Biomedical subjects

Stefan Stevanovic

Publications and source records attributed to Stefan Stevanovic.

At least 19 recordsLinked to original sources

PubMed2006

Assessment of CD4+ T cells specific for the tumor antigen SSX-1 in cancer-free individuals.

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.

Adult↗

PubMed2006

Identification of a highly immunogenic HLA-A*01-binding T cell epitope of WT1.

PURPOSE: The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy. EXPERIMENTAL DESIGN: Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell-depleted PBMC. RESULTS: One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327-reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327-specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327-specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01-negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327-specific T cell responses in two of six patients with HLA-A*01-positive melanoma or lung cancer. CONCLUSION: In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01-restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.

Antigens, Neoplasm↗

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

Identification of a naturally processed T cell epitope derived from the glioma-associated protein SOX11.

The development of T cell-based immunotherapies of cancer depends on the identification of tumor-associated antigens capable of eliciting tumor-directed cytotoxic T cell responses. In malignant glioma the number of well-defined target antigens for cytotoxic T lymphocytes (CTLs) is still very limited. Recently, we demonstrated the abundant and specific overexpression of the transcription factor SOX11 in malignant glioma. Here, we describe the SOX11-derived peptide LLRRYNVAKV which is capable of inducing human leukocyte antigen-A*0201-restricted and tumor-reactive CTLs. This novel CTL epitope may serve as an attractive candidate for a T cell-based immunotherapy of glioma.

Amino Acid Sequence↗

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↗

PubMed2006

Generation of multiple stable dermcidin-derived antimicrobial peptides in sweat of different body sites.

Antimicrobial peptides (AMPs) are effector molecules of innate immunity. Dermcidin (DCD), a recently discovered AMP with broad-spectrum activity, is produced constitutively by the eccrine sweat glands and secreted into sweat. In this study, we investigated the proteolytic processing, site-specific expression, and stability of DCD peptides in eccrine sweat. Using surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and reversed-phase high-pressure liquid chromatography analysis, we identified in eccrine sweat 14 proteolytically processed DCD peptides. Semiquantitative SELDI-TOF-MS analysis indicated that processing of DCD-1L is individually different, but generates a few dominant peptides. At body sites with a high probability for contact with pathogenic microorganisms, a high amount of antimicrobial active DCD peptides was detected in sweat. Furthermore, we show that the secretion rate of DCD is constant during a period of prolonged sweating and that DCD peptides are stable in sweat over several hours. Other known AMPs like the human cathelicidin LL-37 and alpha- or beta-defensins were not detected in significant quantity in eccrine sweat. Owing to the durable and abundant presence, DCD-derived peptides contribute to the first line of defense by building a constant barrier that overlies the epithelial skin.

Adult↗

PubMed2006

Contribution of mass spectrometry-based proteomics to immunology.

Antigen processing forwards various information about the cellular status and the proteome to the cell surface for scrutiny by the cellular immune system. Thus the repertoire of major histocompatibility complex (MHC)-bound peptides and the MHC ligandome, indirectly mirrors the proteome in order to make alterations instantly detectable and, if necessary, to oppose them. Mass spectrometry is the core technology for analysis of both proteome and MHC ligandome and has evoked several strategies to gain qualitative and quantitative insight into the MHC-presented peptide repertoire. After immunoaffinity purification of detergent-solubilized peptide-MHC complexes followed by acid elution of peptides, liquid chromatography-mass spectrometry is applied to determine individual peptide sequences and, thus, allow qualitative characterization of the MHC-bound repertoire. Differential quantification based on stable isotope labeling enables the relative comparison of two samples, such as diseased and healthy tissue. Targeted searches for certain natural ligands, such as the 'predict-calibrate-detect' strategy, include motif-based epitope prediction and calibration with reference peptides. Thus, various approaches are now available for exposing and understanding the intricacies of the MHC ligand repertoire. Analysis of differences in the MHC ligandome under distinct conditions contributes to our understanding of basic cellular processes, but also enables the formulation of immunodiagnostic or immunotherapeutic strategies.

Animals↗

PubMed2005

Cathepsin D is present in human eccrine sweat and involved in the postsecretory processing of the antimicrobial peptide DCD-1L.

