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E De Plaen

Publications and source records attributed to E De Plaen.

51 records · Page 3Linked to original sources
PubMed1991

The gene coding for a major tumor rejection antigen of tumor P815 is identical to the normal gene of syngeneic DBA/2 mice.

We showed previously that mouse mastocytoma P815 expresses several distinct antigens that are recognized by cytolytic T lymphocytes (CTL) of syngeneic DBA/2 mice. Antigens P815A and P815B are usually lost jointly and are targets for immune rejection responses in vivo. We used a cosmid library and a CTL stimulation assay to obtain transfectants expressing tumor rejection antigen P815A. From these transfectants we retrieved gene P1A which transferred the expression of both P815A and B. This gene is unrelated to three previously isolated genes coding for tum-antigens. It encodes a putative protein of 224 amino acids which contains two highly acidic domains showing homology with similar regions of nuclear proteins. The P1A gene expressed by tumor P815 is completely identical to the gene present in normal DBA/2 cells. Expression of the gene was tested by Northern blots. Cells from liver, spleen, and a number of mast cell lines were negative, but mast cell line L138.8A produced a high level of P1A message and was lysed by CTL directed against antigens P815A and B. We conclude that major tumor rejection antigens of P815 are encoded by a gene showing little or no expression in most normal cells of adult mice.

Amino Acid Sequence↗
PubMed1990

Structure of the gene of tum- transplantation antigen P198: a point mutation generates a new antigenic peptide.

Mutagen treatment of mouse tumor cell line P815 produces tum- variants that are rejected by syngeneic mice because they express new transplantation antigens. These tum- antigens are recognized by cytotoxic T lymphocytes (CTL) but induce no detectable antibody response. By transfecting P815 cell line P1.HTR with DNA of tum- variant P198, we obtained transfectants expressing tum- antigen P198 that could be identified on the basis of their ability to stimulate anti-P198 CTL. This was repeated with DNA of a cosmid library derived from variant P198, and a cosmid carrying the sequence encoding antigen P198 was recovered from a transfectant. Gene P198 is 3 kb long and contains eight exons. It shows no homology with previously identified tum- gene P91A, nor with any gene presently recorded in the data banks. The long open reading frame codes for a 23.5-kD protein. The antigenic allele of gene P198 differs from the normal allele by a point mutation located in exon 7. This mutation causes an Ala to Thr change, and was shown by site-directed mutagenesis to be responsible for the expression of the antigen. An 11-amino acid synthetic peptide covering the sequence surrounding the tum- mutation rendered P815 cells sensitive to lysis by anti-P198 CTL. The homologous peptide corresponding to the normal sequence of the gene did not, but it was able to compete for binding to major histocompatibility complex molecule Kd. We conclude that tum- mutation P198 generates a new epitope recognized by syngeneic T cells. As observed with gene P91A, we found that a fragment of gene P198 that contained only exons 3-7, cloned in nonexpression vectors, transferred efficiently the expression of the antigen.

Amino Acid Sequence↗
PubMed1990

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

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.

Alleles↗
PubMed1989

Structure of the gene of tum- transplantation antigen P91A: the mutated exon encodes a peptide recognized with Ld by cytolytic T cells.

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. We found that the gene conferring expression of tum- antigen P91A contains 12 exons, encoding a 60 kd protein lacking a typical N-terminal signal sequence. The sequence shows no significant similarity with sequences in current data bases. A mutation that causes expression of the antigen is located in exon 4; it is the only apparent difference between the normal and the antigenic alleles. A short synthetic peptide corresponding to a region of exon 4 located around this mutation makes P815 cells sensitive to lysis by anti-P91A cytolytic T cells. The mutation creates a strong aggretope enabling the peptide to bind the H-2 Ld molecule. Several secondary tumor cell variants that no longer express tum- antigen P91A were found to carry deletions in the gene.

Amino Acid Sequence↗
PubMed1988

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

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 [Wölfel, T., Van Pel, A., De Plaen, E., Lurquin, C., Maryanski, J. L. & Boon, T. (1987) Immunogenetics 26, 178-187]. 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 lambda 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 tum- forms 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.

Amino Acid Sequence↗
PubMed1988

Identification of genes encoding T cell defined tum- antigens.

Tum- mutants are immunogenic mutants obtained by mutagen treatment of mouse tumor cells. They express new "tum- antigens" recognized by cytolytic T cells (CTL) but not by specific antibodies. We have recently developed a method aimed at cloning the genes coding for such transplantation antigens. It is based on gene transfection and detection of transfectants by their ability to stimulate CTL. Tum- gene P91A has been isolated. It codes for a 60 kDa protein which does not carry a signal sequence at its N-terminus. The tum- allele differs from its normal counterpart by a point mutation. The sequence of this gene and that of two other tum- genes are totally unrelated with each other and with any sequence presently recorded in data banks. We will try to apply the same cloning method to isolate mouse and human tumor-specific transplantation antigens (TSTA).

