FUNCTIONAL EXPRESSION OF HUMAN-CD28 IN MURINE T-CELL HYBRIDOMAS

CD28 is a 44 kDa Ig superfamily cell surface molecule expressed on mos t mature T cells. Through its interaction with the recently identified B7/BB1 counter-receptor, it is believed to play an important role as a co-stimulator of T cells along with the TCR-CD3 complex. Activation of T cells with CD28 mAbs synergizes with TCR-CD3 and CD2 stimulation, resulting in long term T cell proliferation, differentiation of cytot oxic T cells and production of large amounts of cytokines. In order to further delineate the role of CD28 in signal transduction and T cell activation, human CD28 was transfected into CD3+ murine T cell hybrido mas. High levels of cell surface CD28 expression was achieved by proto plast fusion. The transfected molecule retained all the native CD28 mA b epitopes found on human T cells. In these transfectants, CD28 mAbs, similarly to CD3 mAbs, were able to induce Ca2+ mobilization, IL-2 pro moter induction (measured as beta-galactosidase activity in T cells hy bridomas pre-transfected with the IL-2-lac Z reporter gene), IL-2 secr etion, TNF alpha production and apoptosis (observed as growth arrest a nd genome fragmentation). The parental host cells, or cells transfecte d with vector alone, responded only to mAbs to CD3. IL-2 secretion in the transfectants was obtained using either an IgM mAb to CD28 or IgG mAbs presented on the surface of IgG-FcR+ B lymphoma cells. Optimal ac tivation via CD28 was inhibited by suboptimal concentrations of solubl e CD3 mAb, suggesting an interaction between the two pathways. The imm unosuppressive drugs Cyclosporin A and FK506 completely blocked CD28 a nd CD3 mediated IL-2 production in these transfectants whereas rapamyc in had only a partial inhibitory effect. Finally, since the transfecte d human CD28 molecule confers full functional responsiveness to the, m urine T cell hybridomas without the need for costimulators such as PMA , this model is ideal for studying the structure-function relationship s of the CD28 molecule as well as the transmembrane and cytoplasmic as sociations implied in CD28 signaling.