液-质联用分析肝癌细胞HLA-A2类分子递呈的抗原肽

目的：寻找MHCI类分子递呈的、T淋巴细胞识别的肝癌抗原肽。方法：人肝癌细胞系hHCC以枸橼酸－磷酸盐（pH3.3)液洗脱,洗脱物经粗分离,得到相对分子质量（Mr)＜5000的各种多肽组分混合液;取各组分多肽分离液分别与抗原加工缺陷细胞（T2,HLA－A2）进行细胞表位重建,加入特异性细胞毒T淋巴细胞（CTL),^51Cr释放法测杀伤活性;选活性值高的抗原肽组分进行反相高效液相色谱－质谱（RP－HPLC－MS）检测,对活性峰手工读谱分析其一级结构;最后进行氨基酸同源性分析。结果：酸洗10^10hHCC细胞,样品蛋白质浓度2mg/ml;通过RP－HPLC－MS检测,其质谱图经手工解析,氨基酸序列为SXXVHXNEV,X＝1或L;氨基酸同源性分析证明SXXVHXNEV为SLIVHLNEV,是肝细胞生长因子受体的第1075－1083肽段,第1080位点发生突变,由F变为L,由HLA－A2分子递呈。结论：细胞膜温和酸洗技术结合RP－HPLC－MS联用技术,是寻找肝癌抗原肽的有效方法,尚未见相同研究报道。发现的肝癌抗原肽SLIVHLNEV是肝细胞生长因子受体的突变物,可能具有重要生物学意义。     

Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes

A number of cytolytic T lymphocyte (CTL) clones derived from several melanoma patients have been found to recognize a majority of melanomas from HLA-A2 patients. We have reported previously that two such CTL clones recognize a product of the tyrosinase gene that is presented by HLA-A2. Here we show that one of these CTL clones recognizes a peptide encoded by the first nine amino acids of the putative signal sequence of tyrosinase. The other CTL clone recognizes a different tyrosinase peptide corresponding to amino acids 368–376. Both peptides contain consensus motifs of HLA-A2 binding peptides.     

反义HLA-A2 cDNA抑制人成纤维细胞于大鼠脑内移植后的免疫排斥反应

为了探讨人类白细胞抗原(HLA)反义RNA在抑制异种移植免疫排斥反应中的作用,用HLA A2反义cDNA和GDNF共表达的逆转录病毒转导人胚肺成纤维细胞,再移植到Parkinson病大鼠模型纹状体(实验组);同时移植到未感染病毒的人成纤维细胞作对照。移植后每周检测大鼠旋转行为的变化;并于4d、2周、6周灌杀大鼠,进行HLA A、CD4、CD8以及人核糖核蛋白颗粒(RNP)免疫组化染色等以观察宿主的免疫排斥反应和移植物在脑内的存活情况。结果发现:两组间动物的旋转行为变化不明显。实验组HLA A阳性细胞数在移植后的各时间点均明显少于对照组(P<0.05)。移植后4d两组的CD4和CD8阳性细胞数量变化均不明显(P>0.05),CD8细胞较多。2周和6周时实验组的CD4阳性细胞均明显少于对照组(P<0.01),且随时间逐渐下降。2周后实验组的CD8细胞基本消失,而对照组的CD8水平在2周时仍较高, 6周时也可见到。实验组RNP阳性细胞数量在各个时间点都明显多于对照组(P<0.01),到6周时明显下降, 12周时完全消失。反义HLA A2cDNA可以降低异种移植引起的免疫排斥反应程度,延长移植物存活时间,但抑制长期免疫排斥反应方面仍需寻找更好的方法。     

