HLA-A2限制性CTL表位HPV18E7_(7-15)分子修饰的实验研究

目的对人类白细胞抗原A2分子(HLA-A2)限制性细胞毒性T淋巴细胞(CTL)表位HPV18E77-15进行氨基酸置换修饰,并探讨修饰后多肽的免疫原性。方法根据量化模体方案,比较置换后的多肽与HLA-A2分子的结合系数,采用标准Fmoc方案合成并纯化多肽、细胞毒实验(51Cr释放法),观察多肽是否能够诱导特异性CTLs。结果修饰肽TLQDIVLHV符合HLA-A2分子限制性细胞毒性T细胞的表位要求,具有特异性细胞毒性T细胞诱导活性。结论修饰肽TLQDIVLHV具有更好的结合力和较强的抗原性,可以作为高危型人乳头瘤病毒(HPV)感染治疗性肽疫苗分子设计的候选表位。     

人白细胞抗原-A*0201限制性丙型肝炎病毒细胞毒性T淋巴细胞表位的鉴定

目的 鉴定人白细胞抗原(HLA)-A*0201限制性HCV-CTL表位.方法 基于RANKpep和SYFPEITHI细胞表位预测软件预测结果,选择合成6条候选CTL表位.研究候选CTL表位肽与T2细胞表达的HLA-A*0201分子的亲和力,进一步采用酶联免疫斑点实验(ELISPOT)和细胞内细胞因子染色(ICS)实验研究HLA-A*0201高亲和力肽在HLA-A*0201阳性HCV感染者的外周血单个核细胞(PBMC)中刺激CTL反应情况.结果 在6条候选CTL表位肽中,肽C_181(LLSCLTTPV)和NS2_172(VLQAGLIRV)与HLA-A*0201分子有高亲和力,其亲和力随肽浓度增加而升高.在10例HLA-A*0201阳性HCV-1b感染者每1×105PBMC中,肽C_181和NS2_172刺激后,特异性分泌IFN-γ细胞的斑点形成细胞数(SFC)分别为0～19和0～20.肽C_181和NS2_172特异性IFN-γ+CD8+T淋巴细胞占CD8+T淋巴细胞的比例分别为0.006%～0.065%和0.005%～0.080%.结论 肽C_181(LLSCLTTPV)和NS2_172(VLQAGLIRV)为HLA-A*0201 限制性HCV-CTL表位.     

结核分枝杆菌Rv0440 CTL表位肽的免疫原性研究

目的 体外鉴定结核分枝杆菌（Mycobacterium tuberculosis,Mtb）Rv0440蛋白序列中表位肽362 370 aa和369-377 aa的HLA A＊0201限制性CD8＋ CTL表位的免疫原性,为基于表位的结核疫苗研究提供实验依据.方法 根据T2细胞HLA-A＊ 0201分子与多肽结合力分析实验结果,选取结核分枝杆菌Rv0440蛋白质氨基酸序列中对HLA-A＊ 0201分子高亲合力的Rv0440 1（362-370 aa,KLQERLAKL）和Rv0440-2（369 377 aa,KLAGGVAVI）作为候选表位肽.用候选表位肽刺激PPD（＋＋＋）健康志愿者外周血单个核细胞（peripheral blood mononuclear cells,PBMC）检测细胞分泌IFN γ的水平.用候选表位肽诱导特异性CTL细胞,检测特异性CTL细胞对负载表位肽的T2细胞的杀伤活性,观察Rv0440-1和Rv0440 2的HLA-A＊ 0201限制性CD8＋ CTL表位的免疫原性.结果 ELISPOT实验结果显示,表位肽Rv0440-1能够明显诱导HLA-A＊ 0201（＋）、PPD（＋＋＋）健康志愿者PBMC分泌IFN γ（P＜0.05）;且表位肽Rv0440-1负载DC诱导的CTL在效靶比为10：1时对负载相应表位肽的T2细胞的特异性杀伤活性高于对照组（P＜0.05）;与对照组相比,表位肽Rv0440 2没有诱导能力.结论 表位肽Rv0440-1（362-370 aa,KLQERLAKL）具有良好的免疫原性,是有效的结核分枝杆菌的HLA-A＊0201限制性CTL表位.     

