Amino acid substitutions in the putative MHC class II "dimer of dimers" interface inhibit CD4(+) T cell activation

Activation of T lymphocytes is dependent on multiple ligand-receptor intera ctions. The possibility that TCR dimerization contributes to T cell trigger ing was raised by the crystallographic analysis of MHC class II molecules. The MHC class II molecules associated as double dimers, and in such a way t hat two TCR (and two CD4 molecules) could bind simultaneously. Several subs equent studies have lent support to this concept, although the role of TCR cross-linking in T cell activation remains unclear. Using DRA cDNAs modifie d to encode two different C-terminal tags, no evidence of constitutive doub le dimer formation was obtained following immunoprecipitation and Western b lotting from cells transiently transfected with wild-type DRB and tagged DR A constructs, together with invariant chain and HLA-DM, To determine whethe r MHC class II molecules contribute actively to TCR-dependent dimerization and consequent T cell activation, panels of HLA-DR1 beta and H2-E-k cDNAs w ere generated with mutations in the sequences encoding the interface region s of the MHC class II double dimer, Stable DAP.3 transfectants expressing t hese cDNAs were generated and characterized biochemically and functionally. Substitutions in either interface region I or III did not affect T cell ac tivation, whereas combinations of amino acid substitutions in both regions led to substantial inhibition of proliferation or IL-2 secretion by human a nd murine T cells. Because the amino acid-substituted molecules were serolo gically indistinguishable from wild type, bound antigenic peptide with equa l efficiency, and induced Ag-dependent CD25 expression indicating TCR recog nition, the reduced ability of the mutants to induce full T cell activation is most likely the result of impaired double dimer formation. These data s uggest that MHC class II molecules, due to their structural properties, act ively contribute to TCR cross-linking.