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Hans-Georg Rammensee

Publications and source records attributed to Hans-Georg Rammensee.

At least 73 records · Page 4Linked to original sources
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↗
PubMed2003

MAPPP: MHC class I antigenic peptide processing prediction.

MAPPP is a bioinformatics tool for the prediction of potential antigenic epitopes presented on the cell surface by major histocompatibility complex class I (MHC I) molecules to CD8 positive T lymphocytes. It combines existing predictions for proteasomal cleavage with peptide anchoring to MHC I molecules.

Algorithms↗
PubMed2002

Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines.

Despite worldwide eradication of naturally occurring variola virus, smallpox remains a potential threat to both civilian and military populations. New, safe smallpox vaccines are being developed, and there is an urgent need for methods to evaluate vaccine efficacy after immunization. Here we report the identification of an immunodominant HLA-A*0201-restricted epitope that is recognized by cytotoxic CD8(+) T cells and conserved among Orthopoxvirus species including variola virus. This finding has permitted analysis and monitoring of epitope-specific T cell responses after immunization and demonstration of the identified T cell specificity in an A*0201-positive human donor. Vaccination of transgenic mice allowed us to compare the immunogenicity of several vaccinia viruses including highly attenuated, replication-deficient modified vaccinia virus Ankara (MVA). MVA vaccines elicited levels of CD8(+) T cell responses that were comparable to those induced by the replication-competent vaccinia virus strains. Finally, we demonstrate that MVA vaccination is fully protective against a lethal respiratory challenge with virulent vaccinia virus strain Western Reserve. Our data provide a basis to rationally estimate immunogenicity of safe, second-generation poxvirus vaccines and suggest that MVA may be a suitable candidate.

Amino Acid Sequence↗
PubMed2002

The heat shock protein Gp96 binds to human neutrophils and monocytes and stimulates effector functions.

The endoplasmic reticulum (ER)-resident heat shock protein Gp96 is involved in protein folding and is released into the extracellular space after necrotic cell death. In this context, Gp96 has immunostimulatory properties: it activates dendritic cells or macrophages and delivers associated peptides into the antigen presentation pathway, resulting in the induction of specific T-cell responses. The inflammatory response after necrotic tissue damage leads to the recruitment of polymorphonuclear neutrophils (PMNs) and monocytes, allowing them to make their first encounter with Gp96. We therefore investigated whether PMNs and monocytes interact with Gp96. We were able to show that PMNs and monocytes specifically bind fluorescein isothiocyanate (FITC)-conjugated Gp96. The binding of Gp96-FITC was competed by lipopolysaccharide (LPS) or fucoidan, a known inhibitor of scavenger receptors. Interestingly, the binding of LPS-FITC was also competed not only by fucoidan, but by Gp96, suggesting that LPS and Gp96 share a common receptor on PMNs. One important effector function of PMNs is the clearance of an inflammatory site by phagocytosis. We therefore assessed the influence of Gp96 on phagocytic activity using fluorochrome-labeled polystyrene beads. We found a marked enhancement of phagocytosis in the presence of Gp96 and concluded that PMNs not only bind Gp96, but are also activated by it. Additionally, Gp96-stimulated PMNs and especially monocytes release large amounts of interleukin-8, a potent neutrophil-attracting chemokine. In conclusion, we demonstrate that Gp96 specifically binds to and activates PMNs and monocytes, extending the function of Gp96 as a danger signal to additional members of the innate immune system.

Animals↗
PubMed2002

Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding.

The immunoreceptor NKG2D stimulates tumor immunity through activation of CD8 T cells and NK cells. Its ligand MICA has been shown to be broadly expressed on human tumors of epithelial origin. MICA expression correlates with an enrichment of Vdelta1 T cells in tumor tissue. We report that human tumor cells spontaneously release a soluble form of MICA encompassing the three extracellular domains, which is present at high levels in sera of patients with gastrointestinal malignancies, but not in healthy donors. Release of MICA from tumor cells is blocked by inhibition of metalloproteinases, concomitantly causing accumulation of MICA on the cell surface. Shedding of MICA by tumor cells may modulate NKG2D-mediated tumor immune surveillance. In addition, determination of soluble MICA levels may be implemented as an immunological diagnostic marker in patients with epithelial malignancies.

