1．HLA-A2/HPV6E7四聚体的构建和应用 2．FPH致病基因定位于19p13.1-19pter
|
摘要
目的: HPV6对皮肤粘膜的感染往往造成局部组织增生而形成CA,CA的转归与患者的细胞免疫功能密切相关。为了探讨CA患者的T细胞免疫状态,本研究拟:①构建一种能够与HPV6E7抗原特异性T细胞结合的HLA-A2/HPV6E7四聚体;②应用该四聚体来检测不同病程CA患者的抗原特异性T细胞的频率。
方法:①通过原核表达、大量提取HLA-A2-BSP、β2m两种蛋白,通过体外稀释复性、与人工合成的HPV6E7抗原肽(GLHCYEQLV)折叠形成相应的HLA-A2/HPV6E7单体,经过Western-blot和ELISA证实其构象正确。在BirA酶的作用下,将生物素结合到HLA重链的BSP位点,实现HLA-A2/HPV6E7单体的特异位点生物素化。通过生物素与亲合素4:1的特异性结合关系,将4个生物素化的HLA-A2/ HPV6E7单体与PE标记的亲合素结合形成HLA四聚体;②以制备的PE标记的HLA-A2/HPV6E7四聚体和FITC-CD8单克隆抗体对HLA-A2阳性、HPV6感染致CA患者的PBL和疣体组织进行双色荧光染色,经流式细胞仪和荧光显微镜观察。③通过T2细胞(HLA-A2阳性,TAP缺陷)负载HPV6E7抗原肽,作为刺激细胞,与CA患者的PBL进行共培养,获得了特异性CTL,经过LDH法进行了不同病程CA患者CTL的杀伤活性检测。
结果:①成功构建了有生物学功能的HLA-A2/HPV6E7四聚体;②不同病程CA患者PBL中抗原特异性CTL(即CD8+和HLA-A2/HPV6E7四聚体染色阳性细胞)的检测结果显示:缓解期CA患者双阳性的细胞比例是0.11%,反复发作期CA患者双阳性的细胞比例是0.10%,而正常对照是0.02%。③特异性CTL的体外扩增及杀伤活性检测:经过长期混合淋巴细胞培养后,缓解期CA患者CTL的频数明显增高(0.23% vs 1.19%,P<0.05),反复发作期CA患者CTL的频数增高也很明显(0.23% vs 1.16%,P<0.05),利用LDH方法检测抗原特异性CTL的杀伤活性。缓解期CTL杀伤率为(64.5±2.5)%,复发期为(51.5±1.5)%,不同病程CA的CTL的杀伤率比较有显著差异(P<0.05)。④不同病程CA的疣体组织冰冻切片后四聚体原位染色:选取HLA-A2阳性、HPV6感染导致的不同病程CA患者,其疣体组织经手术切除后,进行40μm冰冻切片,经FITC-CD8单抗及PE-HPV6E7四聚体共染后,荧光显微镜观察发现:缓解期CA患者疣体组织中有一群阳性细胞,此即HPV6特异性的CTL;而反复发作期CA患者疣体组织中未能观察到抗原特异性T细胞浸润。
结论:①本研究成功制备了加载HLA-A2限制性HPV6E7抗原肽的四聚体,并用此四聚体对HPV6感染、HLA-A2阳性的患者的PBL进行了HLA-A2/HPV6E7四聚体染色,缓解期CA与反复发作的CA患者的特异性T细胞数量相差不大,即反复发作的CA不存在全身特异性T细胞数量绝对减少。②不同病程CA患者的CTL的杀伤率比较有显著差异(P<0.05)。缓解期CA的CD8+T细胞杀伤活性处于高水平激活状态,从而能够有效地清除HPV,导致疾病的缓解。而反复发作的CA患者的PBL,抗原特异性T细胞虽然数量没有减少,但是杀伤活性不强,即机体不能建立有效的免疫反应,可能是导致CA反复发作的原因。③荧光显微镜观察,缓解期的患者疣体中能观察到CD8+和HLA-A2/HPV6E7四聚体的双阳性细胞浸润,而反复发作的患者疣体中未见此群细胞。提示这些抗原特异性T细胞在疣体中的浸润并发挥抗病毒作用,才导致CA的缓解。而反复发作的CA虽然外周血液中抗原特异性T细胞数量并没有减少,但是这些CTL的杀伤活性较低,并且在疣体局部几乎没有观察到抗原特异性T细胞的浸润。分析原因可能是局部细胞因子、粘附分子分泌障碍,不能有效地将血液中的T细胞趋化到疣体局部组织,所以局部存在免疫缺陷,难以建立有效的细胞免疫反应,造成HPV清除障碍。
目的:家族性进行性色素过度沉着症(familial progressive hyperpigmentation, FPH)是一种单基因、常染色体显性遗传病,其致病基因尚未在染色体上定位。本研究的目的是:①在一个三代共17人的FPH家系的样本中,通过检测STR进行FPH基因定位;②为了永久性地保存该家系的具有特定生物学性状的标本,采用EBV转化外周血B淋巴细胞,获得该FPH家系成员的永生化的B淋巴母样细胞系(B lymphoblastoid cell line,B-LCL);③对该致病基因初步定位的染色体区段进行分析,其中存在着与色素代谢相关的基因丝氨酸/苏氨酸蛋白激酶11(Serine/Threonine protein Kinase 11,STK11),为此进行了FPH家系中病人的STK11基因的克隆和序列鉴定。
方法:①提取该家系成员的外周血基因组DNA,在每条常染色体上选取8个STR位点进行基因筛查,对基因型与疾病表型间进行连锁分析,筛选可疑染色体;在筛出的染色体相应位置上增加6个STR位点以进行单倍型分析;②采用EB病毒(Epstein-Barr virus,EBV)感染,加入环孢霉素A抑制T淋巴细胞的方法,对该家系17人外周血B淋巴细胞进行了转化;③采用分子克隆技术,构建FPH患者的pcDNA3.0-STK11的克隆载体,测序并与NCBI网上(BC007981)进行了序列比对。
结果:①经单倍型分析证实FPH的致病基因定位于19p13.1-19pter。Marshfield遗传图距为45.48cM,UCSC物理图距为17.17Mb,其间有628个基因;
②成功建立该家系成员永生化B-LCL15个;③构建了该FPH家系中患者的STK11克隆载体。经NCBI网上比对,测序结果与正常序列(BC007981)完全一致,而未发现突变。
结论:①将FPH的致病基因定位于19p13.1-19pter,该致病基因与初步定位区段内的色素代谢相关的基因STK11可能无关。在19p13.1-19pter之间可能存在新的致病基因,导致FPH的发生;②成功建立的该家系的永生化B-LCL为后续精细定位和克隆FPH致病基因、进行皮肤色素代谢障碍的生理生化分子机制研究提供了长期的研究材料。
Objectives: MHC tetramer is a powerful tool for detecting the antigen specific T cells with exquisitely antigen-specific and highly sensitive. Since HLA-A*0201 is the most common allele of human MHC I molecules; Human Papillomavirus 6 (HPV6) is the major cause of Condyloma Acuminata (CA), we choose HLA-A*0201 and its restricted peptide HPV6E7 (22-30) to construct sHLA-A2/HPV6E7 tetramer. With the tetramer, we detected the antigen specific T cells in the different courses of CA.
Methods: The two subunits of the human leucocyte antigen (HLA-A2) have been expressed at high levels as insoluble aggregates in bacterial cells. They were induced by IPTG to overexpress the target proteins. In the presence of an HLA-A2 restricted antigenic peptide (HPV6E7), refolding was initiated by dilution of the denaturant in the solution of heavy chain andβ2m. After concentrate and buffer exchange of refolding products, the BirA enzyme was used to biotinylate the refolded complex. Biotinylation was performing at the COOH-terminus of the heavy chain named BirA substrate peptide (BSP). The refolded and biotinylated products were detected by ELISA and Western blot with mAbW6/32 and HRP-streptavidin. sHLA-A2/HPV6E7 tetramer was prepared by mixing the biotinylated peptide-HLA complex with phycoerythrin (PE) labeled avidin at a molar ratio of 4:1.
Peripheral blood was obtained from HLA-A2 positive healthy individual and different course of CA. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll/Paque density gradient centrifugation and then placed in sterile tissue culture dishes for separation of adherent from nonadherent cells by 2h incubation at 37℃. The nonadherent cells (PBLs) were plated into tube. With the PE-tetramer and FITC-CD8 mAb, a group of double positive cells can be observed by FACS.
Here we use the T2 (HLA-A2+) cell pulsed with HPV6E7 as stimulating cells to stimulate the HLA-A2+ CA with HPV6 infected PBLs to generate the peptide-specific CTLs with the long term mixed lymphocytes culture. After culture, the specific CTLs will proliferate, and are used to monitor our sHLA-A2/ HPV6E7 tetramer. To detect the function of these specific CTLs, T2 cells loading peptide act as target cells, we detect their killing activation by LDH assay.
