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Vincent Stroobant

Publications and source records attributed to Vincent Stroobant.

14 recordsLinked to original sources
PubMed2006

An antigen produced by splicing of noncontiguous peptides in the reverse order.

CD8-positive T lymphocytes recognize peptides that are usually derived from the degradation of cellular proteins and are presented by class I molecules of the major histocompatibility complex. Here we describe a human minor histocompatibility antigen created by a polymorphism in the SP110 nuclear phosphoprotein gene. The antigenic peptide comprises two noncontiguous SP110 peptide segments spliced together in reverse order to that in which they occur in the predicted SP110 protein. The antigenic peptide could be produced in vitro by incubation of precursor peptides with highly purified 20S proteasomes. Cutting and splicing probably occur within the proteasome by transpeptidation.

Alleles↗
PubMed2006

Destructive cleavage of antigenic peptides either by the immunoproteasome or by the standard proteasome results in differential antigen presentation.

The immunoproteasome (IP) is usually viewed as favoring the production of antigenic peptides presented by MHC class I molecules, mainly because of its higher cleavage activity after hydrophobic residues, referred to as the chymotrypsin-like activity. However, some peptides have been found to be better produced by the standard proteasome. The mechanism of this differential processing has not been described. By studying the processing of three tumor antigenic peptides of clinical interest, we demonstrate that their differential processing mainly results from differences in the efficiency of internal cleavages by the two proteasome types. Peptide gp100(209-217) (ITDQVPSFV) and peptide tyrosinase369-377 (YMDGTMSQV) are destroyed by the IP, which cleaves after an internal hydrophobic residue. Conversely, peptide MAGE-C2(336-344) (ALKDVEERV) is destroyed by the standard proteasome by internal cleavage after an acidic residue, in line with its higher postacidic activity. These results indicate that the IP may destroy some antigenic peptides due to its higher chymotrypsin-like activity, rather than favor their production. They also suggest that the sets of peptides produced by the two proteasome types differ more than expected. Considering that mature dendritic cells mainly contain IPs, our results have implications for the design of immunotherapy strategies.

Amino Acid Sequence↗
PubMed2005

A new LAGE-1 peptide recognized by cytolytic T lymphocytes on HLA-A68 tumors.

Antigens encoded by genes of the LAGE family, including LAGE-1 and NY-ESO-1, are of interest for cancer immunotherapy because they are tumor-specific and shared by tumors of different histological types. Several clinical trials are in progress with NY-ESO-1 peptides, protein, recombinant poxviruses, and dendritic cells pulsed with peptides. In this study, CD8 T lymphocytes from an individual without cancer were stimulated with dendritic cells infected with a recombinant avian poxvirus encoding a complete LAGE-1 protein. A CTL clone was isolated that recognized a new LAGE-1 peptide, ELVRRILSR, which corresponds to position 103-111 of the protein sequence. It is presented by HLA-A6801 molecules. When tumor cells expressing LAGE-1 were transfected with HLA-A68, they were lysed by the CTL clone, indicating that the peptide is processed in tumor cells. These results indicate that the LAGE-1.A68 peptide can be used for antitumoral vaccination. We observed also that specific T cells could be detected in a blood sample with a high sensitivity by using an A68/LAGE-1 fluorescent multimer.

Amino Acid Sequence↗
PubMed2005

Plant ribulosamine/erythrulosamine 3-kinase, a putative protein-repair enzyme.

