Structure of the gene of tum- transplantation antigen P35B: presence of a point mutation in the antigenic allele.

Abstract

Mutagen treatment of P815 tumour cells produces tum‐ variants that are rejected by syngeneic mice because they express new transplantation antigens. These ‘tum‐’ antigens elicit a cytolytic T lymphocyte (CTL) response but no detectable antibody response. The DNA of tum‐ variant P35 was transfected into P815 cell line P1.HTR. Transfectants expressing tum‐ antigen P35B were identified on the basis of their ability to stimulate anti‐P35B CTL. This was repeated with a cosmid library and a cosmid carrying the sequence encoding antigen P35B was recovered from a transfectant expressing the antigen. Gene P35B is 6 kb long and contains 11 exons. The sequence shows no homology with the previously identified tum‐ gene P91A nor with any gene presently recorded in the data banks. The antigenic allele of gene P35B differs from the normal allele by a point mutation located in exon 5. This mutation, which replaces a Ser by an Asn residue, was shown by site‐directed mutagenesis to be responsible for the expression of the antigen. A synthetic decapeptide covering the sequence surrounding the tum‐ mutation rendered P815 cells sensitive to lysis by anti‐P35B CTL. Surprisingly, the homologous peptide corresponding to the normal sequence of the gene had the same effect, indicating that this tum‐ mutation does not exert its effect by generating the aggretope or the epitope of the antigenic peptide. As observed previously with gene P91A, we found that fragments of gene P35B containing only exons 4 and 5, which were cloned in non‐expression vectors, transferred efficiently the expression of the antigen.

Immunogenic tumor variants induced by drug treatment of the L5178Y lymphoma: Search for serologically defined antigens at the clonal level

“…In addition, a short peptide derived from exon 4 (out of 12 exons in the gene) was able to sensitize parental cells for lysis by anti-P91A CTL. Similar results were obtained for other tum-antigens, but no homology was found among different tum-mutant genes or with genes recorded in data banks (De Plaen et al, 1988;Szikora et al, 1990;Sibille et al, 1990). In our triazene xenogenization system, anti-DMTA antibodies have been used to immunoprecipitate proteins from a clone of the xenogenized L5178Y lymphoma line (Grohmann et al, 1990).…”

A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma.

“…F stP .tg tag GTT TTG GCC CGA ACC GCG ACT TCA ACA AAA AAT AAG AGA AGA AAG GAA TAT TTT CTA GCT GTG CAA ATC CTC TCC CTA GAG GAA AAg tta att gtt gtg ttg ttt taa tac.. (15)(16)(17). In some instances, an amino acid change transforms a peptide that is incapable of binding to a class I molecule into a peptide that binds well.…”

“…A third category of antigens could also be expected to be present on human tumors; namely, those caused by point mutations. We have observed in mouse tumor systems that point mutations can generate potent antigens recognized by syngeneic T lymphocytes (15)(16)(17). Recently, a mouse tumor antigen recognized on Lewis lung carcinoma cells has been shown to result from a mutation in the connexin gene (18).…”

Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes.

“…In the P815 mouse mastocytoma tumor system, nontumorigenic variants (turn-) 1 of tumorigenic P815 cells (turn +) were isolated after chemical mutagenesis of the turn + cells (21). The decrease in tumorigenicity observed for turn-cells correlated with the acquisition of genomic point mutations creating new CTL antigenicities recognized by turn--specific CTL (21)(22)(23)(24)(25). Antigen-loss variant cells were obtained by cocultivating tum-"antigengain" variant cells with CTL specific for the new turn-CTL epitopes.…”