The Journal of Experimental Medicine
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© The Rockefeller University Press, 0022-1007/1997/2/695/ $5.00
The Journal of Experimental Medicine, Volume 185, Number 4, February 17, 1997 695-706

Articles

The Immunodominant Antigen of an Ultraviolet-induced Regressor Tumor Is Generated by a Somatic Point Mutation in the DEAD Box Helicase p68

Purnima Dubey*, Ronald C. Hendrickson{ddagger}, Stephen C. Meredith*, Christopher T. Siegel*, Jeffrey Shabanowitz{ddagger}, Jonathan C.A. Skipper||, Victor H. Engelhard||, Donald F. Hunt{ddagger},§, and Hans Schreiber*

From the * Department of Pathology, The University of Chicago, Chicago, Illinois 60637; and the {ddagger} Department of Chemistry, § Department of Pathology, and || Department of Microbiology and the Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22901

The genetic origins of CD8+ T cell–recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A– 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor–specific mutation in the coding region of a nucleic acid helicase.

Address correspondence to Purnima Dubey, The University of Chicago, Department of Pathology, 5841 South Maryland, MC1089, Chicago, IL 60637. J.C.A. Skipper's present address is University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK. R.C. Hendrickson's present address is Corixa Corp., 1124 Columbia St., Ste 464, Seattle, WA 98104.

The authors deeply appreciate the generous gift of the BPV series of C57BL/6 tumors by Mr. Vijay Sreedhar and Dr. Margaret Kripke. We also thank Dr. Jeffrey Bluestone for his gift of the Y-3 hybridoma and RMA and RMA-S cells. We are grateful to Dr. Paola Rizzo for advice and assistance with the sequence analysis. We are also grateful to Mrs. Helene Auer and Ms. Julianne Liebsohn for excellent technical assistance. We would like to thank Drs. Donald Rowley, Maresa Wick, Paola Rizzo, Paul Monach, and Dominik Mumberg for critical reading of the manuscript.

P. Dubey and R.C. Hendrickson contributed equally to this paper.

 This work was presented in abstract form at the FASEB meeting. June 1996. FASEB J. 10:A1437 (Abstr.).

1 Abbreviations used in this paper: CAD, collision-activated dissociation; CDMEM, complete DMEM; CRPMI, complete RPMI; HFBA, heptafluorobutyric acid; HLF, heart-lung fibroblast; LC-MS, liquid chromatography-mass spectrometry; MLTC, mixed-lymphocyte tumor cell culture; PEC, peritoneal exudate cell; PITC, phenylisothiocyanate; TAP, transporter associated with antigen processing.


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