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P Kourilsky

Publications and source records attributed to P Kourilsky.

At least 19 recordsLinked to original sources
PubMed1992

Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules.

We have tested 21 independent CTL clones for recognition of a single peptide derived from the Plasmodium berghei circumsporozoite protein in the context of 13 mutants of the murine MHC class I molecule H-2Kd. In this series of Kd mutants, amino acid residues located on the upper surface of the alpha-helices were individually substituted by alanine. Remarkably, most clones displayed individual recognition patterns on the Kd mutants. We had previously found that this series of CTL clones was likewise highly diverse in terms of both TCR primary structure and peptide fine specificity. Our data thus reinforce the concept that multiple T cell epitopes are available on the surface of a single peptide-MHC class I complex for recognition by specific TCR.

Amino Acid Sequence↗
PubMed1992

Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses.

The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation.

Animals↗
PubMed1992

Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

Adenovirus E1A Proteins↗
PubMed1992

Altered binding of regulatory factors to HLA class I enhancer sequence in human tumor cell lines lacking class I antigen expression.

Class I antigens encoded in the major histocompatibility complex (MHC) (HLA in man, H-2 in the mouse) play a key role in the recognition of target cells by cytolytic T lymphocytes. Tumor cells frequently do not express class I MHC molecules, which strongly suggests that down-regulation of the latter facilitates escape of tumor cells from immune surveillance. The expression of class I MHC genes is tightly regulated. An enhancer element, conserved in the promoters of mouse and human MHC genes, has been shown to be important for mouse class I MHC gene expression. At least two related regulatory factors (KBF1 and NF-kappa B) bind to this regulatory element. We have analyzed the binding of these factors in cellular extracts of 23 human tumor cell lines displaying various levels of class I mRNA and surface expression. In this panel, combined deficiency of KBF1- and NF-kappa B-like DNA-binding activities was frequent among the class I-negative cell lines and correlated with the absence of class I mRNA. A few cell lines that lack KBF1 binding activity still display NF-kappa B-like activity and express normal levels of MHC class I mRNA. These results suggest (i) that, in the absence of KBF1, NF-kappa B or a related factor promotes MHC class I gene transcription; and (ii) that a combined defect in KBF1/NF-kappa B DNA-binding activity can cause a pleiotropic defect in class I gene expression, which may facilitate tumor progression.

Antibodies, Monoclonal↗
PubMed1992

Tolerance to a self-protein involves its immunodominant but does not involve its subdominant determinants.

We have produced transgenic mice expression hen egg-white lysozyme (HEL) under the control of a ubiquitous promoter, so that in transgenic animals, HEL is presumably present in the serum and thymus throughout the period of establishment of the T-cell repertoire. We show that HEL transgenic H-2d mice with HEL blood levels greater than 10 ng/ml are tolerant to HEL as well as to the immunodominant peptide 108-116. Thus, their T lymphocytes do not proliferate in response to the immunodominant peptide 108-116 after in vivo immunization with HEL or peptide 108-116. In contrast, in transgenic mice tolerant to HEL, the state of tolerance to subdominant peptides 1-18 and 74-96 appears variable and highly depended on HEL blood levels. Complete unresponsiveness is seen when HEL serum levels are high, and this unresponsiveness is reached at a lower HEL concentration for peptide 1-18 than for peptide 74-96. Thus, a hierarchy exists among the three peptides (108-116 much greater than 1-18 greater than 74-96) for induction of a response to HEL and for HEL tolerance induction in T cells specific for these peptides. Persistence in the periphery of autoreactive T cells recognizing subdominant peptides of self-proteins, as shown in this transgenic model, indicates that self-tolerance is limited to a subset of dominant self-peptides and suggests a role for T lymphocytes specific for subdominant determinants in autoimmunity.

Animals↗
PubMed1992

Molecular detection and in vivo analysis of the specific T cell response to a protein antigen.

