Deleterious knock-outs in the HLA class I antigen processing and presentation machinery induce distinct changes in the immunopeptidome

ABSTRACT: The human leukocyte antigen (HLA) processing and presentation machinery (APPM) is altered in various diseases and in response to drug treatments. Defects in the machinery may change presentation levels or alter the repertoire of presented peptides, globally or in an HLA allele restricted manner, with direct implications for adaptive immunity. In this study, we investigated the immunopeptidome landscape across a panel of isogenic HAP1 cell line clones each with a knock-out of a single gene encoding a key protein in the APPM, including B2M, TAP1, TAP2, TAPBP, IRF2, PDIA3, ERAP2, GANAB, SPPL3, CANX, and CALR. We applied immunopeptidomics and proteomics methods to assess the successful gene knock-outs on the protein level, to understand how these proteins participate in antigen presentation, and to contextualize protein expression and antigen presentation. The knocked-out proteins were clearly absent in the respective samples. We find that knocking-out an APPM component leads to the presentation of a subset of peptides that are normally presented on the control wild type cells. We assessed the immunopeptidomes qualitatively and quantitatively, considering factors like peptide diversity, peptide length distribution, and binding affinity to the endogenously expressed HLA alleles in HAP1 cells. We demonstrated a prominent HLA allele-specific alterations in several knock-out conditions. For CALR, CANX, and TAP1, HLA allele-related significant change in presentation level was found for A*02:01 and as well as B*40:01. Overall, this work represents a first systematic analysis of the effect of a panel of single APPM knock-out clones from a single cell line in a controlled environment. This approach could facilitate the creation of predictive tools capable of prioritizing HLA-bound peptides likely to be presented when presentation defects occur, such as in cancer and viral infections.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Michal Bassani-Sternberg  

LAB HEAD: Michal Bassani-Sternberg

PROVIDER: PXD056426 | Pride | 2025-04-04

REPOSITORIES: Pride