Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay

Abstract

Efficient presentation of peptide-MHC class I complexes to immune T cells depends upon stable peptide-MHC class I interactions. Theoretically, determining the rate of dissociation of a peptide-MHC class I complexes is straightforward; in practical terms, however, generating the accurate and closely timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal requirements. To avoid labeling of the highly variable peptide, we labeled the invariant β2m and monitored its dissociation by a scintillation proximity assay, which has no separation steps and allows for real-time quantitative measurement of dissociation. Validating this work-around to create a virtually label-free assay, we showed that rates of peptide-MHC class I dissociation measured in this assay correlated well with rates of dissociation rates measured conventionally with labeled peptides. This assay can be used to measure the stability of any peptide-MHC class I combination, it is reproducible and it is well suited for high-throughput screening. To exemplify this, we screened a panel of 384 high-affinity peptides binding to the MHC class I molecule, HLA-A*02:01, and observed the rates of dissociation that ranged from 0.1h to 46h depending on the peptide used.

Figure 1

Schematic diagram of the dissociation assay. Biotinylated MHC-I heavy chain binds to the surface of streptavidin scintillation microplates (FlashPlates PLUS). In the presence of a binding peptide, radiolabeled β₂m is bound to the MHC-I heavy and the beta-radiation can reach the scintillant embedded in the microplate resulting in a scintillation signal.

Figure 2

Adjusting assay conditions. A) HLA-A*02:01 heavy chain dose-response titration with or without a binding peptide (FLPSDYFPSV). B) Association of radiolabeled β₂m to HLA-A*02:01 at 18°C with 4 different peptides: (▽) SLDQSVVEL, (●) GLYSLPHDL, (▼) RLTRFLSRV and (○) YLNKIQNSL. Steady-state is reached after approximately 10 hours. C) Peptide dose-response titrations of five different peptides: (●) ILYAHLHKL, (▼) ILSDENYLL, (▲) FLTSVINRV, (○) YLIDTTSREL, (◆) QVKDEKLNL. For some peptides an inhibition of the signal was observed at peptide concentrations above 10 μM.

Figure 3

A) Dissociation of four different peptides binding to HLA-A*02:01. The data was fitted to a one-phase decay function, and the calculated half-lifes were (▼, RLTRFLSRV) 26.7 hours, (○, YLNKIQNSL) 7.2 hours, (▽, SLDQSVVEL) 2.7 hours and (●, GLYSLPHDL) 1.4 hours. B) The half-life of 384 different peptides binding to HLA-A*02:01 with K_D < 500nM. The half-lifes ranged from 46 hours to 0.1 hours C) Reproducibility of the assay. The stability of 384 peptides was analyzed for HLA-A*02:01 in one experiment (Experiment 1). The experiment was repeated with new batches of peptide, MHC, ¹²⁵I-β₂m, experiment 2, and the half-lifes for the two experiments were compared.

Figure 4

Comparison of the dissociation rates measured with either ¹²⁵I-β₂m or ¹²⁵I-peptide. Two different peptides (FLPSDYFPSV and RLPAYAPLL) were radiolabeled. pMHC’s were generated with different MHC-I molecules (A*02:01, A*02:02, A*02:03, A*02:04, A*02:05, A*02:11, A*02:19 and A*69:01) using either labeled peptide and excess of β₂m or labeled β₂m and excess peptide. For each pMHC the dissociation rate measured with labeled peptide was compared to the dissociation rate measured with labeled β₂m.