TIL识别的HLA-A2限制性人黑色素瘤特异抗原肽的研究

目的：探讨TIL识别的人黑色素瘤HLA-A2限制性的肿瘤抗原肽特性。方法：通过亲和层析柱从三种肿瘤细胞系（624-Mel、Chap-Mel和JY）中纯化HLA-A2蛋白和结合的多肽分子，经弱酸高温处理和离心超滤后，应用反相．高效液相色谱层析（RT-RPLC）分离各多肽组份，并将其各组份与T2细胞共同孵育、进行重建TIL识别的人黑色素瘤特异表位试验，鉴定肽分子生物活性，通过质谱分析所获活性肽分子特性，参照活性肽序列，制备人工合成肽加以进一步证实。结果：从RT-RPLC层析的624-Mel的肽组份，经特异表位鉴定发现3个活性峰（P19、P25和P31）。其中P31，TIL杀伤率达67％，质谱分析表明肽分子量为948，氨基酸序列为H^＋Ala Lue Trp Lue Phe Phe Gly Val Lue OH^-的9肽分子。经特异表位测定表明人工合成肽具有纯化活性肽的生物活性特征。结论：分离鉴定的这-9肽分子是一种TIL识别的HLA-A2限制性肿瘤抗原肽，为肿瘤防治、合成肽疫苗的研究奠定了可靠的实验基础。     

Natural killer-cell activity in cyclosporine-treated renal allograft recipients

We prospectively studied natural killer (NK)-cell activity in 16 cyclosporine-treated renal transplant recipients. NK function remained intact in the group as a whole in the initial 6 months following transplantation. The percentage of CD16-positive cells within the peripheral blood mononuclear-cell population was highly correlated with NK activity both prior to and following transplantation in the absence of rejection. During rejection, the correlation was poor. A marked increase in NK activity occurred during 9 of 12 rejection episodes; similar increases in NK activity were rarely observed in the absence of rejection. Significant infiltrates of NK cells, as determined by expression of CD16, were not demonstrated in stained biopsy specimens obtained from rejecting allografts. Pretransplant NK activity did not predict clinical outcome of the allograft. Our results indicate that NK cells are activated during allograft rejection in cyclosporinetreated patients, but their exact role in the rejection process is unknown.     

可生物素化HLA-A2-生物素化序列融合基因的构建与表达

本文采用RT PCR技术从人外周血PBMC中克隆HLA A2基因的胞外区 ,拼接上依赖BirA酶的可生物素化序列后 ,装入pET 42b (+)高效表达载体进行诱导表达。并对表达产物进行纯化与复性。结果成功地扩增出HLA A2基因并测序证实 ;构建了融合表达载体pET HLA A2 ,并获表达与纯化。为研究在原核系统中表达、纯化与复性及进一步体外构建MHC I类分子四聚体 ,探讨免疫识别机制奠定了基础。     

The immunodominant influenza matrix T cell epitope recognized in human induces influenza protection in HLA-A2/K(b) transgenic mice

The protective efficacy of the influenza matrix protein epitope 58-66 (called M1), recognized in the context of human HLA-A2 molecules, was evaluated in a HLA-A2/K(b) transgenic mouse model of lethal influenza infection. Repeated subcutaneous immunizations with M1 increased the percentage of survival. This effect was mediated by T cells since protection was abolished following in vivo depletion of all T lymphocytes, CD8(+), or CD4(+) T cells. The survival correlated with the detection of memory CD8(+) splenocytes able to proliferate in vitro upon stimulation with M1 and to bind M1-loaded HLA-A2 dimers, as well as with M1-specific T cells in the lungs, which were directly cytotoxic to influenza-infected cells following influenza challenge. These results demonstrated for the first time that HLA-A2-restricted cytotoxic T cells specific for the major immunodominant influenza matrix epitope are protective against the infection. They encourage further in vivo evaluation of T cell epitopes recognized in the context of human MHC molecules.     

HPV type 16 protein E7 HLA-A2 binding peptides are immunogenic but not processed and presented

Human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies and a high percentage of cervical carcinoma cells express HPV-16 E6 and E7 oncoproteins. These proteins are attractive targets for cytolytic T lymphocyte (CTL) mediated immunotherapy. We screened peptides derived from the HPV-16 E7 protein for binding to HLA-A2 and tested their potential to induce specific CTL responses in chimeric HLA-A2/H2-Kb transgenic mice. From eight potential binding peptides four displayed binding and were tested for immunogenicity. CTL activity was tested using target cells pulsed with peptide or expressing E7 protein. While there was no CTL induction observed with the peptides 7-15, 66-74 and 82-90, CTL from mice immunized with 86-93 lysed targets presenting the peptide in the context of the HLA-A2/H2-Kb molecule or wild-type HLA-A2. In contrast, 86-93 induced CTL showed no cytolytic activity against cells expressing the protein E7 and vaccination with the E7 protein did not lead to cytotoxicity against targets pulsed with the 86-93 peptide. Therefore the peptide 86-93, which binds to HLA-A2, is able to induce CTL responses in context of HLA-A2, but the peptide appears not to be processed or presented by HPV type 16 infected cells.     

