W188 - Quantitative Profiling of HLA Class I and II Antigens and Neoantigens in Tissue and PBMC Samples with an Optimized Mass Spectrometry Approach

Abstract Text: Human leukocyte antigen (HLA) molecules are essential mediators of immune surveillance, presenting antigenic peptides to T cells and enabling immune recognition of pathogenic or abnormal cells, such as cancer. HLA-driven antigen presentation plays a central role in immunology and immuno-oncology, guiding immune responses and informing targeted interventions.

In clinical trials, peripheral blood mononuclear cells (PBMCs) present a practical alternative to tissue biopsies for immunopeptidomics (IMPX) and neoantigen discovery. PBMCs are easily accessible via blood sampling, allow for serial monitoring, and encompass diverse immune cell populations, offering a comprehensive view of immune modulation and pharmacodynamics.

We developed a high-sensitivity, robust workflow for profiling class I and II immunopeptidomes from PBMCs, tissues, and cell lines. Leveraging native lysis and magnetic bead-based immunoprecipitation, we achieved efficient extraction of HLA complexes from minimal starting materials, including as few as 5 million PBMCs. The pipeline integrates optimized LC-MS and AI-driven bioinformatics for reproducible and streamlined analysis.
The workflow demonstrated high reproducibility and specificity across sample types, identifying robust immunopeptide profiles. >60% of class I immunopeptides were 9-mers, with >80% predicted as strong binders, showing expected amino acid enrichment at anchor residues. >50% of class II immunopeptides were 14-to-16-mers, with >50% predicted as strong binders. Input material ramping tests identified >2,800 class I immunopeptides from 2.5 mg of fresh-frozen tissue and 2,000–3,000 from 5 million PBMCs.

In summary, we developed a high-sensitivity, robust pipeline for class I and II immunopeptidome profiling compatible with low-input samples, suitable for target discovery and clinical applications.