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.
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).
Spencer
J S
JS
Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
McCormack
J E
JE
Kubo
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000378:metabolism*; D016393:Lymphoma, B-Cell / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D000949:Histocompatibility Antigens Class II; C024138:I-E-antigen; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D012333:RNA, Messenger