, et al. TGF-beta induces Foxp3 + T-regulatory cells from CD4 + CD25 - precursors. Am J Transplant, 2004, 4:1614-1627.
73. Groux M, O'Garra A, Bigler M, et al. A CD41 T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature, 1997, 389:737-742.
74. Sonoda KH, Faunce DE, Taniguchi M, et al. NK T cell-derived IL-10 is essential for the differentiation of antigen-specific T regulatory cells in systemic tolerance. J Immunol, 2001,166:42-50.
75. Levings MK. Gregori S, Tresoldi E, et al. Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells. Blood, 2005,105:1162-1169.
76. Bacchetta R. Claudia S, Levings MK, et al. Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines. Eur J Immunol, 2002, 32:2237-2245.
77. Cavani A, Nasorri F, Prezzi C, et al. Human CD4+ T lymphocytes with remarkable regulatory functions on dendritic cells and nickel-specific Th1 immune responses. J Invest Dermatol, 2000,114:295-302.
78. Levings MK, Bacchetta R, Schulz U, et al. The role of IL-10 and TGF-beta in the differentiation and effector function of T regulatory cells. Int Arch Allergy Immunol,2002, 129:263-276.
79. Bluestone JA. Tang Q. How do CD4+CD25+ regulatory T cells control autoimmunity? Curr Opin Immunol, 2005, 17:638-642.
80. Yamazaki S. Iyoda T, Tarbell K, et al. Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells. J Exp Med, 2003,198:235-247.
81. Oldenhove G. de Heusch M, Urbain-Vansanten G. et al. CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo. J Exp Med, 2003, 198:259-266.
82. Cong Y, Konrad A, Iqbal N, et al. Generation of antigen-specific. Foxp3-expressing CD4+ regulatory T cells by inhibition of APC proteosome function. J Immunol. 2005,174:2787-2795.
83. Bilsborough J, George TC, Norment A, et al. Mucosal CD8 alpha+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties. Immunology, 2003, 108:481-492.
84. Hall BM, Jelbart ME, Gurley KE, et al. Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine: Mediation of specific suppression by T helper/inducer cells. J Exp Med, 1985, 162: 1683.
85. Van Maurik A, Herber M, Wood KJ, et al. Cutting edge: CD4~+CD25~+ alloantigen-specific immunoregulatory cells that can prevent CD8~+ T cell-mediated graft rejection: implications for anti- CD154 immunotherapy. J Immunol, 2002, 169:5401-5404.
86. Kingsley CI. Karim M, Bushell AR, et al. CD25~+CD4~+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. J Immunol, 2002, 168:1080-1086.
87. Hara M, Kingsley CI, Niimi M, et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol, 2001, 166: 3789-3796.
88. Sanchez-Fueyo A, Weber M, Domenig C, Strom TB, Zheng XX. Tracking the immunoregulatory mechanisms active during allograft tolerance. J Immunol, 2002, 168:2274-2281.
89. Gregori S, Casorati M, Amuchastegui S, et al. Regulatory T cells induced by 1 alpha.25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance. J Immunol, 2001, 167:1945-1953.
90. Yamada, A. et al. Cutting edge: recipient MHC class ###### expression is required to achieve long-term survival of murine cardiac allografts after costimulatory blockade. J Immunol, 2001, 167:5522-5526.
91. Nishimura E. Sakihama T, Setoguchi R, et al. Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells. Int Immunol, 2004, 16:1189-1201.
92. Waldmann H. Cobbold S. Regulating the immune response to transplants, a role for CD4+ regulatory cells? Immunity, 2001, 14:399-406.
93. Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol, 2003, 3:199-210.
94. Belkaid Y, Piccirillo CA, Mendez S, et al. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature, 2002, 420:502-507.
95. Graca L, Cobbold SP, Waldmann H. Identification ofregulatory T cells in tolerated allografts. J Exp Med, 2002, 195:1641-1646.
96. Ludewig B, Odermatt B, Landmann S, et al. Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue. J Exp Med, 1998, 188:1493-1501.
97. Reed AJ, Noorchashm H, Rostami SY, et al. Alloreactive CD4 T cell activation in vivo: an autonomous function of the indirect pathway of alloantigen presentation. J Immunol,2003,171:6502-6509.
98. Ochando JC, Yopp AC, Yang Y, et al. Lymph node occupancy is required for the peripheral development of alloantigen-specific Foxp3+ regulatory T cells. J Immunol,2005,174:6993-7005.