In situ tetramer (IST) staining has opened the door to exploration of antigen specific T cells in tissues. The development of MHC tetramer staining has revolutionized our ability to study antigen specific T cells. This technique permits visualization of antigen-specific T cells in tissue sections along with their spatial relationship to their microenvironment. The quantities and qualities of CTLs infiltrating in tissues reflect the status of cell immunity. All have an effect on prognosis of CA. Condyloma tissues sections were incubated with the tetramers, washed with PBS and frozen in Tissue-Tek OCT compound. To monitor tetramer binding to CD8-positive antigen specific CTL in the cryopreserved tissue, 40μm cryosections were prepared, fixed in acetone for 10 min at RT and pre-incubated for 30 min in 5% (v /v) normal cow serum. Then they were incubated with the relevant mAb CD8+ and analyzed by fluorescence microscope.
Results: After initial purification, the aggregates were dissolved in 8M urea. The yields are 450mg and 350mg for theβ2m and heavy chain from 6 liter saturant baterial cells, respectively. The purity of theβ2m and heavy chain is 65.3% and 69.8%, respectively. The western blot result shows monoclonal antibody (mAb) W6/32, which recognizes a determinant present on the heavy chain only when the heavy chain is associated withβ2m, binds to the refolded component. Sandwich ELISA with mAb W6/32 and rabbit anti-β2m antibody demonstrates the success of refolding. The sHLA-A2/ HPV6E7 tetramer were constructed successfully.
The frequency of double positive cells in PBL of healthy dividual is 0.02%; but it is 0.11% in paramedic CA and is 0.10% in recurrence CA. We apply our prepared sHLA-A2/ HPV6E7 tetramer to stain the cells after long term mixed lymphocytes culture. The results shows our tetramer is able to discriminate the frequencies of specific CTL induced by HLA-A2/HPV6E7. The frequency of specific CTLs in paramedic CA is very high (0.23% vs 1.19%,P<0.05). The frequencies of specific CTLs in recurrence CA is obvious high too (0.23% vs 1.16%,P<0.05). The CTLs killing frequency in paramedic CA is (64.5±2.5) %. But the CTLs killing frequency in recurrence CA is (51.5±1.5) %. The difference is obvious in the CTLs killing frequency different course CA. Additionally, antigen specific T cells infiltrating in tissues in paramedic CA can be observed, but not observed in recurrence CA.
Conclusions: In this study the HLA-A2 / HPV6E7tetramer were successfully constructed. The tetramer can be applied to directly visualize antigen-specific CTLs efficiently and become the critical approach in assessment of T cell immune responses in CA patients.
Objectives: In this study, we undertook a linkage analysis with microsatellite polymorphic markers?short tandem repeat (STR) in a three-generation Familial Progressive Hyperpigmentation (FPH, MIM 145250) family with 17 members, including 6 affected individuals residing in Hubei Province. The immortal B Lymphocyte Cell Lines (B-LCLs) of the family were established by EBV transformation and addition of cyclosporin A (CysA) to inhibit the activity of T cells. Additional, the plasmid pcDNA3.0-STK11 was constructed from 3 affected individuals in this FPH family.
Methods: DNA was isolated from peripheral blood cells from all 17 individuals. Genome screening was performed using 176 STR microsatellite markers from 22 autosomes, 8 for each chromosome, these STR markers were selected for their polymorphism in Chinese population and their physic locations which were able to represent a given chromosome segment. Additional 6 STR markers were chosen from the short arm of 19th chromosome to define the borders of the cosegregating region. These markers were amplified by polymerase chain reaction (PCR) based on the protocol reported previous. PCR products were analyzed by vertical electrophoresis polyacrylamide gels (PAGE) and silver staining. According to the guideline recommended by the DNA Commission of the International Society for Forensic Haemogenetics (ISFH), every allele of STR locus was named by the repeat number of its repetitive sequence. After purification, PCR products of 2 alleles from every STR locus were directly sequenced for their repetitive numbers on an Applied Biosystems 377 automatic sequencer. PCR products were synelectrophoresed with molecular weight standard and then every allele was named by its repeat number. As FPH was demonstrated to be an autosomal dominantly inherited disease, principles for judging linkage between its genotype and phenotype are as follows: (1) If an allele or haplotype is present in all affected individuals and not present in all unaffected individuals, it is suggested to be linked to the pathogenic gene; (2) If an allele or haplotype is present in some, but not all, affected individuals and not present in all unaffected individuals, it is possible that this allele or haplotype links to the pathogenic gene; (3) If an allele or haplotype is present randomly in affected and unaffected individuals, it is excluded to be linked to the pathogenic gene.
The total cell RNA is extracted from the Peripheral blood mononuclear cells; and the cDNA encoding the STK11 was acquired by RT-PCR technique and then inserted into the plasmid pcDNA3.0.
Results: the FPH locus is observed to link to the region covering D19S714 and D19S591 which are markers for 19p13.1-19pter. The genetic distance of region in Marshfield is 45.48 cM and physical distance in UCSC is 17.17Mb, harboring 628 defined genes. 15 immortal cell lines were established. The results showed that cellular volume of these immortal cells was enlarged and their morphology was diversity with glitch. There was increasing development of cell aggregates of proliferative lymphocyte cells. The recombinant plasmid pcDNA3.0-STK11 was identified to be correct by restriction endoniclease digestion and sequence analysis.
Conclusions: the FPH locus is linked to chromosome 19p13.1-19pter. Interestingly, this region harbors STK11 gene, germline mutations in which were shown to be associated with Peutz-Jeghers Syndrome (PJS). PJS and FPH share the disorder of hyperpigmentation. But construcated STK11 sequences were completely coincidence with Genebank. So, the STK11 was not the virulence gene for FPH. There is a new gene not found for FPH in 19p13.1-19pter. For further fine mapping, it is obvious that more familial or/and sporadic FPH cases are required for the further fine mapping. The fine mapping of FPH gene is expected to lead to a better understanding of the etiology for both FPH and PJS.
The immortal B-LCLs acquired successfully provide a long and plentiful DNA material for mapping and cloning causative gene for FPH and studying punctually the molecular mechanism of pigment metabolism in the skin.
方法:①通过原核表达、大量提取HLA-A2-BSP、β2m两种蛋白,通过体外稀释复性、与人工合成的HPV6E7抗原肽(GLHCYEQLV)折叠形成相应的HLA-A2/HPV6E7单体,经过Western-blot和ELISA证实其构象正确。在BirA酶的作用下,将生物素结合到HLA重链的BSP位点,实现HLA-A2/HPV6E7单体的特异位点生物素化。通过生物素与亲合素4:1的特异性结合关系,将4个生物素化的HLA-A2/ HPV6E7单体与PE标记的亲合素结合形成HLA四聚体;②以制备的PE标记的HLA-A2/HPV6E7四聚体和FITC-CD8单克隆抗体对HLA-A2阳性、HPV6感染致CA患者的PBL和疣体组织进行双色荧光染色,经流式细胞仪和荧光显微镜观察。③通过T2细胞(HLA-A2阳性,TAP缺陷)负载HPV6E7抗原肽,作为刺激细胞,与CA患者的PBL进行共培养,获得了特异性CTL,经过LDH法进行了不同病程CA患者CTL的杀伤活性检测。