Animals↗
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↗
PubMed2002

Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy.

We adoptively transferred donor-derived cytomegalovirus (CMV)-specific T-cell lines into 8 stem cell transplant recipients lacking CMV-specific T-cell proliferation. All patients, of whom one was infected by a CMV strain that was genotypically ganciclovir resistant, had received unsuccessful antiviral chemotherapy for more than 4 weeks. CMV-specific lines had been prepared by repetitive stimulation with CMV antigen, which increased the percentage of CMV-specific T cells and ablated alloreactivity completely even against patients mismatched for 1 to 3 HLA antigens. After transfer of 10(7) T cells/m(2) at a median of 120 days (range, 79-479 days) after transplantation, no side effects were noticed. Despite cessation of antiviral chemotherapy, the CMV load dropped significantly in all 7 evaluable patients, with a maximal reduction after a median of 20 days (range, 5-31 days). In 2 patients with high virus load, the antiviral effect was only transient. One of these patients received a second T-cell infusion, which cleared the virus completely. At a median of 11 days after transfer, CMV-specific T-cell proliferation was demonstrated in 6 patients, and an increase in CMV-specific CD4(+) T cells was demonstrated in 5 patients. In 6 patients, 1.12 to 41 CMV-specific CD8(+) T cells/microL blood were detected at a median of 13 days after transfer, with an increase in all patients lacking CMV-specific CD8(+) T cells prior to transfer. Hence, anti-CMV cellular therapy was successful in 5 of 7 patients, whereas in 2 of 7 patients, who received an intensified immune suppression at the time of or after T-cell therapy, only transient reductions in virus load were obtained.

Adoptive Transfer↗
PubMed2002

Human platelets express heat shock protein receptors and regulate dendritic cell maturation.

Immunizations using the endoplasmic reticulum-resident heat shock protein Gp96 induce specific immune responses. Specificity is based on the major histocompatibility complex class I-restricted cross-presentation of Gp96-associated peptides derived from endogenous proteins. Initiation of the immune response depends on the ability of Gp96 to induce the production of proinflammatory cytokines by macrophages and dendritic cells (DCs) and of their maturation in a fashion presumably independent of associated peptide. Both events are mediated by Gp96 receptors on antigen-presenting cells. It is known that Gp96 is released from cells at necrosis induced, for example, by virus infection. Although this event supports the efficient induction of immune responses, it might also interfere with processes that are susceptible to chronic inflammation, such as wound healing after tissue damage. Therefore, Gp96-mediated stimulation of the immune system requires tight regulation. Here we show that human thrombocytes specifically interact with Gp96 and that binding of Gp96 to platelets is enhanced more than 10-fold on activation by thrombin. Gp96 interferes with neither thrombin-induced platelet activation nor platelet aggregation. However, the presence of platelets during Gp96-mediated DC activation reduces the secretion of proinflammatory cytokines and the activation of DCs. This effect is independent of soluble platelet factors and cell-to-cell contact between DCs and thrombocytes. Thus, we provide evidence for a regulatory mechanism that neutralizes Gp96 molecules systemically, especially in the blood. This effect might be of significance in wounds in which chronic inflammation and immune responses against autoantigens have to be prevented.

Antigens, Neoplasm↗
PubMed2002

Sensitive detection of human cytomegalovirus peptide-specific cytotoxic T-lymphocyte responses by interferon-gamma-enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation.

Reconstitution of human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs), predominantly directed against pp65, provides protective immunity for the development of HCMV disease after allogeneic stem cell transplantation (SCT). To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-gamma release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8(+) T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide-specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen-specific CD4(+) T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35->180 days). Peak counts of peptide-specific CD8(+) T cells ranged from 0.14 to 60.6 cells/microL; those of protein-specific CD4(+) T cells ranged from 0.64 to 18.97 cells/microL. Reconstitution of HCMV-peptide-specific CD8(+) T cells and protein-specific CD4(+) T cells was associated with clearance of HCMV infection (r(2) = 0.89, P <.0001 and r(2) = 0.61, P =.0045, respectively). HCMV infection recurred after documentation of HCMV-specific T-cell reconstitution (n = 4) when immunosuppression was intensified. Patients in whom late-onset HCMV disease developed lacked HCMV-protein-specific T cells at 3 months after SCT. In conclusion, prospective monitoring of HCMV-specific CD4(+) and CD8(+) T-cell reconstitution can be performed rapidly by using flow cytometry after specific stimulation with HCMV peptides and proteins and might help to further improve clinical management of HCMV infection after allogeneic SCT.

