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

Immunology. 1991 Nov;74(3):504-10.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Viral / analysis*
  • B-Lymphocytes / immunology*
  • Cells, Cultured
  • Epitopes / analysis
  • Epstein-Barr Virus Nuclear Antigens
  • Humans
  • Immunoglobulin M / immunology*
  • Immunoglobulin mu-Chains / immunology
  • Immunologic Memory / immunology
  • Recombination, Genetic
  • T-Lymphocytes, Cytotoxic / immunology*
  • Vaccinia / immunology*


  • Antigens, Viral
  • Epitopes
  • Epstein-Barr Virus Nuclear Antigens
  • Immunoglobulin M
  • Immunoglobulin mu-Chains