Immunopeptidomics shows how deleterious knockouts in the HLA class I antigen processing and presentation machinery induce distinct changes in the antigen landscape

Antigen presentation is an indispensable trigger for T-cell-based immune responses where T-cell receptors recognize HLA-peptide complexes on the cell surface. In the case of HLA class I, cells constantly sample the cytoplasmic proteome for short peptides to present them as antigens. For antigen presentation, an entire network of proteins act in concert to uphold a molecular machinery that delivers a flow of HLA-peptide complexes to the cell surface to potentially bind T-cell receptors.

Functionally impairing mutations of the antigen processing and presentation machinery (APPM) are a frequently observed immune evasion mechanism in cancer. Our aim is to understand how deleterious knock out of key players in the APPM affects the antigenic landscape, and, consequentially, potential to stimulate T-cells.

HAP1 is a near-haploid leukemia-derived cell line model expressing highly frequent HLA class I alleles among the European population, and, as such, a suitable model to generate and study the immunopeptidome of APPM knock-outs. In total, a series of 11 knock-outs have been previously generated (IRF2, B2M, GANAB, CALR, CALX, TAPBP, PDIA3, ERAP1, TAP1, TAP2, SPPL3), including genes involved in modifying, chaperoning, translocating, and loading the HLA molecules with peptides.

In order to study antigen presentation, we investigated each knock-out"s immunopeptidome with our high-throughput 96-well plate-based immunopeptide enrichment workflow, in triplicates and we further measured the respective proteomes. Data was acquired in DDA and DIA on a HF-X Q Exactive. With help of the FragPipe search engine environment and using chimeric spectra to find PSMs with a database search, we accumulated a library of more than 22"000 unique immunopeptides, mapping to more than 8"000 unique proteins, for a subsequent library search of the DIA data (FDR 1%).

Our data shows varying levels of impact on the overall ability to present antigens, ranging from complete elimination of antigen presentation capability, like in the case of B2M, to sampling depths resembling those of the wildtype controls as in CALR. We determined knock-out specific effects that are directly linked to the biological function, e.g., knocking-out ERAP shifted the peptide length distribution to longer peptides. In addition, due to the quantitative quality of DIA library searches, we also determined an HLA allele-dependent shift in antigen presentation levels in different knock-outs, directly linking the dependencies of HLA-alleles on APPM components regarding the regulation of their presentation. Proteomics data provided insights on the efficiency of sampling source proteins for presentation.

We demonstrate that impairments in the APPM result in distinct features in the antigenic landscape. This research enhances our understanding of the roles played by various APPM actors in shaping the immunopeptidome and could lead to improved HLA binding predictions that account for defects in the machinery.