Project description:HLA class Ι molecules on the cell surface enable CD8+ T lymphocytes to recognize cellular alterations in the form of antigens, including mutations, protein copy number alterations, aberrant post-translational modifications or pathogen proteins. At any given moment, tens of thousands of different self and foreign HLA class Ι peptide ligands may be presented on the cell surface by MHC class Ι complexes. Analysis of the HLA ligandome thrusts therefore unique challenges due to their enormous biochemical diversity and inherently wide range of abundances. Despite advances in enrichment, separation, MS instrumentation and fragmentation, it is still not achievable to cover the HLA class Ι ligandome in sufficient depth to support routine identification of e.g. viral pathogens or immuno-therapeutically important tumor neo-antigens. In this study, we evaluate two pre-fractionation techniques, high pH reversed phase and strong cation exchange for complementary analysis of HLA class Ι peptide ligands, benchmarking them against analyses circumventing pre-fractionation. We observe that pre-fractionation substantially extends the detectable HLA class Ι ligandome, but also creates an identification bias. We advocate a rational choice between no-fractionation, high pH reversed phase or strong cation exchange pre-fractionation for deeper HLA class Ι ligandome analysis depending on the targeted HLA locus, allele or peptide ligand modification
Project description:The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8+ T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface HLA class I molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles remains poorly defined. Here we report the HLA class I ligandome of both cell surface and extracellular vesicles from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226. Utilising HLA class I immunoisolation and mass spectrometry we detected a total of 6574 peptides from the cell surface and 2461 peptides from the extracellular vesicles of the cell lines studied. Within the extracellular vesicle HLA class I ligandome we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T cell responses in previous studies. Our data thus confirms for the first time the presence of clinically relevant tumour-associated antigenic peptides in the HLA class I ligandome present on EV.
Project description:Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, due to the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly modest correlation between the abundance levels of surface-presented peptides and the cellular expression of their source proteins. Important selective sieving from the sampled proteome to the ligandome, was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease.
Project description:Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, due to the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly modest correlation between the abundance levels of surface-presented peptides and the cellular expression of their source proteins. Important selective sieving from the sampled proteome to the ligandome, was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease.
Project description:Immunopeptidome analysis of colorectal adenocarcinoma tissues as well as adjacent benign tissues was performed to characterize the natural HLA class I and class II presented ligandome represented in the context of malignancy as well as in benign tissue of the colon.
Project description:Immunopeptidome analysis of patient-derived colorectal cancer cell lines HROC113 and HROC285 T0 M2 from the HROC collection [Mullins et al. Cancers (Basel). 2019;11(10):1520. doi: 10.3390/cancers11101520] was performed to characterize the natural HLA class I presented ligandome in native as well as IFNγ treated cells.