The protein pattern of healthy human eccrine sweat was investigated and 10 major proteins were detected from which apolipoprotein D, lipophilin B, and cathepsin D (CatD) were identified for the first time in human eccrine sweat. We focused our studies on the function of the aspartate protease CatD in sweat. In vitro digestion experiments using a specific fluorescent CatD substrate showed that CatD is enzymatically active in human sweat. To identify potential substrates of CatD in human eccrine sweat LL-37 and DCD-1L, two antimicrobial peptides present in sweat, were digested in vitro with purified CatD. LL-37 was not significantly digested by CatD, whereas DCD-1L was cleaved between Leu(44) and Asp(45) and between Leu(29) and Glu(30) almost completely. The DCD-1L-derived peptides generated in vitro by CatD were also found in vivo in human sweat as determined by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. Furthermore, besides the CatD-processed peptides we identified additionally DCD-1L-derived peptides that are generated upon cleavage with a 1,10-phenanthroline-sensitive carboxypeptidase and an endoprotease. Taken together, proteolytic processing generates 12 DCD-1L-derived peptides. To elucidate the functional significance of postsecretory processing the antimicrobial activity of three CatD-processed DCD-1L peptides was tested. Whereas two of these peptides showed no activity against Gram-positive and Gram-negative bacteria, one DCD-1L-derived peptide showed an even higher activity against Escherichia coli than DCD-1L. Functional analysis indicated that proteolytic processing of DCD-1L by CatD in human sweat modulates the innate immune defense of human skin.

Amino Acid Sequence↗

PubMed2005

SNEP: SNP-derived epitope prediction program for minor H antigens.

The single nucleotide polymorphism (SNP)-derived Epitope Prediction program (SNEP) is now available to the public. It predicts minor histocompatibility antigens (miHAgs), which are T-cell epitopes containing polymorphic spots, from proteins listed in the SWISS-PROT database. SNEP recognizes polymorphisms (termed VARIANT or CONFLICT in SWISS-PROT) and predicts potential T-cell epitopes within a chosen distance around the polymorphic residue. The prediction algorithm is based on the SYFPEITHI T-cell epitope prediction program. SNEP is able to search for proteins according to their accession numbers, sequence stretches or gene names, for example. The predictions are available for several human leucocyte antigen class I and class II allelic products, which allow for a rapid and precise evaluation of potential miHAgs within polymorphic antigens.

Amino Acid Sequence↗

PubMed2005

Multiple synergizing factors contribute to the strength of the CD8+ T cell response against listeriolysin O.

Immunodominance in CD8+ T cell responses against Listeria monocytogenes is a well-recognized but still not fully understood phenomenon. From listeriolysin, the major virulence factor of L. monocytogenes, only a single epitope, pLLO91-99, is presented by MHC class I molecules in BALB/c mice which dominates the cytotoxic T cell response against this bacterial pathogen. To obtain more insights into the molecular and cellular mechanisms underlying immunodominance of this particular epitope, we compared the various steps involved in the presentation and recognition of pLLO91-99 derived from a wild-type toxin with an equivalent epitope from a mutated toxin. This fully functional variant contains within the pLLO91-99 epitope a conservative isoleucine to alanine replacement at the C-terminal anchor residue which results in loss of antigenicity. The binding properties of the variant peptide to soluble Kd remained unaffected and cytotoxic T cells capable of recognizing the pLLO99A/Kd complex were detectable in BALB/c mice. However, such T cells required higher concentrations of antigen in order to be optimally activated in vitro. A comparison between the TAP translocation efficiency of wild-type and mutant peptide demonstrated that the mutation at the C-terminus leads to a reduced transportation rate. Furthermore, the amino acid substitution changes the in vitro proteasomal cleavage pattern, resulting in a reduced liberation of the correct peptide from a polypeptide precursor. Thus, in all assays employed the immunodominant epitope performs optimally while the variant was found to be inferior. The synergy of all these steps most likely is the decisive factor in the immunodominance of pLLO91-99.

Amino Acid Substitution↗

PubMed2005

Generation of RAGE-1 and MAGE-9 peptide-specific cytotoxic T-lymphocyte lines for transfer in patients with renal cell carcinoma.

