Full CD3/TCR activation through cholesterol-depleted lipid rafts

Exogenous bacterial sphingomyelinase (SMase) and C6-Ceramides (C6-Cer) considerably lower buoyant cholesterol on sucrose density-gradient (at least 55% less cholesterol). In opposition, short C2-Cer fails to displace buoyant cholesterol. Note that neither SMase nor C6-Cer delocalize raft markers (Lck, LAT, CD55, and GM1). They are still anchored in ceramides-rich/cholesterol-poor domains, demonstrating that cholesterol is not necessary for their buoyancy. SMase-treated cells, i.e. cells exhibiting cholesterol-depleted rafts, optimally transmit CD3-induced phosphorylations (tyrosine, threonine, and serine). SMase, that extracts and partially displaces buoyant cholesterol, does not inhibit PLCgamma1-LAT interaction, Vav 1 phosphorylation, the actin polymerization, IL-2 and NF-kappaB (EMSA and luciferase assays) activation, and CD25 up-regulation (RT-PCR and cytometry) at all. Nevertheless, Ca(2+) influx and diacylglycerol (palmitoyl-DAG and arachidonoy-DAG) production are lowered. The drop of CD3-induced Ca(2+) influx is due to a strong plasma membrane depolarization because of Cer. The decreased DAG level is a consequence of the drop of intracellular Ca(2+) that is a cofactor for the PLCgamma1. In conclusion, our study challenges the real role of cholesterol-rich rafts in CD3/TCR signaling and suggests that other membrane domains than cholesterol-rich rafts can optimally transmit CD3/TCR signals.