Cytokine production and cytolytic mechanism of CD4+cytotoxic T lymphocytes in ex vivo expanded therapeutic Epstein-Barr virus–specific T-cell cultures

Ex vivo expanded Epstein-Barr virus (EBV)-specific T cells have been successfully applied clinically for adoptive immunotherapy. However, the role of CD4(+) T cells in the therapeutic T-cell culture has not been established for the reconstitution of EBV-specific immunity. We isolated and characterized CD4(+) T-cell lines from the ex vivo T-cell cultures. Monoclonal line PD-F4 and oligoclonal lines ND-R4 and TD-B4 were CD3(+)CD4(+)CD8(-). Cytolytic tests with targets of mismatched major histocompatibility complex (MHC) and anti-MHC antibodies confirmed that the cytotoxicity of these CD4(+) cells was restricted by MHC class II. Single cells of ND-R4 expressed interferon-gamma (IFN-gamma, or interleukin 4 (IL-4), but rarely coexpressed these 2 cytokines. In contrast, PD-F4 coexpressed IFN-gamma, IL-2, and IL-4. Kinetic studies with PD-F4 showed that expression of the 3 cytokines plateaued 5 hours upon stimulation and was then drastically reduced, with a pattern consistent with independent modulation and differential off-cycle signal requirements. The cytotoxicity of these CD4(+) cells was largely resistant to brefeldin A, an inhibitor for cytolytic pathways by Fas-ligand family molecules. Although sensitive to concanamycin A and ethyleneglycotetraacetic acid, which inhibit cytotoxicity by granule exocytosis, the CD4(+) cytotoxic T lymphocytes (CTLs) did not express perforin, suggesting a cytotoxic mechanism independent of perforin although involving exocytosis. Flow cytometric analysis showed that the CD4(+) CTLs expressed granulysin, a recently identified cytolytic molecule associated with exocytotic cytolytic granules. These data suggested that CD4(+) T cells in the therapeutic B-lymphoblastoid cell lines-primed T-cell culture are diverse in producing T(H)1 and T(H)2 cytokines, and may exert specific cytotoxicity via exocytosis of granulysin.