乙型肝炎相关肝癌患者乙型肝炎病毒不同抗原肽特异性CD3+CD8+人白细胞抗原-A2+细胞表达

目的 探讨乙型肝炎相关肝细胞癌(HCC)患者外周血单个核细胞(PBMC)中HBV不同抗原肽特异性CD3+ CD8+人白细胞抗原( HLA)-A2+细胞的表达.方法 从4例HLA-A2阳性乙型肝炎相关HCC患者外周血中分离PBMC,分别与HBV抗原肽sAg( FLLTRILTI、GLSPTVWLSV、WLSLLVPFV),HBV eore(FLPSDFFPSV)和HBV pol(FLLSLGIHL)及抗-CD3-pacific blue、抗-CD8-异硫氰酸荧光素共育,流式细胞仪分析HBV/HLA-A2-CD3-CD8阳性细胞,克隆培养,筛选出克隆培养的抗HBV T淋巴细胞;再与含有HBV的肝癌细胞株HepG2( HLA-A2+)共育,ELISA法检测其分泌IFN-γ水平.结果 4例患者体内受GLSPTVWLSV肽诱导的特异性抗HBV T淋巴细胞占所有CD8+细胞的1.44％±0.04％,高于FLLTRILTI肽的0.68％±0.08％、FLPSDFFPSV肽的1.06％±0.09％、FLLSLGIHL肽的0.56％±0.04％和WLSLLVPFV肽的0.46％±0.08％(t=0.001,P＜0.05).将GLSPTVWLSV/HLA-A2获得的HBV/HLA-A2 PentamerCD3-CD8阳性细胞克隆,获得2株抗HBV CD8 T淋巴细胞,与负荷GLSPTVWLSV肽段的HepG2 (HLA-A2+)细胞共育,CD8 T淋巴细胞分泌较高水平的IFN-γ.结论 乙型肝炎相关HCC患者外周血中存在针对不同HBV抗原肽的特异性抗HBV CD8 T淋巴细胞,其表达量与抗原肽段有关.     

结核杆菌抗原Ag85A HLA-A*0201限制性CD8+ CTL表位的预测和鉴定

目的预测结核杆菌（Mtb）抗原Ag85A的HLA-A＊0201限制性CD8^＋细胞毒性T细胞（CTL）表位，并对其进行鉴定。方法应用数据库SYFPEITHI预测可能存在的HLA—A＊0201的限制性CD8^＋CTL表位，并经流式细胞术分析抗原肽与HLA-A＊0201的亲合力，经时间荧光分辨法检测结核（TB）患者外周血单个核细胞（PB-MC）对抗原肽的增殖反应，再通过细胞毒实验研究抗原肽诱导的T细胞毒杀伤活性，逐步鉴定Ag85A的HLA—A＊0201限制性CD8^＋CTL表位。结果位于Ag85A氨基酸序列7（48-56aa）和5（242-250aa）的表位与HLA—A＊0201分子具有较高的亲和力，并能刺激HLA-A＊0201阳性结核患者PBMC增殖，诱导产生具有特异性杀伤活性的CTL。结论肽7GLPVEYLQV（48—56aa）和肽5KLIANNTRV（242—250aa）是Mtb抗原Ag85A的HLA—A＊0201限制性CD8^＋CTL表位。     

HLA-肽复合物研究HBV/C区变异对HLA-A2限制性表位的影响

目的 探讨乙型肝炎病毒(HBV)C区热点变异S87G和(或)197L对细胞毒T细胞表位形成的影响。方法以我国C基因型HBV DNA参照序列为标准(EMBL：Y18855-18858)，在计算机预测197L和(或)S87G变异对HLA-A*0201限制性表位影响的基础上，利用基因T程制备的HLA-A*0201轻链、重链多肽在体外只能与相应表位短肽形成稳定复合物的特点，对预测到的可疑表位肽段进行验证。结果197L时，肽段HBcAg96105(KLRQLLWFHI)的DC50比正常(KIRQLLWFHI)时大5倍以上；S87G无影响；197L、S87G没有协同作用。人工合成该短肽在体外不能与HLA-A*0201轻、重链多肽形成稳定复合物。结论HBV／C基因197L和(或)S87G变异时，没有新的HLA-A*0201限制表位产生。     

