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.antigen processing ͉ lysosomal proteases ͉ T helper cells P eptides of foreign and self proteins are presented on MHC-I and MHC-II molecules at the cell surface and can be recognized by CD8ϩ and CD4 ϩ T lymphocytes, respectively (1, 2). From a classical point of view, MHC-I molecules present antigenic peptides derived from intracellular proteins, whereas MHC-II molecules do so for exogenous and membrane proteins (3). This phenomenon is reflected in the two major cellular breakdown pathways for proteins: proteasomal degradation, particularly relevant to the generation of MHC-I peptides (4), and degradation by the endosomal͞lysosomal system, which is responsible for the processing of MHC-II peptides (5). However, the separation of these distinct pools of source proteins is less stringent than originally believed. It is now well established that MHC-I molecules are able to present peptides derived from exogenous antigens (Ag) by a process known as cross presentation (6). On the other hand, intracellular proteins can be presented by MHC-II molecules (7,8), even though the underlying processes are less clear. It has been recently shown that peptides from cytosolic model proteins can be presented on MHC-II molecules through autophagy (9-11). Autophagy plays a role in the endosomal͞lysosomal degradation pathway and is responsible for feeding intracellular components into this pathway. It is thought to be required for normal turnover of cellular components, particularly in response to starvation (12). Against this background, we hypothesized that autophagy might mediate MHC-II presentation of intracellular Ag, meaning the contents of a cell contained within the plasma membrane, excluding large vacuoles and secretory or ingested material (Gene Ontology classifications), in general. Therefore, we performed a detailed characterization of the MHC-II ligand repertoire (ligandome) presented at the cell surface under normal conditions and after increased autophagy, leading to a comprehensive overall picture of changes in peptide processing and presentation. Materials and MethodsCell Culture and Autophagy Inducti...

The major histocompatibility complex (MHC) presents peptides derived from degraded cellular proteins to Tcells 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.

The main part of cytosolic protein degradation depends on the ubiquitin-proteasome system. Proteasomes degrade their substrates into small peptide fragments, some of which are translocated into the endoplasmatic reticulum and loaded onto MHC class I molecules, which are then transported to the cell surface for inspection by CTL. A reliable prediction of proteasomal cleavages in a given protein for the identification of CTL epitopes would benefit immensely from additional cleavage data for the training of prediction algorithms. To increase the knowledge about proteasomal specificity and to gain more insight into the relation of proteasomal activity and susceptibility to prion disease, we digested sheep prion protein with human constitutive and immuno-20S proteasomes. All fragments generated in the digest were quantified. Our results underline the different cleavage specificities of constitutive and immunoproteasomes and provide data for the training of prediction programs for proteasomal cleavages. Furthermore, the kinetic analysis of proteasomal digestion of two different alleles of prion protein shows that even small changes in a protein sequence can affect the overall efficiency of proteasomal processing and thus provides more insight into the possible molecular background of allelic variations and the pathogenicity of prion proteins.

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.

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 ...