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T Boon

Publications and source records attributed to T Boon.

At least 145 records · Page 8Linked to original sources
PubMed1992

Precursor frequency analysis of human cytolytic T lymphocytes directed against autologous melanoma cells.

Limiting numbers of peripheral-blood mononuclear cells (PBMC) from melanoma patients were stimulated with irradiated autologous tumor cells in the presence of interleukins-2 and -4 and in the absence of feeder cells. The responder cells were restimulated every week. After 2 to 4 weeks, the microcultures were tested for their lytic activity against the autologous tumor cells. Significant lysis of the tumor cells was observed with a fraction of these microcultures, whereas no lysis was observed with control microcultures seeded without stimulator melanoma cells. Because our aim was to measure the precursor frequency of CTL showing specificity for the tumor, and not that of NK-like effectors that were also capable of lysing the melanoma cells, we used cold-target inhibition with an excess of NK target K562 to inhibit the NK-like activity. Microcultures whose lysis on the tumor cells was not abolished by K562 competition were observed. The specificity of these CTL clones was confirmed by the absence of lytic activity on autologous T-cell blasts. The numbers of microcultures with anti-tumor CTL activity fitted the zero-order of the Poisson distribution equation, indicating that they resulted from the activity of single T-cell clones. The frequency of anti-tumor CTL precursor cells (CTL-P) of 7 melanoma patients ranged from 1/900 to 1/33,000. Frequencies of anti-tumoral CTL-P were higher and NK-like effectors were less frequent when sorted CD8+ T lymphocytes were used as responder cells.

CD4-CD8 Ratio↗
PubMed1992

Mouse tumor rejection antigens P815A and P815B: two epitopes carried by a single peptide.

Mouse mastocytoma P815 expresses several distinct tumor rejection antigens recognized by syngeneic cytolytic T lymphocytes (CTL). Two of these tumor rejection antigens, P815A and P815B, are encoded by gene P1A, the sequence of which was reported previously. Tumor cell variants having lost one or both of these antigens were isolated by in vitro selection with CTL and also by collecting tumor cells that progressed in vivo after escaping a nearly complete immune rejection process. The structure of gene P1A in these antigen-loss variants was examined. Several A-B- variants presented a complete or partial deletion of the gene. One variant that had lost only antigen A displayed a point mutation in the first exon. Peptides were synthesized that corresponded to the normal sequence located in the region of this point mutation. They sensitized target cells to both anti-A and anti-B CTL. The homologous peptide encoded by the mutated gene of the P815 A-B+ variant sensitized cells only to anti-B CTL. We conclude that anti-A and anti-B CTL recognize on the same peptide two distinct epitopes that are affected differently by the mutation.

Amino Acid Sequence↗
PubMed1992

Efficient expression of tum- antigen P91A by transfected subgenic fragments.

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. The gene encoding tum- antigen P91A comprises 12 exons and a mutation located in exon 4 is responsible for the production of a new antigenic peptide. Transfection experiments showed that the expression of the antigen could be transferred not only by the entire gene but also by gene segments comprising only the mutated exon and parts of the surrounding introns. This was observed with subgenic regions that were not cloned in expression vectors. Antigen expression did not require the integration of the transfected gene segment into a resident P91A gene by homologous recombination. It also occurred when the subgenic segment was transfected without the usual selective gene, which comprises an eucaryotic promoter, and also without plasmid sequences, which are known to contain weak promoters. When a stop codon was introduced at the beginning of exon 4, the expression of the antigen was maintained and evidence was obtained that an ATG codon located in this region served as initiation site for the translation of the antigenic peptide. But we have not obtained evidence indicating that antigenic peptides are direct translation products rather than degradation products of entire proteins.

Amino Acid Sequence↗
PubMed1992

Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes.

Human melanoma line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocytes (CTL). As a first step towards the cloning of the gene coding for one of these antigens, we tried to obtain transfectants expressing the antigen. The DNA recipient cell was a variant of MZ2-MEL which had been selected with a CTL clone for the loss of antigen E. It was cotransfected with genomic DNA of the original melanoma line and with selective plasmid pSVtkneo beta. Geneticin-resistant transfectants were obtained at a frequency of 2 x 10(-4). These transfectants were then screened for their ability to stimulate the production of tumor necrosis factor by the anti-E CTL clone. One transfectant expressing antigen E was identified among 70,000 drug-resistant transfectants. Its sensitivity to lysis by the anti-E CTL was equal to that of the original melanoma cell line. When this transfectant was submitted to immunoselection with the anti-E CTL clone, the resulting antigen-loss variants were found to have lost several of the transfected pSVtkneo beta sequences. This indicated that the gene coding for the antigen had been integrated in the vicinity of pSVtkneo beta sequences, as expected for cotransfected DNA.

