Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV)-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8+ and CD4+ HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean) expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold) compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.

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 ...

Short peptides derived from intracellular proteins and presented on MHC class I molecules on the cell surface serve as a showcase for the immune system to detect pathogenic or malignant alterations inside the cell, and the sequencing and analysis of the presented peptide pool has received considerable attention over the last two decades. In this review, we give a comprehensive presentation of the methods employed for the large-scale qualitative and quantitative analysis of the MHC class I ligandome. Furthermore, we focus on insights gained into the underlying processing pathway, especially involving the roles of the proteasome, the TAP complex, and the peptide specificities and motifs of MHC molecules. The identification of post-translational modifications in MHC ligands and their implications for processing are also considered. Finally, we review the correlations of the ligandome to the proteome and the transcriptome.

The rapamycin-inducible gene regulation system was designed to minimize immune reactions in man and may thus be suited for gene therapy. We assessed whether this system indeed induces no immune responses. The protein components of the regulation system were produced in the human cell lines HEK 293T, D407, and HER 911 following lentiviral transfer of the corresponding genes. Stable cell lines were established, and the peptides presented by major histocompatibility complex class I (MHC I) molecules on transduced and wild-type (wt) cells were compared by differential mass spectrometry. In all cell lines examined, expression of the transgenes resulted in prominent changes in the repertoire of MHC I-presented self-peptides. No MHC I ligands originating from the transgenic proteins were detected. In vitro analysis of immunogenicity revealed that transduced D407 cells displayed slightly higher capacity than wt controls to promote proliferation of cytotoxic T cells. These results indicate that therapeutic manipulations within the genome of target cells may affect pathways involved in the processing of peptide antigens and their presentation by MHC I. This makes the genomic modifications visible to the immune system which may recognize these events and respond. Ultimately, the findings call attention to a possible immune risk.