99. Horvath-Arcidiacono JA, Bloom ET. Characterization of human killer cell reactivity against porcine target cells: differential modulation by cytokines. Xenotransplantation, 2001,8:62-74.
100. Friedman T, Shimizu A, Smith RN. Human CD4+ T cells mediate rejection of porcine xenografts. J Immunol, 1999, 162: 5256-5262.
101. Friedman T, Smith RN, Colvin RB,et al. A critical role for human CD4+ T-cells in rejection of porcine islet cell xenografts. Diabetes, 1999, 48:2340-2348.
102. Trzonkowski P, Szmit E, Mysliwska J, et al. CD4+CD25+ T regulatory cells inhibit cytotoxic activity of T CD8+ and NK lymphocytes in the direct cell-to-cell interaction. Clin Immunol, 2004, 112:258-267.
103. Smyth MJ, Teng MW, Swann J, et al. CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer. J Immunol, 2006, 176:1582-1587.
104. Porter CM, Bloom ET. Human CD4+CD25+ regulatory T cells suppress anti-porcine xenogeneic responses. Am J Transplant, 2005, 5:2052-2057.
105. Cederbom L, Hall H, Ivars F. CD4+CD25+ regulatory T cells downregulate co-stimulatory molecules on antigen-presenting cells. Eur J Immunol, 2000, 30:1538-1543.
106. Yi S, Feng X, Wang Y,et al. CD4+ cells play a major role in xenogeneic human anti-pig cytotoxicity through the Fas/Fas ligand lytic pathway. Transplantation, 1999,67:435-443.
107. Baecher-Allan C, Brown JA, Freeman GJ, et al. CD4+CD25high regulatory cells in human peripheral blood. J Immunol, 2001 ,167:1245-1253.
108. Godfrey WR, Ge YG, Spoden DJ, et al. In vitro expanded human CD4+CD25+ T regulatory cells can markedly inhibit allogeneic dendritic cell stimulated MLR cultures. Blood, 2004, 104:453-461.
109. Bluestone JA, Tang Q. Therapeutic vaccination using CD4+CD25+ antigen-specific regulatory T cells. Proc Natl Acad Sci, 2004, 101 Suppl 2:14622-14626.
2. Sachs DH. The pig as a potential xenograft donor . Vet Immunol Immunopathol, 1994,43:185-1911
3. Shibuya A, Campbell D, Hannum C, et al.DNAM-l,a novel adhesion molecule involved in the cytolytic function of T lymphochtes. Immunity. 1996, 4: 573-581
4. Delia Chiesa M, Romagnani C, Thiel A, et al. Multidirectional interactions are bridging human NK cells with plasmacytoid and monocyte-derived dendritic cells during innate immune responses. Blood, 2006, 108: 3851-3858
5. Pende D, Castriconi R, Romagnani P,et al. Expression of the DNAM-1 ligands, Nectin-2(CD112) and poliovirus receptor(CD155), on dendritic cells:relevance for natural killer-dendritic cell interaction. Blood, 2006, 107: 2030-2036
6. Spaggiari GM,Capobianco A, Becchetti S et al. Mesenchymal stem cell-natural killer cell interactions:evidence that activated NK cells are capable of killing MSCs,whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood, 2006, 107: 1484-1490
7. Bottino C, Castriconi R, Pende D, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule.J Exp Med, 2003, 198:557-67
8. Lopez M, Aoubala M, Jordier F, et al. The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule. Blood, 1998, 92:4602-4611
9. Kozak M. A consideration of alternative models for the initiation of translation in eukaryotes. Crit Rev Biochem Mol Biol, 1992, 27: 385-402
10. Eberle F, Dubreuil P, Mattei MG, et al. The human PRR2 gene, related to the human poliovirus receptor gene(PVR), is the true homolog of the murine MPH gene. Gene,1995. 159:267-272
11. Takahashi K, Nakanishi H, Miyahara M,et al. Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to Cadherin-Based adherens junctions through interaction with afadin,a PDZ domain-containing protein. J Cell Biol, 1999, 145:539-549
