Abstract
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.
Highlights
Soluble fluorochrome-conjugated multimeric pMHC class I (pMHC-I) molecules have revolutionized the field of cellular immunology by enabling the direct ex vivo visualization, enumeration, characterization, and isolation of Ag-specific CD8+ T cell populations [4, 7]
Technical and biological constraints have hindered the development of pMHC class II (pMHC-II) multimers for the ex vivo study of Ag-specific CD4+ T cells [4, 8,9,10]
The MHC class I (MHC-I) and MHC class II (MHC-II) peptide-binding grooves are very similar in overall conformation, both comprising two “sides” formed from a helices and a floor of b sheets, the nature of the bound peptide in each MHC class is distinct
Summary
Our previously reported biophysical data demonstrate that targeted substitutions in the C-terminal PFRs of MHC-II epitopes can substantially enhance TCR binding affinity [35]. To extend these preliminary results and confirm the generality of this observation, we derived a new DR1-restricted CD4+ T cell clone (2C5) specific for the HA3062318 epitope (Flu1, PKYVKQNTLKLAT; core binding P1–P9 nonamer underlined). As predicted previously by structural modeling [35], these data suggest an increase in net electrostatic interactions compared with HA1.7-DR1-Flu1 and offer a thermodynamic explanation for increased affinity TCR binding to an HLA-DR–presented peptide with a substituted PFR.
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