结果:①成功构建了有生物学功能的HLA-A2/HPV6E7四聚体;②不同病程CA患者PBL中抗原特异性CTL(即CD8+和HLA-A2/HPV6E7四聚体染色阳性细胞)的检测结果显示:缓解期CA患者双阳性的细胞比例是0.11%,反复发作期CA患者双阳性的细胞比例是0.10%,而正常对照是0.02%。③特异性CTL的体外扩增及杀伤活性检测:经过长期混合淋巴细胞培养后,缓解期CA患者CTL的频数明显增高(0.23% vs 1.19%,P<0.05),反复发作期CA患者CTL的频数增高也很明显(0.23% vs 1.16%,P<0.05),利用LDH方法检测抗原特异性CTL的杀伤活性。缓解期CTL杀伤率为(64.5±2.5)%,复发期为(51.5±1.5)%,不同病程CA的CTL的杀伤率比较有显著差异(P<0.05)。④不同病程CA的疣体组织冰冻切片后四聚体原位染色:选取HLA-A2阳性、HPV6感染导致的不同病程CA患者,其疣体组织经手术切除后,进行40μm冰冻切片,经FITC-CD8单抗及PE-HPV6E7四聚体共染后,荧光显微镜观察发现:缓解期CA患者疣体组织中有一群阳性细胞,此即HPV6特异性的CTL;而反复发作期CA患者疣体组织中未能观察到抗原特异性T细胞浸润。
结论:①本研究成功制备了加载HLA-A2限制性HPV6E7抗原肽的四聚体,并用此四聚体对HPV6感染、HLA-A2阳性的患者的PBL进行了HLA-A2/HPV6E7四聚体染色,缓解期CA与反复发作的CA患者的特异性T细胞数量相差不大,即反复发作的CA不存在全身特异性T细胞数量绝对减少。②不同病程CA患者的CTL的杀伤率比较有显著差异(P<0.05)。缓解期CA的CD8+T细胞杀伤活性处于高水平激活状态,从而能够有效地清除HPV,导致疾病的缓解。而反复发作的CA患者的PBL,抗原特异性T细胞虽然数量没有减少,但是杀伤活性不强,即机体不能建立有效的免疫反应,可能是导致CA反复发作的原因。③荧光显微镜观察,缓解期的患者疣体中能观察到CD8+和HLA-A2/HPV6E7四聚体的双阳性细胞浸润,而反复发作的患者疣体中未见此群细胞。提示这些抗原特异性T细胞在疣体中的浸润并发挥抗病毒作用,才导致CA的缓解。而反复发作的CA虽然外周血液中抗原特异性T细胞数量并没有减少,但是这些CTL的杀伤活性较低,并且在疣体局部几乎没有观察到抗原特异性T细胞的浸润。分析原因可能是局部细胞因子、粘附分子分泌障碍,不能有效地将血液中的T细胞趋化到疣体局部组织,所以局部存在免疫缺陷,难以建立有效的细胞免疫反应,造成HPV清除障碍。
目的:家族性进行性色素过度沉着症(familial progressive hyperpigmentation, FPH)是一种单基因、常染色体显性遗传病,其致病基因尚未在染色体上定位。本研究的目的是:①在一个三代共17人的FPH家系的样本中,通过检测STR进行FPH基因定位;②为了永久性地保存该家系的具有特定生物学性状的标本,采用EBV转化外周血B淋巴细胞,获得该FPH家系成员的永生化的B淋巴母样细胞系(B lymphoblastoid cell line,B-LCL);③对该致病基因初步定位的染色体区段进行分析,其中存在着与色素代谢相关的基因丝氨酸/苏氨酸蛋白激酶11(Serine/Threonine protein Kinase 11,STK11),为此进行了FPH家系中病人的STK11基因的克隆和序列鉴定。
方法:①提取该家系成员的外周血基因组DNA,在每条常染色体上选取8个STR位点进行基因筛查,对基因型与疾病表型间进行连锁分析,筛选可疑染色体;在筛出的染色体相应位置上增加6个STR位点以进行单倍型分析;②采用EB病毒(Epstein-Barr virus,EBV)感染,加入环孢霉素A抑制T淋巴细胞的方法,对该家系17人外周血B淋巴细胞进行了转化;③采用分子克隆技术,构建FPH患者的pcDNA3.0-STK11的克隆载体,测序并与NCBI网上(BC007981)进行了序列比对。
结果:①经单倍型分析证实FPH的致病基因定位于19p13.1-19pter。Marshfield遗传图距为45.48cM,UCSC物理图距为17.17Mb,其间有628个基因;
②成功建立该家系成员永生化B-LCL15个;③构建了该FPH家系中患者的STK11克隆载体。经NCBI网上比对,测序结果与正常序列(BC007981)完全一致,而未发现突变。
结论:①将FPH的致病基因定位于19p13.1-19pter,该致病基因与初步定位区段内的色素代谢相关的基因STK11可能无关。在19p13.1-19pter之间可能存在新的致病基因,导致FPH的发生;②成功建立的该家系的永生化B-LCL为后续精细定位和克隆FPH致病基因、进行皮肤色素代谢障碍的生理生化分子机制研究提供了长期的研究材料。
Objectives: MHC tetramer is a powerful tool for detecting the antigen specific T cells with exquisitely antigen-specific and highly sensitive. Since HLA-A*0201 is the most common allele of human MHC I molecules; Human Papillomavirus 6 (HPV6) is the major cause of Condyloma Acuminata (CA), we choose HLA-A*0201 and its restricted peptide HPV6E7 (22-30) to construct sHLA-A2/HPV6E7 tetramer. With the tetramer, we detected the antigen specific T cells in the different courses of CA.
Methods: The two subunits of the human leucocyte antigen (HLA-A2) have been expressed at high levels as insoluble aggregates in bacterial cells. They were induced by IPTG to overexpress the target proteins. In the presence of an HLA-A2 restricted antigenic peptide (HPV6E7), refolding was initiated by dilution of the denaturant in the solution of heavy chain andβ2m. After concentrate and buffer exchange of refolding products, the BirA enzyme was used to biotinylate the refolded complex. Biotinylation was performing at the COOH-terminus of the heavy chain named BirA substrate peptide (BSP). The refolded and biotinylated products were detected by ELISA and Western blot with mAbW6/32 and HRP-streptavidin. sHLA-A2/HPV6E7 tetramer was prepared by mixing the biotinylated peptide-HLA complex with phycoerythrin (PE) labeled avidin at a molar ratio of 4:1.
Peripheral blood was obtained from HLA-A2 positive healthy individual and different course of CA. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll/Paque density gradient centrifugation and then placed in sterile tissue culture dishes for separation of adherent from nonadherent cells by 2h incubation at 37℃. The nonadherent cells (PBLs) were plated into tube. With the PE-tetramer and FITC-CD8 mAb, a group of double positive cells can be observed by FACS.
Here we use the T2 (HLA-A2+) cell pulsed with HPV6E7 as stimulating cells to stimulate the HLA-A2+ CA with HPV6 infected PBLs to generate the peptide-specific CTLs with the long term mixed lymphocytes culture. After culture, the specific CTLs will proliferate, and are used to monitor our sHLA-A2/ HPV6E7 tetramer. To detect the function of these specific CTLs, T2 cells loading peptide act as target cells, we detect their killing activation by LDH assay.
In situ tetramer (IST) staining has opened the door to exploration of antigen specific T cells in tissues. The development of MHC tetramer staining has revolutionized our ability to study antigen specific T cells. This technique permits visualization of antigen-specific T cells in tissue sections along with their spatial relationship to their microenvironment. The quantities and qualities of CTLs infiltrating in tissues reflect the status of cell immunity. All have an effect on prognosis of CA. Condyloma tissues sections were incubated with the tetramers, washed with PBS and frozen in Tissue-Tek OCT compound. To monitor tetramer binding to CD8-positive antigen specific CTL in the cryopreserved tissue, 40μm cryosections were prepared, fixed in acetone for 10 min at RT and pre-incubated for 30 min in 5% (v /v) normal cow serum. Then they were incubated with the relevant mAb CD8+ and analyzed by fluorescence microscope.
Results: After initial purification, the aggregates were dissolved in 8M urea. The yields are 450mg and 350mg for theβ2m and heavy chain from 6 liter saturant baterial cells, respectively. The purity of theβ2m and heavy chain is 65.3% and 69.8%, respectively. The western blot result shows monoclonal antibody (mAb) W6/32, which recognizes a determinant present on the heavy chain only when the heavy chain is associated withβ2m, binds to the refolded component. Sandwich ELISA with mAb W6/32 and rabbit anti-β2m antibody demonstrates the success of refolding. The sHLA-A2/ HPV6E7 tetramer were constructed successfully.
The frequency of double positive cells in PBL of healthy dividual is 0.02%; but it is 0.11% in paramedic CA and is 0.10% in recurrence CA. We apply our prepared sHLA-A2/ HPV6E7 tetramer to stain the cells after long term mixed lymphocytes culture. The results shows our tetramer is able to discriminate the frequencies of specific CTL induced by HLA-A2/HPV6E7. The frequency of specific CTLs in paramedic CA is very high (0.23% vs 1.19%,P<0.05). The frequencies of specific CTLs in recurrence CA is obvious high too (0.23% vs 1.16%,P<0.05). The CTLs killing frequency in paramedic CA is (64.5±2.5) %. But the CTLs killing frequency in recurrence CA is (51.5±1.5) %. The difference is obvious in the CTLs killing frequency different course CA. Additionally, antigen specific T cells infiltrating in tissues in paramedic CA can be observed, but not observed in recurrence CA.
Conclusions: In this study the HLA-A2 / HPV6E7tetramer were successfully constructed. The tetramer can be applied to directly visualize antigen-specific CTLs efficiently and become the critical approach in assessment of T cell immune responses in CA patients.
Objectives: In this study, we undertook a linkage analysis with microsatellite polymorphic markers?short tandem repeat (STR) in a three-generation Familial Progressive Hyperpigmentation (FPH, MIM 145250) family with 17 members, including 6 affected individuals residing in Hubei Province. The immortal B Lymphocyte Cell Lines (B-LCLs) of the family were established by EBV transformation and addition of cyclosporin A (CysA) to inhibit the activity of T cells. Additional, the plasmid pcDNA3.0-STK11 was constructed from 3 affected individuals in this FPH family.