12. Irie K. Shimizu K, Sakisaka T, et al. Roles and modes of action of nectins in cell-cell adhesion. Semin Cell Dev Biol, 2004, 15: 643-656
13. Takai Y. Nakanishi H. Nectin and afadin: novel organizers of intercellular junctions. J Cell Sci. 2003, 116:17-27
14. Takai Y, Irie K, Shimizu K, et al. Nectins and nectinlike molecules: roles in cell adhesion, migration, and polarization. Cancer Sci, 2003, 94: 655-667
15．Campadelli-Fiume G，Cocchi F．Menotti L，et al．The novel receptors that mediate the entry of herpes simplex viruses and animal alphaherpesviruses into cells．Rev Med Virol，2000，10：305-319
16．Davoli R，Bigi D，Fontanesi L，et al．Mapping of 14 expressed sequence tags(ESTs)from porcine skeletal muscle by somatic cell hybrid analysis．Animal Genetics，2000，31：400-403
17．Chevalet C，Gouzy J，SanCristobal-Gaudy M．Regional assignment of genetic markers using a somatic cell hybrid panel：a WWW interactive program available for the pig genome．Comput Appl Biosci，1997，13：69-73
18．Hawken RJ，Murtaugh J，Flickinger GH，et al．A first-generation porcine whole-genome radiation hybrid map．Mamm Genome，1999，10：824-830
19．朱正茂．应用cDNA宏阵列技术发现猪某些产肉性状的候选基因．[博士学位论文]．华中农业大学图书馆，2003
20．余梅．猪12号染色体上四个新基因的分离、鉴定和物理定位．[博士学位论文]．华中农业大学图书馆，2002
21．Goureau A，Yerle M，Schmitz A，et al．Human and porcine correspondence of chromosome segments using bidirectional chromosome painting．Genomics，1996，36：252-262
22．Reymond N，Imbert AM，Devilard E，et al．DNAM-1 and PVR Regulate Monocyte Migration through Endothelial Junctions．J Exp Med，2004，199：1331-1341
23．Miller DA，Miller OJ，Dev VG，et al．Human chromosome 19 carries a poliovirus receptor gene．Cell，1974，1：167-173
24．邹文雄，黄明经．蛋白质结构之电脑预测．Chemistry，1997，55：101-109
25．Mart(?)-Renom M A，Stuart A C，Fiser A，et al．Comparative protein structure modeling of genes and genomes．Annu Rev Biophys Biomol Struct，2000，29：291-325
26．He Y，Bowman VD，Mueller S，et al．Interaction of the poliovirus receptor with poliovirus．Proc Natl Acad Sci，2000，97：79-84
27．Du Pasquier L，Zucchetti I，De Santis R．Immunoglobulin superfamily receptors in protochordates：before RAG time．Immunol Rev，2004，198：233-248
28．Fabre S，Reymond N，Cocchi F，et al．Prominent role of the Ig-like V domain in trans-interactions of nectins．Nectin3 and nectin 4 bind to the predicted C-C′-C″-D beta-strands of the nectinl V domain．J Biol Chem，2002，277：27006-27013
1．Yasumi M，Shimizu K，Honda T，et al．Role of each immunoglobulin-like loop of nectin for its cell-cell adhesion activity．Biochem Biophys Res Commun，2003，302：61-66
2．Momose Y，Honda T，Inagaki M，et al．Role of the second immunoglobulin-like loop of nectin in cell-cell adhesion．Biochem Biophys Res Commun，2002，293：45-49
3．John FM，Paul B．Cleavage of Simian Virus 40 DNA at a Unique Site by a Bacterial Restriction Enzyme．Proc Natl Acad Sci，1972，69：3365-3369
4．冯作化．医学分子生物学．人民卫生出版社．2001年9月第1版，139
5．Lin SJ，Wang JY，Klickstein LB，et al．Lack of age-associated LFA-1 up-regulation and impaired ICAM-1 binding in lymphocytes from patients with Down syndrome．Clin Exp Immunol，2001，126：54-63
6．Shreffler WG，Castro RR，Kucuk ZY，et al．The major glycoprotein allergen from Arachis hypogaea．Ara h 1，is a ligand of dendritic cell-specific ICAM-grabbing nonintegrin and acts as a Th2 adjuvant in vitro．J Immunol，2006，177：3677-3685
7．Smyth N，Odenthal U，Merkl B，et al．Eukaryotic expression and purification of recombinant extracellular matrix proteins carrying the strep Ⅱ tag．Methods Mol Biol，2009，522：63-72
