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Recent studies suggest that CLL is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell-based immunotherapy. However, CLL is associated with a profound immune defect, which might represent a critical limitation for mounting clinically effective antitumor immune responses. As several studies have demonstrated that lenalidomide can reinforce effector T-cell responses in CLL, the combination of T-cell-based immunotherapy with the immunomodulatory drug lenalidomide represents a promising approach to overcome the immunosuppressive state in CLL. Antigen-specific immunotherapy also requires the robust presentation of tumor-associated HLA-presented antigens on target cells. We thus performed a longitudinal study of the effect of lenalidomide on the HLA ligandome of primary CLL cells in vitro. We showed that lenalidomide exposure does not affect absolute HLA class I and II surface expression levels on primary CLL cells. Importantly, semi-quantitative mass spectrometric analyses of the HLA peptidome of three CLL patient samples found only minor qualitative and quantitative effects of lenalidomide on HLA class I- and II-restricted peptide presentation. Furthermore, we confirmed stable presentation of previously described CLL-associated antigens under lenalidomide treatment. Strikingly, among the few HLA ligands showing significant modulation under lenalidomide treatment, we identified upregulated IKZF-derived peptides, which may represent a direct reflection of the cereblon-mediated effect of lenalidomide on CLL cells. Since we could not observe any relevant influence of lenalidomide on the established CLL-associated antigen targets of anticancer T-cell responses, this study validates the suitability of lenalidomide for the combination with antigen-specific T-cell-based immunotherapies. [ABSTRACT FROM AUTHOR]

The classical HLA-C and the nonclassical HLA-E and HLA-G molecules play important roles both in the innate and adaptive immune system. Starting already during embryogenesis and continuing throughout our lives, these three Ags exert major functions in immune tolerance, defense against infections, and anticancer immune responses. Despite these important roles, identification and characterization of the peptides presented by these molecules has been lacking behind the more abundant HLA-A and HLA-B gene products. In this study, we elucidated the peptide specificities of these HLA molecules using a comprehensive analysis of naturally presented peptides. To that end, the 15 most frequently expressed HLA-C alleles as well as HLA-E*01:01 and HLA-G*01:01 were transfected into lymphoblastoid C1R cells expressing low endogenous HLA. Identification of naturally presented peptides was performed by immunoprecipitation of HLA and subsequent analysis of HLA-bound peptides by liquid chromatographic tandem mass spectrometry. Peptide motifs of HLA-C unveil anchors in position 2 or 3 with high variances between allotypes, and a less variable anchor at the C-terminal end. The previously reported small ligand repertoire of HLA-E was confirmed within our analysis, and we could show that HLA-G combines a large ligand repertoire with distinct features anchoring peptides at positions 3 and 9, supported by an auxiliary anchor in position 1 and preferred residues in positions 2 and 7. The wealth of HLA ligands resulted in prediction matrices for octa-, nona-, and decamers. Matrices were validated in terms of their binding prediction and compared with the latest NetMHC prediction algorithm NetMHCpan-3.0, which demonstrated their predictive power. [ABSTRACT FROM AUTHOR]

There are limitations in pre-clinical settings using mice as a basis for clinical development in humans. In cancer, similarities exist between humans and dogs; thus, the dog patient can be a link in the transition from laboratory research on mouse models to clinical trials in humans. Knowledge of the peptides presented on MHC molecules is fundamental for the development of highly specific T cell-based immunotherapies. This information is available for human MHC molecules but is absent for the canine MHC. In the present study, we characterized the binding motif of dog leukocyte antigen (DLA) class I allele DLA-88*50101, using human C1R and K562 transfected cells expressing the DLA-88*50101 heavy chain. MHC class I immunoaffinity-purification revealed 3720 DLA-88*50101 derived peptides, which enabled the determination of major anchor positions. The characterized binding motif of DLA-88*50101 was similar to HLA-A*02:01. Peptide binding analyses on HLA-A*02:01 and DLA-88*50101 via flow cytometry showed weak binding of DLA-88*50101 derived peptides to HLA-A*02:01, and vice versa. Our results present for the first time a detailed peptide binding motif of the canine MHC class I allelic product DLA-88*50101. These data support the goal of establishing dogs as a suitable animal model for the evaluation and development of T cell-based cancer immunotherapies, benefiting both dog and human patients. [ABSTRACT FROM AUTHOR]

Direct analysis of HLA-presented antigens by mass spectrometry provides a comprehensive view on the antigenic landscape of different tissues/malignancies and enables the identification of novel, pathophysiologically relevant T-cell epitopes. Here, we present a systematic and comparative study of the HLA class I and II presented, non mutant antigenome of multiple myeloma (MM).Quantification of HLA surface expression revealed elevated HLA molecule counts on malignant plasma cells compared with normal B cells, excluding relevant HLA downregulation in MM. Analyzing the presentation of established myeloma-associated T-cell antigenson the HLA ligandome level, we found a substantial proportion of antigens to be only infrequently presented on primary myelomas or to display suboptimal degrees of myeloma specificity. However, unsupervised analysis of our extensive HLA ligand data set delineated a panel of 58 highly specific myeloma-associated antigens (including multiple myeloma SET domain containing protein) which are characterized by frequent and exclusive presentation on myeloma samples. Functional characterization of these target antigens revealed peptide-specific, preexisting CD81 T-cell responses exclusively in myeloma patients, which is indicative of pathophysiological relevance. Furthermore, in vitro priming experiments revealed that peptide-specific T-cell responses can be induced in response-naive myeloma patients. Together, our results serve to guide antigen selection for T-cell-based immunotherapy of MM. [ABSTRACT FROM AUTHOR]