Establishment and characterization of Epstein-Barr virus-specific human CD4+ T lymphocyte clones

We developed a simple method for establishing Epstein-Barr virus (EBV)-specific, human CD4+ T cell clones. The method originates from our experience that the regression of cell growth in in vitro EBV transformation of B cells occurs when round lymphoid cells appear in the culture. Peripheral blood mononuclear cells (PBMCs) were cultured with EBV, and IL-2 (20 U/ml) was added to the culture on day 17 after the virus addition. The phenotype of the growing cells was CD3+, CD4+, and CD8-. The cells were cytotoxic for autologous lymphoblastoid B cell line (LCL) and EBV-superinfected autologous LCL. The cytotoxic T lymphocytes (CTLs) were confirmed to be CD4+ T cells but not CD8+ T cells in the culture. CTL clones were established by a limiting dilution method. All the CTL clones had the phenotype of CD3+, CD4+ and CD8-, and proliferated in response to autologous LCL. They produced interferon (IFN)-gamma, interleukin 2 (IL-2) and tumour necrosis factor (TNF)-beta but not IL-4. All but one clone responded to both autologous, EBV-superinfected and non-superinfected LCLs. Proliferative and cytotoxic responses to allogenic LCLs were heterogeneous. These results suggest that this method induces heterogeneous, EBV-specific CD4+ CTL clones and is useful for analysis of CD4+ T cells in EBV infections.