8．Taatjes DJ，Chen TH，Ackerstr(o|¨)m B，et al．Streptococcal protein G-gold complex：comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling．Eur J Cell Biol，1987，45：151-159
1．Shibuya A，Campbell D，Hannum C，et al．DNAM-1，a novel adhesion molecule involved in the cytolytic function of T lymphocytes．Immunity，1996，4：573．
2．Tahara-Hanaoka S，Shibuya K，Kai H，et al．Tumor rejection by the poliovirus receptor family ligands of the DNAM-1(CD226)receptor．Blood，2006，107：1491-1496
3．Della Chiesa M，Romagnani C，Thiel A，et al．Multidirectional interactions are bridging human NK cells with plasmacytoid and monocyte-derived dendritic cells during innate immune responses．Blood，2006，108：3851-3858
4．Pende D，Castriconi R，Romagnani P，et al．Expression of the DNAM-1 ligands，Nectin-2(CD112)and poliovirus receptor(CD155)，on dendritic cells：relevance for natural killer-dendritic cell interaction．Blood，2006，107：2030-2036
5．Spaggiari GM，Capobianco A，Becchetti S et al．Mesenchymal stem cell-natural killer cell interactions：evidence that activated NK cells are capable of killing MSCs，whereas MSCs can inhibit IL-2-induced NK-cell proliferation．Blood，2006，107：1484-1490
6. Bottino C. Castriconi R, Pende D, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule.J Exp Med. 2003, 198: 557-567
7. Pende D. Bottino C, Castriconi R, et al. PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in tumor cell lysis. Mol Immunol, 2005, 42: 463-469
8. Lopez M. Aoubala M, Jordier F, et al. The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule. Blood, 1998. 92: 4602-4611
9. Shibuya A, Campbell D. Hannum C, et al. DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes. Immunity, 1996, 4: 573-581
10. Irie K, Shimizu K, Sakisaka T, et al. Roles and modes of action of nectins in cell-cell adhesion. Semin Cell Dev Biol, 2004, 15: 643-656
11. Shibuya A D, Campbell C, Hannum H, et al. DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes. Immunity, 1996, 4: 573-581
12. Ralston KJ, Hird SL, Zhang X, et al.The LFA-1-associated molecule PTA-1(CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large. J Biol Chem, 2004, 279: 33816-33828
13. Shibuya K, Lanier LL, Phillips JH et al. Physical and functional association of LFA-1 with DNAM-1 adhesion molecule. Immunity, 1999, 11: 615-623
14. Chen L, Xin X, Zhang XH. et al. The expression,regulation and adhesion function of a novel CD molecule,CD226,on human endothelial cells. Life Science, 2003.73:2373-2382
15. Jia W, Xu X, Jin B, et al. CD226 is expressed on the megakaryocytic lineage from hematopoietic stem cells/progenitor cells and involved in its polyploidyization. Eur J Haematol, 2005, 74: 228-240
16. Dunn SM, Jin B, Hillam AJ, et al. Characterization of a novel membrane glycoprotein involved in platelet activation. J Bio Chem, 1989, 264: 13475-13482
17. Zuzel M, Walton S, Burns GF, et al. A monoclonal antibody to a 67kD cell membrane glycoprotein directly induces persistent platelet aggregation independently of granule secretion.Brit J Haematol, 1991, 79: 466-473
18. Shirakawa J, Wang Y, Tahara-Hanaoka S, et al. LFA-1-dependent lipid raft recruitment of DNAM-1 (CD226) in CD4+ T cell. Int Immunol, 2006, 18: 951-957
19. Shirakawa J, Shibuya K, Shibuya A. Requirement of the serine at residue 329 for lipid raft recruitment of DNAM-1 (CD226). Int Immunol, 2005, 17: 217-223
20. Tahara-Hanaoka S, Shibuya K, Onoda Y, et al. Functional characterization of DNAM-1 (CD226) interaction with its ligands PVR (CD 155) and nectin-2 (PRR-2/CD112). Int Immunol, 2004, 16: 533-538.