Renal cell carcinomas (RCCs) are supposed to be immunogenic, and several clinical trials of immunotherapy using tumor lysate-pulsed dendritic cells (DCs) have been performed. We report on the generation of RAGE-1 and MAGE-9 peptide-specific CTL lines. RAGE-1 and MAGE-9 are expressed in 56% and 38% of RCCs. Seven MAGE-9- and 13 RAGE-1-derived peptides were found to be immunogenic in the context of the HLA-A*0201 MHC. CTLs were generated by coculture with peptide-pulsed, activated B cells, which were easily generated in great quantities and displayed functional activity for a prolonged period of time. MAGE-9 and RAGE-1 peptide-specific CTL lines were strictly peptide-specific and displayed high cytotoxic activity not only against peptide-loaded T2 cells but also against HLA-A*0201-positive RCC lines, which naturally express MAGE-9, RAGE-1 or both. Thus, B cells are well suited as APCs for the generation of large numbers of tumor peptide-specific CTLs for adoptive transfer. MAGE-9 as well as RAGE-1 may well provide suitable targets for immunotherapy of RCC.

Adoptive Transfer↗

PubMed2005

The kinase insert domain-containing receptor is an angiogenesis-associated antigen recognized by human cytotoxic T lymphocytes.

Antigen-specific cancer immunotherapy directed toward tumor-nourishing angiogenic blood vessels holds the promise of high efficacy, low toxicity, and ease of application. To evaluate whether the human angiogenic kinase insert domain-containing receptor (KDR) can serve as a target for cellular immunotherapy, 19 peptide sequences with HLA-A*0201 motifs were selected by computer-based algorithms. Five peptides (KDR82-90, KDR288-297, KDR766-774, KDR1093-1101, KDR1035-1044) stimulated specific cytotoxic T lymphocytes (CTLs) from peripheral-blood mononuclear cells (PBMCs) of 3 HLA-A*0201 donors. The decapeptide KDR288-297 was efficient in sensitizing target cells for recognition by a CTL clone at a concentration of 10 nM. More important, KDR288-297-specific CTLs lysed target cells transfected with HLA-A2/KDR cDNAs and a range of HLA-matched KDR+ angiogenic endothelial cells (aECs) and also recognized CD34+ endothelial progenitor cells. The specificity of CTLs was further confirmed by tetramer assay and cold-target inhibition assay. In addition, ex vivo exposure of aECs to the inflammatory cytokines enhanced CTL reactivity, which is in keeping with up-regulated KDR and HLA class 1 expression. In Matrigel assays, recognition of aECs by specific CTLs triggered an antivascular effect. These findings provide the first proof of the antigenic property of KDR protein and may be useful for devising new immunotherapeutic approaches to human cancers.

Antigens, CD↗

PubMed2005

Melanoma patients respond to a new HLA-A*01-presented antigenic ligand derived from a multi-epitope region of melanoma antigen TRP-2.

Tyrosinase-related protein-2 (TRP-2) is a known target antigen of spontaneous cytotoxic T cell responses in melanoma patients. Its frequent expression in metastatic tumors suggests that it might be an ideal candidate antigen for T cell-based immunotherapy. To provide knowledge about TRP-2-derived T cell epitopes useful for immunotherapy we applied a "reverse immunology strategy" based on repeated in vitro peptide stimulation of peripheral blood lymphocytes (PBL) from normal donors with predicted HLA-A*01 ligands. This led to the identification of TRP-2(181-190) as the first HLA-A*01-presented TRP-2-derived epitope. T-cell lines specific for peptide TRP-2(181-190) could be established from PBL of 50% of the normal HLA-A*01(+) donors tested. Such T cells responded specifically to autologous dendritic cells transduced virally with TRP-2, as well as to HLA-A*01(+), TRP-2(+) melanoma cells, although tumor cells had to be pretreated with IFN-gamma to become susceptible to T cell recognition. Interestingly, short-term in vitro peptide stimulation of PBL from HLA-A*01(+) melanoma patients showed the presence of TRP-2(181-190)-reactive CD8(+) T cells in some donors, suggesting their in vivo sensitization. Because TRP-2(181-190) overlaps with the known HLA-A*0201-presented epitope TRP-2(180-188), an 11mer peptide encompassing both epitopes might be of specific value for vaccination of a broad population of melanoma patients.

