title: 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.
abstract: 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.
authors.affiliation: Queensland Institute of Medical Research, Herston, Brisbane, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Khanna
authors.forename: C A
authors.initials: CA
authors.lastname: Jacob
authors.forename: S R
authors.initials: SR
authors.lastname: Burrows
authors.forename: M G
authors.initials: MG
authors.lastname: Kurilla
authors.forename: E
authors.initials: E
authors.lastname: Kieff
authors.forename: I S
authors.initials: IS
authors.lastname: Misko
authors.forename: T B
authors.initials: TB
authors.lastname: Sculley
authors.forename: D J
authors.initials: DJ
authors.lastname: Moss
mesh_terms: D000956:Antigens, Viral / Q000032:analysis*; D001402:B-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D000939:Epitopes / Q000032:analysis; D019309:Epstein-Barr Virus Nuclear Antigens; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology*; D007148:Immunoglobulin mu-Chains / Q000276:immunology; D007156:Immunologic Memory / Q000276:immunology; D011995:Recombination, Genetic; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014615:Vaccinia / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D000939:Epitopes; D019309:Epstein-Barr Virus Nuclear Antigens; D007075:Immunoglobulin M; D007148:Immunoglobulin mu-Chains