Biomedical subjects

Oliver Schoor

Publications and source records attributed to Oliver Schoor.

16 recordsLinked to original sources

PubMed2006

Distorted relation between mRNA copy number and corresponding major histocompatibility complex ligand density on the cell surface.

The major histocompatibility complex (MHC) presents peptides derived from degraded cellular proteins to T-cells and is thus crucial for triggering specific immune responses against viral infections or cancer. Up to now, there has been no evidence for a correlation between levels of mRNA (the "transcriptome") and the density of MHC-peptide complexes (the "MHC ligandome") on cells. Because such dependences are of intrinsic importance for the detailed understanding of translation efficiency and protein turnover and thus for systems biology in general and for tumor immunotherapy in practical application, we quantitatively analyzed the levels of mRNA and corresponding MHC ligand densities in samples of renal cell carcinomas and their autologous normal kidney tissues. Relative quantification was carried out by gene chip analysis and by stable isotope peptide labeling, respectively. In comparing more than 270 pairs of gene expression and corresponding peptide presentation ratios, we demonstrate that there is no clear correlation (r = 0.32) between mRNA levels and corresponding MHC peptide levels in renal cell carcinoma. A significant number of peptides presented predominantly on tumor or normal tissue showed no or only minor changes in mRNA expression levels. In several cases, peptides could even be identified despite the virtual absence of the respective mRNA. Thus we conclude that a majority of epitopes from tumor-associated antigens will not be found in approaches based mainly on mRNA expression studies as mRNA expression reflects a distorted picture of the situation on the cell surface as visible for T-cells.

Amino Acid Sequence↗

PubMed2006

Unexpected abundance of HLA class II presented peptides in primary renal cell carcinomas.

PURPOSE: To elicit a long-lasting antitumor immune response, CD8+ and CD4+ T cells should be activated. We attempted to isolate HLA-DR-presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumor-associated antigens (TAA) for their use in peptide-based immunotherapy. EXPERIMENTAL DESIGN: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression. HLA class II peptides were subsequently isolated and identified by mass spectrometry. Gene expression analysis was done to detect genes overexpressed in tumor tissue. Peptides from identified TAAs were used to induce peptide-specific CD4+ T-cell responses in healthy donors and in tumor patients. RESULTS: In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system. To our surprise, we were able to isolate and characterize hundreds of class II peptides directly from primary dissected solid tumors, especially from renal cell carcinomas, and from colorectal carcinomas and transitional cell carcinomas. Infiltrating leukocytes expressed MHC class II molecules and tumor cells, very likely under the influence of IFNgamma. Our list of identified peptides contains ligands from several TAAs, including insulin-like growth factor binding protein 3 and matrix metalloproteinase 7. The latter bound promiscuously to HLA-DR molecules and were able to elicit CD4+ T-cell responses. CONCLUSIONS: Thus, our direct approach will rapidly expand the limited number of T-helper epitopes from TAAs for their use in clinical vaccination protocols.

Antigens, Neoplasm↗

PubMed2006

Autophagy in innate and adaptive immunity against intracellular pathogens.

Autophagy delivers cytoplasmic constituents for lysosomal degradation. Recent studies have demonstrated that this pathway mediates resistance to pathogens and is targeted for immune evasion by viruses and bacteria. Lysosomal degradation products, including pathogenic determinants, are then surveyed by the adaptive immune system to elicit antigen-specific T cell responses. CD4(+) T helper cells have been shown to recognize nuclear and cytosolic antigens via presentation by major histocompatibility complex (MHC) class II molecules after autophagy. Furthermore, some sources of natural MHC class II ligands display characteristics of autophagy substrates, and autophagosomes fuse with late endosomes, in which MHC class II loading is thought to occur. Although MHC class II antigen processing via autophagy has so far mainly been described for professional antigen-presenting cells like B cells, macrophages, and dendritic cells, it might be even more important for cells with less endocytic potential, like epithelial cells, when these express MHC class II at sites of inflammation. Therefore, autophagy might contribute to immune surveillance of intracellular pathogens via MHC class II presentation of intracellular pathogen-derived peptides.

Adaptation, Physiological↗

PubMed2005

MAGED4-expression in renal cell carcinoma and identification of an HLA-A*25-restricted MHC class I ligand from solid tumor tissue.

