Recombinase-deficient T cell development by selective accumulation of CD3 into lipid rafts

The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrangement at the TCR beta chain locus by signaling in a ligand-independent fashion. The TCR beta chain associates with the invariant pre-T alpha (pT alpha) chain, which bears specific charged residues in the extracellular portion mediating pre-TCR self-oligomerization. In recombinase-deficient thymocytes, calnexin (CNX) associated with CD3 chains is inefficiently retained in the endoplasmic reticulum (ER) and weakly expressed in the plasma membrane. Deliberate cross-linking of CNX/CD3 complexes mimics pre-TCR signaling. Here, we show that, analogously to the pT alpha chain, surface CNX is palmitoylated and that CD3 prominently accumulated in lipid rafts upon cross-linking. Mutant CNX isoforms devoid of ER retention determined pre-TCR-like signaling and simulated beta selection only when stably translocating CD3 to lipid rafts. Inclusion of the palmitoylated cytoplasmic tail from the pT alpha chain in recombinant CNX strikingly improved the pre-TCR-like signaling efficiency of CNX/ CD3 in rafts. This study indicates that lipid rafts in the plasma membrane represent proficient microdomains for the initiation of pre-TCR signaling, and supports the view that beta selection by oligomerized pre-TCR is implemented by the pT alpha cytoplasmic tail.