Scaling measurements of peptide-HLA complex stability using user-defined libraries and mass spectrometry 2310029

Mapping the TCR–Peptide-HLA Interface through Hydrogen–Deuterium Exchange Mass Spectrometry

T-cell receptor (TCR)–based therapeutics recognize specific peptides presented by human leukocyte antigens (HLAs) on antigen-presenting cells. The docking orientation of TCRs to peptide–HLA (pHLA) complexes is critical for T-cell activation as it underpins signaling and downstream immune responses. Traditionally, TCR binding geometries with pHLAs have been resolved through X-ray crystallography. Here, we demonstrate that conventional peptide-level hydrogen–deuterium exchange mass spectrometry (HDX-MS) workflows can identify productive TCR engagement with their cognate pHLAs. By quantifying differential deuterium uptake upon complex formation, we show that TCRs that bind centrally within the pHLA groove have distinct uptake profiles compared with those adopting alternative orientations. Comparison of uptake data with information from structure prediction further contextualized the HDX data for distinct TCR–pHLA binding modes, thus highlighting the value of integrating experimental and computational approaches. Collectively, our findings establish existing HDX-MS workflows as rapid and accessible approaches for probing TCR–pHLA interactions, with direct implications for the development of TCR-based biotherapeutics.

Profiling Protein‐Peptide Interactions by Yeast Surface Display

A protocol for discovering protein‐binding peptides using a very large, target‐agnostic yeast surface display library containing approximately 6.1 × 109 unique clones and providing broad coverage of short peptide sequence space is described.

HLA-Shuttle: A system for enhancing antigen presentation in immunologically cold tumors 2258016

Peptides presented by class-I Human Leukocyte Antigen (HLA-I) proteins provide the basis of immune surveillance. Conversely, reduced surface HLA-I expression is a hallmark of immune evasion in cancers, which confounds the identification of peptide antigens and neoantigens. Here, we outline a system (HLA-Shuttle) for in vitro manipulation of cells with engineered components of the HLA-I processing pathway that improves recovery of the immunopeptidome of immunologically “cold” tumors. HLA-Shuttle is comprised of an engineered variant of the HLA class I chaperone tapasin that bypasses its native degradation and ER retention signals, enabling improved expression and escape from the ER. HLA-Shuttle provides a continuum of chaperoning activity for HLA-I complexes from their point of assembly in the ER to the cell surface, improving antigen presentation in those cells. Our data suggest that HLA-Shuttle functions in a multimodal fashion, both enhancing HLA-I complex production in the ER while stabilizing the folded conformation of HLA-I molecules globally. This is evidenced by increased surface expression of HLA-I, while cellular trafficking assays and single particle tracking reveal an extension of their cell-surface lifetimes and microdomain formation, implying an enhancement in their stability. Leveraging this technology, we captured the immunopeptidomes of neuroblastoma cell lines. We observed improved immunoprecipitation of HLA-I complexes, which correlated with a significant expansion of the observable immunopeptidome in immunologically cold neuroblastoma cells. Following bioinformatics analysis to search for therapeutically relevant peptides, we identified multiple novel tumor associated antigens (TAAs) from both known and novel cancer immunotherapy targets. In conclusion, HLA-Shuttle restores antigen presentation in immunologically cold tumor cells, facilitating identification of TAAs with favorable therapeutic potential. T32 Fellowship Classical and Non-Classical Antigen Presenting Cells (APC)

Peptide-HLA II interaction prediction for post-translationally modified peptides

PepChem, a deep learning model utilizing novel, molecular-level peptide representations that enable predictions for sidechain modifications, bridges the critical gap in PTM-aware immune recognition prediction, with immediate applications in autoimmunity, cancer, and infectious disease.

A predictive multiscale framework for post-translational modification-dependent peptide-MHC class I binding.

A unified structure-energy-dynamics model explaining how Post-translational modifications function as atomic-level chemical switches in antigen presentation is established, establishing a unified structure-energy-dynamics model explaining how PTMs function as atomic-level chemical switches in antigen presentation.

RISING STAR ABSTRACTS Beyond Ensemble Measurements: Resolving Antigen Binding and Conformational Heterogeneity in Multispecific Antibodies