TCRαβ-Independent CD28 Signaling and Costimulation Require Non-CD4-Associated Lck

Whether the sequelae of signals generated through CD28 either directly or in circumstances of costimulation require proximal events mediated by p56lck remains contentious. We demonstrate that CD4−, but not CD4+ clonal variants respond to CD28-specific mAb with both early and late indicators of activation. Forced expression of A418/A420-mutated CD4 or wild-type CD4 in the CD4− variant recapitulated the CD28-mediated responses of the CD4− and CD4+ variants, respectively. The implicated involvement of non-CD4-associated Lck is formally demonstrated by overexpressing S20/S23 Lck or wild-type Lck in CD4+ variants. The former, but not latter, rescues direct CD28 signaling, and supports costimulation. The results demonstrate that constitutive levels of non-CD4-associated Lck functionally limit CD28-mediated signaling.