Use of Epstein-Barr virus-transformed, autologous B-lymphoblastoid cells as antigen-presenting cells for establishment and maintenance of dengue virus-specific, human cytotoxic T lymphocyte clones

Abstract

We have been maintaining dengue virus specific CD8+ cytoxic T lymphocyte (CTL) clones by repeated stimulation using autologous peripheral blood mononuclear cells (PBMC) as antigen presenting cells (APCs). In the present study, Epstein-Barr virus (EBV)-transformed autologous lymphoblastoid cell lines (LCL) were compared with autologous PBMC as APCs for long term culture of a dengue virus-specific, HLA class I-restricted CD8+ CTL clone CB2.8. We substituted autologous LCL for autologous PBMC and maintained CB2.8 for several months. CB2.8 cultured using LCL as APCs maintained antigen specific cytolytic activity. No demonstrable difference in the specificity or in the level of cytolytic activity against a panel of target cells was noted between the CB2.8 maintained with LCL and those maintained with PBMC. Lysis of the target cells was blocked by the anti-HLA-class I antibody indicating that HLA class I-restriction was also maintained. We then compared autologous LCL with autologous PBMC in the establishment of CD4 + CTL clones from the PBMC of a dengue-1 immune donor. Dengue 1-specific clones were derived from limiting dilution cultures using either type of APCs. Similar numbers of dengue virus-specific CD4+ CTL clones were established using LCL or PBMC as APCs. These results indicate that autologous LCL act as APCs for long term culture of virus-specific CTL clones and represent a cost effective alternative to repeated collection of PBMC.