HLA class II-restricted proliferative and cytotoxic T cell (CTL) responses to B cell chronic lymphocytic leukaemia (B-CLL) can be generated using autologous dendritic cells (DCs) pulsed with tumour cell lysate. In this study a number of different approaches were used to optimize further the in vitro system. First, the effects of a variety of maturation agents were studied. The addition of TNF-alpha, polyriboinosinic polyribocytidylic acid (Poly(I:C)) and LPS to autologous DCs resulted in the emergence of only a small percentage of CD83+ DCs, IFN-alpha having no demonstrable effect. Only the addition of Poly(I:C) to DCs resulted in modestly increased specific cytotoxicity to B-CLL targets, IFN-alpha and LPS having no effect. Secondly, T cells were pretreated with IL-15, prior to culturing with lysate-pulsed autologous DCs. A significant increase in T cell activation (P = 0.038), IFN-gamma secretion (P = 0.030) and specific cytotoxicity to B-CLL targets (P = 0.006) was demonstrated compared to untreated T cells. Thirdly, monocyte derived DCs electrofused with B-CLL B cells were compared with lysate-pulsed DCs. T cells stimulated by fused DCs generated higher levels of specific cytotoxicity to autologous B-CLL B cell targets than those stimulated by lysate pulsed DCs (P = 0.013). Blocking studies demonstrated inhibition of this cytotoxicity by both anti-CD4 (P = 0.062) and anti-CD8 monoclonal antibodies (P = 0.018), suggesting the generation of both HLA class I- and HLA class II-restricted CTL responses. In summary, in vitro B-CLL-specific T cell responses can be enhanced further by preincubating T cells with IL-15 and using autologous fused DC-B-CLL hybrids instead of autologous lysate-pulsed DCs. These preliminary data require confirmation with larger numbers of patients. Such an approach, however, may eventually provide effective immunotherapy for treatment of B-CLL.