1型糖尿病关键抗原谷氨酸脱羧酶65蛋白表达的研究现状

谷氨酸脱羧酶（GAD）属于内分泌酶,包括GAD65和GAD67两种同工酶,但只有GAD65才是重要的胰岛自身抗原.众多研究认为GADA是诊断和预测1型糖尿病（T1DM）和成人隐匿性自身免疫糖尿病（LADA）最敏感且应用最广泛的指标[1-2].GADA检测方法多样,包括放射配体法和酶联免疫吸附实验（ELISA）法等,但诊断敏感性和特异性与GAD65蛋白纯度和抗原性密切相关.而抗原特异性T细胞更能直接反映胰岛自身免疫状态,可改善抗体诊断T1 DM的效率.研究表明在新诊断T1DM患者中使用酪氨酸磷酸酶（IA-2）、GAD65、胰岛细胞特异性葡萄糖-6-磷酸酶催化亚基相关蛋白（IGRP）和前胰岛素原作为CD8＋T细胞刺激抗原,其诊断敏感性波动在25％～40％,特异性在87％ ～ 100％[3].随后T细胞检测国际标准化工作组证实GAD270-285是人白细胞抗原（HLA）Ⅱ类分子四聚体检测技术的主要刺激片段[4-5].因此,选用合适的GAD65抗原或蛋白片段可改善GADA或自身反应性T细胞的诊断敏感性和特异性.     

1型糖尿病患者人类白细胞抗原Ⅰ类基因与胰岛功能及胰岛自身抗体的关系

目的 探讨1型糖尿病（T1DM）患者胰岛功能、胰岛自身抗体[谷氨酸脱羧酶抗体（GADA）、蛋白酪氨酸磷酸酶抗体（IA-2A）、胰岛素自身抗体（IAA）和阳离子外流锌转运体蛋白抗体（ZnT8A）]与人类白细胞抗原（HLA）Ⅰ类等位基因的关系.方法 选取2008年6月至2012年10月在南京医科大学第一附属医院及其开放课题26家医院（共同组成江苏省T1DM合作组）内分泌科就诊的T1DM患者330例.采用聚合酶链反应-寡核苷酸探针序列特异性引物（PCR-SSO）技术进行T1DM患者HLAⅠ类基因（A、B基因）分型,放射免疫配体法检测胰岛自身抗体（GADA、ZnT8A、IA-2A、IAA）.选择同地区、遗传背景相近的中华骨髓库无关供者HLA分型资料20 248份作为研究对照.通过Arlequin软件进行HL4Ⅰ类等位基因频率及单倍型频率分析.定量资料的组间比较采用独立样本的t检验.结果 与对照组相比,T1DM组HLA-A＊24、B＊08、B＊41、B＊54、B＊58频率明显升高（OR=1.250、2.062、6.927、2.472、1.608,均P＜0.05）;A＊01、B＊07、B＊27、B＊52、B＊57频率显著下降（OR =0.481、0.464、0.372、0.160、0.315,均P＜0.05）;HLA-A＊24-B＊ 54单倍型频率明显升高（OR=4.128,P＜0.05）.携带A＊02基因位点的T1DM患者其空腹C肽水平高于不携带A ＊02的患者[（356±249）比（250±199） pmol/L,t=-3.004,P＜O.05].未发现HLAⅠ类基因与胰岛自身抗体存在统计学关联.结论 江苏地区汉族T1DM患者中,HLAⅠ类等位基因不仅可影响T1DM的发病风险,而且可能影响发病后胰岛功能的衰竭速度.     

HBV/C热点变异对HLA-A2限制性T细胞表位的影响

乙型肝炎病毒(HBV)变异与人体特异性免疫应答的之间的关系,是阐明变异的生物意义的关键之一,但受方法学的限制迄今所知甚微。应用计算机表位预测和人白细胞抗原(HLA)肽复合物技术,分析了我国HBV慢性感染者C基因热点变异I97L和(或)S87G对HLA—A2限制性T细胞表位的影响。     