MAGE derived HLA ligands have repeatedly been shown to elicit T-cell responses against tumor cells. In renal cell carcinoma (RCC), however, only few T-cell epitopes from cancer testis antigens have been described. To identify potential candidates, we applied a combined approach of microarray/qPCR expression analysis and sequencing of HLA ligands from RCC by mass spectrometry. We analyzed the expression of 21 MAGE genes in ten RCC samples and two glioblastoma samples and could identify the first MHC class I ligand NIGDEALIGRW from MAGED4 presented by HLA-A*25 on RCC solid tumor tissue. MAGED4 was expressed in 30% of RCC and both glioblastoma samples. Among the other MAGE family members only MAGEB2 and -C1 and the broadly expressed MAGED1, -D2, -F1 and -H1 were expressed in RCC. Ligands from MAGED4 could thus be interesting tumor-associated antigens in a subset of RCC, even though the identified ligand is presented by a rather rare allele.

Antigens, Neoplasm↗

PubMed2005

Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas.

BACKGROUND: Anti-tumor vaccines targeting the entire tumor antigen repertoire represent an attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the large quantities of mRNA needed for each patient, the Stratagene Creator SMART cDNA library construction method was modified and applied to produce libraries derived from the tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through sequencing and microarray analysis. RESULTS: Random analysis of bacterial clones of the library showed a rate of 95% of recombinant plasmids among which a minimum of 51% of the clones contained a full-Open Reading Frame. In addition, despite a biased amplification toward small abundant transcripts compared to large rare fragments, we could document a relatively conserved gene expression profile between the total RNA of the tumor of origin and the corresponding in vitro transcribed complementary RNA (cRNA). Finally, listing the 30 most abundant transcripts of patient MEL02's library, a large number of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were found. CONCLUSION: Our results show that unlimited amounts of cRNA representing tumor's transcriptome could be obtained and that this cRNA was a reliable source of a large variety of tumor antigens.

Journal Article↗

PubMed2005

Autophagy promotes MHC class II presentation of peptides from intracellular source proteins.

MHC-peptide complexes mediate key functions in adaptive immunity. In a classical view, MHC-I molecules present peptides from intracellular source proteins, whereas MHC-II molecules present antigenic peptides from exogenous and membrane proteins. Nevertheless, substantial crosstalk between these two pathways has been observed. We investigated the influence of autophagy on the MHC-II ligandome and demonstrated that peptide presentation is altered considerably upon induction of autophagy. The presentation of peptides from intracellular and lysosomal source proteins was strongly increased on MHC-II in contrast with peptides from membrane and secreted proteins. In addition, autophagy influenced the MHC-II antigen-processing machinery. Our study illustrates a profound influence of autophagy on the class II peptide repertoire and suggests that this finding has implications for the regulation of CD4(+) T cell-mediated processes.

Amino Acid Sequence↗

PubMed2005

CD8+ T cells specific for a potential HLA-A*0201 epitope from Chlamydophila pneumoniae are present in the PBMCs from infected patients.

Infection with the common pathogen Chlamydophila pneumoniae (Cpn, previously Chlamydia pneumoniae) has a high prevalence in patients suffering from arteriosclerosis and may trigger or contribute to heart disease. In mice, CD8-positive T cells are critical for the eradication of the infection and the development of immune memory against Cpn. Although several H2-class I epitopes have been described, no HLA-class I-associated peptides from Cpn are known. In order to define HLA-A*0201 epitopes from Cpn, we focused on the bacterial heat shock proteins (HSP) 60 and 70 which are known to be recognized by the immune system. Using epitope prediction, peptide binding studies and peptide-specific CTLs from HLA-A2 transgenic mice, we could define a potential HSP-70-derived epitope. The study of PBMCs from Cpn-infected individuals using fluorescent MHC tetramers revealed that some patients have CD8(+) T cells capable of recognizing the Cpn HSP-70 HLA-A*0201 epitope. Our studies pave the way to the immunomonitoring of the anti-Cpn CTL immune response present in patients suffering from different diseases potentially linked to Cpn or anti-Cpn immunity.

Animals↗

PubMed2005

Effects of imatinib on monocyte-derived dendritic cells are mediated by inhibition of nuclear factor-kappaB and Akt signaling pathways.

