In this study we provide evidence that a human breast carcinoma cell line, MDA-MB-231 (MDA), can be made immunogenic following B7 transfection and that full T cell activation is obtained through cooperation of T-B lymphocytes via CD40-CD40L interactions. Tumor cells transfected with either B7 gene (MDAB7), neomycin-resistant gene only (MDAneo), or untransfected (MDA) were used in an allogeneic mixed lymphocyte tumor culture (MLTC) to investigate their ability to stimulate T cell proliferation and generate cytotoxic T lymphocytes (CTL). MDAB7 induced moderate T cell proliferation while MDAneo or MDA did not. Substantial T cell proliferation and de novo generation of cytolytic T cells was obtained only in response to MDAB7 when B cells were present during the MLTC. CD8+-purified T + B cells proliferated to a greater extent than whole T cell populations + B or CD4+ + B in response to MDAB7. Addition of alpha-B7-1 or alpha-CD40 in the MLTC inhibited T cell proliferation by 65 and 40%, respectively, whereas T cell proliferation and generation of CTL was completely abrogated when MLTC was performed in the presence of both antibodies. These data suggest that the engagement of CD40L on T cells with CD40 on B cells provides a costimulatory signal which, in synergism with TCR-dependent MDAB7-T cell recognition (signal 1) and B7/CD28 interactions (signal 2), leads to full T cell activation.