Induction of specific cellular immunity to EBV-positive T- and NK-lymphomas

EBV与几种恶性疾病有关，包括免疫受损宿主中的一组淋巴瘤、鼻咽癌、Burkitt淋巴瘤和免疫母细胞淋巴瘤。我们通过计算机辅助算法、体外MHC稳定化试验和酶联免疫斑点试验(ELISPOT)建立了一种在HLAA^*2402分子背景下确定CTL表位的有效方法。人类白细胞抗原A24是最常见的人类白细胞抗原I类等位基因，其等位基因几乎全部为A^*2402。我们首先通过计算机辅助算法在被报道为特定CD8^+T细胞靶向的EBV蛋白中寻找具有HLAA24结合基序的候选肽。通过对5个裂解蛋白和6个潜伏环蛋白的氨基酸序列分析，选择合成了42个多肽。所有这些化合物都能稳定在多肽转运蛋白缺陷细胞系T2上表达的HLAA^*2402分子。下一步是用ELISPOT方法筛选多肽，以供大量EBV特异性CD8^+T细胞识别，CD8^+T细胞是从HLAA24阳性EBV免疫捐赠者的PBMCs中建立的。我们已经从BRLF1和BMLF1的氨基酸序列中确定了2个可能的主要裂解循环EBV特异性CTL表位，从HLAA^*2402分子提出的LMP2中发现了1个可能的EBV特异性CTL表位。新鉴定的多肽将被证明对检测和/或研究HLAA^*2402阳性人群中EBV特异性CD8^+T细胞反应很有帮助。我们合成了HLAA^*2402四聚体，包括3个多肽和2个已知表位多肽，一个来自EBNA3A(RYSIFFDYM)，另一个来自EBNA3B(TYSAGIVQI)。5个四聚体均能成功染色不同比例的EBV特异性CD8+T细胞。新发现的多肽将被证明对检测和/或研究HLAA^*2402阳性人群中EBV特异性CD8^+T细胞反应很有帮助。

EBV is associated with several malignant diseases, including a subset of lymphomas, nasopharyngeal carcinomas, Burkitt's lymphomas and immunoblastic lymphomas seen in immunocompromised hosts. We established an efficient approach for determination of CTL epitopes in the context of HLA A^*2402 molecules through multiple screenings, consisting of a computer-assisted algorithm, an in vitro MHC stabilization assay and an enzyme-linked immuno-spot (ELISPOT) assay. HLA A24 is the most frequently encountered HLA class I allele and the genotype is almost exclusively A^*2402. We first searched for candidate,peptides with the HLA A24-binding motif by a computer-assisted algorithm among EBV proteins reported to be targeted by specific CD8^+ T cells. We selected and synthesized 42 peptides after analyzing amino acid sequences of the 5 lytic and 6 latent cycle proteins. All of them functionally stabilized HLA A^*2402 molecules which had been expressed on the peptide transporter-deficient cell line T2. Next is screening of the peptides by ELISPOT assay for their recognition by bulk EBV-specific CD8^+ T cells, established from PBMCs of EBV-immune donors positive for HLA A24.-typing. We have identified 2 possible major lytic cycle EBV-specific CTL epitopes from the amino acid sequences of BRLF1 and BMLF1 and 1 from LMP2 presented by HLA A^*2402 molecules. The newly identified peptides should prove useful for detection and/or study of EBV-specific CD8^+ T cell responses among populations positive for HLA A^*2402. We produced HLA A^*2402tetramers-incorporating the 3 peptides and 2 known epitope peptide, one derived from EBNA3A (RYSIFFDYM), and the other derived from EBNA3B (TYSAGIVQI). All the 5 tetramers successfully stained various proportions of EBV-specific CD8+ T cells. The newly identified peptides should prove useful for detection and/or study of EBV-specific CD8^+ T cell responses among populations positive for HLAA^*2402.