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

Abstract

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/ pMHC interactions to be interrogated easily at large scale.

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

“…More and more advanced CITE-seq-related cytometry-by-sequencing platforms are rapidly being developed. However, while these platforms utilize different methods to assure single-cell resolution and use different approaches to label the cells, they all use high-throughput sequencing to count signal from a variety of oligo-conjugated probes (such as antibodies with both surface and intracellular targets, MHC-peptide multimers, and B-cell receptor antigens) ( Stoeckius et al, 2017 ; Peterson et al, 2017 ; Hwang, 2020 ; Setliff et al, 2019 ; O'Huallachain et al, 2020 ; Overall et al, 2020 ; Gaublomme et al, 2019 ; Katzenelenbogen et al, 2020 ). Most of the observations and conclusions from this study will be applicable to tthese platforms, where improving oligo-conjugated probe signal is critical to their utility and economic feasibility.…”

Variations in MHC class I antigen presentation and immunopeptidome selection pathways

“…There is an additional hierarchy among the HLA-A allotypes, and members of the A*02 and A*24 supertypes demonstrate preferential binding to TAPBPR. Interestingly, the addition of soluble TAPBPR to cells facilitates peptide exchange on the surface from selected HLA-I allotypes 83 , which can be used to generate peptide–MHC-I libraries in vitro 86 . TAPBPR binding preferences for a given allotype correlate with the ability of TAPBPR to mediate cell surface peptide exchange on the respective allotype 82 .…”

Immune Literacy: Reading, Writing, and Editing Adaptive Immunity

“…For cells that lack membrane-bound BCR (but secrete antibodies) such as plasma cells, a recent droplet-based microfluidic platform “CelliGO” was developed, which allows antigen-specific sorting and single-cell sequencing within one workflow ( Gérard et al., 2020 ). Similar as for B cells, 10X Genomics can also be utilized for antigen-specific screening of T cells by the use of DNA-barcoded peptide-MHC multimers ( Overall et al., 2020 ).…”