The cytoplasmic domain of CD28 is both necessary and sufficient for costimulation of interleukin-2 secretion and association with phosphatidylinositol 3'-kinase.
T-cell activation requires two signaling events. One is provided by the engagement of the T-cell antigen receptor, and the second represents a costimulatory signal provided by antigen-presenting cells. CD28 mediates a costimulatory signal by binding its ligands, B7-1 and B7-2, on antigen-presenting cells, but the signaling pathway activated by CD28 has not been identified. A homologous molecule, CTLA-4, expressed on activated T cells, also binds to B7-1 and B7-2, but whether it has a signaling function is not known. We performed a structure-function analysis of CD28 to identify the functional domain which activates signal transduction. Truncation of the 40-amino-acid CD28 cytoplasmic tail abrogated costimulatory signaling. Chimeric constructs containing the extracellular and transmembrane regions of CD8 linked to the cytoplasmic region of CD28 had a costimulatory signaling function. Similar chimeras containing the cytoplasmic tail of CTLA-4 did not signal. Thus, the cytoplasmic region of CD28, but not CTLA-4, is sufficient to mediate costimulatory signaling. In addition, after CD28 stimulation, the p85 subunit of phosphatidylinositol 3'-kinase and phosphatidylinositol 3'-kinase activity were found in CD28 immunoprecipitates. The CD8-CD28 chimera, which has a costimulatory signaling function, associates with p85, while the nonfunctioning CD8-CTLA-4 chimera and a CD8-zeta chimera do not associate with p85. These results suggest that phosphatidylinositol 3'-kinase is specifically activated by CD28 and may mediate proximal events in the costimulatory signaling pathway regulated by CD28.
Stein
P H
PH
Howard Hughes Medical Institute, Department of Medicine, University of California, San Francisco 94143.
Fraser
J D
JD
Weiss
A
A
D000069594:Abatacept; D000595:Amino Acid Sequence; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000096:biosynthesis / Q000378:metabolism; D018106:CD28 Antigens / Q000096:biosynthesis / Q000378:metabolism*; D060908:CTLA-4 Antigen; D002460:Cell Line; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D018796:Immunoconjugates; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000378:metabolism; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018106:CD28 Antigens; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D018796:Immunoconjugates; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D000069594:Abatacept; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)