FN3K (fructosamine 3-kinase) is a mammalian enzyme that catalyses the phosphorylation of fructosamines, which thereby becomes unstable and detaches from proteins. The homologous mammalian enzyme, FN3K-RP (FN3K-related protein), does not phosphorylate fructosamines but ribulosamines, which are probably formed through a spontaneous reaction of amines with ribose 5-phosphate, an intermediate of the pentose-phosphate pathway and the Calvin cycle. We show in the present study that spinach leaf extracts display a substantial ribulosamine kinase activity (approx. 700 times higher than the specific activity of FN3K in erythrocytes). The ribulosamine kinase was purified approx. 400 times and shown to phosphorylate ribulose-epsilon-lysine, protein-bound ribulosamines and also, with higher affinity, erythrulose-epsilon-lysine and protein-bound erythrulosamines. Evidence is presented for the fact that the third carbon of the sugar portion is phosphorylated by this enzyme and that this leads to the formation of unstable compounds decomposing with half-lives of approx. 30 min at 37 degrees C (ribulosamine 3-phosphates) and 5 min at 30 degrees C (erythrulosamine 3-phosphates). This decomposition results in the formation of a 2-oxo-3-deoxyaldose and inorganic phosphate, with regeneration of the free amino group. The Arabidopsis thaliana homologue of FN3K/FN3K-RP was overexpressed in Escherichia coli and shown to have properties similar to those of the enzyme purified from spinach leaves. These results indicate that the plant FN3K/FN3K-RP homologue, which appears to be targeted to the chloroplast in many species, is a ribulosamine/erythrulosamine 3-kinase. This enzyme may participate in a protein deglycation process removing Amadori products derived from ribose 5-phosphate and erythrose 4-phosphate, two Calvin cycle intermediates that are potent glycating agents.

Amino Acid Sequence↗
PubMed2005

Monitoring of anti-vaccine CD4 T cell frequencies in melanoma patients vaccinated with a MAGE-3 protein.

Quantitative evaluation of T cell responses of patients receiving antitumoral vaccination with a protein is difficult because of the large number of possible HLA-peptide combinations that could be targeted by the response. To evaluate the responses of patients vaccinated with protein MAGE-3, we have developed an approach that involves overnight stimulation of blood T cells with autologous dendritic cells loaded with the protein, sorting by flow cytometry of the T cells that produce IFN-gamma, cloning of these cells, and evaluation of the number of T cell clones that secrete IFN-gamma upon stimulation with the Ag. An important criterion is that T cell clones must recognize not only stimulator cells loaded with the protein, but also stimulator cells transduced with the MAGE-3 gene, so as to exclude the T cells that recognize contaminants generated by the protein production system. Using this approach it is possible to measure T cell frequencies as low as 10(-6). We analyzed the frequencies of anti-vaccine CD4 T cells in five metastatic melanoma patients who had been injected with a MAGE-3 protein without adjuvant and showed evidence of tumor regression. Anti-MAGE-3 CD4 T cells were detected in one of the five patients. The frequency of the anti-MAGE-3 CD4 T cells was estimated at 1/60,000 of the CD4 T cells in postvaccination blood samples, representing at least an 80-fold increase in the frequency found before immunization. The frequencies of one anti-MAGE-3 CD4 T cell clonotype were confirmed by PCR analysis on blood lymphocytes. The 13 anti-MAGE-3 clones, which corresponded to five different TCR clonotypes, recognized the same peptide presented by HLA-DR1.

Amino Acid Sequence↗
PubMed2004

An antigenic peptide produced by peptide splicing in the proteasome.

CD8 T lymphocytes recognize peptides of 8 to 10 amino acids presented by class I molecules of the major histocompatibility complex. Here, CD8 T lymphocytes were found to recognize a nonameric peptide on melanoma cells that comprises two noncontiguous segments of melanocytic glycoprotein gp100(PMEL17). The production of this peptide involves the excision of four amino acids and splicing of the fragments. This process was reproduced in vitro by incubating a precursor peptide of 13 amino acids with highly purified proteasomes. Splicing appears to occur by transpeptidation involving an acyl-enzyme intermediate. Our results reveal an unanticipated aspect of the proteasome function of producing antigenic peptides.

Animals↗
PubMed2004

Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy.

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.

Amino Acid Sequence↗
PubMed2004

Development of an anti-IL-12 p40 auto-vaccine: protection in experimental autoimmune encephalomyelitis at the expense of increased sensitivity to infection.