21．Moretta A C，Bottino M，Vitale D，et al．Activating receptors and co-receptors involved in human natural killer cell-mediated cytolysis．Annu Rev Immunol，2001，19：197-223
22．Marcusson-Stahl M，Cederbrant K．A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies．Toxicology，2003，193：269-279
23. 王晓祺，谭岩，段秀梅，等．流式细胞术检测细胞毒方法的建立．中国免疫学杂 志．2004，20：704-707
24．Munn DH，Sharma MD，Mellor AL．Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2，3-dioxygenase activity in dendritic cells．J Immunol，2004，172：4100-4110
25．Current Opinion in Organ Transplantation．Current world literature．Curr Opin Organ Transplant，2008，13：664-682
26．Rodig N，Ryan T，Allen JA，et al．Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis．Eur J Immunol，2003，33：3117-3126
27．Grabie N，Gotsman I，DaCosta R，et al．Endothelial programmed death-1 ligand 1(PD-L1)regulates CD8+ T-cell mediated injury in the heart．Circulation，2007，116：2062-2071
1．Dieckmann D，Bruett CH，Ploettner H，et al．Human CD4(+)CD25(+)regulatory．contact-dependent T cells induce interleukin 10-producing，contact-independent type 1-like regulatory T cells．J Exp Med，2002，196：247-253．
2．Liu Z，Tugulea S，Cortesini R，et al．Specific suppression of T helper alloreactivity by allo-MHC class I-restricted CD8+CD28 T cells．Int Immunol，1998，10：775-783．
3．Zhang Z，Yang L，Young K，et al．Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression．Nat Med，2000．6：782-789．
4．Seino KI，Fukao K，Muramoto K，et al．Requirement for natural killer T(NKT)cells in the induction of allograft tolerance．Proc Natl Acad Sci，2001，98：2577-2581．
5. Jordan MS, Boesteanu A, Reed AJ, et al. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat Immunol, 2001, 2:301-306.
6. Bensinger SJ, Bandeira A, Jordan MS, et al. Major histocompatibility complex class ######-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells. J Exp Med, 2001, 194:427-438.
7. Jonuleit H, Schmitt E, Kakirman H, et al. Infectious tolerance: human CD25(+) regulatory T cells convey suppressor activity to conventional CD4(+) T helper cells. J Exp Med, 2002, 196:255-260.
8. Annunziato F, Cosmi L, Liotta F, et al. Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+) human thymocytes, J Exp Med, 2002, 196:379-387.
9. Dieckmann D, Plottner H, Berchtold S, et al. Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood, J Exp Med,2001. 193:1303-1310.
10. Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med, 1998, 188:287-296.
11. Malek TR, Bayer AL. Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol, 2004, 4:665-674.
12. Willerford DM, Chen J, Ferry JA, et al. Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity, 1995, 3:521-530.
13. Lin JX, Leonard WJ. Signaling from the IL-2 receptors to the nucleus. Cytokine Growth Factor Rev, 1997, 8:313-332.
14. Suto A, Nakajima H, Ikeda K, et al. CD4(+)CD25(+) T-cell development is regulated by at least 2 distinct mechanisms. Blood, 2002, 99:555-560.
15. Horak I, Lohler J, Ma A, et al. Interleukin-2 deficient mice: a new model to study autoimmunity and self-tolerance. Immunol Rev, 1995, 148:35-44.
16. de la Rosa M, Rutz S, Dorninger H, et al. Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur J Immunol, 2004, 34:2480-2488.
17. Klein L, Khazaie K,von Boehmer H. In vivo dynamics of antigenspecific regulatory T cells not predicted from behavior in vitro. Proc Natl Acad Sci. 2003, 100: 8886-8891.
18. Furtado GC, Curotto de Lafaille MA, Kutchukhidze N, et al. Interleukin 2 signaling is required for CD4(+) regulatory T cell function. J Exp Med, 2002, 196:851-857.
19. Thornton AM, Donovan EE, Piccirillo CA,et al. Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function. J Immunol, 2004, 172:6519-6523.
20. Barthlott T, Moncrieffe H, Veldhoen M, et al. CD25+CD4+ T cells compete with naive CD4+ Tcells for IL-2 and exploit it for the induction of IL-10 production. Int Immunol, 2005. 17:279-288.
21. Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers. Nat Rev Immunol, 2002, 2: 389-400.
22. Brunkow ME, Jeffery EW, Hjerrild K.A, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet, 2001, 27:68-73.
23. Chatila TA, Blaeser F, Ho N, et al. JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunity-allergic disregulation syndrome. J Clin Invest, 2000,106:R75-81.
24. Bennett CL, Christie J, Ramsdell F, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet, 2001, 27:20-21.
25. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science, 2003, 299:1057-1061.
26. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol, 2003, 4:330-336.
27. Yagi H. Nomura T, Nakamura K, et al. Crucial role of FOXP3 in the development and function of human CD25~+CD4~+ regulatory T cells. Int Immunol, 2004, 16:1643-1656.