Methods: DNA was isolated from peripheral blood cells from all 17 individuals. Genome screening was performed using 176 STR microsatellite markers from 22 autosomes, 8 for each chromosome, these STR markers were selected for their polymorphism in Chinese population and their physic locations which were able to represent a given chromosome segment. Additional 6 STR markers were chosen from the short arm of 19th chromosome to define the borders of the cosegregating region. These markers were amplified by polymerase chain reaction (PCR) based on the protocol reported previous. PCR products were analyzed by vertical electrophoresis polyacrylamide gels (PAGE) and silver staining. According to the guideline recommended by the DNA Commission of the International Society for Forensic Haemogenetics (ISFH), every allele of STR locus was named by the repeat number of its repetitive sequence. After purification, PCR products of 2 alleles from every STR locus were directly sequenced for their repetitive numbers on an Applied Biosystems 377 automatic sequencer. PCR products were synelectrophoresed with molecular weight standard and then every allele was named by its repeat number. As FPH was demonstrated to be an autosomal dominantly inherited disease, principles for judging linkage between its genotype and phenotype are as follows: (1) If an allele or haplotype is present in all affected individuals and not present in all unaffected individuals, it is suggested to be linked to the pathogenic gene; (2) If an allele or haplotype is present in some, but not all, affected individuals and not present in all unaffected individuals, it is possible that this allele or haplotype links to the pathogenic gene; (3) If an allele or haplotype is present randomly in affected and unaffected individuals, it is excluded to be linked to the pathogenic gene.
The total cell RNA is extracted from the Peripheral blood mononuclear cells; and the cDNA encoding the STK11 was acquired by RT-PCR technique and then inserted into the plasmid pcDNA3.0.
Results: the FPH locus is observed to link to the region covering D19S714 and D19S591 which are markers for 19p13.1-19pter. The genetic distance of region in Marshfield is 45.48 cM and physical distance in UCSC is 17.17Mb, harboring 628 defined genes. 15 immortal cell lines were established. The results showed that cellular volume of these immortal cells was enlarged and their morphology was diversity with glitch. There was increasing development of cell aggregates of proliferative lymphocyte cells. The recombinant plasmid pcDNA3.0-STK11 was identified to be correct by restriction endoniclease digestion and sequence analysis.
Conclusions: the FPH locus is linked to chromosome 19p13.1-19pter. Interestingly, this region harbors STK11 gene, germline mutations in which were shown to be associated with Peutz-Jeghers Syndrome (PJS). PJS and FPH share the disorder of hyperpigmentation. But construcated STK11 sequences were completely coincidence with Genebank. So, the STK11 was not the virulence gene for FPH. There is a new gene not found for FPH in 19p13.1-19pter. For further fine mapping, it is obvious that more familial or/and sporadic FPH cases are required for the further fine mapping. The fine mapping of FPH gene is expected to lead to a better understanding of the etiology for both FPH and PJS.
The immortal B-LCLs acquired successfully provide a long and plentiful DNA material for mapping and cloning causative gene for FPH and studying punctually the molecular mechanism of pigment metabolism in the skin.
引文
1.洪少林,王家璧,刘跃华.北京地区尖锐湿疣患者皮损中人乳头瘤病毒的检测和分型[J].中国医学科学院学报,2002, 24(4): 397-400.
2. Garboczi DN, Hung DT, Wiley DC, et al. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides [J]. Proc Natl Acad Sci USA, 1992, 89(8): 3429-3433.
3. Altman J, PAH Moss P, Goulder D, et al. Phenotypic analysis of antigen-specific T lymphocytes [J]. Science, 1996, 274(5284): 94-96.
4. McMichael AJ, Callaghan CA. A new look at T cells [J]. J Exp Med, 1998, 187(9):1367-1371.
5. Boniface JJ, Rabinowitz JD, Wulfing C, et al. Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [J]. Immunity, 1998, 9:459-466.
6. Fernandez-Vina MA, Falco M, Sun Y, et al. DNA typing for HLA class I alleles: I. Subsets of HLA-A2 and of -A28 [J]. Hum Immunol, 1992, 33(3):163-73.
7. Tarpey I, Stacey S, Hickling J, et al. Human cytotoxic T lymphocytes stimulated by endogenously processed human papillomavirus type 11 E7 recognize a peptide containing a HLA-A2(A*0201)motif [J]. Immunology, 1994, 81(2):222-227.
8. Parker, KC, Bednarek MA, and Coligan JE. 1994. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. Available at: http://www-bimas.cit.nih.gov/molbio/hla_bind/. Accessed 12 February 2005.
9.陈浩,翁秀芳,吴雄文,等.人β2m基因的克隆及表达[J].华中科技大学学报(医学版),2004,33(1): 1-4.
10. Maziarz RT, Fraser J, Srominger JL, et al. The human HLA-specific monoclonal antibody W6/32 recognizes a discontinuous epitope within the alpha 2 domain of murine H-2Db [J]. Immunogenetics, 1986, 24(3):206 -208.
11. Tran TM, Ivanyi P, Hilgert I, et al. The epitope recognized by pan-HLA class I-reactive monoclonal antibody W6/32 and its relationship to unusual stability of theHLA-B27/beta2-microglobulin complex [J]. Immunogenetics, 2001, 53(6): 440-446.
12. Ladasky JJ, Shum BP, Canavez F, et al. Residue 3 of beta2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody [J]. Immunogenetics, 1999, 49(4):312-320.
13.沈关心,周汝麟主编.现代免疫学实验技术(第二版).湖北科学技术出版社.1998.
14.吴雄文,梁智辉.实用免疫学实验技术(第一版).湖北武汉:湖北科学技术出版社, 2002.
15. Taswell C. Limiting dilution assays for determination of immunocompetent cell frequencies [J]. J Immunol, 1981, 126:1614-1619
16. Czerkinsky C, andersson G, Ekre HP, et al. Reverse ELISPOT assay for clonal analysis of cytokine production [J]. J Immunol Methods, 1988, 110:29-36
17. Maino VC, Picker LJ. Identification of function subsets by flow cytometry: intracellular detection of cytokine expression [J]. Cytometry, 1998, 34:207-215
18. Michelle S, Longworth, Laimonis A. Laimins Pathogenesis of Human Papillomaviruses in Differentiating Epithelia [J]. Microbiology and molecular biology reviews, 2004, 68(2). 362–372
19. Parker KC, Bednarek MA, Hull LK, et al. Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2 [J]. J Immunol, 1992, 149:3580-3587.
20.周玉英,胡牧,刘翠苓.鼻息肉病中人类乳突瘤病毒检测[J]。临床耳鼻咽喉科杂志,2001, 15(5): 212-13.
21. Hosken NA, Bevan MJ. Defective presentation of endogenous antigen by a cell line expressing class I molecules [J]. Science, 1990, 248:367-370
22. Rener M, Claesson MH, Bregenholt S, et al. An improved method for detection of peptide-induced upregulation of HLA-A2 molecules on TAP-deficient T2 cells [J]. Exp Clin Immunogenet, 1996, 13:30-35.
23.房崇芸,吴雄文,韩军艳等.长期混合淋巴细胞培养-细胞毒实验模型的建立[J].中华器官移植杂志,2001,22(5):277-279.
24. Skinner PJ, Daniels MA, Schmidt CS, et al. Cutting edge: in situ tetramer stainingof antigen-specific T cells in tissues [J]. J Immunol, 2000, 165(2), 613-7.
25. Vyth-Dreese FA, Kim YH, Dellemijn TA, et al. In situ visualization of antigen-specific T cells in cryopreserved human tissues [J]. J Immunol Methods, 2006, 310(1-2), 78–85.
26. Kobayashi A, Miaskowski C, Wallhagen M, et al. Recent development in understanding the immune response to human papillomavirus infection and cervical neoplasia [J]. Oncol Nurs Forum, 2000, 27 (4):643 - 651.
27. Evans M, Borysiewicz L K, Evans AS, et al. Antigen p rocessing defects in cervical carcinomas limit t he p resentation of a CTL epitope from human papillomavi rus16 E6 [J]. J Immunol, 2001, 167 (9) :5420 - 5428.
28. Trizna Z, Evans T, Bruce S, et al. A randomized p haseⅡstudy comparing four diff erent interf eron t herapies in patients with recalcit rant condylomata acuminata [J]. Sex Transm Dis, 1998, 25 (7) :361 - 365.
29. Pious D, Krangel MS, Dixon LL, et al. HLA Antigen Structural Gene Mutants Selected with an Allospecific Monoclonal Antibody [J]. Proc Natl Acad Sci, 1982, 79(24): 7832-36
30. Greer, CE, Wheeler CM, Ladner MB, et al. Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts [J]. J Clin Microbiol, 1995, 33(8): 2058–2063.
31. Liu F, Wang JB, Liu YH, et al. Comparison between immunofluorescence and PCR in detecting human papilloma virus in condyloma acuminate [J]. Chin Med Sci J, 2006, 21(2):125-7.
32. Coleman N, Birley HD, Renton AM, et al. Immunological events in regressing genital warts [J]. Am J Clin Patho, 1994, 102(6):768-74.