Adult↗
PubMed2002

Identification of CD70-mediated apoptosis of immune effector cells as a novel immune escape pathway of human glioblastoma.

Interactions of CD70, a tumor necrosis factor-related cell surface ligand and its receptor, CD27, are thought to play an important role for T-, B-, and natural killer-cell activation. However, ligation of CD27 can also induce apoptosis. Human glioblastoma is paradigmatic for cancer-associated immunosuppression. We identified CD70 as a radioinducible gene in U87 MG glioma cells. A screening of a panel of human glioma cell lines revealed that 11 of 12 cell lines expressed CD70 mRNA and protein. Two human neuroblastoma cell lines did not express CD70. CD70 mRNA expression was enhanced by irradiation in 8 of 12 glioma cell lines in a p53-independent manner. No alteration in CD70 expression was observed after glioma cell exposure to cytotoxic drugs such as lomustine. CD70 protein was also detected by immunocytochemistry in 5 of 12 glioblastomas and 3 of 4 anaplastic astrocytomas in vivo. CD27 expression was not detected in any glioma cell line, and there was no evidence for autocrine or backward signaling of the CD70 system in human glioma cells. Unexpectedly, CD70 expressed on glioma cells did not increase the immunogenicity of glioma cells in vitro. In contrast, CD70-positive glioma cells induced apoptosis in peripheral blood mononuclear cells (PBMCs) in a CD70-dependent manner. Neutralization of CD70 expressed on glioma cells prevented apoptosis and enhanced the release of tumor necrosis factor-alpha in cocultures of glioma cells and PBMCs. The effects of CD70-expressing glioma cells on PBMCs were mimicked by agonistic CD27 antibodies. Conversely, the shedding of CD27 by PBMCs was identified as a possible escape mechanism from glioma cell-induced CD70-dependent apoptosis. Thus, induction of B-cell and T-cell apoptosis via interactions of CD70 expressed on glioma cells and CD27 expressed on B and T cells may be a novel way for the immune escape of malignant gliomas.

Antigens, CD↗
PubMed2002

The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway.

The heat shock protein Gp96 has been shown to induce specific immune responses. On one hand, this phenomenon is based on the specific interaction with CD91 that mediates endocytosis and results in major histocompatibility complex class I-restricted representation of the Gp96-associated peptides. On the other hand, Gp96 induces activation of professional antigen-presenting cells, resulting in the production of pro-inflammatory cytokines and up-regulation of costimulatory molecules by unknown mechanisms. In this study, we have analyzed the consequences of Gp96 interaction with cells expressing different Toll-like receptors (TLRs) and with bone marrow-derived dendritic cells from mice lacking functional TLR2 and/or TLR4 molecules. We find that the Gp96-TLR2/4 interaction results in activation of nuclear factor kappaB-driven reporter genes and mitogen- and stress-activated protein kinases and induces IkappaBalpha degradation. Bone marrow-derived dendritic cells of C3H/HeJ and more pronounced C3H/HeJ/TLR2(-/-) mice fail to respond to Gp96. Interestingly, activation of bone marrow-derived dendritic cells depends on endocytosis of Gp96 molecules. Our results provide, for the first time, the molecular basis for understanding the Gp96-mediated activation of antigen-presenting cells by describing the simultaneous stimulation of the innate and adaptive immune system. This feature explains the remarkable ability of Gp96 to induce specific immune responses against tumors and pathogens.

Animals↗
PubMed2002

Identification of known and novel immunogenic T-cell epitopes from tumor antigens recognized by peripheral blood T cells from patients responding to IL-2-based treatment.