Dendritic cells are the most powerful antigen-presenting cells playing a decisive role for the initiation and maintenance of primary immune responses. However, signaling pathways involved in the differentiation of these cells have not been fully determined. Imatinib is a novel tyrosine kinase inhibitor effective against Abl kinases, c-Kit, and platelet-derived growth factor receptor. Using this compound, we show that human monocyte-derived dendritic cells generated in the presence of therapeutic concentrations of imatinib show a reduced expression of CD1a, MHC class I and II, and costimulatory molecules as well as decreased secretion of chemokines and cytokines resulting in an impaired capacity of dendritic cells to elicit primary T-cell responses. Using Western blot analyses, we found that these effects are mediated by inhibition of phosphatidylinositol 3-kinase/Akt pathways and a pronounced down-regulation of nuclear localized protein levels of nuclear factor-kappaB family members. Importantly, using blocking antibodies and tyrosine kinase inhibitors, we show that the inhibitory effects of imatinib on dendritic cell differentiation are not mediated via platelet-derived growth factor receptor and c-Kit. Taken together, our study reveals that imatinib inhibits dendritic cell differentiation and function via Akt and nuclear factor-kappaB signal transduction. Importantly, we show that imatinib can inhibit the function of normal, nonmalignant cells that may result in immunosuppression of these patients.

Antineoplastic Agents↗

PubMed2004

Lessons to be learned from primary renal cell carcinomas: novel tumor antigens and HLA ligands for immunotherapy.

The lack of sufficient well-defined tumor-associated antigens is still a drawback on the way to a cytotoxic T-lymphocyte-based immunotherapy of renal cell carcinoma (RCC). We are trying to define a larger number of such targets by a combined approach involving HLA ligand characterization by mass spectrometry and gene expression profiling by oligonucleotide microarrays. Here, we present the results of a large-scale analysis of 13 RCC specimens. We were able to identify more than 700 peptides, mostly from self-proteins without any evident tumor association. However, some HLA ligands derived from previously known tumor antigens in RCC. In addition, gene expression profiling of tumors and a set of healthy tissues revealed novel candidate RCC-associated antigens. For several of them, we were able to characterize HLA ligands after extraction from the tumor tissue. Apart from universal RCC antigens, some proteins seem to be appropriate candidates in individual patients only. This underlines the advantage of a personalized therapeutic approach. Further analyses will contribute additional HLA ligands to this repertoire of universal as well as patient-individual tumor antigens.

Antigens, Neoplasm↗

PubMed2004

The non-classical HLA class I molecule HFE does not influence the NK-like activity contained in fresh human PBMCs and does not interact with NK cells.

In humans, four beta2-microglobulin-associated non-classical class I molecules are encoded in the MHC: HLA-E, -F, -G and -H. Three of them (HLA-E, -F and -G) were shown to inhibit NK activity. On the contrary, the fourth one, HLA-H, named HFE after it was found to be mutated in patients suffering from inherited hemochromatosis, has been shown to be involved only in the regulation of iron uptake. We tested the capacity of HFE to affect (enhance or reduce) specifically the NK activity contained in non-manipulated fresh human PBMCs. We showed that HFE expression by target cells does not affect their killing by the NK-like activity contained in PBMCs. Moreover, using fluorescent HFE tetramers, we could confirm that blood NK cells as well as blood gammadelta T cells do not bind HFE. Altogether, our data indicate that HFE does not affect the NK activity contained in the PBMCs.

Animals↗

PubMed2004

Induction of adipophilin-specific cytotoxic T lymphocytes using a novel HLA-A2-binding peptide that mediates tumor cell lysis.

Identification of tumor-associated antigens and advances in tumor immunology resulted in the development of vaccination strategies to treat patients with malignant diseases. Using a novel approach that combines DNA chip analysis of tumor samples with isolation of peptides on the surface of tumor cells, a HLA-A*0201-binding peptide derived from the adipophilin protein was identified. Adipophilin is involved in lipid storage and was thought to be expressed only in adipocytes, but it can be found in other cell types such as macrophages or tumor cells. In the present study, we analyzed the possible use of this peptide as a T-cell epitope presented by malignant cells. To accomplish this, we induced CTL responses using this HLA-A*0201-binding peptide. The in vitro-induced CTLs efficiently lysed cells pulsed with the adipophilin peptide and HLA-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Finally, the induced CTLs recognized autologous dendritic cells (DCs) pulsed with the antigenic peptide or transfected with tumor RNA purified from an adipophilin-expressing tumor cell line. To further analyze the possible use of this peptide in immunotherapies of human malignancies, we induced adipophilin-specific CTLs using peripheral blood mononuclear cells and DCs from HLA-A*0201-positive patients with chronic lymphatic leukemia and plasma cell leukemia. The in vitro-generated CTLs recognized autologous chronic lymphatic leukemia cells and malignant plasma cells, whereas they spared nonmalignant resting or activated B and T lymphocytes, monocytes, or DCs. Our results demonstrate that this peptide might represent an interesting candidate for the development of cancer vaccines designed to target adipophilin-derived epitopes in a wide range of malignancies.

