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A Toubert

Publications and source records attributed to A Toubert.

At least 55 records · Page 3Linked to original sources
PubMed1997

[Antigen presentation and macrophages].

T-cells recognize antigenic peptides associated with HLA molecules belonging to Class I (HLA-A, -B, or -C) or Class II (HLA-DP, -DQ, -DR). Roughly, Class I HLA molecules represent antigens to cytotoxic CD8+ T-cells and Class II HLA molecules to helper CD4+ T-cells. Class II HLA molecules primarily present "exogenic" peptides that penetrate within cells by endocytosis, whereas Class I HLA molecules present "endogenic" peptides produced within cells. In both cases, the mechanisms of antigen presentation are closely liked to the biosynthesis of HLA molecules and to the expression of other molecules including proteasome (LMP) and the peptide transporters (TAP) needed to transport peptides through the endoplasmic reticulum in the case of Class I molecules, and invariant chain (Ii) and HLA-DM antigens in the case of Class II molecules. In addition, "exogenous" presentation by Class I molecules has recently been described, and may be relatively specific of phagocytic cells such as macrophages.

Cysteine Endopeptidases↗
PubMed1996

Differences in endogenous peptides presented by HLA-B*2705 and B*2703 allelic variants. Implications for susceptibility to spondylarthropathies.

The association between HLA-B27 and spondylarthropathies is currently being reinvestigated in the light of HLA-B27 subtyping. At least 11 different subtypes have been described among which B*2703, B*2706, and B*2709 could be less closely associated with disease at the population level. Differences in the presentation of antigenic peptides by these subtypes could be related to differences in disease susceptibility. We focused our work on the comparison of B*2705 and B*2703 which differ at a single position at residue 59 in pocket A of the peptide binding groove. Endogenous peptides from the human C1R line transfected by B*2705 or B*2703 were acid-eluted and separated by HPLC. Major individual fractions were sequenced by Edman NH2-terminal degradation. Differences observed between B*2705 versus B*2703 individual ligands were confirmed in an in vitro stabilization assay with T2-B*2705 or B*2703 transfected cells in the presence of synthetic peptides. One B*2705 associated peptide is derived from the sequence 169-179 in the second extracellular domain of several HLA class I molecules including HLA-B27. This sequence (RRYLENGKETL) is highly homologous to a previously reported sequence (LRRYLENGK) sharing similarities with proteins from enteric bacteria. We show here that it is naturally presented as a major endogenous peptide by B*2705 and B*2702 disease-associated subtypes and not by B*2703.

Amino Acid Sequence↗
PubMed1996

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.

Alleles↗
PubMed1995

T-cell repertoires in healthy and diseased human tissues analysed by T-cell receptor beta-chain CDR3 size determination: evidence for oligoclonal expansions in tumours and inflammatory diseases.

Many examples of oligoclonal T-cell expansion in infiltrated diseased tissues have been reported. However, it remains to be established whether such observations can be generalized and to what extent oligoclonal patterns obtained after in vitro culture of T-cell infiltrates reflect in vivo situations. Using new high resolution analysis which requires no in vitro cellular expansion, we detected such oligoclonal T-cell expansions in 7/7 melanoma tumour biopsies, 3/3 biopsies of inflammatory skin during acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation (alloBMT) and 7/7 synovial membranes from patients with rheumatoid arthritis. Thus, oligoclonal T-cell expansions are readily observed when a sufficiently sensitive detection method is used, suggesting that similar expansions are the rule among T-cell infiltrates in different diseases. This observation and the monitoring of the in vivo evolution of such expansion during the course of the disease and during in vitro culture should have important clinical implications.

Acute Disease↗
PubMed1995

Characterization of two human IgM monoclonal antibodies reactive with HLA-B27.

We describe here the generation and characterization of two human monoclonal IgM antibodies (UL-4F11 and UL-F6) reactive with HLA-B27. The monoclonal antibody (mAb) UL-4F11 is cytotoxic for peripheral mononuclear cells and, therefore, useful as typing reagent for HLA-B27 and HLA-B38. Protein chemistry showed that the mAb UL-4F11 precipitates HLA-B27 molecules. Epitope mapping analysis suggests that the amino acids 45, 67, 82 and 83 (alpha-1 domain) of the HLA-B27 sequence are necessary for mAb UL-4F11 reactivity. The mAb UL-F6 is suitable for complement dependent lysis of lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells with HLA-B27 (B*2701, B*2702, B*2703, B*2705, B*2707), B13, B40 (60,61), B47 and B48 specificities. Its reactivity indicates that the amino acid valine in position 152 and glutamic acid in position 163 of the alpha-2 domain are crucial for the binding epitope.

Amino Acid Sequence↗
PubMed1993

A peptide substitution in HIV-1 gp120 first hypervariable domain enhances its immunogenicity in mice.