IL-12 and IL-23, which share the IL-12 p40 subunit, have been ascribed central roles in many autoimmune disorders. We describe here an anti-IL-12 (alphaIL-12) auto-vaccine that potentially blocks both factors in vivo. Immunization of mice with mouse IL-12 coupled to OVA or Pan DR epitope (PADRE) peptide induced Ab directed against the IL-12 p40 subunit, which prevented IFN-gamma production in response to IL-12 administration in vivo. Experimental autoimmune encephalomyelitis, an IL-23-dependent disease model, induced in SJL mice with a proteolipid protein (PLP) peptide was almost undetectable after alphaIL-12 vaccination. Myelin oligodendrocyte glycoprotein (MOG)-induced disease in C57BL/6 mice was also significantly inhibited. This protection correlated with inhibited Th1 cytokine responses in vitro and with an increase in the IgG1/IgG2a anti-PLP Ab balance. Detrimental consequences of alphaIL-12 vaccination were evaluated in C57BL/6 mice infected with Leishmania major (L.m.). While delayed-type hypersensitivity (DTH) suppression and immunoglobulin as well as interleukin production patterns reflected a major shift toward a Th2-type response, L.m. growth was still significantly retarded as compared to that seen in susceptible BALB/c mice. However, vaccinated animals ultimately failed to control parasite expansion. These results suggest that some chronic autoimmune diseases may benefit from alphaIL-12 vaccination at the expense of reduced, but not completely abrogated, cell-mediated immunity.

Animals↗
PubMed2003

Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase.

T lymphocytes undergo proliferation arrest when exposed to tryptophan shortage, which can be provoked by indoleamine 2,3-dioxygenase (IDO), an enzyme that is expressed in placenta and catalyzes tryptophan degradation. Here we show that most human tumors constitutively express IDO. We also observed that expression of IDO by immunogenic mouse tumor cells prevents their rejection by preimmunized mice. This effect is accompanied by a lack of accumulation of specific T cells at the tumor site and can be partly reverted by systemic treatment of mice with an inhibitor of IDO, in the absence of noticeable toxicity. These results suggest that the efficacy of therapeutic vaccination of cancer patients might be improved by concomitant administration of an IDO inhibitor.

Animals↗
PubMed2003

A MAGE-3 peptide presented by HLA-DR1 to CD4+ T cells that were isolated from a melanoma patient vaccinated with a MAGE-3 protein.

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific Ags, which have been used in therapeutic vaccination trials of cancer patients. MAGE-3 is expressed in 74% of metastatic melanoma and in 50% of carcinomas of esophagus, head and neck, bladder, and lung. We report here the identification of a new MAGE-3 peptide, which is recognized by three different CD4(+) T cell clones isolated from a melanoma patient vaccinated with a MAGE-3 protein. These clones, which express different TCRs, recognize an HLA-DR1 peptide ACYEFLWGPRALVETS, which corresponds to the MAGE-3(267-282) and the MAGE-12(267-282) protein sequences. One of the T cell clones, which expresses LFA-1 at a high level, lysed tumor cells expressing DR1 and MAGE-3. Another of these DR1-restricted CD4(+) clones recognized not only the MAGE-3/12 peptide but also homologous peptides encoded by genes MAGE-1, 2, 4, 6, 10, and 11.

Amino Acid Sequence↗
PubMed2003

A mammalian protein homologous to fructosamine-3-kinase is a ketosamine-3-kinase acting on psicosamines and ribulosamines but not on fructosamines.