28. Khattri R, Cox T, Yasayko SA, et al. An essential role for Scurfin in CD4~+CD25~+ T regulatory cells. Nat Immunol, 2003, 4:337-342.
29. Walker MR, Kasprowicz DJ, Gersuk VH, et al. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4~+CD25~- T cells. J Clin Invest, 2003,112: 1437-1443.
30. Salomon B, Lenschow DJ, Rhee L,et al. B7/CD28 costimulation is essential for the homeostasis of the CD4~+CD25~+ immunoregulatory T cells that control autoimmune diabetes. Immunity, 2000,12:431-440.
31. Read S. Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med, 2000, 192:295-302.
32. Takahashi T, Tagami T, Yamazaki S, et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med, 2000, 192:303-310.
33. Manzotti CN, Tipping H, Perry LC,et al. Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25+ regulatory T cells. Eur J Immunol, 2002,32:2888-2896.
34. Fallarino F, Grohmann U, Hwang KW, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol. 2003, 4:1206-1212.
35. Munn DH, Sharma MD, Mellor AL. Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J Immunol, 2004, 172:4100-4110.
36. Paust S, Lu L, McCarty N, et al. Engagement of B7 on effector T cells by regulatory T cells prevents autoimmune disease. Proc Natl Acad Sci, 2004, 101:10398-10403.
37. Tang Q, Boden EK, Henriksen KJ, et al. Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function. Eur J Immunol, 2004, 34:2996-3005.
38. Salomon B, Bluestone JA. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu Rev Immunol, 2001, 19:225-252.
39. Tang Q, Henriksen KJ, Boden EK. et al. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J Immunol, 2003, 171:3348-3352.
40. Setoguchi R, Hori S, Takahashi T,et al. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med, 2005, 201:723-735.
41. Takahashi T, Kuniyasu Y, Toda M, et al. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol, 1998, 10:1969-1980.
42. Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol, 2002, 3: 135-142.
43. McHugh RS, Whitters MJ, Piccirillo CA, et al. CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity, 2002, 16:311-323.
44. Ji HB, Liao G, Faubion WA, Cutting edge: the natural ligand for glucocorticoid-induced TNF receptor-related protein abrogates regulatory T cell suppression. J Immunol, 2004, 172:5823-5827.
45. Ronchetti S, Zollo O, Bruscoli S,et al. GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur J Immunol, 2004,34:613-622.
46. Esparza EM, Arch RH. Signaling triggered by glucocorticoid-induced tumor necrosis factor receptor family-related gene: regulation at the interface between regulatory T cells and immune effector cells. Front Biosci, 2006, 11:1448-1465.
47. Stephens GL, McHugh RS, Whitters MJ, et al. Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J Immunol, 2004, 173:5008-5020.
48. Tone M, Tone Y, Adams E, et al. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc Natl Acad Sci, 2003,100:15059-15064.
49. Iellem A, Mariani M, Lang R, et al. Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells. J Exp Med, 2001. 194:847-853.
50. Iwasaki ARM. Toll-like receptor control of the adaptive immune responses. Nat. Immunol, 2004, 5: 987
51. Caramalho I, Lopes-Carvalho T, Ostler D, et al. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med. 2003,197:403-411.
52. Pasare C, Medzhitov R. Toll Pathway-Dependent Blockade of CD4_CD25_ T Cell-Mediated Suppression by Dendritic Cells. Science, 2003, 299: 1033.
53. Pasare C, Medzhitov R. Toll-Dependent Control Mechanisms of CD4 T Cell Activation. Immunity, 2004, 21: 733.
54. Peng G, Guo Z, Kiniwa Y. Toll-Like Receptor 8-Mediated Reversal of CD4~+Regulatory T Cell Function. Science, 2005, 309:1380-1384.
55. Obhrai J, Goldstein DR. The role of toll-like receptors in solid organ transplantation. Transplantation, 2006, 81:497-502.
56. Kumanogoh A, Wang X, Lee I, et al. Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development. J Immunol, 2001, 166: 353-360.
57. Serra P, Amrani A, Yamanouchi J, et al. CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells. Immunity, 2003, 19:877-889.
58. Grossman WJ, Verbsky JW, Barchet W, et al.Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity, 2004, 21:589-601.
