Journal: Nature

Article Title: Deciphering the immunopeptidome in vivo reveals novel tumor antigens

doi: 10.1038/s41586-022-04839-2

Figure Lengend Snippet: a) Experimental illustration of samples used for immunopeptidome comparison in PDAC. b) Multiplexed immunofluorescence of a representative autochthonous PDAC tumor. White arrows indicate cancer cell nests. c) High magnification multiplexed immunofluorescence image depicting the specificity of StrepTagII staining on cancer cells. d) Schematic illustration of the traditional method of peptide extraction (top) and the method used for competitive elution of MHC-I complexes with biotin used in this study (bottom). e) Immunoblot demonstrating efficient precipitation of the MHC-I heavy and light chain with acid (1% TFA) prior to peptide clean-up with C18 ziptip. f) Representative immunoblot demonstrating purification of MHC-I specifically from KP/KbStrep tumor bearing mice. g) Number of unique peptides identified in each sample type after filtering for length (8-11 amino acids) and NetMHCPan predicted affinity (<1000 nM). h) Amino acid length distribution of all peptides identified from 2D, Ortho, Auto, or WT samples. i) Peptide motifs of 8- and 9-mers isolated from 2D, orthotopic, and autochthonous tumors. j) Venn diagram comparison of peptides found in 2D, Ortho, or Auto samples. Peptides only found in vivo are outlined in red. k) Venn diagram comparing MHC-I peptides derived from normal pancreas in Schuster et. al. (gray) versus orthotopic transplant (light blue) or autochthonous PDAC (dark blue) in this study. l) UMAP embedding of reanalyzed pancreatic scRNAseq data from the Tabula Muris. For clarity of presentation in Extended Data Figure 2m, cells from a specific lineage were collapsed into a single cluster if they originally separated into multiple clusters (i.e. Alpha 1 and Alpha 2 → Alpha). m) Expression of gene signatures derived from genes encoding for normal pancreas peptides (gray), orthotopic PDAC peptides (light blue), and autochthonous PDAC peptides (dark blue) in all cell types of the normal pancreas as measured by scRNAseq from Tabula Muris.

Article Snippet: The fluffy white precipitate was then pelleted by centrifugation at 20,000 x g for 10 min. Supernatants containing liberated MHC-I peptides were then directly aspirated into 8 ug binding capacity C18 solid phase extraction pipette tips (Pierce).

Techniques: Immunofluorescence, Staining, Western Blot, Purification, Isolation, In Vivo, Derivative Assay, Expressing