The cytoplasmic domain of CD28 is both necessary and sufficient for costimulation of interleukin-2 secretion and association with phosphatidylinositol 3'-kinase.

Abstract

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-; 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.

A functional role for CD28 costimulation in tumor recognition by single-chain receptor-modified T cells

“…26,27,31 Nevertheless, the signaling events associated with the YMNM motif in CD28 costimulation remain controversial, and we did not detect any changes in PI3 kinase activity between mock-transduced or scFv-expressing T cells (data not shown). Several groups have investigated the effect of the CD28 Y170F mutation on cytokine secretion.…”

“…To determine whether the latter was true, mutant scFv receptors were expressed in primary mouse T cells by retroviral transduction, including those containing mutations within the cytoplasmic PXXP or YMNM signaling motifs of CD28, known to be critical for CD28 signal transduction. [26][27][28] In this study, mouse T lymphocytes expressing the scFv-CD28-(P187/190)-z or scFv-CD28-(Y170F)-z mutant receptors demonstrated reduced proliferation and a significant reduction in both IFN-g and GM-CSF secretion after antigen ligation in vitro. Consistent with an important role for IFN-g in vivo, such T cells demonstrated a decreased ability to eradicate tumor in vivo compared to T cells expressing the wild-type scFv-CD28-z receptor.…”

CD28 and Inducible Costimulatory Protein Src Homology 2 Binding Domains Show Distinct Regulation of Phosphatidylinositol 3-Kinase, Bcl-xL, and IL-2 Expression in Primary Human CD4 T Lymphocytes

“…Ligation of this molecule augmented PMA/ionomycin-stimulated IL-2 expression in Jurkat as reported for similar molecules (48), however this construct completely failed to costimulate CD4 T lymphocytes, suggesting that the identity of the extracellular domain is more important in primary T cells than Jurkat. We have confirmed that the mCD28/hCD28 chimera accurately models native human CD28 in terms of both signal transduction and costimulation of cytokine production, demonstrating the validity of this approach.…”

Ras/PI3Kinase/cofilin‐independent activation of human CD45RA+ and CD45RO+ T cells by superagonistic CD28 stimulation

“…Uncontrolled PI3Kinase signaling contributes to autoimmunity [26]. In human T cells, the costimulatory receptor CD28 associates with PI3Kinase through the YXXM motif [27][28][29][30]. It was recently demonstrated that, following costimulation, the binding of PI3Kinase to CD28 is essential for IL-2 gene transcription, but IL-2 mRNA stability is mediated via a PI3Kinase-independent pathway [31].…”