Immunogenic (tum-) variants of mouse tumor P815: cloning of the gene of tum- antigen P91A and identification of the tum- mutation.

Abstract

Mutagen treatment of mouse P815 tumor cells produces tum-variants that are rejected by syngeneic mice because these variants express new surface antigens. These "tum antigens" are recognized by cytolytic T lymphocytes but induce no detectable antibody response. Transfection of P815 cell line P1.HTR with DNA of tum-variant P91 yielded transfectants expressing tum antigen P91A. They were detected by their ability to stimulate proliferation of cytolytic T lymphocytes [Wolfel, T., Van Pel, A., De Plaen, E., Lurquin, C., Maryanski, J. L. & Boon, T. (1987) Immunogenetics 26,[178][179][180][181][182][183][184][185][186][187]. A cosmid library of a cell line expressing antigen P91A was transfected into P1.HTR. Transfectants expressing the antigen were obtained. By packaging directly the DNA of a transfectant with A phage extracts, we obtained a small cosmid population containing as major component a cosmid that transferred the expression of P91A. The assay of various restriction fragments of this cosmid led to the isolation of an 800-base-pair fragment containing the P91A sequence required for transfection. Comparison with a homologous cDNA showed that this fragment contained only one of the several exons of the P91A gene. The normal and the tumforms of the gene differ by one nucleotide located in this 137-base-pair exon. The essential role of this mutation, which produces an amino acid change, was confirmed by site-directed mutagenesis. No significant sequence similarity was found between the 800-base-pair fragment and any recorded gene.In vitro mutagen treatment of mouse tumor cell lines generates at high frequency immunogenic variants that are rejected by syngeneic mice (1). Since they fail to form tumors, these stable variants have been named "tum-" as opposed to the original "tumr+" cell, which forms progressive tumors. Most tum-variants express transplantation antigens not found on the tum+ cell. The existence of these "tumantigens" can be demonstrated in vivo by cross-immunization experiments or in vitro with cytolytic T lymphocytes (CTLs) (2).We have studied a series of tum-variants derived from mastocytoma P815, a tumor induced iq a DBA/2 mouse with methylcholanthrene (3). For most P815 tum-variants, stable CTL clones have been obtained that show a strict specificity for the immunizing variant and thus define one or several tum-antigens (4). Despite numerous attempts, we failed to obtain antibodies against these variaqt-specific antigens.The diversity of tum-antigens is considerable: the analysis of 15 P815 tum-variants demonstrated the existence of a different antigen on each of them (5). To understand the underlying genetic mechanism, we undertook the cloning of the "tum-genes" that determine the expression of tumantigens. As a first step, we attempted DNA-mediated transfer of antigen P91A, one of the two tum-antigens of P815 variant P91. Other workers have described the production of transfectants for surface antigens (6, 7), but in these instances, the antigen-expressing transfectants were detected ...

Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine

“…Identification of tumor-associated antigens (TAA) recognized by T lymphocytes makes it possible to test the function of recombinant immunogens in the treatment of cancer (1)(2)(3)(4)(5). Optimization of the design and delivery of these immunogens is facilitated by a growing understanding of the molecular signals involved in the activation and proliferation of T lymphocytes and a knowledge of how antigens are processed and presented for recognition.…”

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.…”

Fever therapy revisited

“…This exciting observation published in 1956, however, did not lead into any research activity aimed at strengthening the defence capacities of the body, but rather was overshadowed by hopes and hypes offered by the emerging chemotherapy. The first molecular identification of tumour antigens was described in 1988 in mice (Plaen et al, 1988) and 1991 in humans (VanderBruggen et al, 1991). Today, it is certain that tumour-specific T-cells can be found in high numbers among tumour-infiltrating lymphocytes (TIL) (Romero et al, 1998), but this T-cell response usually does not translate into healing progress.…”