Enhanced Detection of Antigen-Specific CD4(+) T Cells Using Altered Peptide Flanking Residue Peptide–MHC Class II Multimers

Fluorochrome-conjugated peptide–MHC (pMHC) class I multimers are staple components of the immunologist’s toolbox, enabling reliable quantification and analysis of Ag-specific CD8(+) T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4(+) T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II–bound peptides, can enhance TCR–pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4(+) T cells, highlighting an unappreciated feature of TCR–pMHC-II interactions.