Antigen Presentation↗

PubMed2004

A conserved sequence in the mouse variable T cell receptor alpha recombination signal sequence 23-bp spacer can affect recombination.

Although the V-gene segments coding for the TCR alpha and delta chains are mixed together in the alpha delta locus and are recombined by the same processes, some gene segments (TRAV) are rearranged only with TCR Jalpha gene segments, some (TRDV) only with TCR Ddelta gene segments and some (TRADV) with both. To date, no molecular signal is known that can characterize these three different types of gene segments. Studying the recombination signal sequences (RSS) of all mouse TCR V-gene segments we observed that 80% of the TRAV contain a palindrome sequence (CTGCAG) or its related variant CTGTAG in their 23-bp spacer. Using gel-shift assays we show that these sequences are specifically recognized by some nuclear proteins that are expressed by fresh thymocytes, fresh lymphocytes and tumor cells. Recombination assays on plasmid substrates in a pre-B cell line showed that RSS containing the CTGCAG sequence can impair recombination. From the protein fractions containing the CTGCAG-binding activity, three proteins were identified: G3BP1 (a nucleic-acid-binding protein with a proposed helicase activity) and two proteins from the high-mobility group (HMG) family--HMGB2 and HMGB3. We hypothesize that these proteins can affect recombination at the TCR alpha delta locus.

Animals↗

PubMed2004

Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry.

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.

Cyclin D1↗

PubMed2003

Cutting edge: predetermined avidity of human CD8 T cells expanded on calibrated MHC/anti-CD28-coated microspheres.

Cytotoxic CD8 T cells are key effectors in the immunotherapy of malignant and viral diseases. However, the lack of efficient methods for their in vitro priming and expansion has become a bottleneck to the development of vaccines and adoptive transfer strategies. Synthetic artificial APCs (aAPCs) are now emerging as an attractive tool for eliciting and expanding CTL responses. We show that, by controlling the MHC density on aAPCs, high- or low-avidity tumor-directed human CTL lines can be raised effectively in vitro if costimulation via CD28 and IL-12 is provided. Compared with low-avidity CTL lines, high-avidity CTLs need 100- to 1000-fold less peptide for activation, bind more MHC tetramers, and, as expected, are superior in recognizing tumor cell lines expressing Ag. We believe that the possibility to raise Ag-specific T cells with predetermined avidity will be crucial for the future use of aAPCs in immunotherapeutical settings.

Antigen-Presenting Cells↗

PubMed2003

Moderate degradation does not preclude microarray analysis of small amounts of RNA.

Gene expression analysis by microarrays using small amounts of RNA is becoming more and more popular against the background of advances and increasing importance of small-sample acquisition methods like laser microdissection techniques. The quality of RNA preparations from such samples constitutes a frequent issue in this context. The aim of this study was to assess the impact of different extents of RNA degradation on the expression profile of the samples. We induced RNA degradation in human tumor and healthy tissue samples by endogeneous ribonucleases. Next, we amplified 20 ng total RNA degraded to different extents by two rounds of in vitro transcription and analyzed them using Affymetrix oligonucleotide microarrays. Expression differences for some genes were independently confirmed by real-time quantitative PCR. Our results suggest that gene expression profiles obtained from partially degraded RNA samples with still visible ribosomal bands exhibit a high degree of similarity compared to intact samples and that RNA samples of suboptimal quality might therefore still lead to meaningful results if used carefully.

Carcinoma, Renal Cell↗

PubMed2002

Integrated functional genomics approach for the design of patient-individual antitumor vaccines.

Our aim is to identify as many candidates as possible for tumor-associated T-cell epitopes in individual patients. First, we performed expression profiling of tumor and normal tissue to identify genes exclusively expressed or overexpressed in the tumor sample. Then, using mass spectrometry, we characterized up to 77 different MHC ligands from the same tumor sample. Several of the MHC ligands were derived from overexpressed gene products, one was derived from a proto-oncogene, and another was derived from a frameshift mutation. At least one was identified as an actual T-cell epitope. Thus, we could show that by combining these two analytic tools, it is possible to propose several candidates for peptide-based immunotherapy. We envision the use of this novel integrated functional genomics approach for the design of antitumor vaccines tailored to suit the needs of each patient.

Antigens, Neoplasm↗