A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3

The human MAGE-3 gene is expressed in many tumors of several histological types but it is silent in normal tissues, with the exception of testis. Antigens encoded by MAGE-3 may, therefore, be useful targets for specific anti-tumor immunization of cancer patients. We reported previously that MAGE-3 codes for an antigenic peptide recognized on a melanoma cell line by autologous cytolytic T lymphocytes (CTL) restricted by HLA-A1. Here we report that the MAGE-3 gene also codes for another antigenic peptide that is recognized by CTL restricted by HLA-A2. MAGE-3 peptides bearing consensus anchor residues for HLA-A2 were synthesized and tested for binding. T lymphocytes from normal individuals were stimulated with autologous irradiated lymphoblasts pulsed with each of three peptides that showed strong binding to HLA-A2. Peptide FLWGPRALV was able to induce CTL. We obtained CTL clones that recognized not only HLA-A2 cells pulsed with this peptide but also HLA-A2 tumor cell lines expressing the MAGE-3 gene. The proportion of melanoma tumors expressing this antigen should be approximately 32 % in Caucasian populations, since 49 % of individuals carry the HLA-A2 allele and 65 % of melanomas express MAGE-3.     

Severe graft rejection, increased immunosuppression, and active CMV infection in renal transplantation

Associations between active cytomegalovirus (CMV) infection, graft rejection, rejection therapy, and clinical signs and symptoms have been shown repeatedly. However, the causes and the sequence of events remain an area of debate. Two hundred twenty five patients with cadaveric renal transplant were included in the present study. Clinical signs and symptoms, and the development of active CMV infections were recorded during the first 3 months after renal transplantation. CMV monitoring by pp65-antigenemia was performed followed by preemptive antiviral therapy. Delayed graft function and severe graft rejection followed by anti T-cell antibody therapy was associated with the development of active CMV infection. In contrast, the induction therapy with anti-T-cell antibodies was not associated with more active CMV infections. Post-transplant morbidity determined by fever, pneumonia, and duration of hospital stay was increased significantly in patients with active CMV infection. However, in times of preemptive antiviral therapy an increased morbidity occurred in association with severe graft rejection and not with active CMV infection alone. In patients with renal transplantation and preemptive antiviral therapy, the morbidity was no more influenced by the CMV serostatus although the prevalence of active CMV infection was obviously different between CMV exposed (D+/R+,D+/R-, D-/R+) and unexposed (D-/R-) patients. Severe graft rejection and increased immunosuppression could stimulate cooperatively active CMV infections whereas immunosuppression alone may not be as effective. Prevention of severe graft rejection may be important to decrease early post-transplant morbidity and also the development of active CMV infections after renal transplantation.     

A simplified PCR-SSP method for HLA-A2 subtype in a population of Wuhan, China

HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the HLA-A2 subtypes and to understand the profile of HLA-A2 allellc distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers （PCR-SSP） to discriminate 17 HLA-A2 subtypes which cover the most HLA-A2 alleles （〉 99% allele frequency） reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was HLA-A2 positive, meanwhile the subtypes of HLA-A＊0209 and HLA-A＊0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of HLA-A2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random HLA-A2 positive individuals for their HLA-A2 subtypes. Those samples were screened for HLA-A2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis （FCM） with HLA-A2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry HLA-A2, HLA-A＊0201 ranks the first （allele frequency = 15.5%）, followed by A＊0207 （5.8%）, A＊0206 （4.7%）, A＊0203 （2.6%）, A＊0210 （0.7%）, and these 5 alleles account for 99.0% HLA-A2 subtypes of allele frequency. Our study indicates that the developed typing method is simple and reliable for HLA-A2 subtyping in Chinese, and the profile of allelic distribution of HLA-A2 subtypes is revealed in the population of Wuhan, China.     