33. Selvakumar R, Borenstein LA, Lin YL, et al. Immunization with nonstructural proteins E1 and E2 of cottontail rabbit papillomavirus stimulates regression of virus-induced papillomas [J]. J Virol, 1995, 69(1):602-5.
34. Selvakumar R, Schmitt A, Iftner T, et al. Regression of papillomas induced by cottontail rabbit papillomavirus is associated with infiltration of CD8+ cells and persistence of viral DNA after regression [J]. J Virol, 1997, 71(7): 5540-8.
35. Dorothee G, Vergnon I, El Hage F, et al. In situ sensory adaptation of tumor-infiltrating T lymphocytes to peptide-MHC levels elicits strong antitumor reactivity [J]. J Immunol, 2005, 174(11): 6888-97.
36. Tully G, Kortsik C, Hohn H, et al. Highly focused T cell responses in latent human pulmonary Mycobacterium tuberculosis infection [J]. J Immunol, 2005, 174(4):2174-84.
37. Masopust D, Vezys V, Wherry EJ, et al. Cutting edge: gut microenvironment promotes differentiation of a unique memory CD8 T cell population [J]. J Immunol, 2006, 176(4):2079-83.
38. Chernosky ME, Anderson DE, Chang JP, et al. Familial progressive hyperpigmentation [J]. Arch Derm, 1971, 103: 581-591.
39. Rebora A, Parodi A. Universal inherited melanodyschromatosis: a case of melanosis universalis hereditaria [J]? Arch Derm, 1989, 125: 1442-1443.
40. Debao L, Ting L.Familial progressive hyperpigmentation: a family study in China [J]. Brit J Derm 1991; 125: 607.
41.罗挺,凌淑清,刘若英,等.进行性色素过度沉着症一个汉族家系的研究.中华医学遗传学杂志[J],1994;11(3):174-5.
42. The Cooperative Human Linkage Center. CHLC Marker Maps. Available at: http://lpgws.nci.nih.gov/html-chlc/Chlc Marker Maps. Html. Accessed 12 October 2004.
43. Taiwan Polymorphic Marker Database.TPMD Map Display. Available at: http://tpmd.nhri.org.tw/php-bin/graphic.php. Accessed 12 October 2004.
44. National Center for Biotechnology Information. NCBI Map Viewer. Available at: http://www.ncbi.nlm.nih.gov/mapview/maps.cgi Accessed 15 July 2005.
45. Walsh PS, Fildes NJ, Reynolds R. Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA [J]. Nucleic Acids Res, 1996, 24:2807- 2812.
46. Bar W, Brinkmann B, Budowle B, et al. DNA recommendations. Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. International Society for Forensic Haemogenetics [J]. Int J Legal Med 1997; 110:175-176.
47. DNA commission of the international society for forensic haemogenetics report concerning further recommendations of the DNA Commission of the ISFH regarding PCR-based polymorphisms in STR [J]. Int J Legal Med 1994; 107:159.
48. Neitzel H. A routine method of the establishment of permanent growing lymphoblastoid cell lines [J]. Hum Genet, 1986, 73(4):320-326.
49. Schlee M, Krug T, Gires O, et al. Identification of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) target proteins by proteome analysis: activation of EBNA2 in conditionally immortalized B cells reflects early events after infection of primary B cells by EBV [J]. J Virol, 2004, 78(8):3941-3952.
50. Aman P, Ehlin-Henriksson B, Klein G. Epstein-Barr virus susceptibility of normal human B lymphocyte populations [J]. J Exp Med, 1984, 159:208-220.
51. Brack C, Mattaj IW, Gautschi J, et al. Cyclosporin A is a differential inhibitor of eukaryotic RNA polymerases [J]. Exp Cell Res, 1984, 151:314-321.
52. Litt M, Luty JA. A hypervariable microsatellite revaled by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene [J]. Am J Hum Genet, 1989, 44(3):397-401.
53. Edwards A, Civitello A, Hammond Ha, et al. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats [J]. Am J Hum Genet, 1991, 49:746-756.
54. Hochmeister MN. Molecular aspects of medicine-DNA technology in forensic application. Printed in Great Britain by Nuffield Press Ltd. 1995,400.
55. Ricci U, Klintschar M, Neuhuber F, et al. Study on the STR TPOX in an Italian and an Austrian population using two different prime pairs and three different electrophoretic methods [J]. Int J Leg Med. 1998, 111:212-214
56. Zhang Caie, Deng Yunhua, Chen Xingping, et al. Linkage of a locus determining Familial Progressive Hyperpigmentation (FPH) to chromosome 19p13.1-pter in a Chinese family [J]. Eur J Dermatol, 2006; 16(3):1-6.
57. Jenne DE, Reimann H, Nezu J, et al. Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase [J]. Nat Genet, 1998; 18:38-43.
58. Qiu W, Schonleben F, Thaker HM, et al. A novel mutation of STK11/LKB1 geneleads to the loss of cell growth inhibition in head and neck squamous cell carcinoma.Oncogene. 2006 ;25(20):2937-42.
59. Launonen V. Mutations in the human LKB1/STK11 gene [J]. Hum Mutat, 2005, 26: 291-297.
60. Marignani PA. LKB1, the multitasking tumour suppressor kinase [J]. J Clin Pathol, 2005, 58:15-19.
61.金冬雁,黎梦枫译.分子克隆实验指南. (第二版).北京:科学技术出版社.1992.
62. Hemminki A, Tomlinson I , Markie D , et al . Locatigation of a susceptibility locus for Peutg-Jeghers Syndrome to 19p using comparative genomic hybridigation and targeted linkage analysis [J]. Nat Genet, 1997, 15 :526.
63. Hemminki A, Markie D, Tomlinson I, et al. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome [J]. Nature, 1998; 391(6663):184-187.
1 Altman J D ,Moss P A H , Goulter P J R , et al. Phenotypic analysis of antigen specific T lymphocytes [J] . Science ,1996 ;274 :94-96.
2 Appay V, Nixon DF, Donahoe SM, et al. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function [J]. J Exp Med, 2000; 192(1), 63-75.
3 Dunbar PR, Ogg GS, Chen J, et al. Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood [J]. Curr Biol, 1998; 8(7), 413-16.
4万瑛,吴玉章.特异性细胞毒性T细胞活化的研究进展[J].第三军医大学学报,2000;22(10):992-997.
5赵建平,吴玉章. CTL表位预测的方法学进展[J].第三军医大学学报,2000;22(10):988-992.
6 Xiao-Ning Xu , Gavin R , Screaton et al . MHC/peptide tetramer-based studies of T cell function [J]. J of Immunological Methods ,2002 ;268 (1) :21-28.
7 Doherty P C. The numbers game for virus-specific CD8 + T cell [J]. Science ,1998 ;280(5361) :227-31.
8 Grakoui A, Bromley SK, Sumen C, et al.The immunological synapse: a molecular machine controlling T cell activation [J]. Science, 1999; 285(5425), 221-27.
9 Boniface JJ, Rabinowitz JD, Wulfing C, et al. Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [J]. Immunity, 1998;9, 459.
10 Xu XN, Purbhoo MA, Chen N, et al . A novel approach to antigen specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex [J]. Immunity, 2001; 14,591.
11 Tough DF, Borrow P, Sprent J. Induction of bystander T cell proliferation by viruses and type I interferon in vivo [J]. Science, 1996; 272(5270), 1947-50.
12 Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution [J]. J Immunol Methods, 2000; 243(1-2), 147-54.
13 Doherty PC, Christensen JP. Accessing complexity: the dynamics of virus-specific T cell responses [J]. Annu Rev Immunol , 2000; 18, 561-92.
14 Sakai JA, Nagai M, Brennan MB, et al. In vitro spontaneous lymphoproliferation in patients with human T-cell lymphotropic virus type I-associated neurologic disease: predominant expansion of CD8(+) T cells [J]. Blood, 2001; 98(5), 1506-11.
15 Kaech SM, Ahmed R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells [J]. Nat Immunol , 2001; 2(5), 381-2.
16 Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis [J]. Semin Hematol , 2001;38,179.
17 Gerdes J, Lemke H, Baisch H, et al. Cell cycle analysis of a cellproliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67 [J]. J Immunol, 1984; 133(4), 1710-5.
18 Oxenius A, Gunthard HF, Hirschel B, et al. Direct ex vivo analysis reveals distinct phenotypic patterns of HIV-specific CD8(+) T lymphocyte activation in response to therapeutic manipulation of virus load [J]. Eur J Immunol, 2001; 31(4), 1115-21.
19 Purbhoo MA, Boulter JM, Price DA, et al . The human CD8 coreceptor effects cytotoxic T cells activation and antigen sensitivity primary by mediating complete phosphorylation of the T cell receptor zeta chain [J] . J Biol Chem, 2001 ;276 :32786.
20 Takahashi M, Osono E, Nakagawa Y, et al . Rapid induction of apoptosis in CD8 ( + ) HIV-1 envelope-specific murine CTLs by short exposure to antigenic peptide [J] . J Immunol , 2002 ;169 (11) :6588-93.
