Expression of Epstein-Barr virus nuclear antigens in anti-IgM-stimulated B cells following recombinant vaccinia infection and their recognition by human cytotoxic T cells

• Published in: Immunology Vol. 74; no. 3; pp. 504 - 510
• Main Authors: KHANNA, R, JACOB, C. A, BURROWS, S. R, KURILLA, M. G, KIEFF, E, MISKO, I. S, SCULLEY, T. B
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01-11-1991
• Subjects: Antigens, Viral - analysis; B-Lymphocytes - immunology; Biological and medical sciences; Cells, Cultured; Epitopes - analysis; Epstein-Barr Virus Nuclear Antigens; Fundamental and applied biological sciences. Psychology; Humans; Immunoglobulin M - immunology; Immunoglobulin mu-Chains - immunology; Immunologic Memory - immunology; Microbiology; Recombination, Genetic; T-Lymphocytes, Cytotoxic - immunology; Techniques used in virology; Vaccinia - immunology; vaccinia virus; Virology; Gammaherpesvirinae; Human; Chordopoxvirinae; Antigenic determinant; Recombinant virus; Herpesviridae; Orthopoxvirus; Cell nucleus; Cytotoxicity; B-Lymphocyte; Epstein Barr virus; Virus; Antigen; Vaccinia virus; Cell line; Poxviridae; T-Lymphocyte; Infected cell
• ISSN: 0019-2805; 1365-2567

Cytotoxic T lymphocytes (CTL) recognizing Epstein-Barr virus (EBV) nuclear antigens (EBNA) are an important host defence mechanism in restricting the proliferation of EBV-infected B cells. Previously, B-type lymphoblastoid cell lines (LCL) infected with vaccinia recombinants encoding for the EBNA proteins have been used to identify A-type-specific CTL epitopes. However, to localize the CTL epitopes encoded by both A- and B-type transformants, B-type LCL are an inappropriate host for vaccinia. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-IgM (mu-chain specific)-stimulated human B cells allowed vaccinia virus to replicate more efficiently than either phytohaemagglutinin-stimulated lymphocytes (PHA blasts) or CTL and expressed EBNA proteins following recombinant vaccinia infection. Furthermore, the presentation and recognition of target epitopes expressed on vaccinia-infected anti-mu-stimulated B cell blasts were comparable to that on similarly infected LCL. Anti-mu-stimulated B cells were used to define the CTL epitopes recognized by a panel of CTL clones from an EBV-immune donor. Using recombinant vaccinia-infected anti-mu-stimulated B cells, the CTL response from this donor was mapped to the EBNA6 protein. Most importantly, in vitro stimulation of unfractionated mononuclear cells with vaccinia-infected anti-mu B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 localized the epitope for the majority of the EBNA6-specific CTL clones to the sequence EENLLDFVRFM, apparently in association with HLA-B44. This work clearly demonstrates that anti-mu-stimulated B cells not only provide an efficient model for localizing the CTL epitope(s) but also raises the possibility of reactivating a memory T-cell response to any gene product expressed by recombinant vaccinia.