Ex vivo EXPANSION AND CHARACTERIZATION OF EPSTEIN-BARR VIRUS-SPECIFIC CD4-POSITIVE BULK CYTOTOXIC T LYMPHOCYTES

Epstein-Barr virus (EBV) -specific cytotoxic T lymphocytes (CTLs) were induced from PBMCs of a sero-positive healthy donor by stimulation with an autologous EBV-transformed B-lymphoblastoid cell line (EBV-LCL). CD4+ lymphocytes with high cytotoxic activities were found in the cultured fraction. The purified CD4+ bulk CTLs were expanded with immobilized anti-CD3 and anti-CD28 antibodies in the presence of IL-2. The CD4+ CTLs showed vigorous proliferation, up to 6, 000-fold cumulative expansion. They maintained high killing activity against autologous target cells while exhibiting no NK and LAK activities, even after expansion. These data indicate that ex vivo expansion with immobilized antibodies is useful to obtain a large number of CTLs without losing their specific activities. The cytotoxity mediated by the CD4+ bulk CTLs seemed to depend mainly on the perforin/granzyme pathway because a H+-ATPase inhibitor, concanamycin A, strongly inhibited the killing. The CD 4+ CTLs proliferated in response to autologous monocyte-derived dendritic cells pulsed with one of the EB viral proteins, EBNA 1. This finding suggests the existence of EBNA 1-recognizing clone (s) in the cell fractions. When stimulated with autologous EBV-LCL or activated with PMA and ionomycin, the CD4+ CTLs predominantly produced IFN-γ, not IL-4, indicating that they belong to the T helper 1 (Th1) type.