TCR interactions with cognate peptide-MHC (pepMHC) ligands are generally low affinity. This feature, together with the requirement for CD8/CD4 participation, has made it difficult to dissect relationships between TCR-binding parameters and T cell activation. Interpretations are further complicated when comparing different pepMHC, because these can vary greatly in stability. To examine the relationships between TCR-binding properties and T cell responses, in this study we characterized the interactions and activities mediated by a panel of TCRs that differed widely in their binding to the same pepMHC. Monovalent binding of soluble TCR was characterized by surface plasmon resonance, and T cell hybridomas that expressed these TCR, with or without CD8 coexpression, were tested for their binding of monomeric and oligomeric forms of the pepMHC and for subsequent responses (IL-2 release). The binding threshold for eliciting this response in the absence of CD8 (KD = 600 nM) exhibited a relatively sharp cutoff between full activity and no activity, consistent with a switchlike response to pepMHC on APCs. However, when the pepMHC was immobilized (plate bound), T cells with the lowest affinity TCRs (e.g., KD = 30 μM) responded, even in the absence of CD8, indicating that these TCR are signaling competent. Surprisingly, even cells that expressed high-affinity (KD = 16 nM) TCRs along with CD8 were unresponsive to oligomers in solution. The findings suggest that to drive downstream T cell responses, pepMHC must be presented in a form that supports formation of appropriate supramolecular clusters.

Interactions between Ag-specific receptors (TCRs) on CTL and peptide-MHC (pepMHC)4 class I complexes on other cells underlie the ability of these T cells to distinguish an individual’s normal cells from viral-infected or cancer cells, as well as from genetically dissimilar tissue transplants (allografts). Because TCRs and their pepMHC ligands are integral membrane proteins, measurements of the affinity and kinetic constants for these fundamental, membrane-proximal interactions have had to resort to a variety of different maneuvers to obtain one or the other reactant, or both, in soluble form and to study their interactions under diverse conditions, sometimes involving native TCRs on intact CTL and often not involving any cells at all, as with surface plasmon resonance (SPR) (1, 2, 3). Perhaps as a result of the varying conditions, values reported for binding parameters of a given TCR:pepMHC interaction have differed, sometimes considerably (1). Nevertheless, there is general agreement that TCR:pepMHC affinity constants are relatively low, falling, for example, orders of magnitude below what is commonly found for reactions of Ags with affinity-matured IgG Abs, but within the range seen for Ag reactions with primary response Abs, especially IgM (4), whose encoding gene segments, like those for TCRs, are not subject to somatic cell mutation.

The presence of the CD8 coreceptor on most CTL enhances the apparent sensitivity of TCR:pepMHC reactions (5, 6, 7), in part at least because CD8 binds, albeit with low affinity and rapid kinetics (8), to an invariant region on MHC-I molecules (9, 10). Besides helping to stabilize TCR:pepMHC complexes (11), CD8 helps recruit the kinase Lck to TCR complexes that are bound to pepMHC-I and thereby enhances signal transduction from the liganded TCR and consequent T cell responsiveness (11, 12, 13). Some evidence suggests that CD8 participation may vary depending on the nature of the TCR:CD8 or TCR:pepMHC interaction (14, 15, 16).

Given all of these complexities, it is perhaps not surprising that there has been considerable debate over which parameter(s) of the TCR:pepMHC interaction is most important for initiating a T cell’s response to pepMHC. Some evidence indicates that affinity (equilibrium-binding constant, KD) is critical (17, 18, 19), but other studies suggest that it is not affinity, but dissociation rate (t1/2 or lifetime) of the TCR:pepMHC complex that plays the pivotal role (20, 21, 22, 23, 24). Other data have implicated the importance of molecular flexibility of the interaction (25, 26). Another unresolved parameter involves the form of the cell surface TCR complex that is necessary for triggering the T cell (e.g., monomeric TCR/CD8, oligomeric TCR/CD8, or supramolecular TCR/CD8 and adhesion molecules). Although early studies suggested that binding of soluble pepMHC monomers could stimulate T cell activity with the participation of CD8 (27), subsequent evidence has revealed that multivalent binding is essential (28, 29, 30, 31), and that observations of triggering by pepMHC monomers may be an artifact due to transfer of stimulatory peptides from the monomers to MHC on the surface of cells for re-presentation (32, 33).

