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

Abstract

Ligation of either CD28 or inducible costimulatory protein (ICOS) produces a second signal required for optimal T cell activation and proliferation. One prominent difference between ICOS- and CD28-costimulated T cells is the quantity of IL-2 produced. To understand why CD28 but not ICOS elicits major increases in IL-2 expression, we compared the abilities of these molecules to activate the signal transduction cascades implicated in the regulation of IL-2. Major differences were found in the regulation of phosphatidylinositol 3-kinase activity (PI3K) and c-jun N-terminal kinase. ICOS costimulation led to greatly augmented levels of PI3K activity compared with CD28 costimulation, whereas only CD28 costimulation activated c-jun N-terminal kinase. To examine how these differences in signal transduction affected IL-2 production, we transduced primary human CD4 T cells with a lentiviral vector that expressed the murine CD28 extracellular domain with a variety of human CD28 and ICOS cytoplasmic domain swap constructs. These domains were able to operate as discrete signaling units, suggesting that they can function independently. Our results show that even though the ICOS Src homology (SH) 2 binding domain strongly activated PI3K, it was unable to substitute for the CD28 SH2 binding domain to induce high levels of IL-2 and Bcl-xL. Moreover, the CD28 SH2 binding domain alone was sufficient to mediate optimal levels of Bcl-xL induction, whereas the entire CD28 cytoplasmic tail was required for high levels of IL-2 expression. Thus, differences within their respective SH2 binding domains explain, at least in part, the distinct regulation of IL-2 and Bcl-xL expression following ICOS- or CD28-mediated costimulation.

The p110δ Isoform of Phosphoinositide 3-Kinase Controls Clonal Expansion and Differentiation of Th Cells

“…Anti-CD28 ligation using Abs in vitro can stimulate signals that cannot be induced by the physiological ligands (32). Although the capacity of CD28 to rescue some signals appears to be PI3K independent, the PI3K signaling pathway may be required by other costimulatory receptors such as ICOS and OX40, and these can contribute to clonal expansion and differentiation in vivo (33,34). A failure of p110␦ D910A/D910A T cells to form functional conjugates with APCs or a state of anergy in a population of p110␦ D910A/D910A T cells may also help explain the reduced response to stimulation with APCs.…”

Intracellular Signals of T Cell Costimulation

“…The CD28 cytoplasmic domain that binds with a cytosolic protein called translationally controlled tumor protein (TCTP), a novel multifunctional antiapoptotic bcl-xL-interacting protein to regulate cell survival (72,73). In particular, one tyrosine (Y170 in mouse CD28, Y173 in human CD28) that is important in PI3K activation permits CD28 to recruit SH2-containing signaling molecules, including PI3K, Grb2, and Gads that control the regulation of bcl-xL (21,74). However, whether or not this regulation was dependent on mammalian target of rapamycin pathway is still controversial (13,75).…”

The role of apoptosis in the development and function of T lymphocytes

“…In vitro studies have shown that CD28 signaling specifically upregulates the expression of the anti-apoptotic protein Bcl-x L , but not Bcl-2 [222]. Point mutation analysis has shown that tyrosine residue Y170 on the cytoplasmic tail of CD28 is essential for the recruitment of PI-3K, which induces the expression of Bcl-x L [223][224][225]. CD28 carrying a mutation at Y170 can promote T cell activation, proliferation and IL-2 production.…”