Profound enhancement of T cell activation mediated by the interaction between the TCR and the D3 domain of CD4

CD4 plays an important role in the activation and development of CD4(+) T c ells, This is mediated via its bivalent interaction with MHC class II, mole cules and the TCR:CD3 complex through p56(lck). Recent studies have implica ted a third site of interaction between the membrane-proximal extracellular domains of CD4 and the TCR, Due to these multiple interactions, direct evi dence for the functional importance of this extracellular association has r emained elusive. Furthermore, the residues that mediate this interaction ar e unknown. In this study, we analyzed the function of 61 CD4 mutants. Alani ne substitution of just 2 residues, either Q114/F182 or F182/F201, which ar e partially buried and located close to the D2/D3 interface, completely abr ogated CD4 function. Direct evidence for the functional importance of TCR:C D4,D3 interaction was obtained using an anti-CD3fos:anti-CD4jun-bispecific Ab, Surprisingly, it induced strong T cell activation in hybridomas transfe cted,vith cytoplasmic-tailless CD4, despite the lack of association with ei ther p56(ick) or MHC class II molecules. However, this effect was completel y abrogated with the CD4 mutants Q114A/F182A or F182A/F201A, These data dem onstrate that TCR:CD4,D3 interaction can have a profound effect on T cell a ctivation and obviates the need for receptor oligomerization.