Functional expression of human CD28 in murine T cell hybridomas.
CD28 is a 44 kDa Ig superfamily cell surface molecule expressed on most 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 cytotoxic 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 hybridomas. High levels of cell surface CD28 expression was achieved by protoplast fusion. The transfected molecule retained all the native CD28 mAb epitopes found on human T cells. In these transfectants, CD28 mAbs, similarly to CD3 mAbs, were able to induce Ca2+ mobilization, IL-2 promoter induction (measured as beta-galactosidase activity in T cells hybridomas pre-transfected with the IL-2-lac Z reporter gene), IL-2 secretion, TNF alpha production and apoptosis (observed as growth arrest and genome fragmentation). The parental host cells, or cells transfected 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 activation via CD28 was inhibited by suboptimal concentrations of soluble CD3 mAb, suggesting an interaction between the two pathways. The immunosuppressive drugs Cyclosporin A and FK506 completely blocked CD28 and CD3 mediated IL-2 production in these transfectants whereas rapamycin had only a partial inhibitory effect. Finally, since the transfected human CD28 molecule confers full functional responsiveness to the murine T cell hybridomas without the need for costimulators such as PMA, this model is ideal for studying the structure-function relationships of the CD28 molecule as well as the transmembrane and cytoplasmic associations implied in CD28 signaling.
Couez
D
D
Unité 119 de l'Institut National de la Santé et de la Recherche Médicale, Marseille, France.
Pagès
F
F
Ragueneau
M
M
Nunès
J
J
Klasen
S
S
Mawas
C
C
Truneh
A
A
Olive
D
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D017209:Apoptosis; D018106:CD28 Antigens / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D018076:DNA, Complementary; D006801:Humans; D006825:Hybridomas; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D017252:CD3 Complex; D018076:DNA, Complementary; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D002118:Calcium