Claudia Lemmel

Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling

Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin–derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)*6801. Guanidination/nicotinylation was applied to keratin 18–transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A*0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.

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.

Lessons to be learned from primary renal cell carcinomas: novel tumor antigens and HLA ligands for immunotherapy

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.

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.

The use of HPLC-MS in T-cell epitope identification

The hunt for T-cell epitopes is going on because hopes are set on such peptide sequences for diagnosis and vaccine development in the fight against infectious and tumor diseases. In addition to a variety of other techniques used in T-cell epitope identification, mass spectrometers coupled to microcapillary liquid chromatography have now become an important and sensitive tool in separation, detection, and sequence analysis of highly complex natural major histocompatibility complex (MHC) ligand mixtures. In this article, we review the basics of mass spectrometric techniques and their on-line coupling to microcapillary liquid chromatography (microcap-LC). Furthermore, we introduce current strategies for the identification of new T-cell epitopes using microcapillary liquid chromatography-mass spectrometry (microcap-LC-MS).