Structure-function analysis of the A(β)^bm12 mutation using site-directed mutagenesis and DNA-mediated gene transfer

The B6.C-H-2^bm12 (bm 12) mouse possesses a naturally occurring mutation in its class II MHC A(β) gene. The three amino acid substitutions at positions 67, 70, and 71 that comprise this mutation lead to changes in both Ia expression and immune recognition of the resultant A(β)A(α) molecule. The experiments reported here utilize a combination of oligonucleotide-mediated site-directed mutagenesis and DNA-mediated gene transfer to explore the roles played by each of the three mutant residues in these various phenotypic changes. A(β) genes comprising all permutations of the residues distinguishing A(β)^b from A(β)^bm12 were created and were individually co-transfected with A(α)^b into mouse L cells. Sublines expressing high levels of membrane Ia were selected by preparative flow cytometry and were studied for reactivity with a panel of monoclonal anti-Ia antibodies, or for their ability to act as antigen-presenting cells (APC) for the stimulation of T cell hybridomas. During the generation of these transfectant lines, it was noted that expression of a high level of A(β)^bm12 A(α)^b was more difficult to achieve than a similar level of A(α)^bA(α)^b. Northern blot analysis of specific A(β) and A(α) mRNA levels in these various lines indicated that more class II mRNA, and presumably more A(β) and A(α) chains, were required to achieve expression of A(β)^bm12 A(α)^b equal to that of A(β)^bA(α)^b, suggesting that the previously noted reduction of Ia expression on cells from bm 12 mice reflects a decreased ability of A(β)^bm12 A(α)^b chains to pair, or to reach the membrane. Staining of the panel of transfectants with monoclonal antibodies revealed that antibodies which did not distinguish A(β)^bA(α)^b from A(β)^bm12 A(α)^b also reacted equally well with all molecules involving in vitro mutant A(β) chains. Monoclonal antibodies reactive with A(β)^bA(α)^b but not A(β)^bm12 A(α)^b were specific for an epitope primarily determined by the presence or absence of Arg 70 in A(β)^b. In striking contrast, all three mutant positions were found to play crucial roles in T cell recognition, because all substitutions led to significant or complete loss of antigen-presenting function with all but one of the T hybridomas tested. The few cases of residual reactivity with the mutant Ia molecules appeared to correlate with L3T4 expression by the hybridoma cells, and it was shown that responses to mutant APC were more easily inhibited by anti-L3T4 than responses to parental APC, suggesting that the A(β) mutations directly affect the avidity of the T cell receptor's interaction wit Ia-antigen, rather than antigen-Ia interaction itself. These latter findings indicate that the region of the bm 12 mutation is a critical one for the general process of Ia-restricted T cell recognition of antigen, most likely by serving a relatively direct role in the binding events involved in formation of the ternary complex of receptor, antigen, and Ia.