Limits...
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.

Show MeSH

Related in: MedlinePlus

The anti-A and anti-B CTL clones are uniquely specific for the  8101 tumor lineage. The anti-A CTL clone ( A) lyses only the 8101-RE  tumor cell but not five other C57BL/6-derived UV-induced tumors, or  the H-2b haplotype lymphoma RMA. The anti-B CTL clone (B) lyses  the 8101-RE and the 8101-PRO tumor cells, but not three other  C57BL/6-derived UV-induced tumors, or RMA cells. Targets were  tested for lysis in a 51Cr-release assay.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196148&req=5

Figure 2: The anti-A and anti-B CTL clones are uniquely specific for the 8101 tumor lineage. The anti-A CTL clone ( A) lyses only the 8101-RE tumor cell but not five other C57BL/6-derived UV-induced tumors, or the H-2b haplotype lymphoma RMA. The anti-B CTL clone (B) lyses the 8101-RE and the 8101-PRO tumor cells, but not three other C57BL/6-derived UV-induced tumors, or RMA cells. Targets were tested for lysis in a 51Cr-release assay.

Mentions: 8101 was the first tumor isolated from a group of C57BL/6 mice that received chronic UV-irradiation. The tumor arose on the back of the mouse after 11 mo of irradiation. At this time the mouse was 14 mo old. As usually observed for UV-induced tumors, 8101 readily adapted to culture and the primary culture was cloned to study the antigenic diversity of the cells that adapted. CD8+ cytotoxic T lymphocyte (CTL) clones were generated from mixed lymphocyte tumor cell cultures (MLTCs) of spleen cells from mice immunized by i.p. injection of live uncloned tumor cells. The resulting T cell clones identified two types of tumor cell clones: one which expressed two antigens designated A and B, and a second that only expressed the B antigen. The parental uncloned cell line, designated 8101-PAR, expressed both antigens and was lysed by anti-A as well as anti-B CTL clones (Fig 1, A and B). The anti-A (Fig. 1 A) and anti-B (Fig. 1 B) CTL clones lysed only 8101 lineage tumor cells and did not lyse autochthonous normal fibroblasts, or RMA cells. We injected the original uncloned 8101-PAR tumor cell line and 8101 A+B+ and 8101 A−B+ clones into C57BL/6 nude mice to generate tumor fragments. The developing tumors were transplanted as fragments into naive normal C57BL/6 mice. Table 1 shows that tumors derived from the A+ clone were regularly rejected and thus were designated 8101-RE. Tumors derived from the A− clone grew progressively and were therefore designated as 8101-PRO. These results strongly suggested that the expression of the A antigen correlated with rejection of the A+ tumor in naive mice, and that absence of the A antigen led to progressive tumor growth. The B antigen was expressed on all 8101 lineage tumors but not other C57BL/6 UV tumors (Fig. 2 B), indicating that the 8101RE and 8101-PRO tumors were of the same clonal origin. The parental cell line 8101-PAR which, as suggested by clonal analysis, contained A+ as well as A− tumor cells, grew progressively when fragments were transplanted into naive normal mice. The reisolated tumor cells when readapted to culture were resistant to anti-A CTL, unlike the 8101-PAR tumor cells which had been used for the challenge (Fig. 1 A). These data indicated that normal mice usually selected against expression of the A antigen. Only one re-isolated tumor still showed sensitivity to lysis by anti-A CTL; this tumor may have been able to grow as A+ because of the simultaneous challenge with A−B+ progressor tumor cells which were also present in the 8101-PAR tumor. These A−B+ tumor cells may have prevented the establishment of an effective anti-A response. This suggestion is in agreement with our previous observation that progressor tumors can sometimes prevent an immune response to highly antigenic tumor cells (36).


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 anti-A and anti-B CTL clones are uniquely specific for the  8101 tumor lineage. The anti-A CTL clone ( A) lyses only the 8101-RE  tumor cell but not five other C57BL/6-derived UV-induced tumors, or  the H-2b haplotype lymphoma RMA. The anti-B CTL clone (B) lyses  the 8101-RE and the 8101-PRO tumor cells, but not three other  C57BL/6-derived UV-induced tumors, or RMA cells. Targets were  tested for lysis in a 51Cr-release assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196148&req=5

Figure 2: The anti-A and anti-B CTL clones are uniquely specific for the 8101 tumor lineage. The anti-A CTL clone ( A) lyses only the 8101-RE tumor cell but not five other C57BL/6-derived UV-induced tumors, or the H-2b haplotype lymphoma RMA. The anti-B CTL clone (B) lyses the 8101-RE and the 8101-PRO tumor cells, but not three other C57BL/6-derived UV-induced tumors, or RMA cells. Targets were tested for lysis in a 51Cr-release assay.
Mentions: 8101 was the first tumor isolated from a group of C57BL/6 mice that received chronic UV-irradiation. The tumor arose on the back of the mouse after 11 mo of irradiation. At this time the mouse was 14 mo old. As usually observed for UV-induced tumors, 8101 readily adapted to culture and the primary culture was cloned to study the antigenic diversity of the cells that adapted. CD8+ cytotoxic T lymphocyte (CTL) clones were generated from mixed lymphocyte tumor cell cultures (MLTCs) of spleen cells from mice immunized by i.p. injection of live uncloned tumor cells. The resulting T cell clones identified two types of tumor cell clones: one which expressed two antigens designated A and B, and a second that only expressed the B antigen. The parental uncloned cell line, designated 8101-PAR, expressed both antigens and was lysed by anti-A as well as anti-B CTL clones (Fig 1, A and B). The anti-A (Fig. 1 A) and anti-B (Fig. 1 B) CTL clones lysed only 8101 lineage tumor cells and did not lyse autochthonous normal fibroblasts, or RMA cells. We injected the original uncloned 8101-PAR tumor cell line and 8101 A+B+ and 8101 A−B+ clones into C57BL/6 nude mice to generate tumor fragments. The developing tumors were transplanted as fragments into naive normal C57BL/6 mice. Table 1 shows that tumors derived from the A+ clone were regularly rejected and thus were designated 8101-RE. Tumors derived from the A− clone grew progressively and were therefore designated as 8101-PRO. These results strongly suggested that the expression of the A antigen correlated with rejection of the A+ tumor in naive mice, and that absence of the A antigen led to progressive tumor growth. The B antigen was expressed on all 8101 lineage tumors but not other C57BL/6 UV tumors (Fig. 2 B), indicating that the 8101RE and 8101-PRO tumors were of the same clonal origin. The parental cell line 8101-PAR which, as suggested by clonal analysis, contained A+ as well as A− tumor cells, grew progressively when fragments were transplanted into naive normal mice. The reisolated tumor cells when readapted to culture were resistant to anti-A CTL, unlike the 8101-PAR tumor cells which had been used for the challenge (Fig. 1 A). These data indicated that normal mice usually selected against expression of the A antigen. Only one re-isolated tumor still showed sensitivity to lysis by anti-A CTL; this tumor may have been able to grow as A+ because of the simultaneous challenge with A−B+ progressor tumor cells which were also present in the 8101-PAR tumor. These A−B+ tumor cells may have prevented the establishment of an effective anti-A response. This suggestion is in agreement with our previous observation that progressor tumors can sometimes prevent an immune response to highly antigenic tumor cells (36).

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