21 Globerson A, Effros RB. Ageing of lymphocytes and lymphocytes in the aged [J]. Immunol Today, 2000; 21(10), 515-21.
22 Effros RB, Pawelec G. Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion [J]? Immunol Today, 1997; 18(9), 450-4.
23 Rufer N, Dragowska W, Thornbury G, et al. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry [J]. Nat Biotechnol, 1998; 16(8), 743-7.
24 Rufer N, Brummendorf TH, Kolvraa S, et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood [J]. J Exp Med, 1999; 190, 157.
25 Plunkett FJ, Soares MV, Annels N, et al. The flow cytometric analysis of telomere length in antigen-specific CD8+ T cells during acute Epstein–Barr virus infection [J]. Blood, 2001; 97(3), 700-7.
26 Trapani JA, Sutton VR, Smyth MJ. CTL granules: evolution of vesicles essential for combating virus infections [J]. Immunol Today , 1999; 20, 351.
27 Liu CC, Walsh CM, Young JD. Perforin: structure and function [J]. Immunol Today, 1995; 16(4), 194-201.
28 Smyth MJ, Trapani JA. Granzymes: exogenous proteinases that induce target cellapoptosis [J]. Immunol Today, 1995; 16(4), 202-6.
29 Chen G, Shankar P, Lange C, et al. CD8 T cells specific for human immunodeficiency virus, Epstein– Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection [J]. Blood, 2001; 98(1), 156-64.
30 Gamadia LE, Rentenaar RJ, Remmerswaal EB, et al. CMV-specific CD8 (pos) T lymphocyte differentiation in latent CMV infection [J]. Transplant Proc, 2001; 33(1-2), 1802-3.
31 Bossi G, Griffiths GM. Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells [J]. Nat Med, 1999; 5(1), 90-96.
32 Guidotti LG, Chisari FV. To kill or to cure: options in host defense against viral infection [J]. Curr Opin Immunol, 1996; 8(4), 478-83.
33 Pala P, Hussell T, Openshaw PJ. Flow cytometric measurement of intracellular cytokines [J]. J Immunol Methods, 2000; 243(1-2), 107-24.
34 Gea-Banacloche JC, Migueles SA, Martino L, et al. Maintenance of large numbers of virusspecific CD8+ T cells in HIV-infected progressors and long-term nonprogressors [J]. J Immunol, 2000(2); 165, 1082-92.
35 Kostense S, Ogg GS, Manting EH, et al. High viral burden in the presence of major HIV-specific CD8(+) T cell expansions: evidence for impaired CTL effector function [J]. Eur J Immunol, 2001; 31(3), 677-86.
36 Sandberg JK, Fast NM, Nixon DF. Functional heterogeneity of cytokines and cytolytic effector molecules in human cd8(+) t lymphocytes [J]. J Immunol, 2001; 167(1), 181-7.
37 Skinner PJ, Daniels MA, Schmidt CS, et al. Cutting edge: in situ tetramer staining of antigen-specific T cells in tissues [J]. J Immunol, 2000; 165(2), 613-7.
38 Vyth-Dreese FA, Kim YH, Dellemijn TA,et al. In situ visualization of antigen-specific T cells in cryopreserved human tissues [J]. J Immunol Methods 2006; 310(1-2), 78–85.
39 Zhang D, Shankar P , Xu Z, et al. Most antiviral CD8 T cells during chronic viral infection do not express high levels of perforin and are not directly cytotoxic[J] .Blood, 2003 ; 101 (1) :226-235.
40 Geroge W, Bertoletti .Quantity and quality of virus2specific CD8 cell response :relevance to the design of a therapeutic vaccine for chronic HBV Infection [J] .Mol Immunol , 2001 ; 38(6) :467-473.
41 Welsh R M. Assessing CD8T cell number and dysfunction in the presence of antigen [J] . J Exp Med , 2001 ; 193(5) :F19-22.
42 Hirano N, Butler MO, Xia Z, et al. Efficient presentation of naturally processed HLA class I peptides by artificial antigen-presenting cells for the generation of effective antitumor responses [J]. Clin Cancer Res, 2006; 12(10): 2967-75.
43 Wang M, Stepkowski SM, Wang ME, et al. Induction of specific allograft immunity by soluble class I MHC heavy chain protein produced in a baculovirus expression system [J]. Transplantation, 1996; 61(3), 448-57.
44 O’Herrin SM, Slansky JE, Tang Q, et al. Antigen-specific blockade of t cells in vivo using dimeric MHC peptide [J]. J Immunol, 2001; 167(5), 2555-60.
45 Maile R, Wang B, Schooler W, et al. Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers [J]. J Immunol, 2001; 167(7), 3708-14.
46 Aichele P, Brduscha-Riem K, Oehen S, et al. Peptide antigen treatment of naive and virus-immune mice: antigen-specific tolerance versus immunopathology [J]. Immunity, 1997; 6(5), 519-29.
47 Combadiere B, Sousa CR, Trageser C, et al. Differential TCR signaling regulates apoptosis and immunopathology during antigen responses in vivo [J].Immunity, 1998; 9(3), 305-13.
48 Romero P, Dunbar PR, Valmori D, et al. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes [J].J Exp Med, 1998; 188(9), 1641-50.
49 Levy R. A perspective on monoclonal antibody therapy: where we have been and where we are going [J]. Semin Hematol, 2000; 37(4 suppl 7), 43-6.
50 Guo YJ, Che XY, Shen F, et al. Effective tumor vaccines generated by in vitromodification of tumor cells with cytokines and bispecific monoclonal antibodies [J]. Nat Med, 1997; 3(4), 451-5.
51 Ogg GS, Dunbar PR, Cerundolo V, et al. Sensitization of tumour cells to lysis by virus-specific CTL using antibody-targeted MHC class I/peptide Complexes [J]. Br J Cancer, 2000; 82(5), 1058-62.
52 Robert B, Guillaume P, Luescher I, et al. Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes [J]. Eur J Immunol, 2000; 30(11), 3165-70.
2. Garboczi DN, Hung DT, Wiley DC, et al. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides [J]. Proc Natl Acad Sci USA, 1992, 89(8): 3429-3433.
3. Altman J, PAH Moss P, Goulder D, et al. Phenotypic analysis of antigen-specific T lymphocytes [J]. Science, 1996, 274(5284): 94-96.
4. McMichael AJ, Callaghan CA. A new look at T cells [J]. J Exp Med, 1998, 187(9):1367-1371.
5. Boniface JJ, Rabinowitz JD, Wulfing C, et al. Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [J]. Immunity, 1998, 9:459-466.
6. Fernandez-Vina MA, Falco M, Sun Y, et al. DNA typing for HLA class I alleles: I. Subsets of HLA-A2 and of -A28 [J]. Hum Immunol, 1992, 33(3):163-73.
7. Tarpey I, Stacey S, Hickling J, et al. Human cytotoxic T lymphocytes stimulated by endogenously processed human papillomavirus type 11 E7 recognize a peptide containing a HLA-A2(A*0201)motif [J]. Immunology, 1994, 81(2):222-227.
8. Parker, KC, Bednarek MA, and Coligan JE. 1994. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. Available at: http://www-bimas.cit.nih.gov/molbio/hla_bind/. Accessed 12 February 2005.
9.陈浩,翁秀芳,吴雄文,等.人β2m基因的克隆及表达[J].华中科技大学学报(医学版),2004,33(1): 1-4.
10. Maziarz RT, Fraser J, Srominger JL, et al. The human HLA-specific monoclonal antibody W6/32 recognizes a discontinuous epitope within the alpha 2 domain of murine H-2Db [J]. Immunogenetics, 1986, 24(3):206 -208.
11. Tran TM, Ivanyi P, Hilgert I, et al. The epitope recognized by pan-HLA class I-reactive monoclonal antibody W6/32 and its relationship to unusual stability of theHLA-B27/beta2-microglobulin complex [J]. Immunogenetics, 2001, 53(6): 440-446.
12. Ladasky JJ, Shum BP, Canavez F, et al. Residue 3 of beta2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody [J]. Immunogenetics, 1999, 49(4):312-320.
13.沈关心,周汝麟主编.现代免疫学实验技术(第二版).湖北科学技术出版社.1998.
14.吴雄文,梁智辉.实用免疫学实验技术(第一版).湖北武汉:湖北科学技术出版社, 2002.
15. Taswell C. Limiting dilution assays for determination of immunocompetent cell frequencies [J]. J Immunol, 1981, 126:1614-1619
16. Czerkinsky C, andersson G, Ekre HP, et al. Reverse ELISPOT assay for clonal analysis of cytokine production [J]. J Immunol Methods, 1988, 110:29-36
17. Maino VC, Picker LJ. Identification of function subsets by flow cytometry: intracellular detection of cytokine expression [J]. Cytometry, 1998, 34:207-215
18. Michelle S, Longworth, Laimonis A. Laimins Pathogenesis of Human Papillomaviruses in Differentiating Epithelia [J]. Microbiology and molecular biology reviews, 2004, 68(2). 362–372
19. Parker KC, Bednarek MA, Hull LK, et al. Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2 [J]. J Immunol, 1992, 149:3580-3587.
20.周玉英,胡牧,刘翠苓.鼻息肉病中人类乳突瘤病毒检测[J]。临床耳鼻咽喉科杂志,2001, 15(5): 212-13.
21. Hosken NA, Bevan MJ. Defective presentation of endogenous antigen by a cell line expressing class I molecules [J]. Science, 1990, 248:367-370
22. Rener M, Claesson MH, Bregenholt S, et al. An improved method for detection of peptide-induced upregulation of HLA-A2 molecules on TAP-deficient T2 cells [J]. Exp Clin Immunogenet, 1996, 13:30-35.
23.房崇芸,吴雄文,韩军艳等.长期混合淋巴细胞培养-细胞毒实验模型的建立[J].中华器官移植杂志,2001,22(5):277-279.
24. Skinner PJ, Daniels MA, Schmidt CS, et al. Cutting edge: in situ tetramer stainingof antigen-specific T cells in tissues [J]. J Immunol, 2000, 165(2), 613-7.
25. Vyth-Dreese FA, Kim YH, Dellemijn TA, et al. In situ visualization of antigen-specific T cells in cryopreserved human tissues [J]. J Immunol Methods, 2006, 310(1-2), 78–85.
26. Kobayashi A, Miaskowski C, Wallhagen M, et al. Recent development in understanding the immune response to human papillomavirus infection and cervical neoplasia [J]. Oncol Nurs Forum, 2000, 27 (4):643 - 651.
27. Evans M, Borysiewicz L K, Evans AS, et al. Antigen p rocessing defects in cervical carcinomas limit t he p resentation of a CTL epitope from human papillomavi rus16 E6 [J]. J Immunol, 2001, 167 (9) :5420 - 5428.
28. Trizna Z, Evans T, Bruce S, et al. A randomized p haseⅡstudy comparing four diff erent interf eron t herapies in patients with recalcit rant condylomata acuminata [J]. Sex Transm Dis, 1998, 25 (7) :361 - 365.
29. Pious D, Krangel MS, Dixon LL, et al. HLA Antigen Structural Gene Mutants Selected with an Allospecific Monoclonal Antibody [J]. Proc Natl Acad Sci, 1982, 79(24): 7832-36
30. Greer, CE, Wheeler CM, Ladner MB, et al. Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts [J]. J Clin Microbiol, 1995, 33(8): 2058–2063.
31. Liu F, Wang JB, Liu YH, et al. Comparison between immunofluorescence and PCR in detecting human papilloma virus in condyloma acuminate [J]. Chin Med Sci J, 2006, 21(2):125-7.
32. Coleman N, Birley HD, Renton AM, et al. Immunological events in regressing genital warts [J]. Am J Clin Patho, 1994, 102(6):768-74.
33. Selvakumar R, Borenstein LA, Lin YL, et al. Immunization with nonstructural proteins E1 and E2 of cottontail rabbit papillomavirus stimulates regression of virus-induced papillomas [J]. J Virol, 1995, 69(1):602-5.
34. Selvakumar R, Schmitt A, Iftner T, et al. Regression of papillomas induced by cottontail rabbit papillomavirus is associated with infiltration of CD8+ cells and persistence of viral DNA after regression [J]. J Virol, 1997, 71(7): 5540-8.
35. Dorothee G, Vergnon I, El Hage F, et al. In situ sensory adaptation of tumor-infiltrating T lymphocytes to peptide-MHC levels elicits strong antitumor reactivity [J]. J Immunol, 2005, 174(11): 6888-97.
36. Tully G, Kortsik C, Hohn H, et al. Highly focused T cell responses in latent human pulmonary Mycobacterium tuberculosis infection [J]. J Immunol, 2005, 174(4):2174-84.
37. Masopust D, Vezys V, Wherry EJ, et al. Cutting edge: gut microenvironment promotes differentiation of a unique memory CD8 T cell population [J]. J Immunol, 2006, 176(4):2079-83.
38. Chernosky ME, Anderson DE, Chang JP, et al. Familial progressive hyperpigmentation [J]. Arch Derm, 1971, 103: 581-591.
39. Rebora A, Parodi A. Universal inherited melanodyschromatosis: a case of melanosis universalis hereditaria [J]? Arch Derm, 1989, 125: 1442-1443.
40. Debao L, Ting L.Familial progressive hyperpigmentation: a family study in China [J]. Brit J Derm 1991; 125: 607.
41.罗挺,凌淑清,刘若英,等.进行性色素过度沉着症一个汉族家系的研究.中华医学遗传学杂志[J],1994;11(3):174-5.
42. The Cooperative Human Linkage Center. CHLC Marker Maps. Available at: http://lpgws.nci.nih.gov/html-chlc/Chlc Marker Maps. Html. Accessed 12 October 2004.
43. Taiwan Polymorphic Marker Database.TPMD Map Display. Available at: http://tpmd.nhri.org.tw/php-bin/graphic.php. Accessed 12 October 2004.
44. National Center for Biotechnology Information. NCBI Map Viewer. Available at: http://www.ncbi.nlm.nih.gov/mapview/maps.cgi Accessed 15 July 2005.
45. Walsh PS, Fildes NJ, Reynolds R. Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA [J]. Nucleic Acids Res, 1996, 24:2807- 2812.
46. Bar W, Brinkmann B, Budowle B, et al. DNA recommendations. Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. International Society for Forensic Haemogenetics [J]. Int J Legal Med 1997; 110:175-176.
47. DNA commission of the international society for forensic haemogenetics report concerning further recommendations of the DNA Commission of the ISFH regarding PCR-based polymorphisms in STR [J]. Int J Legal Med 1994; 107:159.
48. Neitzel H. A routine method of the establishment of permanent growing lymphoblastoid cell lines [J]. Hum Genet, 1986, 73(4):320-326.
49. Schlee M, Krug T, Gires O, et al. Identification of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) target proteins by proteome analysis: activation of EBNA2 in conditionally immortalized B cells reflects early events after infection of primary B cells by EBV [J]. J Virol, 2004, 78(8):3941-3952.
50. Aman P, Ehlin-Henriksson B, Klein G. Epstein-Barr virus susceptibility of normal human B lymphocyte populations [J]. J Exp Med, 1984, 159:208-220.
51. Brack C, Mattaj IW, Gautschi J, et al. Cyclosporin A is a differential inhibitor of eukaryotic RNA polymerases [J]. Exp Cell Res, 1984, 151:314-321.
52. Litt M, Luty JA. A hypervariable microsatellite revaled by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene [J]. Am J Hum Genet, 1989, 44(3):397-401.
53. Edwards A, Civitello A, Hammond Ha, et al. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats [J]. Am J Hum Genet, 1991, 49:746-756.
54. Hochmeister MN. Molecular aspects of medicine-DNA technology in forensic application. Printed in Great Britain by Nuffield Press Ltd. 1995,400.
55. Ricci U, Klintschar M, Neuhuber F, et al. Study on the STR TPOX in an Italian and an Austrian population using two different prime pairs and three different electrophoretic methods [J]. Int J Leg Med. 1998, 111:212-214
56. Zhang Caie, Deng Yunhua, Chen Xingping, et al. Linkage of a locus determining Familial Progressive Hyperpigmentation (FPH) to chromosome 19p13.1-pter in a Chinese family [J]. Eur J Dermatol, 2006; 16(3):1-6.
57. Jenne DE, Reimann H, Nezu J, et al. Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase [J]. Nat Genet, 1998; 18:38-43.
58. Qiu W, Schonleben F, Thaker HM, et al. A novel mutation of STK11/LKB1 geneleads to the loss of cell growth inhibition in head and neck squamous cell carcinoma.Oncogene. 2006 ;25(20):2937-42.
59. Launonen V. Mutations in the human LKB1/STK11 gene [J]. Hum Mutat, 2005, 26: 291-297.
60. Marignani PA. LKB1, the multitasking tumour suppressor kinase [J]. J Clin Pathol, 2005, 58:15-19.
61.金冬雁,黎梦枫译.分子克隆实验指南. (第二版).北京:科学技术出版社.1992.
62. Hemminki A, Tomlinson I , Markie D , et al . Locatigation of a susceptibility locus for Peutg-Jeghers Syndrome to 19p using comparative genomic hybridigation and targeted linkage analysis [J]. Nat Genet, 1997, 15 :526.
63. Hemminki A, Markie D, Tomlinson I, et al. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome [J]. Nature, 1998; 391(6663):184-187.
1 Altman J D ,Moss P A H , Goulter P J R , et al. Phenotypic analysis of antigen specific T lymphocytes [J] . Science ,1996 ;274 :94-96.
2 Appay V, Nixon DF, Donahoe SM, et al. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function [J]. J Exp Med, 2000; 192(1), 63-75.
