T cell receptor binding kinetics required for T cell activation depend on the density of cognate ligand on the antigen-presenting cell

Abstract

CD8(+) T cells recognize peptides of eight to nine amino acid residues long in the context of MHC class I molecules on the surface of antigen-presenting cells (APCs). This recognition event is highly sensitive, as evidenced by the fact that T cells can be activated by cognate peptide/MHC complex (pMHC) at extremely low densities (1-50 molecules). High sensitivity is particularly valuable for detection of antigens at low density, such as those derived from tumor cells and intracellular pathogens, which can down-modulate cognate pMHCs from the surface of APCs to evade recognition by the adaptive immune system. T cell activation is only triggered in response to interactions between the T cell receptor (TCR) and the pMHC ligand that reach a specific half-life threshold. However, interactions with excessively long half-lives result in impaired T cell activation. Thus, efficient T cell activation by pMHC on the surface of APCs requires an optimal dwell time of TCR-pMHC interaction. Here, we show that, although this is a requirement at low cognate pMHC density on the APC surface, at high epitope density there is no impairment of T cell activation by extended TCR-pMHC dwell times. This observation was predicted by mathematical simulations for T cell activation by pMHC at different densities and supported by experiments performed on APCs selected for varied expression of cognate pMHC. According to these results, effective T cell activation depends on a complex interplay between inherent TCR-pMHC binding kinetics and the epitope density on the APC.

Fig. 1.

Calculated levels of TCR internalization as a function of cognate pMHC density (P) and TCR-pMHC t_1/2. Results shown were generated with parameter values described in Table 1 and supposing a 5-h interaction between the T cell and the APC. TCR internalization is an increasing function of presented pMHC density on the APC surface. An optimal TCR-pMHC interaction t_1/2 for TCR internalization exists only for low pMHC densities. At higher pMHC densities this peak no longer exists and a significant increase in TCR internalization can be seen for those TCRs with prolonged TCR-pMHC interaction t_1/2.

Fig. 2.

T cell hybridomas expressing TCR mutants with prolonged TCR-pMHC interaction half-lives are activated only at high pMHC density on the APC surface. Data shown are units/ml IL-2 released by T cell hybridomas expressing the WT or one of the mutant TCRs in response to VSV peptide-pulsed APCs (either R8, L-K^b-low, or L-K^b-high cells). VSV-pulsed APCs were mixed with an equal number of T cell hybridomas expressing either the N30.7 TCR or one of the CDR3β mutants. After 24 h, supernatants were collected for measurement of IL-2 release. Data are means of three to five independent experiments ± SD.

Fig. 3.

L-K^b-high cells express higher surface levels of pMHCs as compared with R8 cells. Surface H-2K^b and H-2K^b/SIINFEKL complex expression was determined by flow cytometry. (a) For surface H-2K^b expression, R8, L-K^b-high, or L-K^b-low APCs were stained with a H-2K^b-specific mAb. (b) For surface H-2K^b/SIINFEKL complex expression, R8, L-K^b-high, or L-K^b-low APCs were cultured overnight in the presence of SIINFEKL-peptide and then stained with a mAb specific for the H-2K^b/SIINFEKL complex. Surface expression data were standardized by assigning a relative expression value of 1 to R8 cells on each measurement. Data are means of three independent experiments ± SD.

Fig. 4.

Plate-bound purified pMHCs restore activation of T cell hybridomas expressing TCR mutants with prolonged TCR-pMHC interaction half-lives. IL-2 release in response to plate-bound H-2K^b/VSV complexes by T cell hybridomas expressing the WT or one of the CDR3β mutant TCRs. T cell hybridomas were cultured over increasing amounts of plate-bound H-2K^b/VSV complexes. After 24 h, supernatants were collected for measurement of IL-2 release. Data are means of four independent experiments ± SD.

Fig. 5.

TCR down-modulation is also modulated by the TCR-pMHC interaction half-life and cognate pMHC density. Surface TCR down-modulation was measured for T cell hybridomas expressing the WT or one of the CDR3β mutant TCRs in response to VSV peptide-pulsed R8 cells, L-K^b-high cells, or plate-bound H-2K^b/VSV complexes. T cell hybridomas were cocultured with an equal number of VSV peptide-pulsed APCs or 1 μg of plate-bound H-2K^b/VSV complexes. After 5 h, FACS analysis was performed on harvested cells staining for CD8α and TCR-β. Data are means of three independent experiments ± SD (^*, P < 0.05; ^**, P ≤ 0.01; ^***, P < 0.001).

Fig. 6.

T cell activation as a function of TCR-pMHC dwell time is modulated by cognate pMHC density on the APC surface. (a) A Gaussian distribution is obtained for T cell activation and t_1/2 data points at low pMHC density on the APC surface. (b) A hyperbolic distribution is obtained for T cell activation and t_1/2 data points at high pMHC density on the APC surface. Plotted T cell activation data points were obtained from IL-2 release results in response either to R8 or L-K^b-high APCs pulsed with 10 μM VSV-peptide. An arbitrary value of 1 was assigned to the IL-2 release of N30.7 hybridoma in response to R8 cells pulsed with 10 μM VSV-peptide.