A SINGLE AMINO-ACID SUBSTITUTION IN THE H-2K(B) MOLECULE GENERATES A DEFINED ALLOGENEIC EPITOPE

Using Mitomycin C mutagenesis and negative and positive selection with monoclonal antibodies specific for H-2K(b) and H-2K(bm10), respective ly, a mutant cell line clone, Mitc-182, was isolated. Direct sequencin g of uncloned cDNA as well as PCR based cloning and sequencing of the H-2K(b182) transcript from this mutant revealed a single G-->T transve rsion resulting in the substitution of Trp167 by cysteine. Serological ly, the mutant K-b182 and K-bm10 are almost identical as each has lost at least five Kb specific mAb epitopes and gained several new epitope s. Interestingly, the mutant cell line, Mitc-182, is efficiently recog nized by alloreactive CTLs raised in reciprocal combinations, e.g. CB6 anti Cbm10 and Cbm10 anti CB6, indicating that K-b182 contains both K -b and K-bm10 specific epitopes. The mutation has not affected the abi lity of K-b182 to present K-b restricted antigenic peptides of Sendai and vesicular stomatitis viruses. In addition to underscoring the impo rtance of amino acid residue 167 in alloreactivity, these results indi cate a positive correlation between the gain of both an mAb epitope an d a defined alloreactive CTL epitope.