59. Marski M, Kandula S, Turner JR, et al. CD 18 is required for optimal development and function of CD4+CD25+ T regulatory cells. J Immunol, 2005, 175:7889-7897.
60. Bacchetta R, Bigler M, Touraine JL, et al. High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells. J Exp Med, 1994, 179:493-502.
61. Hara M, Kingsley CI, Niimi M, et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol, 2001, 166: 3789-3796.
62. VanBuskirk A, Burlingham W, Jankowska-Gan E, et al. Human allograft acceptance is associated with immune regulation. J Clin Invest, 2000, 106:145.
63. Suri-Payer E, Cantor H. Differential cytokine requirements for regulation of autoimmune gastritis and colitis by CD4(+)CD25(+) T cells. J Autoimmun, 2001,16:115-23.
64. Jiang S, Lechler RI. Regulatory T cells in the control of transplantation tolerance and autoimmunity. Am J Transplant, 2003, 3:516-524.
65. Alpan O, Bachelder E, Isil E, et al. 'Educated' dendritic cells act as messengers from memory to naive T helper cells. Nat Immunol, 2004,5:615-622.
66. Horwitz DA, Zheng SG, Gray JD. The role of the combination of IL-2 and TGF-beta or IL-10 in the generation and function of CD4+ CD25+ and CD8+ regulatory T cell subsets. J Leukoc Biol, 2003, 74: 471-478.
67. Yamagiwa S, Gray JD, Hashimoto S, et al. A role for TGF-beta in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J Immunol,2001, 166:7282-7289.
68. Zheng SG, Gray JD, Ohtsuka K, et al. Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors. J Immunol, 2002, 169:4183-4189.
69. Madisen L, Webb NR, Rose TM et al. Transforming growth factor-beta 2: cDNA cloning and sequence analysis. DNA, 1988, 7:1-8.
70. Marie JC, Letterio JJ, Gavin M,et al. TGF-betal maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. J Exp Med, 2005,201:1061-1067.
71. Coombes JL. Robinson NJ, Maloy KJ, et al. Regulatory T cells and intestinal homeostasis. Immunol Rev, 2005, 204:184-194.
72. Fu S, Zhang N, Yopp AC, et al. TGF-beta induces Foxp3 + T-regulatory cells from CD4 + CD25 - precursors. Am J Transplant, 2004, 4:1614-1627.
73. Groux M, O'Garra A, Bigler M, et al. A CD41 T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature, 1997, 389:737-742.
74. Sonoda KH, Faunce DE, Taniguchi M, et al. NK T cell-derived IL-10 is essential for the differentiation of antigen-specific T regulatory cells in systemic tolerance. J Immunol, 2001,166:42-50.
75. Levings MK. Gregori S, Tresoldi E, et al. Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells. Blood, 2005,105:1162-1169.
76. Bacchetta R. Claudia S, Levings MK, et al. Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines. Eur J Immunol, 2002, 32:2237-2245.
77. Cavani A, Nasorri F, Prezzi C, et al. Human CD4+ T lymphocytes with remarkable regulatory functions on dendritic cells and nickel-specific Th1 immune responses. J Invest Dermatol, 2000,114:295-302.
78. Levings MK, Bacchetta R, Schulz U, et al. The role of IL-10 and TGF-beta in the differentiation and effector function of T regulatory cells. Int Arch Allergy Immunol,2002, 129:263-276.
79. Bluestone JA. Tang Q. How do CD4+CD25+ regulatory T cells control autoimmunity? Curr Opin Immunol, 2005, 17:638-642.
80. Yamazaki S. Iyoda T, Tarbell K, et al. Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells. J Exp Med, 2003,198:235-247.
81. Oldenhove G. de Heusch M, Urbain-Vansanten G. et al. CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo. J Exp Med, 2003, 198:259-266.
82. Cong Y, Konrad A, Iqbal N, et al. Generation of antigen-specific. Foxp3-expressing CD4+ regulatory T cells by inhibition of APC proteosome function. J Immunol. 2005,174:2787-2795.
83. Bilsborough J, George TC, Norment A, et al. Mucosal CD8 alpha+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties. Immunology, 2003, 108:481-492.
84. Hall BM, Jelbart ME, Gurley KE, et al. Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine: Mediation of specific suppression by T helper/inducer cells. J Exp Med, 1985, 162: 1683.