We have analyzed in detail the repertoire of transcripts encoding the V beta chains of the T cell receptor and investigated the T cell response of B10.A mice to pigeon cytochrome c. We were thus able to follow the specific T cell response in vivo after immunization with this protein antigen. The response is first detectable in the draining lymph nodes, then in the spleen and in the blood. It is qualitatively similar in individual animals. It is dominated by a major category of specific T cells harboring a V beta 3-J beta 1.2 rearrangement, and a limited and well-defined set of nucleotide sequences, previously found in several specific T cell hybridomas and clones. This predominance is observed from the onset of the immune response strongly suggesting the notion that there is no variation and, therefore, no maturation of the T cell response in the course of immunization.

Animals↗
PubMed1992

The characterization of the promoter of the gene encoding the p50 subunit of NF-kappa B indicates that it participates in its own regulation.

In order to characterize the regulation of the gene encoding the p50 subunit of the transcription factor NF-kappa B, we have isolated a human genomic clone, and sequenced the promoter of this gene. By in situ hybridization we have mapped the gene encoding the p50 subunit of NF-kappa B to the 4q23-4q25 region of the human genome and the H1-H3 region of the murine chromosome 3. The p50 promoter lacks TATA and CAAT elements, but contains NF-kappa B, AP-1 and HIP-1 binding sequence. The kappa B motif binds NF-kappa B, KBF1, and heterodimers of p50 and c-rel, suggesting that the gene is regulated by members of this family. Co-transfection experiments demonstrate that the p50 promoter can be activated by either of the two subunits of NF-kappa B (p50 and p65), and more strongly by the combination of both. The promoter of p50 can be activated by phorbol esters and tumor necrosis factor alpha but not by forskolin and these responses are mediated through the NF-kappa B binding sequence. The involvement of NF-kappa B in the regulation of the p50 gene is also supported by the inhibition of the PMA activation of the promoter by an NF-kappa B transdominant negative mutant, as well as the product of the v-rel oncogene.

Animals↗
PubMed1992

Evolution of the major histocompatibility complex: a hundred-fold amplification of MHC class I genes in the African pigmy mouse Nannomys setulosus.

The genome of the African murine rodent Nannomys setulosus was found to harbor several thousand major histocompatibility complex (MHC) class I genes instead of the 30-40 genes found in conventional laboratory mice, which are mostly of Mus musculus domesticus origin. Other genes of N. setulosus, either functionally or physically linked to class I genes, are not amplified. Amplified genes derive from as few as three ancestors and amplification has likely occurred after the divergence of the two Nannomys species, N. setulosus and N. minutoides, which took place about three million years ago. Amplified genes are mostly pseudogenes. Statistical analysis of dinucleotide frequencies leads us to propose that inactivation of the genes has occurred through the repeat induced mutation process, a possible "newcomer" in the evolution of the MHC.

Amino Acid Sequence↗
PubMed1992

NF-kappa B and related proteins: Rel/dorsal homologies meet ankyrin-like repeats.

Molecular cloning of the subunits of the transcription factor NF-kappa B and its inhibitor l kappa B revealed regions of sequence homology with two different classes of proteins: the Rel/dorsal family and a heterogeneous group of proteins containing ankyrin-like repeats. Both the Rel/dorsal homology domain and the ankyrin-like repeats appear to play important roles in protein-protein interactions that regulate localization and activity of the NF-kappa B subunits.

Amino Acid Sequence↗
PubMed1992

Inhibition of tumor growth by histoincompatible cells expressing interleukin-2.

Murine tumor cells engineered to express IL-2 have been shown to be rejected by the syngeneic host, which is then protected against a subsequent tumorigenic challenge. To assess whether IL-2 has to be produced by the tumor cells themselves, or whether its local delivery would be sufficient to promote such beneficial effects, the syngeneic tumor cells were co-inoculated with allogeneic or xenogeneic cells secreting IL-2, selected after gene transfection. In several murine systems, it was observed that this is an efficient approach for controlling the growth of the syngeneic tumor. However, animals which rejected the tumor were not protected against a subsequent challenge. Several lines of evidence indicate that NK cells play a major role in tumor rejection induced by the IL-2 expressing histoincompatible vector cells. Thus, while local delivery of IL-2 in the vicinity of a tumor might not be sufficient to promote a systemic long-term specific antitumor immune response, it can control the growth of the primary syngeneic tumor. These experiments demonstrate the feasibility of using genetically engineered histoincompatible cells (which are rejected by the host's immune system) as a transient delivery system in vivo.

