Comprehensive epitope mapping of the Epstein-Barr virus latent membrane protein-2 in normal, non tumor-bearing individuals

Abstract

Latent membrane protein (LMP)-2 is one of the Epstein-Barr virus (EBV)-encoded proteins consistently expressed by nasopharyngeal carcinoma (NPC). EBV-transformed lymphoblastoid cell lines (LCL) have been used in patients with NPC to induce LMP-2-recognizing T cell lines which have been in turn utilized for protein-wide mapping of T cell epitopes. However, comprehensive mapping of naturally recognized LMP-2 epitopes in non tumor-bearing individuals has not been reported. Here, we applied a low sensitivity epitope-defining technique for the identification of LMP-2 CTL responses detectable ex vivo in EBV-experienced individuals. This screening tool has been previously validated by analyzing memory CTL responses to Flu, cytomegalovirus (CMV), and the melanoma associated antigen gp100/Mel17. Peripheral blood monocytes (PBMC) from ten Caucasian and ten Chinese individuals were stimulated ex vivo with pools of nonamer (9-mer) peptides overlapping in a stepwise fashion each single amino acid of the LMP-2 sequence. No obvious differences were observed between the immune response of the two ethnic groups save for those related to the divergence in the ethnic prevalence of HLA haplotypes. Several novel and known LMP-2 epitopes were identified. Reactivity toward at least one LMP-2 epitope was detected in 18 of the 20 donors but no prevalent human leukocyte antigen (HLA)/epitope combination was observed confirming that LMP-2 reactivity in the context of common HLA alleles is more pleiotropic than that of FLU and CMV. We believe that the usefulness of these epitopes occurring naturally in non-cancer bearing patients as reagents for the immunization of patients with early or advanced stage NPC deserves further evaluation.

Fig. 1

Eisen’s clustering analysis of individual peptide pools and PBMC. Normalized (n) log-₂ IFN-γ/β-actin ratios are presented according to the central normalization method [83] for the three sub-categories of pool matrixes (A: including pools 1–12 and A1-A10, B: including pools 13–24 and B1–B10). Top panel Matrix A; PBMC from Chinese subjects are underlined by orange circles. Clusters of PBMC predominantly expressing HLA-A2 are outlined by blue dashed vertical bars and the cluster containing PBMC predominantly not expressing HLA-A2 is outlined by a dashed green bar. Bottom panel Matrix B

Fig. 2

Example of ex vivo testing of reactivity by ICS. PBMC from donor #6 were stimulated ex vivo and analyzed after 6 h with pool C4 (a), pool 30 (b) or peptide 294 (c). d Negative control (stimulation with gp100:209–217). In the upper right corner is shown the percent of CD3+CD8+ and IFN-γ+ cells

Fig. 3

Example of epitope validation applying ICS after IVS to increase the frequency of epitopes-specific T cells. PBMC from donor #8 were tested after IVS at day 1 with pool A4 (a). At day 7 the same culture was stimulated with several overlapping pools of which pool 11 tested positive (b). Based on this result, at day 9 the same culture was tested with the corresponding single peptide corresponding according to the two dimensional matrix to LMP2-104 (c). d Negative control (stimulation with gp100:209–217) of the same culture at day 7. In the upper right corner the percent of CD3+CD8+ and IFN-γ+ cells is shown

Fig. 4

Example of assignment of HLA/epitope association based on parallel stimulation of T cell culture with HLA partially matched and mismatched heterologous LCL exogenously loaded with the target epitope. In this case, PBMC from donor 1 underwent IVS with pool B5. After 7 days the cultures were tested by ICS for reactivity toward a panel of partially matched and unmatched LCL pulsed with LMP:145 peptide. Shown is the response to LCL CL049 pulsed with LMP: 145 (a) or with an irrelevant peptide (b) which shares the HLA-A*0205/B*58 alleles with donor 1. No response could be elicited using other LCL including the unmatched CL274 pulsed with LMP:145 (c) or an irrelevant epitope (d)