肾移植术后血清IL-18的动态监测及临床意义

目的:观察肾移植术后发生急性排斥反应、感染、环孢霉素A(CsA)中毒时血清白介素-18(IL-18)的变化, 探讨IL-18在急性排斥反应中的早期诊断及鉴别诊断的意义.方法:采用 ELISA法对90例肾移植患者手术前后的血清IL-18水平进行动态监测.结果:肾移植患者术前血清IL-18水平与健康对照组比较明显升高(P<0.01), 术后第1天明显升高, 10 d左右基本降至术前水平.发生急性排斥反应时, 血清IL-18持续升高, 经甲基强的松龙(MP)冲击有效后迅速下降, 治疗无效者, 血清IL-18持续在高水平.并发感染时, IL-18也显著升高, 与急性排斥反应组相比差别无显著性意义;而CsA中毒时, IL-18变化不明显.结论:动态监测IL-18有助于用于急性排斥反应的早期诊断和鉴别诊断.     

Identification of a novel gp100/pMel17 peptide presented by HLA-A*6801 and recognized on human melanoma by cytolytic T cell clones

Melanoma-associated peptides recognized by cytolytic T lymphocytes (CTL) in the context of several histocompatibility leukocyte antigens (HLA) are required for the development of specific immunotherapies. Using a transient transfection assay into COS-7 cells, we identified the gp100/pMel17 melanosomal protein as the shared antigen recognized by three independent CD8+ CTL clones in HLA-A*6801-restricted fashion. This finding was confirmed by the correlation between lack of gp100/pMel17 protein in a number of HLA-A*6801-positive melanomas and their resistance to lysis/cytokine production by the specific effectors. The gp100/pMel17 antigenic epitope was identified based on recognition of subfragments and on a computer-based prediction algorithm. Among a panel of gp100/pMel17-derived synthetic peptides only the 10-mer HTMEVTVYHR (gp100/pMel17182-191) induced tumor necrosis factor (TNF) release by CTL clones when pulsed on suitable target cells whereas both the 10-mer and the shorter 9-mer gp100/pMel17183-191 sensitized the same antigen-pulsed cells to lysis. In conclusion, the identification of the HTMEVTVYHR peptide will extend to HLA-A*6801 melanoma patients the possibility to exploit gp100/pMel17 melanosomal protein for experimental and clinical studies.     

Identification of ribosomal proteins S2 and L10a as tumor antigens recognized by HLA-A26-restricted CTL

Recent identification of cytotoxic T lymphocyte (CTL)-directed peptides binding to the HLA-A2 and -A24 alleles has opened the door to peptide-based cancer immunotherapies. However, subsequent studies have succeeded in identifying no more than a few CTL-directed peptides that bind to alleles other than HLA-A2 and -A24, thus hampering development of immunotherapies directed at other alleles. We have shown in this study that two genes coding for ribosomal proteins (S2 and L10a) encoded tumor antigens recognized by HLA-A26-restricted CTLs. The S2 mRNA was expressed in all of the cancer cells and non-malignant cell lines tested, but was not expressed in normal tissues except for the testis, muscle, and peripheral mononuclear leukocyte cell (PBMC). In contrast, the L10a mRNA was expressed in all of these cancer and non-malignant cell lines, and also normal tissues, although the expression levels in normal tissues were mostly low. One S2-derived peptide and two L10a-derived peptides had the ability to induce HLA-A26-restricted and peptide-specific CTLs reactive to tumor cells in PBMCs of cancer patients, respectively. These ribosomal protein-derived peptides, and particularly the S2-derived peptide, could be suitable for use in peptide-based immunotherapy for HLA-A26+ cancer patients.     

HLA-A*02 allele frequencies and haplotypic associations in Koreans

We have investigated the frequencies of HLA-A*02 alleles and their haplotypic associations with HLA-B and -DRB1 loci in 439 healthy unrelated Koreans, including 214 parents from 107 families. All of the 227 samples (51.7%) typed as A2 by serology were analyzed for A*02 alleles using polymerase chain reaction (PCR)-low ionic strength-single-strand conformation polymorphism (LIS-SSCP) method. A total of six different A*02 alleles were detected (A*02 allele frequency 29.6%): A*0201/9 (16.6%), *0203 (0.5%), *0206 (9.3%), *0207 (3.0%), and one each case of *0210 and *02 undetermined type. Two characteristic haplotypes showing the strongest linkage disequilibrium were A*0203-B38-DRB]*1502 and A*0207-B46-DRB1*0803. Besides these strong associations, significant two-locus associations (P<0.001) were observed for A*0201 with B61, DRB1*0901 and DRB1*1401, and for A*0206 with B48 and B61. HLA haplotypes carrying HLA-A2 showed a variable distribution of A*02 alleles, and all of the eight most common A2-B-DR haplotypes occurring at frequencies of > or =1% were variably associated with two different A*02 alleles. These results demonstrate that substantial heterogeneity is present in the distribution of HLA-A*02 alleles and related haplotypes in Koreans.     