Animals↗
PubMed1992

Binding of low concentration of peptide to H-2Kd produced in insect cells requires mouse beta 2-microglobulin co-expression.

A recombinant baculovirus expressing the murine class I MHC heavy chain H-2Kd cDNA under the transcriptional control of Autografa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter has been isolated and used to infect Sf9 lepidopteran cells either alone or in association with a previously isolated virus expressing mouse beta 2-microglobulina (beta 2-ma). When infected with the heavy chain-encoding virus alone, H-2Kd was produced in a beta 2-m-free conformation detected on the surface of infected cells by conformation-independent antibodies. When Sf9 cells were co-infected with both viruses, approximately 10% of the heavy chain pool was engaged in the formation of native heterodimeric MHC class I molecules, which were glycosylated and transported to the cell surface as demonstrated by radio-binding experiments and flow cytometry. The assembly of the recombinant class I molecule was dependent on peptide, since heterodimer formation was brought about by H-2Kd-specific peptide ligands both in vivo, upon incubation with dually infected cells, and in vitro, in cell-free detergent extracts. In addition, a change in heavy chain conformation was brought about upon incubation with high concentrations (100 microM) of an H-2Kd-restricted octapeptide epitope from Plasmodium berghei. Furthermore, using low concentrations (3 nM) of a photoaffinity label derivative of this peptide, we show direct binding to cells co-expressing class I heavy chain and mouse beta 2-m but not to cells expressing free heavy chain only.

Amino Acid Sequence↗
PubMed1992

Most residues on the floor of the antigen binding site of the class I MHC molecule H-2Kd influence peptide presentation.

A panel of 15 single alanine substitutions on the floor of the peptide binding groove of the murine class I histocompatibility molecule H-2Kd has been analyzed. All but two mutant molecules were expressed on the cell surface, and were tested for peptide binding and presentation to specific cytotoxic T lymphocytes. Eleven out of 13 mutant molecules appeared to be functionally altered. Five of the substituted residues were involved in the presentation of all peptides tested. Three participated in the presentation of certain peptides but not others. Three other residues participated in epitope formation through indirect interactions. Only two mutations had no detectable effect.

Alanine↗
PubMed1992

Co-expression of T-cell receptor beta and delta mRNA detected at high frequency in hybridomas derived from adult thymus.

Two different hybridoma collections from adult C3H/HeJ thymus were generated in order to analyse T-cell receptor (TcR) rearrangements, surface expression of T-cell receptors and differentiation markers as well as lymphokine production. Large, low density thymocytes were either directly fused to the thymoma BW 5147 alpha-beta- variant, or fused after stimulation with Concanavalin A in the presence of interleukin-2 for 48 h. The hybrids obtained from Concanavalin A-stimulated cells represented rather mature thymocytes, with regard to TcR rearrangements and surface T-cell receptor expression. The collection of hybrids derived from freshly isolated large thymocytes contained cells in various stages of T-cell development. An unexpectedly large number of hybrids (46 out of 84) from this group expressed full-length C beta together with full-length, or shorter, C delta mRNA. This finding suggests that a considerable proportion of alpha beta T cells proceeds through a stage in development where delta genes are being rearranged and transcribed.

Animals↗
PubMed1992

[Potentiation of the antitumoral effect of electrochemotherapy by immunotherapy with allogeneic cells producing interleukin 2].

Electrochemotherapy (ECT) is a new antitumour treatment which consists of delivering electric pulses to the tumour a few minutes after an intravenous injection of bleomycin. The antitumour efficacy of ECT is increased by local injections of interleukin 2 (IL2) in the oedema which appears at the site of the treated tumours. We have shown that tumour cells inoculated in syngeneic mice are rejected if IL2 secreting allo- or xenogeneic cells are co-injected with tumour cells. We report here the large increase of ECT therapeutical efficacy when allogeneic cells secreting IL2 are injected into the peri-tumoural oedema.

Animals↗