The typical strategy followed to study the relevant binding parameters in TCR-pepMHC interactions involves testing T cells that express a particular TCR against panels of APC or target cells that display diverse pepMHC, whose binding parameters to that TCR differ. Such studies can be difficult to interpret because pepMHC complexes formed from different peptides may differ considerably in their stability. In addition, the binding affinities of TCR-pepMHC interactions are typically quite low, making precise binding measurements with altered peptide ligands more difficult (3). Furthermore, cell surface coreceptor CD8, with its rapid kinetics (8), may influence cellular interactions between TCR and pepMHC to varying extents (6, 16), complicating attempts to correlate TCR:pepMHC-binding parameters that are measured in vitro with CD8+ T cell responses.

In this study, we have approached these problems with a different strategy: using yeast display (34) and genetic engineering, we created a panel of TCRs that bind the same pepMHC complex (SIYRYYGL-Kb, called SIYR/Kb) with affinities that vary ∼1000-fold and on- and off-rate constants that vary ∼60 and 90-fold, respectively. We measured the IL-2 release by transduced CD8 coreceptor-negative T cell hybridomas, each expressing a different member of the TCR panel, in response to the same pepMHC (SIYR/Kb) on the same APCs (T2-Kb). Although these hybridomas lacked CD8, they could be made to express this coreceptor by transfection. Thus, we have been able to evaluate the TCR-mediated responses of these T cells in the absence and presence of CD8. In parallel with the cellular responses, the binding parameters of the various TCR:pepMHC interactions were measured by SPR, using recombinant TCR and SIYR/Kb. Furthermore, to more closely approximate the multivalent TCR:pepMHC interactions at the T cell-APC interface, we examined the binding of SIYR/Kb tetramers to the T cell hybridomas. We thereby established a ruler for tetramer-binding titrations, in the absence of complexities associated with CD8 binding, that can be used to compare results of the same approach used widely in the analysis of CD8-positive cells.

Our findings show that the responses of the CD8-negative T cells to the pepMHC complex on APC were strongly and equally correlated with affinity (KD) and off-rate (kd); and a sharp threshold in these parameters separated responsive from nonresponsive cells. Surprisingly, cells that expressed the highest affinity TCR (KD, 16 nM) and that bound the pepMHC tetramers very strongly (off-rate with t1/2 10 min) were virtually unresponsive to either pepMHC tetramers or pepMHC-Ig dimers in solution, but responded strongly to the pepMHC immobilized on plastic as monomers or tetramers. These and other differences between soluble, APC-bound, and plate-bound forms of the pepMHC suggest that to drive downstream T cell responses (IL-2 production), the pepMHC has to be presented in a form that allows the appropriate supramolecular clustering on the T cell.

SIYR (SIYRYYGL), OVA (SIINFEKL), and QL9 (QLSPFPFDL) peptides were synthesized by the Macromolecular Core Facility of the Section of Research Sources, Penn State College of Medicine. Peptides were purified by reverse-phase chromatography using a C-18 column; mass was confirmed by MALDI. Peptide quantification by amino acid analysis was performed at the Molecular Structure Facility, University of California. Ecopak 2-293 (BD Clontech) retroviral packaging cell line maintained in DMEM supplemented with 10% FCS, l-glutamine, penicillin, and streptomycin. T2-Kb, a TAP-deficient lymphoblastoid cell line transfected with mouse Kb, and the αβ-negative 58−/− T cell hybridoma were maintained in RPMI 1640 complete medium (supplemented with 10% FCS, l-glutamine, penicillin, and streptomycin). The 58−/− T cell hybridomas transfected with a plasmid to express the 2C TCR or the high-affinity m6 TCR that reacts with QL9-Ld (35), with or without cotransfection of plasmids to express CD8α and CD8β (17), were maintained in RPMI 1640 complete medium plus additional resistance marker antibiotics (G418 to select for TCRαβ, hygromycin to select for CD8α, and puromycin to select for CD8β). The 2C T cells were obtained by isolation of splenocytes from 2C TCR transgenic (tg) mice and stimulated in vitro at 4 × 106 cells/ml in RPMI 1640 complete medium containing 1 μM SIYR peptide and 5% rat Con A supernatant. Cells were used in various pepMHC activation assays on day 4 after stimulation.

Single-chain Vβ-linker-Vα TCR constructs were cloned into the pET28a expression plasmid and transformed into BL21(DE3) (Stratagene) (36). Proteins were purified from inclusion bodies, which were added to 400 ml of denaturing solution (3 M urea, 2 mM reduced glutathione, 0.2 mM oxidized glutathione (pH 8)) at 4°C. Dilution buffer (200 mM NaCl, 50 mM Tris (pH 8)) was added dropwise to the stirring mixture over a 36-h period. Ni-NTA agarose beads (Qiagen) were added to the refolding mixture for 24 h and collected using a scintered glass funnel. Elution buffer (500 mM imidizole, 10 mM HEPES, 150 mM NaCl, 2.5 mM EDTA (pH 7.4)) was added to beads, and eluted TCR was purified over a Superdex 200 gel filtration column (GE Healthcare).