肝素酶HLA-A2限制性CTL表位肽的筛选及其活性鉴定

目的 预测并鉴定新的人乙酰肝素酶(Hpa)抗原的HLA-A2限制性CTL表位,为恶性肿瘤多肽疫苗的免疫治疗提供依据. 方法 采用SYFPEITHI和BIMAS软件预测方法,对肝素酶HLA-A2限制性CTL表位进行预测,合成候选表位肽;利用T2细胞特点,对合成的候选肽与HLA-A2分子进行亲和力分析;利用乳酸脱氢酶释放试验检测待检肽特异性CTL诱导活性;利用ELISPOT检测T细胞活性. 结果 在所筛选的6条候选CTL表位肽中,Hpa(310～318)FLNPDVLDI与HLA-A2分子具有高亲和力,在体外可有效诱导肝素酶特异性CTL的产生,对肝素酶阳性且HLA-A2限制性的HCC-LM6肝癌细胞及SW-480结肠癌细胞具有明显的杀伤效应,且能有效诱导IFN-γ分泌,增强免疫活性. 结论 首次发现Hpa(310～318)FLNPDVLDI可能是肿瘤肝素酶抗原的HLA-A2限制性CTL表位.     

肾移植术后血清sICAM-1和sVCAM-1的动态监测及临床意义

目的：探讨肾移植术后监测血清可溶性细胞问粘附分子-1(sICAM-1)和可溶性血管细胞粘附分子-1(sVCAM-1)的临床意义。方法：采用ELISA法，动态监测86例肾移植患者手术前后血清sICAM-l和sVCAM-1的变化。结果：移植术前sICAM-1、sVCAM-l与对照组无显著性差别，术后均明显升高，于第3天时达到高峰，l周至2周后降至术前水平。发生急性排斥反应前l-3天血清sICAM-1、sVCAM-1即开始升高，抗排斥治疗有效后逐渐下降。并发感染时sICAM-1、sVCAM-l显著升高，CsA中毒时无明显变化。结论：动态监测血清sICAM-1、sVCAM-l可做为早期辅助诊断急性排斥反应的免疫学指标，有助于急性排斥反应与CsA肾中毒的鉴别。     

Arteriolitis in renal transplant biopsies is associated with poor graft outcome

AIMS: The Banff 1997 classification of renal allograft pathology identifies arteriolitis as a finding of uncertain significance. We sought to improve our understanding of arteriolitis by correlating its occurrence with histopathological and clinical parameters. METHODS AND RESULTS: Twenty allograft kidney biopsies from 19 patients, showing arteriolitis, were identified. Arterioles were defined as small vessels with: (1) wall thickness of 1-3 myocytes; (2) diameter less than one-third of an adjacent glomerulus; and (3) discontinuous or absent elastica. Arteriolitis was defined as mural infiltration by lymphocytes. Other histological findings were categorized according to the Banff 1997 working formulation. Ten biopsies (50%) showed type IIA rejection, seven (35%) showed type I rejection, and three (15%) showed borderline change. Two patients with borderline change had acute rejection in the next biopsy. None of the seven patients with type I rejection had previous or subsequent type II rejection on biopsy. A total 11/20 biopsies (10/19 patients) showing arteriolitis had type IIA rejection in the index or next biopsy. On follow-up, graft loss due to rejection occurred in 5/19 (26%) patients (median 126 days); all had shown type IIA rejection on a previous biopsy. Chronic allograft nephropathy developed in a further 4/19 (21%) patients (median 157 days), of whom three had shown only type I rejection on biopsy. CONCLUSION: Arteriolitis is associated with acute rejection, often type II rejection, and is associated with poor graft outcome. Other causes of arteriolitis were not encountered in this series.