人端粒酶逆转录酶抗原HLA-A0201限制性CTL表位预测及鉴定

目的应用生物信息学方法对人端粒酶逆转录酶(h TERT)HLA-A2+限制性细胞毒性T细胞(CTL)表位进行预测和鉴定,寻找诱导机体特异性杀伤肺癌肿瘤细胞的抗原表位。方法应用生物信息学软件BIMAS、SYFPEITHI对h TERT蛋白进行HLA-A0201限制性CTL抗原表位预测,筛选优势表位;应用肽亲和力实验、乳酸脱氢酶(LDH)释放实验及人干扰素γ(IFN-γ)ELISPOT实验验证表位,筛选出激发机体产生特异性免疫反应的表位。结果生物信息学软件筛选出优势表位为:ILAKFLHWL、ELLRSFFYV及ILSTLLCSL;肽亲和力实验得到优势表位荧光系数(FI)为:ILAKFLHWL0.67、ELLRSFFYV0.66及ILSTLLCSL0.90;LDH释放实验显示ILAKFLHWL所诱导CTLs的杀伤率明显高于其它各表位,也明显高于阴性表位,差异均具有统计学意义(P0.05);人IFN-γELISPOT实验证明ILAKFLHWL所诱导的CTLs产生的IFN-γ斑点数多于其他表位,差异具有统计学意义(P0.05)。结论ILAKFLHWL的免疫原性强,可用于后续制备肺癌多肽疫苗。     

T cell reactivity to DR*0401- and DQ*0302-binding peptides of the putative autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is thought to be an autoimmune disease mediated by T lymphocytes recognizing critical islet cell antigens. Recently, the tyrosine phosphatase like protein IA-2 was suggested as a putative autoantigen in type 1 diabetes since autoantibodies are detected in sera of diabetic patients and prediabetic subjects. Similarly, T cell responses of peripheral blood lymphocytes of type 1 diabetic patients to this protein have been described. Only very few data is available about immunodominant epitopes of IA-2 recognized by T cells. We have studied T cell responses in type 1 diabetic patients and age and partly HLA matched controls to IA-2 peptides designed to bind HLA risk alleles of IDDM as DR*0401 and DQ*0302. Both diabetic patients and controls responded to IA-2ic and some of the peptides. Three peptides of the C-terminal region of IA-2 were recognised by T cells of a fraction of diabetic patients but at least two of these peptides triggered also T cell responses in DR*0401/DQ*0302-matched controls. Most peptides bound to different HLA alleles ("promiscous binders"). The identification of autoantigenic epitopes may offer clues to related sequences e.g. of viral origin what relates to models of diabetes pathogenesis ("molecular mimicry"). Secondly, the design of antigen- or even epitope-specific immune intervention strategies aiming at tolerization of disease specific T cells in type 1 diabetes may profit from the knowledge of immunodominant T cell epitopes of a putative autoantigen.     

Hemagglutinin protein is a primary target of the measles virus-specific HLA-A2-restricted CD8+ T cell response during measles and after vaccination

To characterize the measles virus (MV)-specific T cell responses important for evaluation of measles vaccines, human leukocyte antigen (HLA)-A2-positive and -negative adults immunized with measles-mumps-rubella vaccine were studied. Both groups developed increases in antibody and in interferon (IFN)- gamma -producing cells in response to pooled hemagglutinin (H) and fusion peptides. HLA-A2-binding peptides were predicted for all MV-encoded proteins and confirmed by T2 cell stabilization. Twenty-nine peptides were tested, and 19 (6 from H) stimulated increased IFN- gamma secretion in a majority of vaccinees. Peptide-loaded HLA-A2 tetramers or immunoglobulin dimers documented MV-specific CD8+ T cell responses after vaccination and during measles and confirmed new A2 epitopes in H (250-259 and 516-525 aa) and matrix (M; 50-58 aa) protein and previously described epitopes in H (30-38 aa), M (211-219 aa), and nonstructural protein C (84-92 aa). No single peptide dominated the response. We conclude that H is an important stimulus for CD8+ T cell as well as for antibody responses in HLA-A2-positive individuals.     

