# Validation of novel conditional ligands and large-scale detection of antigen-specific T cells for H-2Dd and H-2Kd

## TL;DR

This paper introduces new methods to detect antigen-specific T cells in mice, improving preclinical immunological research.

## Contribution

Design and validation of novel conditional ligands for H2Dd and H2Kd MHC class I alleles in mice.

## Key findings

- Conditional ligands for H2Dd and H2Kd were experimentally validated.
- Fluorescent peptide-H2 tetramers were successfully used to stain antigen-specific CD8+ T cells.
- DNA-barcode labeling enabled high-throughput analysis of T cell specificity in murine samples.

## Abstract

The UV-mediated peptide exchange has enabled the generation of multiple different MHC multimer specificities in parallel, surpassing tedious individual refolding of MHC molecules with peptide ligands. Murine models are acknowledged as an effective tool for preclinical research to advance our understanding of immunological mechanisms, with the potential translatability of key learnings from mouse models to the clinic. The common inbred mouse strain BALB/c is frequently used in immunological research. However, for the BALB/c histocompatibility (H)-2 alleles availability of conditional ligand has been limited. To overcome this challenge, we design and experimentally validate conditional ligands restricted to murine MHC class I alleles H2Dd and H2Kd. In addition, we demonstrate the ability of the three H2d molecules and two additional C57BL/6 H2b molecules folded in-house with conditional ligands to generate fluorescently labeled peptide-H2 tetramers that allow staining of antigen-specific CD8+ T cells in splenocyte samples. Finally, we generate large peptide-H-2 multimer libraries with a DNA-barcode labeling system for high-throughput interrogation of CD8+ T cell specificity in murine splenocyte samples. Consequently, the described techniques will contribute to our understanding of the antigen-specific CD8+ T cell repertoire in murine preclinical models of various diseases.