Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs

Abstract

The binding of oligomeric peptide-MHC (pMHC) complexes to cell surface TCR can be considered to approximate TCR-pMHC interactions at cell-cell interfaces. In this study, we analyzed the equilibrium binding of streptavidin-based pMHC oligomers (tetramers) and their dissociation kinetics from CD8(pos) T cells from 2C-TCR transgenic mice and from T cell hybridomas that expressed the 2C TCR or a high-affinity mutant (m33) of this TCR. Our results show that the tetramers did not come close to saturating cell-surface TCR (binding only 10-30% of cell-surface receptors), as is generally assumed in deriving affinity values (K(D)), in part because of dissociative losses from tetramer-stained cells. Guided by a kinetic model, the oligomer dissociation rate and equilibrium constants were seen to depend not only on monovalent association and dissociation rates (k(off) and k(on)), but also on a multivalent association rate (μ) and TCR cell-surface density. Our results suggest that dissociation rates could account for the recently described surprisingly high frequency of tetramer-negative, functionally competent T cells in some T cell responses.

Figure 1. MHC Oligomer Staining of Retrovirally-Transduced T Cell Hybridomas

(A) Distribution of oligomeric species in oligomer (tetramer) preparations. Reduced, unboiled gradient SDS-PAGE gel of reaction of different molar ratios of biotinylated H2-K^b and streptavidin. The K^b is biotinylated site-specifically on the heavy chain concurrently with translation by biotin ligase-expressing E. coli, with a modification efficiency of approximately 50%. Following experiments carried out using reagent corresponding to 16:1 MHC:streptavidin ratio. (B) Steady-state oligomer staining of T cell hybridomas with and without CD8αβ expression—corrected by subtracting fluorescence of 58^−/− cells without TCR or CD8 at the same conditions. (C) Steady-state staining of 2C transgenic T cells carried out at 4°C using various concentrations of SIY/K^b:streptavidin oligomer. Binding of SIY/K^b:streptavidin oligomer to C57BL/6 T cells was subtracted from binding to 2C transgenic T cells. Quantification of molecules per cell indicates number of fluorescent streptavidins associated with the cell; actual number of MHC-bound TCRs may be up to three times higher.

Figure 2. Detection of T Cell Receptor and CD8 Epitopes on T cell hybridomas and transgenics

Quantification of molecules per cell is derived from flow cyotometry staining data using calibrated, fluorescein-labeled antibodies and comparing to two independent batches of fluorescein-labeled beads. (A-D) Staining of cell-surface epitopes including (A,C) TCR Cβ (clone H57-597), TCR Vβ8 (clone F23.1), CD3ε (clone 145-2C11), the 2C TCR specifically (clone 1B2), and (B,D) CD8α (clone 53-6.7) and CD8β (clone 53-5.8) on (A,B) T cell hybridomas or (C,D) mouse T cells. Error bars represent the standard deviation for at least 2 and up to 8 repetitions for each individual measurement. (E,F) Contrast images showing the size differences between (E) mouse T cells (2C TCR transgenic) and (F) T cell hybridomas (m33 TCR).

Figure 3. MHC Oligomer Dissociation from T Cells

(A-B) Remaining associated fluorescent pMHC oligomer measurements were made for (A) 2C hybridomas with (open triangles) or without (closed circles) CD8 (highlighted in inset), and m33 with (open squares) or without (closed diamonds) CD8. (B) 2C TCR transgenic T cells (closed circles) and m33 hybridomas (CD8 negative, open diamonds). Dissociation data for (A-B) were fit by the equation describing a first order exponential decay. (C) Simulated effect of dissociation of bound oligomers on washing the cells over a 4, 8, or 12 min period in the cold (4°-10°) prior to flow cytometry, showing the effect of k_off,app on the level of persisting oligomers. Vertical bars indicate approximate k_off,app values calculated for different 2C receptor mutants.