The envelope (Env) protein from HIV-1 is the focus of several vaccine trials in humans. It could be considered for the optimization of Env vaccinal preparations to add within the molecule defined epitopes, for instance epitopes conserved among viral isolates from HIV-1, such as from Gag or Nef proteins. As a first step to this approach, we have constructed by in vitro mutagenesis HIV-1(LAI) Env gp120 molecules in which a 12-amino acid sequence in the first (V1) or the third (V3) hypervariable region was substituted by the hemagglutinin (HA) 307-318 peptide from the influenza virus, a dominant T helper cell epitope in humans. The proteins were produced by recombinant vaccinia viruses. They had kept their structural properties in terms of serological recognition and binding to CD4. Of note, we observed that the gp120 protein substituted in the V1 domain elicited a stronger serological immune response in mice compared to native or V3 substituted gp120. This indicates that gp120 can accommodate large substitutions without major structural perturbations and that, on the contrary, some of them could prove beneficial in terms of immunogenicity.

Amino Acid Sequence↗
PubMed1992

Mapping of an epitope defined by a human hybridoma antibody (TrD3): a new HLA-B supertype associated with a subset of HLA-Bw6.

The new human-human hybridoma TrD3 secretes a cytotoxic IgM mAb, which reacted with 28 of a panel of 56 HLA-typed lymphoblastoid cells. All 28 TrD3+ cells expressed the HLA-B supertype Bw6, whereas 10 Bw6+ cells were not recognized by the mAb. None of the 17 Bw4 homozygous cells were positive with TrD3. Thus, TrD3 divided the Bw6+ HLA-B specificities of the cell lines into two subgroups, namely, Bw6+TrD3+ and Bw6+TrD3-, and therefore defines a new HLA-B supertype. TrD3 reacted strongly with some B8+ cell lines and weakly or not at all with others, suggesting a new split of HLA-B8. Compared with cell lines, TrD3 reacted more weakly with freshly isolated T cells from blood. The Bw6-specific rat mAb SFR8-B6 partially blocked the binding of 125I-labeled TrD3 to a Bw6+ cell line. By using cell lines transfected with hybrid genes between HLA-B7 (Bw6+) and HLA-B27 (Bw6-) as targets in flow cytometry, critical residues for the TrD3 epitope could be mapped to the amino acid region 24-62 of the HLA class-I alpha 1 domain. Comparison of deduced amino acid sequences of TrD3-positive and -negative cells indicated that a tryptophane residue at position 95 destroyed the TrD3 epitope, and that one or more of the residues in positions 24, 45, and 46 may be critical, suggesting that it is a discontinuous epitope. It is notable that none of these residues are located on alpha-helixes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

The defective in vitro expression of interferon-gamma messenger RNA in rheumatoid arthritis is recovered by cycloheximide.

Circulating T lymphocytes from patients with rheumatoid arthritis (RA) were found to produce low concentrations of interferon-gamma (IFN-gamma) and express low amounts of this messenger RNA (mRNA) in response to stimulation with phytohemagglutinin (PHA). In this study, we have examined the superinduction effect of cycloheximide (CHX) on the IFN-gamma mRNA expression in PHA-stimulated T lymphocytes from nine patients with active RA compared to three healthy individuals. When CHX was added 6 hr postactivation, the expression of IFN-gamma mRNA in RA patients increased significantly at 24 and 48 hr (P less than 0.01) and reached levels similar to those observed in controls. We suggest that posttranscriptional events could play a role in the defective production of IFN-gamma observed in RA.

Arthritis, Rheumatoid↗
PubMed1991

Epitope mapping of human monoclonal antibodies to HLA-B27 by using natural and mutated antigenic variants.

The epitopes defined by three human monoclonal antibodies (mAbs) (Tr3B6, TrCG10, TrBH12) against HLA-B27 have been mapped by flow cytometry. For this purpose we used murine transfected cells expressing at their surface hybrid antigens between HLA-B7 and -B27 and, in addition, Epstein-Barr virus cell lines expressing the six HLA-B27 alleles B*2701 to B*2706. The results indicated that the mAbs are domain specific. TrBH12 recognizes the first external (alpha-1) domain. Residues critical for the TrBH12 epitope are located in the alpha-1 helix and include the polypeptide stretch 63-76 plus a critical amino acid at position 77. Tr3B6 binds the second external (alpha-2) domain, and one mutation (VAL152----GLU152) destroyed its epitope. TrCG10 also binds the alpha-2 domain.

Alleles↗
PubMed1990

Interferon-gamma mRNA expression upon in vitro T lymphocyte activation is decreased in rheumatoid arthritis patients.

A decreased production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) upon in vitro stimulation by phytohemagglutinin (PHA) peripheral blood leucocyte in active rheumatoid arthritis (RA) patients has been described as an important immunological abnormality in this disease. However, the molecular level at which this defect might occur has not been fully documented. We have investigated the kinetics of IFN-gamma, interleukin-2 receptor (IL-2 R), and interleukin-1 beta (IL-1 beta) messenger RNA (mRNA) expression in RA patients (n = 9) and normal controls (n = 5) after PHA leucocyte activation. We demonstrate here a significantly decreased expression of IFN-gamma mRNA in RA patients without modification of its kinetics associated with a similar IL-1 beta mRNA expression.

