Table 1.

Strategies for resolving the recognition motif of T-cell receptors (TCRs)

Method	Readout	Require a pre-established TCR epitope	HLA availability	Reflects natural peptide–HLA binding	Disadvantages	Advantages	First reported
Combinatorial peptide library	Functional readout that reflects the amino acid requirement one position at a time	No	Independent	Yes	No direct measure of the relative importance of each peptide position	Can include variables and synthetic molecules, e.g. peptide mimetics	[92]
Yeast displaya	Quantitative readout based on mutual epitope competition. All positions analysed in one reaction	No	New yeast display library required for every new HLA. Currently only developed for a few HLAs	Moderately	Peptide positions may not be equally well represented. Method restricted to a few specialized laboratories	Unbiased screening based on direct pMHC recognition	[90]
DNA barcode-labeled MHC multimers	Quantitative readout based on mutual epitope competition. All positions analysed in one reaction	Yes	Possible for all foldable HLAs. Currently ∼35	Marginally	New peptide library required for each analysed pMHC specificity	Easy to implement in laboratories working with MHC multimers	[67]

The methods listed have been applied to understand the molecular interaction points of TCRs, which can be used to estimate their cross-recognition potential.

HLA, human leukocyte antigen; pMHC, peptide–major histocompatibility complex.

^aThe strategies reported in References 93–96, which have been applied to discover the antigen specificities of TCRs, also have the potential to resolve their recognition motif. Such implementation will share many characteristics with the yeast display method.