Characterization of defective I-A surface expression in a mixed isotype expressing murine B cell lymphoma: continued expression of E alpha d A beta d despite competition from restored A alpha d A beta d pairs

Abstract

The BALB/c-derived mouse B cell lymphoma line, 2PK3, expresses mixed isotype E alpha dA beta d and classical I-E class II molecules on its surface, but normal surface I-A expression is not detectable. Northern blot analysis showed comparable amounts of A alpha mRNA in 2PK3 as compared to another Iad expressing B cell lymphoma, A20, which predominantly expresses I-A and I-E. Sequence analysis of 2PK3 A alpha cDNA revealed a single nucleotide difference in the signal sequence that would result in a proline for leucine substitution at position - 12. In vitro translation of 2PK3 A alpha mRNA gave results suggesting that the signal peptide mutation prevented translocation of the A alpha protein across the rough endoplasmic reticulum which would provide an explanation for the lack of I-A expression in 2PK3. I-A expression was restored by transfecting a functional A alpha d gene into 2PK3. Although I-A was expressed at high levels in some transfectants, in all cases significant levels of mixed isotype were still detected. Functional studies performed using antigen-specific I-A(d)-restricted and E alpha d-A beta d-specific T cell hybridomas confirmed the levels of expression of I-A(d) and E alpha dA beta d respectively on the transfectants and showed that these molecules were functional. An interesting observation from this study is the continued expression of significant levels of E alpha dA beta d in spite of competition from restored expression of I-A(d).