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The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper JC, Engelhard VH, Hunt DF, Schreiber H - J. Exp. Med. (1997)

Bottom Line: The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Chicago, Illinois 60637, USA.

ABSTRACT
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.

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The A antigen is immunodominant in vivo. Each panel shows  a different C57BL/6 mouse immunized i.p. on day 0, 3, 6, and 9 with  2–5 × 106 A+B+ 8101-RE tumor cells. The PEC were harvested on day  11 and directly tested in a 51Cr-release assay for lytic activity. Only the  A+B+ 8101-RE cells, but not the A−B+ 8101-PRO cells, or RMA cells  are lysed by the PEC.
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Figure 3: The A antigen is immunodominant in vivo. Each panel shows a different C57BL/6 mouse immunized i.p. on day 0, 3, 6, and 9 with 2–5 × 106 A+B+ 8101-RE tumor cells. The PEC were harvested on day 11 and directly tested in a 51Cr-release assay for lytic activity. Only the A+B+ 8101-RE cells, but not the A−B+ 8101-PRO cells, or RMA cells are lysed by the PEC.

Mentions: Since the expression of the A antigen correlated with rejection of the tumor challenge, we further characterized this antigen. Fig. 2 A shows that the anti-A CTL clone recognized only 8101-RE cells, but not any of 5 other UV-induced tumors of C57BL/6 origin that were tested. This result suggests that the 8101 A antigen is unique, i.e., individually distinct, for the 8101 tumor, as has been previously shown in our laboratory for other UVinduced tumors (31). To determine how commonly this antigen is recognized in vivo, four mice were injected repeatedly with 8101-RE cells, which express both the A and the B antigen. Peritoneal exudate cells (PEC) were isolated and directly tested for lytic activity (Fig. 3). PECs from all four mice lysed the A+B+ 8101-RE tumor cells but not the A−B+ 8101-PRO tumor cells, suggesting that the A antigen is regularly recognized and is immunodominant. Lysis by the anti-A CTL clone was inhibited by an anti-Kb but not an anti-Db antibody indicating that the A antigen was H-2Kb restricted (data not shown). To examine the possibility of biochemical identification of the A antigen, we determined whether peptides eluted from the H-2Kb molecules immunoprecipitated from 8101-RE cells would sensitize RMA-S cells to become a specific target for lysis by anti-A CTL. As little as 0.2% of an immunoprecipitate from 1 × 109 8101-RE cells contained sensitizing activity (data not shown).


The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper JC, Engelhard VH, Hunt DF, Schreiber H - J. Exp. Med. (1997)

The A antigen is immunodominant in vivo. Each panel shows  a different C57BL/6 mouse immunized i.p. on day 0, 3, 6, and 9 with  2–5 × 106 A+B+ 8101-RE tumor cells. The PEC were harvested on day  11 and directly tested in a 51Cr-release assay for lytic activity. Only the  A+B+ 8101-RE cells, but not the A−B+ 8101-PRO cells, or RMA cells  are lysed by the PEC.
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Related In: Results  -  Collection

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Figure 3: The A antigen is immunodominant in vivo. Each panel shows a different C57BL/6 mouse immunized i.p. on day 0, 3, 6, and 9 with 2–5 × 106 A+B+ 8101-RE tumor cells. The PEC were harvested on day 11 and directly tested in a 51Cr-release assay for lytic activity. Only the A+B+ 8101-RE cells, but not the A−B+ 8101-PRO cells, or RMA cells are lysed by the PEC.
Mentions: Since the expression of the A antigen correlated with rejection of the tumor challenge, we further characterized this antigen. Fig. 2 A shows that the anti-A CTL clone recognized only 8101-RE cells, but not any of 5 other UV-induced tumors of C57BL/6 origin that were tested. This result suggests that the 8101 A antigen is unique, i.e., individually distinct, for the 8101 tumor, as has been previously shown in our laboratory for other UVinduced tumors (31). To determine how commonly this antigen is recognized in vivo, four mice were injected repeatedly with 8101-RE cells, which express both the A and the B antigen. Peritoneal exudate cells (PEC) were isolated and directly tested for lytic activity (Fig. 3). PECs from all four mice lysed the A+B+ 8101-RE tumor cells but not the A−B+ 8101-PRO tumor cells, suggesting that the A antigen is regularly recognized and is immunodominant. Lysis by the anti-A CTL clone was inhibited by an anti-Kb but not an anti-Db antibody indicating that the A antigen was H-2Kb restricted (data not shown). To examine the possibility of biochemical identification of the A antigen, we determined whether peptides eluted from the H-2Kb molecules immunoprecipitated from 8101-RE cells would sensitize RMA-S cells to become a specific target for lysis by anti-A CTL. As little as 0.2% of an immunoprecipitate from 1 × 109 8101-RE cells contained sensitizing activity (data not shown).

Bottom Line: The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Chicago, Illinois 60637, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus