An Epstein-Barr virus-specific cytotoxic T cell epitope in EBV nuclear antigen 3 (EBNA 3).

Abstract

All individuals previously infected with Epstein-Barr virus (EBV) harbor life-long a population of virus-infected B cells whose number appears to be controlled by EBVspecific, HLA-restricted (class I and class II) CTLs (CD4 and CD8) that recognize a functionally defined lymphocyte-determined membrane antigen, LYDMA (1, 2). There are two recognized types of EBV (A and B) that show sequence divergence within the reading frames encoding EBV nuclear antigens (EBNAs) 2, 3, 4, and 6 (3, 4) and differences in the molecular weight of these proteins (4, 5).Our general approach to identifying CTL epitopes has been to isolate EBVspecific T cell clones that recognize autologous A-type transformant but fail to lyse the corresponding B-type transformant (6). The present report defines the first target epitope recognized by EBVspecific CTLs. Our approach to identifying this epitope has been to screen selected peptide sequences from the EBNA proteins using specific CTLs and the autologous B-type transformant.Volume 171 January 1990 [345][346][347][348][349] Materials and Methods Brief Definitive ReportCellLines. EBV transformed cell lines (LCLs) were established either spontaneously (i.e., by transformation with endogenous virus) or by the addition ofexogenous virus (7) . A spontaneous LCL from the EBV seropositive donor LC was established using cyclosporin A (8) . The type of endogenous virus (A or B) in the spontaneous cell lines was determined using Southern analysis (6) and was used to define donor LC as an A-type donor.Cell lines were established using exogenous A-or B-type virus by infecting B lymphocytes from an EBV seropositive donor Lc, with virus recovered from two A-type cell lines, IARC-BL74 (8) and IARC-BL36, (4) and two B-type cell lines, Ag876 (3) and L4 (4) . Southern and Western analyses were used to confirm the presence of A-or B-type virus in the respective LC transformants (designated LC/BL74, LC/BL36, LC/Ag876, and LC/L4) as previously described (6) .CTL Activation and 5/ Cr-release Assay. Lymphocytes from donor LC (HLA Al, B8, B18) were cocultivated with the irradiated autologous A-type LCL, LC/BL74 for 3 d and the activated T cells were cloned in agar and expanded in the presence of IL-2 and specific stimulating cells (2) . T cell clones were screened in the standard 5 'Cr release assay (E/T ratio of 10 :1) against a panel of cell lines including autologous and allogeneic A-and B-type LCLs .Peptide Selection. To identify the CTL epitope expressed on the A-type transformants but not on the autologous B-type transformants, a series of peptides (20-25 amino acids) deduced from the known sequences of the A-type EBNAs 2, 3, 4, 5, and 6 proteins were synthesized (9) and screened on the autologous B-type transformant LC/Ag876 . Peptides selected were

Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation.

“…Allele-specific motifs have been proposed for several mouse MHC types and the HLA-A2 and B27 alldes (47--49, 52). Comparison of the HLA-B8-restricted gp41 epitope 586-593 with peptides containing other HLA-B8-restricted CTL epitopes (8,9,30,53,54) reveals several potential alignments with shared characteristics at selected positions within an eight-to nine-aa region, including a hydrophobic residue (Ala, Leu, Iso, and Val) at position two, a positively charged aa at position 3, and aa with nonpolar or small polar side chains at the two COOH-terminal residues (Table 5). Definitive assignment of an HLA-B8-specific motif will require the determination of minimum effective epitopes for other HLA-BS-restricted epitopes and the use of competition experiments with variant peptides to define aa that interact with either the TCR or HLA molecule (55)(56)(57)(58).…”

A Positive Correlation between the Precursor Frequency of Cytotoxic Lymphocytes to Autologous Epstein‐Barr Virus‐Transformed B Cells and Antibody Titer Level against Epstein‐Barr Virus‐Associated Nuclear Antigen in Healthy Seropositive Individuals

“…Namely, specific killer T cells recognize the membrane antigen that is expressed on the surface of EBNApositive B cells and prevent proliferation of the virus-infected cells. Recently, transformation and recombinant vaccinia virus experiments have revealed that a significant proportion of the killer T cells recognize some members of the EBNA family such as EBNA 2, 3A, 3B, and 3C (5,8,14,25,26). Although there was no direct evidence for the recognition of these EBNA family antigens by the cytotoxic effector cells in our study, the present result may be interpreted that our PF assay reflects the degree of continuous EBNA-positive cell destruction followed by anti-EBNA antibody production in the latent stage of EBV infection.…”

Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy

“…Antigen-specific CTLs were generated from pre-expanded and fresh PBMCs stimulated with autologous mature peptide-pulsed DCs (50 g/ml for 2 h at 37°C; DC/responder ratio 1:10) in 1% ABS Ex-Vivo medium with 10 ng/ml IL7 (day 0; R&D Systems) and 20 IU/ml IL2 (starting on day 7), and harvested for analysis on day 12. The immunodominant HLA-A2-restricted NLVPMVATV peptide from the pp65 protein of CMV (CMVpp65 NLV ) 40 and HLA-B8-restricted FLRGRAYGL peptide from the latent cycle protein EBNA3A of EBV (EBV-EBNA3A FLR ) 41 were selected for this analysis. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 .…”