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

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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.