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.

Ever since it was discovered that central tolerance to self is imposed on developing T cells in the thymus through their interaction with self-peptide major histocompatibility complexes on thymic antigen-presenting cells, immunologists have speculated about the nature of these peptides, particularly in humans. Here, to shed light on the so-far unknown human thymic peptide repertoire, we analyse peptides eluted from isolated thymic dendritic cells, dendritic cell-depleted antigen-presenting cells and whole thymus. Bioinformatic analysis of the 842 identified natural major histocompatibility complex I and II ligands reveals significant crosstalk between major histocompatibility complex-class I and II pathways and differences in source protein representation between individuals as well as different antigen-presenting cells. Furthermore, several autoimmune-and tumour-related peptides, from enolase and vimentin for example, are presented in the healthy thymus. 302 peptides are directly derived from negatively selecting dendritic cells, thus providing the first global view of the peptide matrix in the human thymus that imposes self-tolerance in vivo.

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.

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.Key words: Antigen presentation/processing . CD8 T cells . Immunotherapy . Mass spectrometry . MHC Supporting Information available online IntroductionPeptides presented on human leukocyte antigen (HLA) class I molecules are the final result of antigen processing and are usually generated under the participation of the cytosolic proteasomes and aminopeptidases. About 1% of peptides produced in the cytosol are translocated to the ER via TAP [1].Upon undergoing further N-terminal trimming, the peptides are finally loaded onto HLA class I molecules. Thus, peptides presented on the cell surface have succeeded in following various rules along the pathway of antigen processing. They are determined by the cleavage specificities of the proteasome and by cytosolic aminopeptidase activity. The transport via TAP shows strong preferences for hydrophobic or charged residues in both the C-terminal and the second position [2]. Selectivity also applies to trimming, as peptide bonds between any amino acid and Pro cannot be cleaved by peptidases of the ER, and HLA class I molecules themselves serve as templates for their to-be-ligands Eur. J. Immunol. 2008. 38: 2993-3003 DOI 10.1002 Antigen processing 2993 [3]. Finally, peptides build stable complexes with HLA molecules only if they fit into the binding groove, which depends on the nature of the so-called pockets. The allele-specific amino acid composition of these pockets determines both their polarity and stereochemistry and, consequently, also the residues of the peptide that are allowed to protrude into these pockets. The peptide motif describes such primary anchors of the peptide, which have the strongest effect on ligand binding as well as less constricted but still nonetheless important auxiliary anchors. The extensive polymorphism of HLA genes provides the basis for similar variability among peptide motifs. With an increasing number of HLA alleles characterized in more detail these were classified into several groups. So-called supertypes can be defined according to either structure or function [4]. The latter approach groups HLA ...

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.

Predicting the T-cell-mediated immune response is an important task in vaccine design and thus one of the key problems in computational immunomics. Various methods have been developed during the last decade and are available online. We present EpiToolKit, a web server that has been specifically designed to offer a problem-solving environment for computational immunomics. EpiToolKit offers a variety of different prediction methods for major histocompatibility complex class I and II ligands as well as minor histocompatibility antigens. These predictions are embedded in a user-friendly interface allowing refining, editing and constraining the searches conveniently. We illustrate the value of the approach with a set of novel tumor-associated peptides. EpiToolKit is available online at www.epitoolkit.org.

This is the first molecular analysis of HLA class I and II ligands ex vivo from human lung cancer tissues which reveals known and novel tumor antigens able to elicit a CTL response.

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