Antigens, Neoplasm↗
PubMed1992

Mapping of the genes encoding tum- transplantation antigens P91A, P35B, and P198.

Tum- comprises a class of genes, mutation of which in P815 tumor cells has led to the acquisition of new cytotoxic T cell-recognized epitopes. The cells carrying the mutant alleles have impaired tumorigenicity compared with their progenitors due to in vivo induction of a cytotoxic T-cell response specific for tum- antigens. Two tum- genes, P91A and P35B, were found to be single copy loci mapping to chromosomes 11 and 15 respectively. A third, P198, was found to map to chromosome 7 and to be a member of a small gene family with other members on chromosomes 13, 14, and 15. Multiple P198-related sequences were found in other mammalian species suggesting the P198 related gene family is a general feature of mammalian genomes.

Animals↗
PubMed1992

Identification and quantification of a naturally presented peptide as recognized by cytotoxic T lymphocytes specific for an immunogenic tumor variant.

The target antigen recognized by H-2Kd-restricted cytotoxic T lymphocytes (CTLs) specific for a mutagen-induced antigen on DBA/2-derived tumor P815 was identified as the product of a normal cellular gene encompassing a point mutation. Using synthetic peptides, the epitope recognized by these CTLs was narrowed down to be contained within the undecamer KYQAVTTTLEE, incorporating the point mutation. The allele-specific peptide motif for H-2Kd molecules allowed us to predict the peptide naturally presented by the tumor cells to be the nonamer KYQAVTTTL. Isolation of the natural tum(-)-specific peptide from P198.3 tumor cells and biochemical comparison with the synthetic nonamer confirmed the prediction. This natural nonapeptide is represented by approximately 100 copies per tumor cell.

Amino Acid Sequence↗
PubMed1992

Identification of tumour rejection antigens recognized by T lymphocytes.

On the basis of the results reviewed here, there are two major mechanisms whereby tumour rejection antigens may arise. The first mechanism is mutational. Point mutations occurring in a large variety of genes may produce new antigenic peptides, either by providing them with the ability to bind to MHC class I molecules or by providing them with a new epitope (Fig. 2). The second mechanism is the activation of a gene that is silent in normal tissues and for which no strong natural tolerance has been established. Plausible candidates for the mutational mechanism are the "tumour specific transplantation antigens" observed on methylcholanthrene induced tumours and tumours induced by ultraviolet light. The diversity of these antigens appears to be very large, like that of the tum- antigens. Moreover, these tumours have been obtained with high doses of carcinogens, which are proven mutagens. On the other hand, a P815 tumour rejection antigen appears to arise through the activation of a silent gene, and it may turn out that this is the rule for most tumour rejection antigens. It is our hope that other genes coding for mouse and human tumour rejection antigens will soon be identified, so that it will become clear whether the activational mechanism is the rule or the exception. In our view, this is a crucial issue. Insofar as tumour rejection antigens result from mutations, they may be highly specific for every individual tumour. The tumour specific nature of these antigens would then be easily ascertained. However, active immunization of cancer patients would require that a tumour cell line be obtained from each patient, a most unpractical prospect. If, on the other hand, production of tumour rejection antigens results from the activation of a normal gene, then there is a good probability that the same gene may be activated in many different tumours, being perhaps preferentially shared by tumours of the same histological type. This would probably not result in the expression of the same antigen in all these tumours, because the patients would differ in their presenting molecules, which are determined by their HLA haplotype. However, a subset of the tumours expressing the same "tumour rejection" gene should share the same class I restricting element, so that all of these patients could be immunized with a cell that would express the gene and carry the appropriate HLA molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.

Many human melanoma tumors express antigens that are recognized in vitro by cytolytic T lymphocytes (CTLs) derived from the tumor-bearing patient. A gene was identified that directed the expression of antigen MZ2-E on a human melanoma cell line. This gene shows no similarity to known sequences and belongs to a family of at least three genes. It is expressed by the original melanoma cells, other melanoma cell lines, and by some tumor cells of other histological types. No expression was observed in a panel of normal tissues. Antigen MZ2-E appears to be presented by HLA-A1; anti-MZ2-E CTLs of the original patient recognized two melanoma cell lines of other HLA-A1 patients that expressed the gene. Thus, precisely targeted immunotherapy directed against antigen MZ2-E could be provided to individuals identified by HLA typing and analysis of the RNA of a small tumor sample.

Antigens, Neoplasm↗
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↗
PubMed1991

The identification of tyrosine as a common key residue in unrelated H-2Kd restricted antigenic peptides.