85. Van Maurik A, Herber M, Wood KJ, et al. Cutting edge: CD4~+CD25~+ alloantigen-specific immunoregulatory cells that can prevent CD8~+ T cell-mediated graft rejection: implications for anti- CD154 immunotherapy. J Immunol, 2002, 169:5401-5404.
86. Kingsley CI. Karim M, Bushell AR, et al. CD25~+CD4~+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. J Immunol, 2002, 168:1080-1086.
87. Hara M, Kingsley CI, Niimi M, et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol, 2001, 166: 3789-3796.
88. Sanchez-Fueyo A, Weber M, Domenig C, Strom TB, Zheng XX. Tracking the immunoregulatory mechanisms active during allograft tolerance. J Immunol, 2002, 168:2274-2281.
89. Gregori S, Casorati M, Amuchastegui S, et al. Regulatory T cells induced by 1 alpha.25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance. J Immunol, 2001, 167:1945-1953.
90. Yamada, A. et al. Cutting edge: recipient MHC class ###### expression is required to achieve long-term survival of murine cardiac allografts after costimulatory blockade. J Immunol, 2001, 167:5522-5526.
91. Nishimura E. Sakihama T, Setoguchi R, et al. Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells. Int Immunol, 2004, 16:1189-1201.
92. Waldmann H. Cobbold S. Regulating the immune response to transplants, a role for CD4+ regulatory cells? Immunity, 2001, 14:399-406.
93. Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol, 2003, 3:199-210.
94. Belkaid Y, Piccirillo CA, Mendez S, et al. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature, 2002, 420:502-507.
95. Graca L, Cobbold SP, Waldmann H. Identification ofregulatory T cells in tolerated allografts. J Exp Med, 2002, 195:1641-1646.
96. Ludewig B, Odermatt B, Landmann S, et al. Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue. J Exp Med, 1998, 188:1493-1501.
97. Reed AJ, Noorchashm H, Rostami SY, et al. Alloreactive CD4 T cell activation in vivo: an autonomous function of the indirect pathway of alloantigen presentation. J Immunol,2003,171:6502-6509.
98. Ochando JC, Yopp AC, Yang Y, et al. Lymph node occupancy is required for the peripheral development of alloantigen-specific Foxp3+ regulatory T cells. J Immunol,2005,174:6993-7005.
99. Horvath-Arcidiacono JA, Bloom ET. Characterization of human killer cell reactivity against porcine target cells: differential modulation by cytokines. Xenotransplantation, 2001,8:62-74.
100. Friedman T, Shimizu A, Smith RN. Human CD4+ T cells mediate rejection of porcine xenografts. J Immunol, 1999, 162: 5256-5262.
101. Friedman T, Smith RN, Colvin RB,et al. A critical role for human CD4+ T-cells in rejection of porcine islet cell xenografts. Diabetes, 1999, 48:2340-2348.
102. Trzonkowski P, Szmit E, Mysliwska J, et al. CD4+CD25+ T regulatory cells inhibit cytotoxic activity of T CD8+ and NK lymphocytes in the direct cell-to-cell interaction. Clin Immunol, 2004, 112:258-267.
103. Smyth MJ, Teng MW, Swann J, et al. CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer. J Immunol, 2006, 176:1582-1587.
104. Porter CM, Bloom ET. Human CD4+CD25+ regulatory T cells suppress anti-porcine xenogeneic responses. Am J Transplant, 2005, 5:2052-2057.
105. Cederbom L, Hall H, Ivars F. CD4+CD25+ regulatory T cells downregulate co-stimulatory molecules on antigen-presenting cells. Eur J Immunol, 2000, 30:1538-1543.
106. Yi S, Feng X, Wang Y,et al. CD4+ cells play a major role in xenogeneic human anti-pig cytotoxicity through the Fas/Fas ligand lytic pathway. Transplantation, 1999,67:435-443.
107. Baecher-Allan C, Brown JA, Freeman GJ, et al. CD4+CD25high regulatory cells in human peripheral blood. J Immunol, 2001 ,167:1245-1253.
108. Godfrey WR, Ge YG, Spoden DJ, et al. In vitro expanded human CD4+CD25+ T regulatory cells can markedly inhibit allogeneic dendritic cell stimulated MLR cultures. Blood, 2004, 104:453-461.
109. Bluestone JA, Tang Q. Therapeutic vaccination using CD4+CD25+ antigen-specific regulatory T cells. Proc Natl Acad Sci, 2004, 101 Suppl 2:14622-14626.