Generation of Human B-Cell Lines Dependent on CD40-Ligation and Interleukin-4

WHERE WE STARTED The description of factor-dependent cytotoxic T-cell lines in the late 1970s transformed T-cell biology (1). Among other events, it led to the cloning of a cDNA encoding IL-2 (2). It also led to the identification of T-cell subsets and formulation of the Th1/Th2 concept in the 80s (3). However, comparable advances in B-cell biology were lacking, partly because of the lack of availability of factor-dependent Bcell lines. This was the case despite the fact that B-cell-specific trophic factors, including BSF (B-cell stimulation Factor), BCGF (B-cell growth factor), and BCDF (B-cell differentiation factor) had been described in the supernatants of activated T cells. The cloning at DNAX, our sister institute acquired by Schering–Plough, of a cDNA encoding BSF-1, later renamed IL4, in mouse (4) and in human (5) was a first step forward to the definition of the molecules controlling B-cell growth and differentiation. In our laboratory, based in Dardilly near Lyon (France), we found that cultured purified human B-cells triggered with anti-B-cell receptor (BCR) and IL-4 resulted in significant B-cell proliferation as measured by tritiated thymidine counts, a common way of measuring Bcell proliferation in the 1980–1990s (6). These cultures yielded more B-cells than did naive cultures or those exposed to antiBCR alone or IL-4 alone. Yet, these cultures established with anti-BCR plus IL-4 yielded less viable B-cells than were input. Thus, we, B-cell biologists had not yet been able to reproduce with B-cells the factor-dependent growth of T cells that our colleagues T-cell biologists have been able to achieve. FEEDER CELLS AND NEW MONOCLONAL ANTIBODIES YIELD MORE ROBUST B-CELL CULTURES A possible explanation for our lack of success was the absence of feeder cells, which had become part of the T-cell culture system and proved necessary to allow for the expansion of human T-cell lines and clones. Meanwhile, Kevin Moore and his colleagues at DNAX, cloned a human cDNA coding for FcγRII/CD32 and found that FcγRII/CD32-transfected fibroblast cell lines could present monoclonal antibodies in a manner that allowed for cross-linking of the target molecule of the relevant cell (7, 8). More specifically, antibodies to the T-cell CD3 complex presented by these transfected cells together with IL-2 could induce prolonged T-cell proliferation (9). Thus, we wondered whether the presentation of monoclonal antibodies specifically directed at B-cell surface molecules in the presence of B-cell tropic cytokines would lead to the proliferation and expansion of Bcells. By the end of the 80s, we, investigators from Schering–Plough/DNAX had cloned cDNAs encoding human GM-CSF (10), IL-4, IL-5 (5, 6), and FcgR/CD32 (8). We had also generated a number of monoclonal antibodies that would recognize Bcells including a CD40 antibody (11) and an anti-B7 antibody now known as CD86 (12). When Paolo de Paoli came to our lab to perfect his flow cytometry skills, he took a side project to refine methods for culturing sorted B-cells using both classical and new approaches, including the addition of a feeder-layer of CD32/FcγR-transfected cells as discussed above (9). To this end, 96-well-plate microwells were first seeded with the irradiated fibroblast line. A few thousand B-cells were then added along with a few selected monoclonal antibodies with or without IL-2 or IL-4. Cultures were harvested 3–5 days later after a brief pulse with tritiated thymidine. It very quickly became apparent that the combination of the CD40 antibody Mab 89 (11) and IL-4 could induce unusually strong B-cell proliferation. The wellknown CD40 antibody G28-5 made by Ed Clark and Jeff Ledbetter also proved highly effective in this system (13, 14). Curiously, IL-2 was unable to enhance CD40induced B-cell proliferation, although it did enhance the proliferation of B-cells activated through their BCR. Furthermore, the fibroblast layer provided some feeder effect, as cross-linking the CD40 antibody on plastic was never as effective in inducing prolonged B-cell proliferation as presenting it with the CD32-transfected fibroblast.