3 Dunbar PR, Ogg GS, Chen J, et al. Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood [J]. Curr Biol, 1998; 8(7), 413-16.
4万瑛,吴玉章.特异性细胞毒性T细胞活化的研究进展[J].第三军医大学学报,2000;22(10):992-997.
5赵建平,吴玉章. CTL表位预测的方法学进展[J].第三军医大学学报,2000;22(10):988-992.
6 Xiao-Ning Xu , Gavin R , Screaton et al . MHC/peptide tetramer-based studies of T cell function [J]. J of Immunological Methods ,2002 ;268 (1) :21-28.
7 Doherty P C. The numbers game for virus-specific CD8 + T cell [J]. Science ,1998 ;280(5361) :227-31.
8 Grakoui A, Bromley SK, Sumen C, et al.The immunological synapse: a molecular machine controlling T cell activation [J]. Science, 1999; 285(5425), 221-27.
9 Boniface JJ, Rabinowitz JD, Wulfing C, et al. Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [J]. Immunity, 1998;9, 459.
10 Xu XN, Purbhoo MA, Chen N, et al . A novel approach to antigen specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex [J]. Immunity, 2001; 14,591.
11 Tough DF, Borrow P, Sprent J. Induction of bystander T cell proliferation by viruses and type I interferon in vivo [J]. Science, 1996; 272(5270), 1947-50.
12 Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution [J]. J Immunol Methods, 2000; 243(1-2), 147-54.
13 Doherty PC, Christensen JP. Accessing complexity: the dynamics of virus-specific T cell responses [J]. Annu Rev Immunol , 2000; 18, 561-92.
14 Sakai JA, Nagai M, Brennan MB, et al. In vitro spontaneous lymphoproliferation in patients with human T-cell lymphotropic virus type I-associated neurologic disease: predominant expansion of CD8(+) T cells [J]. Blood, 2001; 98(5), 1506-11.
15 Kaech SM, Ahmed R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells [J]. Nat Immunol , 2001; 2(5), 381-2.
16 Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis [J]. Semin Hematol , 2001;38,179.
17 Gerdes J, Lemke H, Baisch H, et al. Cell cycle analysis of a cellproliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67 [J]. J Immunol, 1984; 133(4), 1710-5.
18 Oxenius A, Gunthard HF, Hirschel B, et al. Direct ex vivo analysis reveals distinct phenotypic patterns of HIV-specific CD8(+) T lymphocyte activation in response to therapeutic manipulation of virus load [J]. Eur J Immunol, 2001; 31(4), 1115-21.
19 Purbhoo MA, Boulter JM, Price DA, et al . The human CD8 coreceptor effects cytotoxic T cells activation and antigen sensitivity primary by mediating complete phosphorylation of the T cell receptor zeta chain [J] . J Biol Chem, 2001 ;276 :32786.
20 Takahashi M, Osono E, Nakagawa Y, et al . Rapid induction of apoptosis in CD8 ( + ) HIV-1 envelope-specific murine CTLs by short exposure to antigenic peptide [J] . J Immunol , 2002 ;169 (11) :6588-93.
21 Globerson A, Effros RB. Ageing of lymphocytes and lymphocytes in the aged [J]. Immunol Today, 2000; 21(10), 515-21.
22 Effros RB, Pawelec G. Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion [J]? Immunol Today, 1997; 18(9), 450-4.
23 Rufer N, Dragowska W, Thornbury G, et al. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry [J]. Nat Biotechnol, 1998; 16(8), 743-7.
24 Rufer N, Brummendorf TH, Kolvraa S, et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood [J]. J Exp Med, 1999; 190, 157.
25 Plunkett FJ, Soares MV, Annels N, et al. The flow cytometric analysis of telomere length in antigen-specific CD8+ T cells during acute Epstein–Barr virus infection [J]. Blood, 2001; 97(3), 700-7.
26 Trapani JA, Sutton VR, Smyth MJ. CTL granules: evolution of vesicles essential for combating virus infections [J]. Immunol Today , 1999; 20, 351.
27 Liu CC, Walsh CM, Young JD. Perforin: structure and function [J]. Immunol Today, 1995; 16(4), 194-201.
28 Smyth MJ, Trapani JA. Granzymes: exogenous proteinases that induce target cellapoptosis [J]. Immunol Today, 1995; 16(4), 202-6.
29 Chen G, Shankar P, Lange C, et al. CD8 T cells specific for human immunodeficiency virus, Epstein– Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection [J]. Blood, 2001; 98(1), 156-64.
30 Gamadia LE, Rentenaar RJ, Remmerswaal EB, et al. CMV-specific CD8 (pos) T lymphocyte differentiation in latent CMV infection [J]. Transplant Proc, 2001; 33(1-2), 1802-3.
31 Bossi G, Griffiths GM. Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells [J]. Nat Med, 1999; 5(1), 90-96.
32 Guidotti LG, Chisari FV. To kill or to cure: options in host defense against viral infection [J]. Curr Opin Immunol, 1996; 8(4), 478-83.
33 Pala P, Hussell T, Openshaw PJ. Flow cytometric measurement of intracellular cytokines [J]. J Immunol Methods, 2000; 243(1-2), 107-24.
34 Gea-Banacloche JC, Migueles SA, Martino L, et al. Maintenance of large numbers of virusspecific CD8+ T cells in HIV-infected progressors and long-term nonprogressors [J]. J Immunol, 2000(2); 165, 1082-92.
35 Kostense S, Ogg GS, Manting EH, et al. High viral burden in the presence of major HIV-specific CD8(+) T cell expansions: evidence for impaired CTL effector function [J]. Eur J Immunol, 2001; 31(3), 677-86.
36 Sandberg JK, Fast NM, Nixon DF. Functional heterogeneity of cytokines and cytolytic effector molecules in human cd8(+) t lymphocytes [J]. J Immunol, 2001; 167(1), 181-7.
37 Skinner PJ, Daniels MA, Schmidt CS, et al. Cutting edge: in situ tetramer staining of antigen-specific T cells in tissues [J]. J Immunol, 2000; 165(2), 613-7.
38 Vyth-Dreese FA, Kim YH, Dellemijn TA,et al. In situ visualization of antigen-specific T cells in cryopreserved human tissues [J]. J Immunol Methods 2006; 310(1-2), 78–85.
39 Zhang D, Shankar P , Xu Z, et al. Most antiviral CD8 T cells during chronic viral infection do not express high levels of perforin and are not directly cytotoxic[J] .Blood, 2003 ; 101 (1) :226-235.
40 Geroge W, Bertoletti .Quantity and quality of virus2specific CD8 cell response :relevance to the design of a therapeutic vaccine for chronic HBV Infection [J] .Mol Immunol , 2001 ; 38(6) :467-473.
41 Welsh R M. Assessing CD8T cell number and dysfunction in the presence of antigen [J] . J Exp Med , 2001 ; 193(5) :F19-22.
42 Hirano N, Butler MO, Xia Z, et al. Efficient presentation of naturally processed HLA class I peptides by artificial antigen-presenting cells for the generation of effective antitumor responses [J]. Clin Cancer Res, 2006; 12(10): 2967-75.
43 Wang M, Stepkowski SM, Wang ME, et al. Induction of specific allograft immunity by soluble class I MHC heavy chain protein produced in a baculovirus expression system [J]. Transplantation, 1996; 61(3), 448-57.
44 O’Herrin SM, Slansky JE, Tang Q, et al. Antigen-specific blockade of t cells in vivo using dimeric MHC peptide [J]. J Immunol, 2001; 167(5), 2555-60.
45 Maile R, Wang B, Schooler W, et al. Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers [J]. J Immunol, 2001; 167(7), 3708-14.
46 Aichele P, Brduscha-Riem K, Oehen S, et al. Peptide antigen treatment of naive and virus-immune mice: antigen-specific tolerance versus immunopathology [J]. Immunity, 1997; 6(5), 519-29.
47 Combadiere B, Sousa CR, Trageser C, et al. Differential TCR signaling regulates apoptosis and immunopathology during antigen responses in vivo [J].Immunity, 1998; 9(3), 305-13.
48 Romero P, Dunbar PR, Valmori D, et al. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes [J].J Exp Med, 1998; 188(9), 1641-50.
49 Levy R. A perspective on monoclonal antibody therapy: where we have been and where we are going [J]. Semin Hematol, 2000; 37(4 suppl 7), 43-6.
50 Guo YJ, Che XY, Shen F, et al. Effective tumor vaccines generated by in vitromodification of tumor cells with cytokines and bispecific monoclonal antibodies [J]. Nat Med, 1997; 3(4), 451-5.
51 Ogg GS, Dunbar PR, Cerundolo V, et al. Sensitization of tumour cells to lysis by virus-specific CTL using antibody-targeted MHC class I/peptide Complexes [J]. Br J Cancer, 2000; 82(5), 1058-62.
52 Robert B, Guillaume P, Luescher I, et al. Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes [J]. Eur J Immunol, 2000; 30(11), 3165-70.