Nanoscale organization of two-dimensional multimeric pMHC reagents with DNA origami for CD8⁺ T cell detection

Peptide-MHC (pMHC) multimers have excelled in the detection of antigen-specific T cells and have allowed phenotypic analysis using other reagents, but their use for detection of low-affinity T cells remains a challenge. Here we develop a multimeric T cell identifying reagent platform using two-dimensional DNA origami scaffolds to spatially organize pMHCs (termed as dorimers) with nanoscale control. We show that these dorimers enhance the binding avidity for low-affinity antigen-specific T cell receptors (TCRs). The dorimers are able to detect more antigen-specific T cells in mouse CD8⁺ T cells and early-stage CD4⁺CD8⁺ double-positive thymocytes that express less dense TCRs, compared with the equivalent tetramers and dextramers. Moreover, we demonstrate dorimer function in the analysis of autoimmune CD8⁺ T cells that express low-affinity TCRs, which are difficult to detect using tetramers. We anticipate that dorimers could contribute to the investigation of antigen-specific T cells in immune T cell function or immunotherapy applications.