Amino Acid Sequence↗

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

Identification of an epitope derived from the cancer testis antigen HOM-TES-14/SCP1 and presented by dendritic cells to circulating CD4+ T cells.

Because of their frequent expression in a wide spectrum of malignant tumors but not in normal tissue except testis, cancer testis antigens are promising targets. However, except for HOM-TES-14/SCP1, their expression in malignant lymphomas is rare. SCP1 (synaptonemal complex protein 1) has been shown to elicit antibody responses in the autologous host, but no T-cell responses against HOM-TES-14/SCP1 have been reported. Using the SYFPEITHI algorithm, we selected peptides with a high binding affinity to major histocompatibility complex class 2 (MHC 2) molecules. The pentadecamer epitope p635-649 induced specific CD4+ T-cell responses that were shown to be restricted by HLA-DRB1*1401. The responses could be blocked by preincubation of T cells with anti-CD4 and antigen-presenting cells with anti-HLA-DR, respectively, proving the HLA-DR-restricted presentation of p635-649 and a CD4+ T-cell-mediated effector response. Responding CD4+ cells did not secrete interleukin-5 (IL-5), indicating that they belong to the T(H)1 subtype. The natural processing and presentation of p635-649 were demonstrated by pulsing autologous and allogeneic dendritic cells with a protein fragment covering p635-649. Thus, p635-649 is the first HOM-TES-14/SCP1-derived epitope to fulfill all prerequisites for use as a peptide vaccine in patients with HOM-TES-14/SCP1-expressing tumors, which is the case in two thirds of peripheral T-cell lymphomas.

Amino Acid Sequence↗

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

Antigen processing is predictable: From genes to T cell epitopes.

In order to induce a cellular immune response, antigens have to be processed in intracellular compartments, transported, and presented by HLA molecules prior to recognition by specific T cells. Many of the events that contribute to antigen processing have been thoroughly investigated during the past years and are now well understood, which lead to a number of prediction programs. "Reverse immunology" has been used for about 10 years in order to identify T cell epitopes from pathogens or tumor-associated antigens. The advantages and pitfalls of T cell epitope prediction compared to classical experimental procedures such as epitope mapping and cloning experiments have been discussed many times. In this presentation, a number of internet programs that offer help in T cell epitope prediction (or prediction of antigen processing) will be discussed in the light of transfusion medicine. Some databases are listing published HLA ligands and T cell epitopes, others offer epitope prediction for many HLA class I or class II restrictions. In addition, a number of established programs will be demonstrated which are freely accessible at no cost in the world wide web for the prediction of either HLA-peptide binding, proteasomal processing of antigens, or both. Epitope prediction and processing prediction programs will be applied to minor histocompatibility antigens (miHAgs) and compared. This reflects the actual possibilities and limitations of such computer-aided work not only in cellular immunology, but also in transplantation immunology.

Animals↗

PubMed2005

CD4+ T cell responses to SSX-4 in melanoma patients.

Genes of the synovial sarcoma X breakpoint (SSX) family are expressed in different human tumors, including melanomas, but not in adult somatic tissues. Because of their specific expression at the tumor site, SSX-encoded Ags are potential targets for anticancer immunotherapy. In this study, we have analyzed CD4+ T cell responses directed against the Ag encoded by SSX-4. Upon in vitro stimulation of PBMC from four melanoma patients bearing Ag-expressing tumors with a pool of long peptides spanning the protein sequence, we detected and isolated SSX-4-specific CD4+ T cells recognizing several distinct antigenic sequences, mostly restricted by frequently expressed HLA class II alleles. The majority of the identified sequences were located within the Krüppel-associated box domain in the N-terminal region of the protein, indicating a high potential immunogenicity of this region. Together our data document the existence of CD4+ T cells specific for multiple SSX-4 derived sequences in circulating lymphocytes from melanoma patients and encourage further studies to assess the impact of SSX-4-specific T cell responses on disease evolution in cancer patients.

Adult↗