Abstract 2062: Identification of functional MAIT-specific TCRs using MR1 dCODE Dextramer® reagents and evaluating antigen presentation

Abstract

Abstract Objectives: To support TCR discovery and enable evaluation of TCR target expression on the surface of cells, we have established a streamlined workflow allowing TCR Discovery, validation and evaluation of MHC-antigen expression on target cells. As proof of concept, MR1 dCODE Dextramer® reagents were used to identify antigen-specific MAIT cells using single-cell sequencing, TCR sequences were extracted from the resulting data and then cloned and expressed as TCR monomers. The specificity of the TCRs was subsequently validated as TCR Dextramer® reagents and used to evaluate target MHC-peptide presentation on cell surface. Methods: Mixed PBMC samples from three healthy donors were stained with antibodies targeting relevant phenotypic markers as well as MR1 dCODE Dextramer® reagents carrying the MAIT cell-specific ligand 5-OP-RU. Following staining, sorting, and partitioning on a BD Rhapsody™ Single-Cell Analysis System, the cells underwent sequencing for a characterization of gene and surface marker expression as well as paired alpha-beta TCR sequences. Single alpha and beta chains were individually expressed, refolded together and purified. Refolded TCR monomer functionality was validated using an artificial cell assay. The functional TCRs can be multimerized on a Dextramer® backbone and used to evaluate expression of target on cell surface. Results: The majority of the identified CD161+ MAIT cells (~70%) expressed typical TCRs consisting of TRAV1-2 and TRAJ33 as well as TRBV6/20, TRBD1/2, and TRBJ2 gene segments. Three alpha-beta TCR sequences were selected: Two TCRs having common TRAV+TRAJ, TRBV+TRBJ combinations and one having a rare TRAV+TRAJ one. TCR alpha and beta chains were successfully expressed for all selected TCRs but only 2 TCRs showed a correctly refolded TCR as demonstrated by proper recognition of MR1/5-OP-RU. Based on these two functional TCRs, TCR Dextramer® reagents were generated and used to evaluate expression of target antigen on cell surface. Conclusion: We demonstrate a workflow allowing: (i) Identification of MAIT cells and their corresponding TCR sequences. (ii) Generation of soluble TCR molecules based on the identified sequences and validation of their specificity. (iii) Generation of TCR Dextramer® reagents allowing identification of target expression on surface of cells. This workflow is also useful for identification of other antigen-specific T cells and creates a new tool for evaluation of antigen-expression by MHC on surface of cells. By combining the high sensitivity of dCODE Dextramer® technology with TCR sequencing and creation of TCR Dextramer® reagents, both low-affinity interactions and rare cell types can be detected and studied in unique detail. Citation Format: Kevin Lenogue, Bjarke Hansen, Kivin Jacobsen, Liselotte Brix, Thomas Holberg Blicher. Identification of functional MAIT-specific TCRs using MR1 dCODE Dextramer® reagents and evaluating antigen presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2062.