Study of EBNA1-specific cellular immune responses in EB-virus associated diseases in chilhood

EB病毒(Epstein-Barr Virus，EBV)编码的核抗原1(EBNA1)在所有EBV阳性细胞中均有表达，是治疗EBV相关恶性肿瘤的理想靶点。虽然CD8+细胞毒性T淋巴细胞(CTL)的独特结构域在很大程度上阻止了其表位的呈递，但使用mRNA转导的树突状细胞(DC)将提供启动EBNA1特异性CTL的优势。在EBNA1转导的单核细胞来源的DC刺激下，我们成功地从一名健康供体中分离到两个EBNA1特异性OTL克隆B5和C6。这些油分别识别人类白细胞抗原-B*3501和人类白细胞抗原-CW*0303背景下的多肽。然后我们鉴定了一个新的表位FVYGGSKTSL，它由人类白细胞抗原Cw*0303和Cw*0304提出，40%的日本人表达这两个表位。我们制作了荧光标记的MHC-四聚体，用流式细胞术检测EBNA1特异性CTL。淋巴细胞-多肽混合培养法可以估计EBNA1特异性CTL的频率，结果显示FVYGGSKTSL特异性CTL前体在HLACW0303或CW0304阳性供者中的频率介于1×10^-5到1×10^-4 CD^8+T细胞之间。此外，这两个CTL克隆在体外都能抑制BEV转化的B淋巴细胞的生长，而B5 CTL在HLACW*0303的背景下对表达EBNA1的胃癌细胞产生了干扰素-γ反应。我们研究了Cd^4+的吸附能力。用覆盖EBNA1的重叠多肽诱导T细胞克隆，并鉴定了最小表位及其限制的MEIC II类分子。其中，一个克隆识别了DRB1*0401、0403和0406提出的一个新的表位。

Epstein-Barr virus (EBV) -encoded nuclear antigen I (EBNA1) is an attractive target for immunotherapy against EBV-associated malignancies because it is expressed in all EBV-positive cells. Although CD^8+ cytotoxic T-lymphocyte (CTL) epitope presentation is largely prevented by its unique domain, the use of mRNA-transduced dendritic cells (DCs) would offer the advantage of priming EBNAl-specific CTLs. After stimulation with EBNA1 transduced monocyte-derived DCs, we successfully isolated two EBNA1-specific OTL clones B5 and C6 from a healthy donor. These Oils recognize peptides in the context of HLA-B*3501 and HLA-Cw*0303, respectively. We then identified a novel epitope FVYGGSKTSL presented by both HLA-Cw*0303 and Cw*0304, which are expressed by 40 % of Japanese. We made fluorescently labelled MHC-tetramers to detect EBNA1-specific CTLs by flow cytometry. The mixed lymphocyte-peptide culture method, which enabled us to estimate frequencies of EBNA1-specific CTLs, revealed that FVYGGSKTSL-specific CTL precursor frequencies in HLA-Cw*0303 or Cw*0304-positive donors were between 1 x 10^-5 and 1 x 10^-4 CD^8+ T cells. Moreover, both CTL clones inhibited growth of HLA-matched BEV-transformed B lymphocytes in vitro and B5 CTLs produced an IFN-y response to EBNA1-expressing gastric carcinoma cells in the context of HLA-Cw*0303.Accumulating current evidence indicates that CD^4+ T cells,as well as CTLs, are required for effective antitumor immunity. We investigated the ability of CD^4+. T cell clones induced with overlapping peptides covering EBNA1, and identified minimal epitopes and their restricted MEIC class II molecules. Of these, one clone recognized a novel epitope being presented by DRB1*0401, 0403, and 0406.