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

EBV与几种恶性疾病相关，包括淋巴瘤、鼻咽癌、伯基特淋巴瘤和免疫低下宿主中观察到的免疫母细胞淋巴瘤的子集。我们建立了一个有效的方法来确定CTL表位的背景下，HLA A^*2402分子通过多重筛选，包括一个计算机辅助算法，在体外的MHC稳定化试验和酶联免疫斑点（ELISPOT）试验。HLA A24是最常见的HLA I类等位基因，基因型几乎完全是A^*2402。我们首先通过计算机辅助算法在被报道为特异性CD 8 ^+ T细胞靶向的EBV蛋白中搜索具有HLA A24结合基序的候选肽。通过对5个裂解周期蛋白和6个潜伏周期蛋白的氨基酸序列分析，筛选并合成了42个多肽。它们都能稳定在肽转运蛋白缺陷细胞系T2上表达的HLA A^*2402分子。接下来是通过ELISPOT测定筛选肽，以确定它们被大量EBV特异性CD 8 ^+ T细胞识别，所述T细胞从HLA A24阳性的EBV免疫供体的PBMC建立。输入.我们已经从BRLF 1和BMLF 1的氨基酸序列中鉴定了2个可能的主要裂解周期EBV特异性CTL表位，从HLA A^*2402分子呈递的LMP 2中鉴定了1个。新发现的肽段应该可以用于检测和/或研究HLA A^*2402阳性人群中EBV特异性CD 8 ^+ T细胞应答。我们产生了HLA A * 2402四聚体，其掺入了3种肽和2种已知的表位肽，一种来自EBNA 3A（RYSIFFDYM），另一种来自EBNA 3B（TYSAGIVQI）。所有5种四聚体成功地染色了各种比例的EBV特异性CD 8 + T细胞。新发现的肽段应该可以用于检测和/或研究HLAA^*2402阳性人群中EBV特异性CD 8 ^+ T细胞应答。

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.