Two preferentially expressed proteins protect vascular endothelial cells from an attack by peptide-specific CTL.

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.

HLA ligand profiles of primary renal cell carcinoma maintained in metastases.

In recent years, several approaches have been taken in the peptide-based immunotherapy of metastatic renal cell carcinoma (RCC), although little is known about HLA presentation on metastases compared to primary tumor and normal tissue of RCC. In this study we compared primary tumor, normal tissue and metastases with the aim of identifying similarities and differences between these tissues. We performed this comparison for two RCC patients on the level of the HLA ligandome using mass spectrometry and for three patients on the level of the transcriptome using oligonucleotide microarrays. The quantitative results show that primary tumor is more similar to metastasis than to normal tissue, both on the level of HLA ligand presentation and mRNA. We were able to characterize a total of 142 peptides in the qualitative analysis of HLA-presented peptides. Six of them were significantly overpresented on metastasis, among them a peptide derived from CD151; fourteen were overpresented on both primary tumor and metastasis compared to normal tissue, among them an HLA ligand derived from tumor protein p53. Thus, we could demonstrate that peptide-based immunotherapy might affect tumor as well as metastasis of RCC, but not healthy kidney tissue. Furthermore we were able to identify several peptides derived from tumor-associated antigens that are suitable for vaccination of metastatic RCC.

Structure of a bifunctional DNA primase-polymerase.

Genome replication generally requires primases, which synthesize an initial oligonucleotide primer, and DNA polymerases, which elongate the primer. Primase and DNA polymerase activities are combined, however, in newly identified replicases from archaeal plasmids, such as pRN1 from Sulfolobus islandicus. Here we present a structure-function analysis of the pRN1 primase-polymerase (prim-pol) domain. The crystal structure shows a central depression lined by conserved residues. Mutations on one side of the depression reduce DNA affinity. On the opposite side of the depression cluster three acidic residues and a histidine, which are required for primase and DNA polymerase activity. One acidic residue binds a manganese ion, suggestive of a metal-dependent catalytic mechanism. The structure does not show any similarity to DNA polymerases, but is distantly related to archaeal and eukaryotic primases, with corresponding active-site residues. We propose that archaeal and eukaryotic primases and the prim-pol domain have a common evolutionary ancestor, a bifunctional replicase for small DNA genomes.

Identification of HLA-A*01- and HLA-A*02-restricted CD8+ T-cell epitopes shared among group B enteroviruses.

Acute enteroviral infections ranging from meningitis, pancreatitis to myocarditis are common and normally well controlled by the host immune system comprising virus-specific CD8+ cytotoxic T lymphocytes (CTL). However, in some patients enteroviruses and especially coxsackieviruses of group B are capable of inducing severe chronic forms of diseases such as chronic myocarditis. Currently, it is not known whether divergences in the CTL-related immune response may contribute to the different outcome and course of enterovirus myocarditis. A pre-requisite for the study of CTL reactions in patients with acute and chronic myocarditis is the identification of CTL epitopes. In order to define dominant enterovirus CTL epitopes, we have screened, by using gamma interferon (IFN-gamma) ELISPOT, 62 HLA-A*01- and 59 HLA-A*02-positive healthy blood donors for pre-existing CTL reactions against 12 HLA-A*01 and 20 HLA-A*02 predicted CTL epitopes derived from coxsackieviruses of group B. Positive CTL reactions were verified by FACS analysis in a combined major histocompatibility complex-tetramer IFN-gamma staining. A total of 14.8% of all donors reacted against one of the three identified epitopes MLDGHLIAFDY, YGDDVIASY or GIIYIIYKL. The HLA-A*02-restricted epitope ILMNDQEVGV was recognized by 25% of all tested blood donors. For this peptide, we could demonstrate specific granzyme B secretion, a strong cytolytic potential and endogenous processing. All four epitopes were homologous in 36-92% of group B enteroviruses, providing a strong basis for monitoring the divergence of T-cell-based immune responses in enterovirus-induced acute and chronic diseases.

Antibodies and CD4(+) T-cells mediate cross-protection against H5N1 influenza virus infection in mice after vaccination with a low pathogenic H5N2 strain.

A H5N2 low pathogenic avian influenza virus (LPAIV) was isolated from a natural reservoir in Bavaria during a routine screen and was used as a vaccine strain to scrutinize the immune response involved in cross-protection after challenge infection with a H5N1 highly pathogenic avian influenza virus (HPAIV). The challenge virus was also isolated from a natural reservoir in Bavaria. Wild type, antibody deficient (muMT), CD4(-/-) and CD8(-/-) mice were infected with the apathogenic H5N2 vaccine strain and challenge infection with a 100-fold MLD(50) of the H5N1 strain was performed 80 days later. While 100% of the wild type and 100% of the CD8(-/-) mice stayed healthy, only 50% of the CD4(-/-) and none of the antibody deficient mice were protected. These results support the view that the humoral immune response and to certain extends the CD4(+) T helper cells are a prerequisite for cross-protective immunity against H5 influenza virus.