In previous studies CD8+ T cells specific for melanocyte antigens have been frequently found in melanoma patients responding to interleukin-2 (IL-2)-based therapies. In our study we analyzed the suitability of using circulating T cells from melanoma patients with clinical response after IL-2-based therapy to identify novel T-cell epitopes from defined tumor antigens. Using unstimulated peripheral blood mononuclear cells and the interferon-gamma (IFN-gamma) ELISPOT assay, we studied CD8(+) T-cell responses against 5 peptides from the tumor antigen tyrosinase (Tyr) selected by epitope prediction using an HLA-A1-binding computer algorithm. T cells specifically secreting IFN-gamma in response to 3 of these 5 peptides, namely, Tyr (454-463), Tyr (146-156) and Tyr (243-251), could be detected in 4 of 4 HLA-A1-positive patients with clinical response. In contrast, no T-cell responses against these peptides were seen in 6 HLA-A1-positive melanoma patients with progressive disease and in 8 healthy subjects. We could generate specific cytotoxic T lymphocytes (CTL) against Tyr (454-463) using peptide-pulsed autologous dendritic cells as antigen-presenting cells. The induced CTLs efficiently killed melanoma cells that express HLA-A1 and tyrosinase. The peptides Tyr (146-156) and Tyr (243-251) had recently been identified as CTL epitopes by other groups. Further ex vivo characterization of the T cells reactive against the novel epitope Tyr (454-463) in 1 patient by multicolor flow cytometry showed specific CD3+/CD8+/IFN-gamma+ T cells with frequencies of up to 0.41% of the CD3+/CD8+ T-cell population. Most of this T-cell population also expressed granzyme B. Our data confirm that in patients with tumor regressions induced by immunotherapy or chemoimmunotherapy circulating T cells reactive with tyrosinase epitopes can frequently be detected. Peripheral blood T cells from such patients are a valuable source for screening peptides selected by epitope prediction This strategy facilitates the rapid identification of immunogenic T-cell epitopes that are probable targets of immune-mediated tumor rejection.

Antigens, Neoplasm↗
PubMed2002

Possible association of non-binding of HSP70 to HLA-DRB1 peptide sequences and protection from rheumatoid arthritis.

The beta-chains of HLA-DR molecules associated with susceptibility to rheumatoid arthritis (RA) share a common amino acid sequence in their third hypervariable region at position 70-74. This shared epitope could either contribute to preferential binding of a given disease-associated peptide, be involved in disease-induction by molecular mimicry or, by binding to heat shock proteins, influence antigen presentation. It is known that the Escherichia coli M(r)70,000 heat shock protein DnaK can bind peptides from the shared epitope. Using a highly sensitive method, we show that peptides covering the third hypervariable region of associated, but also most of the non-associated HLA-DR alleles, bind to DnaK. Similar binding specificities could be found for the constitutively expressed mammalian M(r)70,000 heat shock protein Hsc73 and the inducible mammalian Hsp72. However, peptides containing the amino acid sequence DERAA, found in HLA-DR alleles and strongly associated with protection from RA, did not bind any HSP70. Thus, our results suggest a possible association of non-binding of HSP70 to HLA-DR molecules or its 70-74 fragments and protection from RA.

Alleles↗
PubMed2002

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma.

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.

Amino Acid Sequence↗
PubMed2002

Peptide analysis, stability studies, and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7).

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.

Amino Acid Motifs↗
PubMed2002

Towards patient-specific tumor antigen selection for vaccination.

In this review, we discuss the possibilities for combining the power of molecular analysis of the antigens expressed in a given individual tumor with the design of a tailored vaccine containing defined antigens. Step 1 is a differential gene expression analysis of tumor and corresponding normal tissue. Step 2 is the analysis of human leukocyte antigen (HLA) ligands on tumor cells. Step 3 is data mining with the aim to select those antigens that might be suitable for tumor attack by the adaptive immune system. Step 4 is the on-the-spot clinical grade production of the constituents of the patient tailored vaccine, e.g. peptides. Step 5 is then vaccination and monitoring. Although it will not be possible to cover all relevant antigens expressed in a tumor, the antigens that can be identified with our present technical possibilities might be enough for improved immunotherapy. The scope of the present review is to explore the possibilities and the formidable technical and logistical challenge for such individual patient-oriented antigen definition to be used for therapeutic immunization.

Algorithms↗