Fructosamine-3-kinase (FN3K) is an enzyme that appears to be responsible for the removal of fructosamines from proteins. In this study, we report the sequence of human and mouse cDNAs encoding proteins sharing 65% sequence identity with FN3K. The genes encoding FN3K and FN3K-related protein (FN3K-RP) are present next to each other on human chromosome 17q25, and they both have a similar 6-exon structure. Northern blots of mouse tissues RNAs indicate a high level of expression of both genes in bone marrow, brain, kidneys, and spleen. Human FN3K-RP was transfected in human embryonic kidney (HEK) cells, and the expressed protein was partially purified by chromatography on Blue Sepharose. Unlike FN3K, FN3K-RP did not phosphorylate fructoselysine, 1-deoxy-1-morpholino-fructose, or lysozyme glycated with glucose. In a more systematic screening for potential substrates for FN3K-RP, we found, however, that both enzymes phosphorylated ketosamines with a D-configuration in C3 (psicoselysine, 1-deoxy-1-morpholino-psicose, 1-deoxy-1-morpholino-ribulose, lysozyme glycated with allose-the C3 epimer of glucose, or with ribose). Tandem mass spectrometry and nuclear magnetic resonance analysis of the product of phosphorylation of 1-deoxy-1-morpholino-psicose by FN3K-RP indicated that this enzyme phosphorylates the third carbon of the sugar moiety. These results indicate that FN3K-RP is a ketosamine-3-kinase (ketosamine-3-kinase 2). This enzyme presumably plays a role in freeing proteins from ribulosamines or psicosamines, which might arise in a several step process, from the reaction of amines with glucose and/or glycolytic intermediates. This role is shared by fructosamine-3-kinase (ketosamine-3-kinase 1), which has, in addition, the unique capacity to phosphorylate fructosamines.

Amino Acid Sequence↗
PubMed2002

Culture filtrate specific H-2(b) restricted CD8+ T cells activated in vivo by Mycobacterium tuberculosis or bovis BCG recognize a restricted number of immunodominant peptides.

We previously demonstrated that Bacillus Calmette-Guerin (BCG) immunization activated D(b) restricted CD8+ cytolytic T lymphocyte (CTL) recognizing target cells incubated with mycobacterial culture filtrate. Here, we show that in vitro restimulation of spleen cells from BCG vaccinated or Mycobacterium tuberculosis infected mice with culture filtrate antigens leads to the appearance of a high percentage of D(b) restricted IFNgamma synthesizing CD8+ T cell blasts. Transporter associated protein-2 mutated RMA-S cells incubated with soluble culture filtrate proteins had their MHC class I D(b) but not K(b) molecules stabilized at the surface indicating that only D(b) ligands might be generated by antigen presenting cells. MHC class I bound peptides were acid eluted from the surface of RMA-S cells incubated with M. tuberculosis culture filtrate proteins. The crude peptide preparation was able to sensitize RMA-S cells for recognition by culture filtrate-specific cytolytic T cells. Peptides were subsequently fractionnated by reverse-phase high performance liquid chromatography and the main biological activity was identified in two fractions. These results provide a further evidence that the processing of exogenous culture filtrate proteins in vitro leads to the presentation of a restricted number or even a single immunodominant peptide to culture filtrate-specific CD8+ T cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Tumor-specific shared antigenic peptides recognized by human T cells.

The first tumor-specific shared antigens and the cancer-germline genes that code for these antigens were identified with antitumor cytolytic T lymphocytes obtained from cancer patients. A few HLA class I-restricted antigenic peptides were identified by this 'direct approach'. A large set of additional cancer-germline genes have now been identified by purely genetic approaches or by screening tumor cDNA expression libraries with the serum of cancer patients. As a result, a vast number of sequences are known that can code for tumor-specific shared antigens, but most of the encoded antigenic peptides have not yet been identified. We review here recent 'reverse immunology' approaches for the identification of new antigenic peptides. They are based on in vitro stimulation of naive T cells with dendritic cells that have either been loaded with a cancer-germline protein or that have been transduced with viruses carrying cancer-germline coding sequences. These approaches have led to the identification of many new antigenic peptides presented by class I or class II molecules. We also describe some aspects of the processing and presentation of these antigenic peptides.

Amino Acid Sequence↗