We have compared the activity of several Kd- or Ld-restricted antigenic peptides as competitors in a functional competition assay using cytolytic T lymphocyte (CTL) clones. All of four unrelated Kd-restricted peptides tested could compete with each other but not with the Ld-restricted peptide P91A-. 12-24 (P91A). Moreover, the P91A peptide failed to compete with the four Kd-restricted peptides. In contrast, another Ld-restricted peptide [mouse cytomegalovirus (MCMV) pp89 167-176] could clearly compete with both Kd- and Ld-restricted peptides. The comparison of a series of modified MCMV pp89 peptides suggested that distinct structural features allow the interaction of the peptide with the two different MHC class I molecules. We showed previously that the competitor activity of two different Kd-restricted antigenic peptides was reduced substantially upon Ala substitution of the single Tyr residues present in these peptides. We now show a similar effect for two additional Kd-restricted peptides. Our results thus suggest that Tyr may function as an 'anchor' residue for many antigenic peptides that bind to the Kd molecule. Molecular modeling of the presumed antigen-binding site of the Kd molecule revealed the presence of two deep cavities that may be involved in binding peptide amino acid side chains. A model illustrating one possible interaction of a Tyr-containing peptide with the Kd molecule is presented.

Alanine↗
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

Antigens recognized on a melanoma cell line by autologous cytolytic T lymphocytes are also expressed on freshly collected tumor cells.

Peripheral blood lymphocytes of a melanoma patient were stimulated in vitro with a permanent cell line derived from the autologous tumor. Stable cytolytic T lymphocyte (CTL) clones were obtained that lysed the melanoma cell line and did not lyse autologous Epstein-Barr virus-transformed B lymphocytes or K-562 cells. These CTL clones were directed against two distinct antigens on the melanoma line. In view of the possibility that these antigens could be culture artefacts, we tested the stimulatory ability of tumor cells that had been freshly collected from metastatic relapses on the CTL clones. A considerable CTL proliferation was observed and it appeared to be specific. We conclude that the antigens recognized by the autologous CTL clones on the permanent melanoma cell line were expressed by the tumor cells in the patient.

Antigens, Neoplasm↗
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↗
PubMed1990

Leishmania major: nature of immunity induced by immunization with a mutagenized avirulent clone of the parasite in mice.

A chemically mutagenized avirulent form of Leishmania major was used to immunize BALB/c and C57B1/6 mice against challenge with virulent L. major. Immunity was elicited when the avirulent parasite was injected intravenously or intraperitoneally, but not subcutaneously. In fact, the latter route of immunization sometimes resulted in exacerbation of a subsequent infection with virulent L. major. Mice immunized with avirulent L. major developed upon challenge with virulent L. major cutaneous lesions which were significantly smaller and contained substantially fewer parasites than lesions on control nonimmune animals. Finally, the protection conferred by immunization with avirulent L. major could be adoptively transferred with T cells of the CD4+ lineage but not the CD8+ lineage.

Animals↗
PubMed1990

Intraprostatic distribution of lomefloxacin following multiple-dose administration.

The ability of lomefloxacin to penetrate and distribute within the human prostate was assessed in 20 patients undergoing elective prostate surgery (18 transurethral prostatic resections and 2 prostatectomies). Subjects were middle-aged to elderly (mean age +/- standard deviation, 69.8 +/- 8.2 years) with normal hepatic function and with creatinine clearances ranging from 35.8 to 141 ml/min/1.73 m2. Lomefloxacin was administered in 400-mg doses orally every 24 h. Its disposition was characterized following the third dose by obtaining multiple serum samples and intraoperative paired central zone and peripheral zone prostate tissue samples. Lomefloxacin concentrations were determined by a validated high-performance liquid chromatography method with fluorescence detection. Concentrations in serum during the perioperative period declined from 3.1 +/- 1.0 mg/liter (mean +/- standard deviation) at 1 h postdose to 2.3 +/- 0.7 mg/liter at 6 h postdose. The time of tissue extraction ranged from 0.1 to 7.1 h postdose. Intraoperative serum lomefloxacin concentrations ranged from 0.5 to 4.8 (median, 2.4) mg/liter, while prostate tissue concentrations ranged from 1.1 to 10.1 (median, 5.4) mg/kg of tissue for the central zone and 0.9 to 6.5 (median, 5.2) mg/kg for the peripheral zone. Intraindividual paired prostate concentrations (central zone versus peripheral zone) were not statistically different. The partition coefficient (ratio of concentration in prostate to concentration in serum) for the central zone was 2.2 +/- 0.6 (range, 1.2 to 3.1), and for the peripheral zone it was 2.1 +/- 0.7 (range, 1.2 to 4.2). Lomefloxacin exhibited good penetration into the human prostate with homogeneous intraprostatic distribution following multiple-dose administration.

4-Quinolones↗