Comprehensive mapping of HLA-A0201-restricted CD8 T-cell epitopes on PDC-E2 in primary biliary cirrhosis

Growing evidence has implicated the involvement of autoreactive T lymphocytes in the pathogenesis of primary biliary cirrhosis (PBC). We have recently taken advantage of motif prediction analysis of HLA-A*0201 and identified the first major histocompatibility complex (MHC) class I restricted epitope, amino acids 159 to 167 on E2 components of pyruvate dehydrogenase complexes (PDC-E2), the major mitochondrial antigens in PBC. The mechanisms involved in the selection of epitope peptide(s) that comprise the PDC-E2-specific autoreactive cytotoxic T lymphocytes (CTLs) are unknown and likely involve other epitopes on PDC-E2 restricted by MHC class I molecules. To address this issue, a comprehensive mapping of the CTL epitope repertoire on the PDC-E2 molecule that binds HLA-A*0201 was performed to provide further clues regarding the role of CTLs. We used the T2 cell line to screen 79 overlapping 15mer peptides, spanning the entire PDC-E2 molecule. Six of the 79 peptides exhibited significantly higher binding activity to HLA-A*0201 than the other 15mer peptides. Two of these 6 peptides induced CTL lines from patients with PBC. Fine mapping with N-terminus or C-terminus truncated peptides identified 10mer peptide, PDC-E2 amino acids 165 to 174, which is a novel CD8 epitope restricted by HLA-A*0201. In conclusion, using a combination of the 15mer peptide library screening with the T2 binding assay and also the induction of CTL lines with candidate peptides, we have defined a novel HLA-A*0201-restricted epitope PDC-E2 165 to 174 in patients with PBC. These data will become important in the development of altered peptide ligands to modulate disease activity.     

A rapid method to identify cytotoxic T-lymphocyte peptide epitopes from HLA-A2 (+) donors.

It would be useful to develop a method to rapidly identify peptide epitopes for vaccine development. We present an algorithm that can predict sequences that have a high binding activity for HLA-A2. These sequences were able to induce specific cytolytic cells from human peripheral blood lymphocytes (PBMC). A computer-assisted algorithm was constructed to predict binding activity for HLA-A2, according to anchoring amino acid combinations. The human papillomavirus (HPV) type 18 E7 oncoprotein was used to test the algorithm. Peptides predicted to bind were synthesized and binding activity was determined by using the T2 cell assay. T2 cells pulsed with HPV-18 peptides were incubated with PBMC. Cytotoxicity assays were performed. From 110 possible sequences, four peptides were found to have a high binding activity. One of these peptides was able to induce significant lysis. Using this selection process only 3.6% of the total number of possible sequences was synthesized to identify an immunogenic peptide. Our algorithm with the T2 binding assay allows a rapid method to detect peptide epitopes.     

Identification and characterization of HLA-A*3303-restricted,HIV type 1 Pol- and Gag-derived cytotoxic T cell epitopes

HLA-A*3303 is one of the common HLA alleles in East and Southeast Asia. Identification of HLA-A*3303-restricted HIV-1 cytotoxic T lymphocyte (CTL) epitopes is therefore required to investigate the immunopathogenesis of AIDS and vaccine development in these areas, where AIDS is rapidly expanding. We attempted to identify HLA-A*3303-restricted CTL epitopes derived from relatively conserved proteins Pol, Gag, and Nef of HIV-1 clade B, using reverse immunogenetics. Ninety-nine 8-mer to 11-mer peptides corresponding to the HLA-A*3303-binding peptide motif were selected from the HIV-1 SF2 sequence. Fifty-two of these 99 peptides bound to HLA-A*3303. Six of these binding peptides induced peptide-specific CTLs in PBMCs from at least one of two HIV-1-seropositive individuals. CTL clones specific for three Pol peptides and one Gag peptide killed HLA-A*3303-restricted target cells infected with HIV-1 recombinant vaccinia, indicating that these peptides were naturally processed HLA-A*3303-restricted CTL epitopes. SF2-Pol 594-602 (FYVDGAANR) and SF2-Gag 144-152 (MVHQAISPR) induced specific CTLs in 5 and 4 of 10 chronically HIV-1-infected individuals, respectively, whereas SF2-Pol 60-70 (TLWQRPLVTIR) and SF2-Pol 934-943 (KIQNFRVYYR) induced specific CTLs in 2 and 1 of 10 chronically HIV-1-infected individuals, respectively. Thus, the former are immunodominant epitopes whereas the latter are not. These epitopes are useful for studies of AIDS immunopathogenesis and vaccine development.     

