GM-CSF Culture Revisited: Preparation of Bulk Populations of Highly Pure Dendritic Cells from Mouse Bone Marrow.

Dendritic cells (DC) loaded with specific peptides are strongly immunogenic for T cells and can be used for cancer immunotherapy. For immunogenic tumors such as melanoma, injection of autologous DC loaded with tumor cell extracts or peptides can induce tumor regression but in only a small proportion of patients. Nevertheless, recent studies on the efficacy of checkpoint blockade for boosting antitumor immunity plus advances in defining tumor neoantigens are stimulating renewed interest in DC immunotherapy. Despite intensive investigation, however, preparation of bulk populations of mature DC has proved difficult, and most preparations contain a significant proportion of potentially tolerogenic immature DC. In this study, we have modified the well-established GM-CSF culture system to prepare substantial quantities of highly pure (>95%) mature DC from mouse bone marrow cells and defined their progenitors. We show that obtaining high yields and purity of DC are heavily dependent on cell density in the cultures and the tempo of addition of growth and maturation stimuli. When loaded with specific peptide, the DC are strongly immunogenic for CD4 and CD8 T cells in vivo and elicit effective antitumor immunity.

SAPrIm, a semi-automated protocol for mid-throughput immunopeptidomics.

Human leukocyte antigen (HLA) molecules play a crucial role in directing adaptive immune responses based on the nature of their peptide ligands, collectively coined the immunopeptidome. As such, the study of HLA molecules has been of major interest in the development of cancer immunotherapies such as vaccines and T-cell therapies. Hence, a comprehensive understanding and profiling of the immunopeptidome is required to foster the growth of these personalised solutions. We herein describe SAPrIm, an Immunopeptidomics tool for the Mid-Throughput era. This is a semi-automated workflow involving the KingFisher platform to isolate immunopeptidomes using anti-HLA antibodies coupled to a hyper-porous magnetic protein A microbead, a variable window data independent acquisition (DIA) method and the ability to run up to 12 samples in parallel. Using this workflow, we were able to concordantly identify and quantify ~400 - 13000 unique peptides from 5e5 - 5e7 cells, respectively. Overall, we propose that the application of this workflow will be crucial for the future of immunopeptidome profiling, especially for mid-size cohorts and comparative immunopeptidomics studies.

Engineering Cell Lines for Specific Human Leukocyte Antigen Presentation.

Epitope-specific immunotherapies have enabled the targeted treatment of a variety of diseases, ranging from cancer, infection, and autoimmune disorders. For CD8+ T cell-based therapies, the precise identification of immunogenic peptides presented by human leukocyte antigen (HLA) class I is essential which can be achieved by immunopeptidomics. Here, using lentivirus-mediated transduction and cell sorting approaches, we present a method to engineer a cell line that does not express its native HLA but instead expresses an HLA of interest (in this instance HLA-A*02:01). This technique can be used to elucidate the immunopeptidome of cell lines expressing different HLAs.