High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Peptide exchange technologies are crucial for generating peptide-MHC (pMHC) multimer libraries.
• These libraries are used to study T cell receptor (TCR) repertoires and their interactions with antigens.

Purpose of the Study:

• To develop a robust method for generating stable, empty MHC-I molecules for high-throughput peptide loading.
• To enable large-scale interrogation of TCR/pMHC interactions and T cell transcription profiles.

Main Methods:

• Utilized the molecular chaperone TAPBPR to capture stable, empty MHC-I molecules (murine H2 and human HLA alleles).
• Developed high-throughput assays for peptide binding validation on empty MHC-I/TAPBPR complexes.
• Integrated the system with tetramer-barcoding and ECCITE-seq for multi-modal cellular indexing.

Main Results:

• Successfully generated stable, empty MHC-I molecules amenable to high-throughput tetramerization and peptide loading.
• Demonstrated the utility of TAPBPR for efficient peptide exchange with high-affinity peptides.
• Enabled combined analysis of TCR repertoires, T cell transcription, and antigen specificities in a single experiment.

Conclusions:

• The developed TAPBPR-based method offers a robust and scalable approach for pMHC-multimer library generation.
• This facilitates comprehensive analysis of T cell responses and antigen recognition at an unprecedented scale.