Essential differences in ligand presentation and T cell epitope recognition among HLA molecules of the HLA-B44 supertype.

Human leukocyte antigens (HLA) have long been grouped into supertypes to facilitate peptide-based immunotherapy. Analysis of several hundreds of peptides presented by all nine antigens of the HLA-B44 supertype (HLA-B*18, B*37, B*40, B*41, B*44, B*45, B*47, B*49 and B*50) revealed unique peptide motifs for each of them. Taking all supertype members into consideration only 25 out of 670 natural ligands were found on more than one HLA molecule. Further direct comparisons by two mass spectrometric methods--isotope labeling as well as a label-free approach--consistently demonstrated only minute overlaps of below 3% between the ligandomes of different HLA antigens. In addition, T cell reactions of healthy donors against immunodominant HLA-B*44 and HLA-B*40 epitopes from EBV lacked promiscuous T-cell recognition within the HLA-B44 supertype. Taken together, these results challenge the common paradigm of broadly presented epitopes within this supertype.

A cryptic vascular endothelial growth factor T-cell epitope: identification and characterization by mass spectrometry and T-cell assays.

Vascular endothelial growth factor (VEGF) is involved in various physiologic processes, such as angiogenesis or wound healing, but is also crucial in pathologic events, such as tumor growth. Thus, clinical anti-VEGF treatments have been developed that could already show beneficial effects for cancer patients. In this article, we describe the first VEGF-derived CD8(+) T-cell epitope. The natural HLA ligand SRFGGAVVR was identified by differential mass spectrometry in two primary renal cell carcinomas (RCC) and was significantly overpresented on both tumor tissues. SRFGGAVVR is derived from a cryptic translated region of VEGF presumably by initiation of translation at the nonclassic start codon CUG(499). SRFGGAVVR-specific T cells were generated in vitro using peptide-loaded dendritic cells or artificial antigen-presenting cells. SRFGGAVVR-specific CD8(+) T cells, identified by HLA tetramer analysis after in vitro stimulation, were fully functional T effector cells, which were able to secrete IFN-gamma on stimulation and killed tumor cells in vitro. Additionally, we have quantitatively analyzed VEGF mRNA and protein levels in RCC tumor and normal tissue samples by gene chip analysis, quantitative reverse transcription-PCR, in situ hybridization, and bead-based immunoassay. In the future, T cells directed against VEGF as a tumor-associated antigen may represent a possible way of combining peptide-based anti-VEGF immunotherapy with already existent anti-VEGF cancer therapies.

Features of TAP-independent MHC class I ligands revealed by quantitative mass spectrometry.

TAP is responsible for transferring cytosolic peptides into the ER, where they can be loaded onto MHC molecules. Deletion of TAP results in a drastic reduction of MHC class I surface expression and alters the presented peptide pattern. This key molecule in antigen processing is tackled by several viruses and lost in some tumors, rendering the altered cells less vulnerable to T cell-based immune surveillance. Using the TAP-deficient cell line LCL721.174 and its TAP-expressing progenitor cell line LCL721.45, we identified and quantified more than 160 HLA ligands, 50 of which were presented TAP-independently. Peptides which were predominantly presented on the TAP-deficient LCL721.174 cell line had a decreased MHC binding affinity according to their SYFPEITHI and BIMAS score. About half of the identified TAP-independently presented peptides were not derived from signal sequences and may partly be generated by the proteasome. Furthermore, we have excluded the possibility that differences in HLA ligand presentation between LCL721.45 and LCL721.174 were due to varying expression of the source proteins or due to changes in the antigen loading complex. Features of peptides presented independently of TAP as well as proteasomal contribution to their generation provide an insight into basic immunological mechanisms.

Alteration of NF-kappaB activity leads to mitochondrial apoptosis after infection with pathological prion protein.

Nuclear factor kappa B (NF-kappaB) is a key regulator of the immune response, but in almost the same manner it is involved in induction of inflammation, proliferation and regulation of apoptosis. In the central nervous system activated NF-kappaB plays a neuroprotective role. While in some neurodegenerative disorders the role of NF-kappaB is well characterized, there is poor knowledge on the role of NF-kappaB in prion disease. We found binding but no transcriptional activity of the transcription factor in vitro. Characterizing the mechanism of cell death after infection with pathological prion protein increased caspase-9 and caspase-3 activity was detected and the lack of NF-kappaB activity resulted in the inability to activate target genes that usually play an important role in neuroprotection. Additionally, we investigated the role of NF-kappaB after prion infection of Nfkb1(-/-), Nfkb2(-/-) and Bcl3(-/-) mice and central nervous system-specific p65-deleted mice revealing an accelerated prion disease in NF-kappaB2- and Bcl-3-deficient mice, which is in line with a reduced neuroprotective activity in prion infection. Based on our findings, we propose a model whereby the alteration of NF-kappaB activity at the early stages of infection with pathological prion protein leads to neuronal cell death mediated by mitochondrial apoptosis.

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.