Therapeutic polypeptides based on HBcAg18-27 CTL epitope can induce antigen-specific CD8＋ CTL-mediated cytotoxicity in HLA-A2 transgenic mice

AIM: To explore how to trigger an HLAI-restricted CD8(+) T cell response to exogenously synthesized polypeptides in vivo. METHODS: Three mimetic therapeutic polypeptides based on the immunodominant CTL epitope of HBcAg, the B- epitope of HBV PreS(2) region and a common T helper sequence of tetanus toxoid were designed and synthesized with Merrifield's solid-phase peptide synthesis method. Their immunological properties of inducing T( H1) polarization, CD8(+) HBV-specific CTL expansion and CD8(+) T cell mediated cytotoxicity were investigated in HLA-A2 transgenic mice. RESULTS: Results demonstrated that the mimetic polypeptides comprised of the immunodominant CTL, B-, and T helper epitopes could trigger specifically and effectively vigorous CD8(+) HBV-specific CTL-mediated cytotoxicity and T(H1) polarization of T cells in HLA-A2 transgenic mice. CONCLUSION: A designed universal T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of CTL epitopes in vivo. And that the mimetic therapeutic peptides based on the reasonable match of the above CTL, B- and T helper epitopes could be a promising therapeutic peptide vaccine candidate against HBV infection.     

Identification of a new HLA-A2-restricted T-cell epitope within HM1.24 as immunotherapy target for multiple myeloma

OBJECTIVE: The aim of this study was identification of human leukocyte antigen (HLA)-A2-restricted T-cell epitopes within the HM1.24 antigen as target for multiple myeloma (MM)-directed specific peptide-based immunotherapy. METHODS: The HM1.24 sequence was scanned for immunogenic peptides using the HLA-binding prediction software SYFPEITHI and BIMAS. Peripheral blood mononuclear cells from HLA-A2(+) healthy volunteers/blood donors (ND) were stimulated with autologous HM1.24-peptide-loaded dendritic cells, and expanded in vitro. Activation of T cells was assessed by ELISpot and cytotoxicity by (51)Chromium ((51)Cr)-release assays. T2-cells pulsed with irrelevant peptide, the HM1.24(-)/HLA-A2(+) breast carcinoma cell line MCF-7 and the HM1.24(+)/HLA-A2(-) myeloma cell line RPMI-8226 were used as controls. Expression of the HM1.24 gene (BST2) was assessed using purified plasma cells and Affymetrix-U133A+B microarrays. Frequency of peptide-specific CD8(+) T cells was detected using the flow-cytometric tetramer technique. RESULTS: Of eight nona-peptides with the highest probability of binding to HLA-A2, the HM1.24 aa22-30 peptide (LLLGIGILV) showed the most frequent activation of CD8(+) T cells in healthy volunteers (specific activation in 8 of 11 [73%] ND; compared with 5-19% for the 7 other HM1.24 peptides). Antigen recognition by the HM1.24 aa22-30-specific CD8(+) T cells was HLA-A2-restricted (ELISpot with HLA-A2-blocking antibodies: median, 15; range, 14-18 spots/well; isotype-control antibodies: median, 47; range, 44-48). HM1.24-aa22-30-specific CD8(+) T cells lysed HLA-A2(+) myeloma-derived cell lines ((51)Cr-release assay: XG-1 vs MCF-7, 91% vs 0%; U266 vs MCF-7, 38% vs 4.2%; IM-9 vs RPMI-8226, 22% vs 0%). Using the cross-reactive Neisseria meningitidis peptide LLSLGIGILV-specific CD8(+) T cells recognizing target cells loaded with the HM1.24 aa22-30 peptide (LLLGIGILV) as well as the myeloma-derived cell line U266 could be expanded from MM patients. The HM1.24 gene was expressed at comparable levels by plasma cells from 65 MM patients, 7 patients with monoclonal gammopathy of undetermined significance, and 7 ND. CONCLUSIONS: HM1.24 aa22-30 is a newly identified HLA-A2-restricted T-cell epitope that is processed and presented by major histocompatibility complex class I. Specifically activated CD8(+) T cells are able to lyse MM cell lines. We conclude that HM1.24 aa22-30 represents a suitable candidate target for a specific peptide-based immunotherapy of MM.