Arthritis, Rheumatoid↗
PubMed1990

Epitope mapping of an HLA-B27 monoclonal antibody that also reacts with a 35-kD bacterial outer-membrane protein.

Ye-3 is an HLA-B27-specific murine monoclonal antibody recognizing the heat-modifiable protein of Enterobacteriaceae. Here we used recombinant hybrid molecules between the HLA-B7 and HLA-B27 antigens to delineate the epitope recognized by Ye-3. Results of these experiments indicated that the segment of HLA-B*2705 spanning residues 77-81 was critical to the reactive epitope. It is known to be a major serological and functional recognition site of HLA-B*2705 and our data give support for its involvement also in the serological cross-reactivity with bacterial antigens.

Alleles↗
PubMed1989

Delineation of determinants on HLA-B7 and HLA-B27 that are necessary for cytolytic T cell recognition by using inter- and intra-domain recombinants.

We have used bulk culture HLA-B7 and HLA-B27 specific CTL lines derived from 11 donors, and a series of rHLA-B7/HLA-B27 genes transfected into and expressed on the surface of the murine cell P815, to determine the amino acid residues on these HLA class I molecules that are critical for allospecific CTL recognition. The results obtained indicate that for four of six HLA-B7-specific CTL lines the alpha-1 domain for CTL recognition. Furthermore, we found that residues 77 and/or 80 had a critical effect on recognition for all of the CTL lines tested. The region 97-156 in the alpha-2 domain was also important for some of these CTL lines. Furthermore, by using five bulk culture HLA-B27-specific CTL lines we were able to show that residues 77 and/or 80 and residue 152 are also essential for recognition of HLA-B27 by HLA-B27-specific CTL. The strong influence exerted by these residues is discussed in terms of the three-dimensional structure of class I molecules. Finally, a selection was regularly observed in the bulk cultures such that the CTL that were preferentially influenced by either the alpha-1 or the alpha-2 domain were lost after 4 to 7 wk of culture resulting in CTL cell lines which were extremely sensitive to sequence modifications of HLA-B7 or HLA-B27. The possible reasons for this selection, which we have previously observed with both anti-HLA-A2 and anti-HLA-A3 cell lines and is therefore not unique to HLA-B7 or HLA-B27, are discussed.

Cell Line↗
PubMed1988

Epitope mapping of HLA-B27 and HLA-B7 antigens by using intradomain recombinants.

To study the HLA-B7 and HLA-B27 antigenic determinants, hybrid genes between these two alleles were constructed by in vivo recombination in Escherichia coli. After transfection of these genes into P815 (high transfection efficiency recipient) murine cells, the bindings of Bw6, HLA-B7, and HLA-B27 allele-specific mAb were studied, as well as that of human anti-HLA-B7 and anti-HLA-B27 monospecific alloantisera. Most of the HLA-B7 antigenic determinants were assigned to the first external domain of the molecule. Four different epitopic areas could be defined: the Bw6 epitope was associated with residues 82 and 83; the BB7.1 epitope to amino acids 63, 67, and 70; the MB40.2 and MB40.3 epitope to amino acid sequence 177-180, and human alloantisera identified as an epitope associated with residue 9. HLA-B27 antigenicity studied by TM-1 mAb was found to involve residues 77 and 80 in the alpha-1 domain. Results obtained with human monospecific alloantisera allowed the definition of an additional allospecific site associated with the NH2 terminal part on the alpha-1 domain of HLA-B27. Epitope mapping fits with data obtained by sequence comparisons and is discussed with reference to the crystallographic three-dimensional structure of the HLA-A2 molecule.

Amino Acid Sequence↗
PubMed1988

Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants.

HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination. In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences. Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences. The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases. No double recombinants were observed. These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa. These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells. Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152). The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161). But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2.

Antibodies, Monoclonal↗
PubMed1988

The chromosome region containing the highly polymorphic HLA class I genes displays limited large scale variability in the human population.

The large-scale organization and polymorphism of the HLA class I region was investigated by pulsed field gel (PFG) fractionation of DNA from various HLA-typed cell lines cleaved by different 'rare cutter' restriction enzymes, followed by hybridization with 'general' and locus-specific HLA probes. Results indicate that (i) most HLA class I sequences are contained in a 340 kb MluI DNA fragment which also carries the HLA-A gene; (ii) HLA-A, -B and -C genes are present on different fragments bounded by 'HTF islands' (CpG-rich, unmethylated DNA regions containing multiple sites for 'rare cutter' enzymes) which generally coincide with the 5' regions of expressed genes; and (iii) very little fragment size polymorphism is seen, implying that expansion/contraction events in the HLA class I region due to unequal crossing over (as documented in the mouse class I system) are infrequently found in the human population.

Chromosome Mapping↗