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1.
Nature ; 607(7917): 149-155, 2022 07.
Article in English | MEDLINE | ID: mdl-35705813

ABSTRACT

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.


Subject(s)
Antigens, Neoplasm , Peptides , Proteomics , Alveolar Epithelial Cells/immunology , Animals , Antigen Presentation , Antigens, Neoplasm/analysis , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Pancreatic Ductal/chemistry , Carcinoma, Pancreatic Ductal/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Lung Neoplasms/chemistry , Lung Neoplasms/immunology , Mice , Pancreatic Neoplasms/chemistry , Pancreatic Neoplasms/immunology , Peptides/analysis , Peptides/chemistry , Peptides/immunology , RNA, Messenger
2.
Biochimie ; 150: 31-36, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29709509

ABSTRACT

Human adipose-derived stromal/stem cells (hASCs) have tremendous therapeutic potential and the ability to offer insight into human development and disease. Here we subject human ASCs to siRNA-mediated knockdown of Notch3 cultured under both self-renewing and adipogenic differentiation conditions. Self-renewal was monitored by assessing viability and proliferation rates through staining and alamarBlue assays, respectively. Adipogenesis was measured through Oil-Red O staining, western blot, and quantitative real-time RT-PCR that determined expression levels of multipotency and adipogenic markers over time. Notch3 was expressed in self-renewing hASCs but knockdown, as validated by qRT-PCR and western blot, showed no impact on cell viability, as measured through live-dead staining, or cell proliferation rates, as measured through alamarBlue assays. However, although Notch3 expression was observed to increase during adipogenesis, in the absence of Notch3 there was a significant increase in hASC adipogenesis as demonstrated through an increased number of lipid vesicles, and increased expression of adipogenic markers ppar-γ, adiponectin, fabp4, and plin2. Although Notch3 is only one of four Notch receptors expressed on the surface of hASCs, this receptor appears important for proper regulation of adipogenic differentiation, possibly serving as a negative regulator to prevent inappropriate adipogenesis or promote other lineage commitments of ASCs.


Subject(s)
Adipogenesis/physiology , Adipose Tissue/cytology , Receptor, Notch3/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Adipocytes/cytology , Adipocytes/metabolism , Adipogenesis/genetics , Blotting, Western , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Proliferation/genetics , Cell Proliferation/physiology , Cell Survival/genetics , Cell Survival/physiology , Cells, Cultured , Humans , Receptor, Notch3/genetics , Receptors, Notch/metabolism , Signal Transduction/genetics , Signal Transduction/physiology
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