T cell activation is regulated by a balance between positive and negative signals mediated by a series of costimulatory ligand–receptor pairs. While costimulatory pathways involving molecules such as CD28, inducible costimulator (ICOS), 4–1BB, and CD40L are essential coactivators of proliferation, cytokine production, and migration, CTLA-4 and PD-1, homologues of CD28 family of cell surface receptors, provide strong negative signals (1
). In particular, CTLA-4 coligation with TCR has been shown to inhibit IL-2 production, cell cycle progression, and proliferation. In vivo, CTLA-4 knockout mice manifest fatal lymphoproliferative phenotype and die within 4 wk of life (2
). In spite of the importance of CTLA-4 in regulation of the immune system, however, the molecular basis for this inhibitory function is still largely unknown.
The interface between the T cell and APC membranes form the hot spot for T cell activation, a highly organized ultra structure which is called the immunological synapse (4
). The membrane lipid raft is biochemically characterized as a detergent insoluble glycosphingolipid enriched microdomain that is considered as an essential component of the immunologic synapse (6
). After TCR engagement, molecules critical for mediating activation signals, such as Lck, Fyn, protein kinase C (PKC)θ, phospholipase C (PLC)γ, and linker for activation of T cells (LAT), are all recruited to the raft aggregates at the T cell–APC contact area. More importantly, the TCR itself dynamically associates with raft upon receptor cross-linking, allowing the accessibility of signaling molecules to TCR facilitating the biochemical signals of activation. As such, pharmacological disruption of the rafts abrogate early signal events in T cell activation such as calcium influx, supporting the essential role of raft integrity for T cell activation (7
Recently, Darlington et al. (8
) reported that CTLA-4 is recruited to the raft during negative signaling. However, the functional importance of this association was unclear. In the present study, we analyzed the biochemical consequences of the colocalization of CTLA-4 to the rafts. We report that CTLA-4 forms a molecular complex with phosphorylated TCRζ within the rafts. Furthermore, the overall levels of TCRζ, most prominently phosphorylated TCRζ, in the rafts is regulated by CTLA-4 as assessed in CTLA4KO T cells and wild-type T cells after CTLA-4 cross-linking. Together, these results support a proximal role for CTLA-4 in attenuating TCR-mediated signal transduction which emphasizing that both positive and negative signaling events impact on the early events of T cell activation.