Biogenesis of HLA Ligand Presentation in Immune Cells Upon Activation Reveals Changes in Peptide Length Preference

Abstract

Induction of an effective tumor immunity is a complex process that includes the appropriate presentation of the tumor antigens, activation of specific T cells, and the elimination of malignant cells. Potent and efficient T cell activation is dependent on multiple factors, such as timely expression of co-stimulatory molecules, the differentiation state of professional antigen presenting cells (e.g., dendritic cells; DCs), the functionality of the antigen processing and presentation machinery (APPM), and the repertoire of HLA class I and II-bound peptides (termed immunopeptidome) presented to T cells. So far, how molecular perturbations underlying DCs maturation and differentiation affect the in vivo cross-presented HLA class I and II immunopeptidomes is largely unknown. Yet, this knowledge is crucial for further development of DC-based immunotherapy approaches. We applied a state-of-the-art sensitive MS-based immunopeptidomics approach to characterize the naturally presented HLA-I and -II immunopeptidomes eluted from autologous immune cells having distinct functional and biological states including CD14 + monocytes, immature DC (ImmDC) and mature DC (MaDC) monocyte-derived DCs and naive or activated T and B cells. We revealed a presentation of significantly longer HLA peptides upon activation that is HLA allotype specific. This was apparent in the self-peptidome upon cell activation and in the context of presentation of exogenously loaded antigens, suggesting that peptide length is an important feature with potential implications on the rational design of anti-cancer vaccines.

A complete mass spectrometry-based immunopeptidomics pipeline for neoantigen identification and validation

“…Application of DeepNovo Peptidome to expand the reference HLA Ligand Atlas of benign human tissues As neoantigens mostly arise from non-canonical sources which are challenging for standard database search approaches, we built DeepNovo Peptidome, a de novo sequencing-based workflow for HLA peptide identification and neoantigen discovery. We trained the de novo sequencing model 13,14 on a very large, carefully curated dataset from 20 previous MS-based immunopeptidomics studies 5,15,16,[24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] . The dataset included nearly 3,000 runs on two major MS instruments Orbitrap and timsTOF, >26M PSMs and >1.1M unique HLA peptides, and covering more than 90 alleles for each HLA class.…”

HLA class I peptidome analysis of synthetic long peptide-fed human dendritic cells for therapeutic vaccine design

“…For the design of next-generation SLP-based therapeutic vaccines it is crucial to know which SLPderived CTL epitopes are effectively cross-presented by DCs 14,49 . We here describe the most exhaustive DC HLA-I ligandome study on SLP-loaded HLA-A-matched DCs to-date.…”

Immunopeptidomics-Guided Warehouse Design for Peptide-Based Immunotherapy in Chronic Lymphocytic Leukemia

“…Most of the recurrent CLL-associated mutations are located in "dark spots" of the immunopeptidome, defined as protein regions without any detectable HLA-presented peptides, explaining the rare detection of neoepitopes especially in low-mutational burden entities. The underlying reasons for the occurrence of such hotspots and corresponding dark spots still remain ambiguous but might include differential proteasomal cleavage, peptide processing, and HLA-binding (16,56,57). Thus, the role of neoantigen-based T cell responses in tumor entities with lowmutational burden remains obscure, calling for the application of alternative targets in peptide-based immunotherapy approaches (1)(2)(3).…”