Animals↗
PubMed1987

Immunogenic (tum-) variants obtained by mutagenesis of mouse mastocytoma P815. VIII. Detection of stable transfectants expressing a tum- antigen with a cytolytic T cell stimulation assay.

Mutagen treatment of mouse mastocytoma P815 produces highly immunogenic "tum-" variants. Most of these variants express potent transplantation antigens which are not present on the original P815 tumor cells. These tum- antigens, which appear to be specific for each variant, elicit a strong cytolytic T lymphocyte (CTL) response, but do not seem to induce a specific antibody response. As a first step in the isolation of the gene of a tum- antigen, we attempted DNA-mediated gene transfer. As a DNA recipient cell we used P1.HTR, a highly transfectable P815 cell line, whose selection has been previously described. For the detection of antigen-expressing cells in transfected populations we developed a procedure that relies on the ability of these cells to stimulate the proliferation of the relevant CTL. Using DNA from tum- variant P91 mixed with a plasmid carrying an antibiotic resistance gene, we obtained several independent transfectants expressing a tum- antigen, at a frequency of approximately 1 in 13,000 antibiotic-resistant transfectants. These transfectants express only one of the two tum- antigens that were identified on P91, suggesting that these tum- antigens correspond to different genes. We expect that the detection procedure described here will be suitable for the identification of transfectants for any gene that determines the expression of an antigen recognized by CTL.

Animals↗
PubMed1985

A simple panning method for the selection of cell surface antigen transfectants.

Mouse Ltk- (H-2k) cells that became reactive with anti-H-2d monoclonal antibodies (McAb) after transfection with DNA from H-2d-positive cells were isolated by an indirect panning method. The cells were transfected with DNA from a plasmid containing the selective thymidine kinase (tk) gene in addition to total cell DNA, so that a first selection could be carried out in hypoxanthine/aminopterin/thymidine (HAT) medium. The HAT-selected tk+ transfectants were incubated with anti-H-2d McAb, washed and transferred to dishes coated with purified anti-immunoglobulin (anti-Ig). This 2-step method of panning has the advantage that only the anti-Ig reagent requires purification. Transfected cells clearly reactive with either anti-Ld/Rd or anti-Kd McAb were isolated after only 1 or 2 cycles of panning.

Animals↗
PubMed1985

Human T cell recognition of cloned HLA class I gene products expressed on DNA transfectants of mouse mastocytoma P815.

Cloned genes for human major histocompatibility complex (MHC) class I antigens were introduced by DNA-mediated gene transfer into a high-efficiency transfection recipient (HTR) cell line previously derived from mouse mastocytoma P815. Cell surface expression of HLA-A3, AW24 and CW3 gene products on P815 transfectants was demonstrated by radioimmune assay and by flow cytometry. The human MHC class I gene products were apparently expressed on P815 transfectants in a form recognized by human cytolytic T lymphocytes (CTL). Human CTL generated in unidirectional mixed lymphocyte culture against AW24+ donor lymphocytes clearly lysed P815-HLA-AW24+ transfectant target cells, but not untransfected P815(HTR) controls. Moreover, P815-HLA transfectants could stimulate in vitro a significant alloreactive human CTL response. Lysis of P815-HLA transfectant target cells by human CTL was inhibited by a monoclonal antibody directed against human MHC class I gene products. These mouse cell transfectants may be useful for the study of human T cell responses.

Animals↗
PubMed1985

A neomycin-resistant cell line for improved production of monoclonal antibodies to cell surface antigens.

Overgrowth of hybridomas by proliferating spleen T cells often hinders the production of monoclonal antibodies to certain antigens. To overcome this problem we derived neomycin-resistant fusion partner SP2 neoR.1 by transfection of SP2/0-Ag14 cells with plasmid pSVTK neo beta. Hybridomas obtained with SP2 neoR.1 grew optimally in the presence of the neomycin analog G418 at concentrations which blocked the proliferative response of T cells to mitogenic stimuli. The advantage of using SP2 neoR.1 and G418 under conditions where spleen cell proliferation occurs after fusion was demonstrated with hybridomas derived from a rat immunized with mouse helper T cell lines.

Animals↗
PubMed1985

Selection of highly transfectable variant from mouse mastocytoma P815.

A tk- cell line derived from mouse mastocytoma P815 was transfected with a plasmid carrying a thymidine kinase gene. The tk+ cells were obtained at a frequency of 10(-6). From some of these tk+ transfectants it was possible to select tk- cells with BrdU so that these cells could be submitted again to transfection with the thymidine kinase gene. By repeating cycles of transfection and tk+ selection followed by reverse selection with BrdU, it was possible to obtain a stable variant having a 100-fold increase in transfection efficiency. This HTR (high transfection) variant shows a high efficiency of transfection (10(-4)) for the neomycin-resistance gene as well as for the thymidine kinase gene.

Animals↗