SINGLE AMINO-ACID MUTATIONS IN THE MURINE MHC CLASS-II A-BETA CYTOPLASMIC DOMAIN ABROGATE ANTIGEN PRESENTATION

Class II MHC molecules are heterodimeric transmembrane glycoproteins t hat function in the presentation of Ag to CD4(+) T cells. Deletion of the cytoplasmic domains of the murine class II A alpha- and A beta-cha ins has previously been shown to diminish Ag presentation and abrogate rejection of class Il-transfected tumor cells. To examine the contrib utions of individual amino acid residues of the A beta cytoplasmic dom ain to Ag presentation and tumor rejection, we have produced a series of cell lines expressing A beta class II molecules with site-directed mutations. An A beta(k) cDNA was constructed with mutations in the fiv e conserved amino acid residues, Q224, K225, L235, L236, and Q237 (Del ta 5). In addition, cDNA were produced in which alanine was individual ly substituted for A beta(k) cytoplasmic domain residues 224 through 2 37 or doubly substituted at residues G226 and P227 or L235 and L236. T hese mutant cDNAs were individually cotransfected with wild-type A alp ha cDNA into the class Ii-negative M12.C3 B lymphoma and Sal sarcoma c ell lines. As was previously reported for transfectants lacking the en tire A beta(k) cytoplasmic domain, the Delta 5 M12.C3 transfectant cou ld not effectively present Ag to an autoreactive A(k)-restricted T cel l hybrid, and the Delta 5 Sal transfectant was not rejected when inocu lated into syngeneic hosts. A finer analysis revealed that alteration of the individual residue Q224 or the two residues G226 and P227 abrog ated Ag presentation in vitro, while mutation of G226 diminished tumor rejection in vivo. Thus, the function of the A beta cytoplasmic domai n in Ag presentation both in vitro and in vivo can be disturbed by mut ation of single amino acid residues.