Using a translation-inhibition flow cytometry assay for CRISPR-Cas9 screening of 281 genes highly expressed in cDC1s, but not cDC2s, Rodriquez-Silvestre et al. identified perforin-2 (Mpeg1) as an APC-restricted pore-forming protein that regulates antigen transit from endosomes to cytosol. In vitro, endocytic escape was impaired in Mpeg1-/- DCs and enabled in non-immune cells by ectopic perforin-2 expression. Cell-associated antigen priming of CD8+ T cells was impaired in Mpeg1-/- mice. Perforin-2 was shown to reside mostly in lysosomes at steady state, transit through endosomes, and undergo proteolytic processing in low-pH antigen-containing compartments.
Contributed by Paula Hochman
ABSTRACT: During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1(-/-) mice failed to efficiently prime CD8(+) T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.
Author Info: (1) MRC Laboratory of Molecular Biology, Cambridge, UK. (2) MRC Laboratory of Molecular Biology, Cambridge, UK. (3) MRC Laboratory of Molecular Biology, Cambridge, UK. (4) Departme
Author Info: (1) MRC Laboratory of Molecular Biology, Cambridge, UK. (2) MRC Laboratory of Molecular Biology, Cambridge, UK. (3) MRC Laboratory of Molecular Biology, Cambridge, UK. (4) Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany. (5) Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany. (6) MRC Laboratory of Molecular Biology, Cambridge, UK. (7) MRC Laboratory of Molecular Biology, Cambridge, UK. UK Dementia Research Institute at the University of Cambridge, Department of Clinical Neurosciences, Cambridge, UK. (8) UK Dementia Research Institute at the University of Cambridge, Department of Clinical Neurosciences, Cambridge, UK. (9) Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany. (10) MRC Laboratory of Molecular Biology, Cambridge, UK.
