Site Directed Mutagenesis Identifies Allo-Antigenic Epitopes of an H-2 Antigen Recognized by Antibodies and by Cytotoxic T-Lymphocytes

Abstract

To test our previous hypothesis that the segment between amino acid position 63 to 73 of the H-2D^d antigen forms a major allo-antigenic site, mutations were introduced into the H-2L^d gene in a sequential fashion, which replaced the codons for amino acid position 63, 65, 66, 70 and 73 of the H-2L^d antigen with those of the H-2D^d antigen. Gain and loss of serological and CTL epitopes specific for the H-2D^d and H-2L^d antigens were examined. The amino acid substitutions at position 63, 65, and 66 led to the acquisition of multiple serological H-2D^d specificities which were expressed in the mutant H-2L^d antigen. A further amino acid substitution at position 70 resulted in the gain of additional H-2D^d specificities, allowing to localize more than half of all the relevant H-2D^d serological epitopes to position 63 to 70. An alto CTL epitope of the H-2D^d antigen was also localized to this stretch of amino acid sequence, as one of several H-2D^d specific CTL clones reacted with the mutant molecule in which amino acids were replaced at position 63 to 70. Further, some H-2L^d specific alto CTL clones lost reactivity to the mutant molecules, demonstrating the presence of CTL epitopes in this region of the H-2L^d antigen. From these results we conclude that the amino acid sequence encompassing from position 63 to 70 of the H-2D^d and H-2L^d molecules forms major alto- antigenic epitopes recognized by multiple antibodies and CTLs.