H2-Kb H chain containing a C-terminal biotinylation signal peptide and mouse or human β2-microglobulin light chains were expressed in Escherichia coli. H2-Kb H chain was biotinylated in vivo by coinduction of biotin ligase, so that the H chain carried a biotin tag (37). Both chains were expressed as inclusion bodies, solubilized in urea, and refolded in vitro in the presence of excess peptide. Folded complexes were purified by anion exchange chromatography using HiTrap Q columns (GE Healthcare) and size exclusion chromatography. MHC complexes were incorporated into fluorescent tetramers for staining and dissociation experiments by adding streptavidin-PE (BD Pharmingen) stepwise to the purified, biotinylated MHCs to a final 1:4 molar ratio.

BD dimerX H-2Kb and H-2Ld IgG1 dimers (BD Pharmingen) were loaded with 40-fold molar excess peptide (SIYR and QL9, respectively) at 37°C overnight. T cell activation assays using these proteins, either immobilized on plastic or in solution, were conducted in the presence of 10 μM excess peptide.

Kinetic and equilibrium-binding data were obtained by SPR using a BIAcore 3000. Biotinylated SIYR/Kb and OVA/Kb monomers were immobilized on a neutravidin-coated CM5 sensor chip on different flow cells to 400 response units. Soluble single-chain TCRs (scTCRs), purified by size-exclusion chromatography no more than 24 h before measurements to avoid aggregates, at various concentrations in Biacore buffer (20 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20 (pH 7.4)), were flowed over the SIYR/Kb and OVA/Kb flow cells at 30 μl/min. Binding of scTCRs to the null complex OVA/Kb was subtracted from TCR binding to SIYR/Kb to correct for bulk shift and any nonspecific binding. All measurements were made at 25°C. On-rate, off-rate, and kinetic-based KD analyses were performed using BIAEvaluation 3.0 software. Equilibrium KD values were calculated by Scatchard analysis. All measurements were performed two to five times.

Restriction sites were added to the ends of 2Cα, m33α, and 2Cβ cDNAs by PCR, and the products were cloned into the retroviral vector murine stem cell virus (MSCV) at BglII/BsrGI and BstXI/MluI sites in a bicistronic configuration of 2Cα-IRES-2Cβ or m33α-IRES-2Cβ. Single-site alanine mutations were cloned into m33 MSCV using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Transfection of the retroviral packaging cell line was conducted using the CalPhos Mammalian Transfection Kit (BD Clontech). EcoPak 2-293 (BD Clontech) packaging cells were transfected with 4 μg of MSCV DNA added to the CalPhos Mammalian Transfection Kit. Forty-eight hours posttransfection, retroviral-containing supernatants were collected, filtered, and added to 58−/− T cell hybridoma cells with 8 μg/ml polybrene (Sigma-Aldrich). Cells were centrifuged at 1200 × g for 45 min, placed in a 37°C 5% CO2 incubator for 3 days, and assayed for TCR Cβ expression using biotinylated H57-597 (BD Pharmingen) and streptavidin:PE (BD Pharmingen). Positive TCR Cβ populations were sorted using a Cytomation MoFlo (DakoCytomation).

TCR-transduced 58−/− cells (105) were incubated with one of various stimuli: immobilized SIYR/Kb or QL9/Ld protein complexes, either as monomers, BD DimerX IgG1 dimeric fusions, or streptavidin-linked tetramers, immobilized anti-CD3 Ab (BD Pharmingen) at 5 μg/ml, or 105 T2-Kb cells with various concentrations of SIYR peptide. Transduced cells were incubated with the various stimuli for 24 h at 37°C 5% CO2, and then supernatants were collected. For IL-2 detection, 96-well plates (Immulon 2HB) were coated with 2.5 μg/ml anti-murine IL-2 (BD Pharmingen) in 0.1 M Na2HPO4 (pH 9) for 2 h at room temperature, then blocked with 1% BSA in PBS for 12 h at 4°C. Wells were washed once with PBS containing 0.05% Tween 20 (PBST) before addition of 50 μl of cell supernatant and 12 h of incubation at 4°C. Wells were washed three times with PBST, and then 6.7 μg/ml biotinylated anti-murine IL-2 (BD Pharmingen) in PBS was added for 1 h at room temperature. Wells were washed three times with PBST, and then incubated with a 1/10,000 dilution of streptavidin-HRP (BD Pharmingen) for 0.5 h at room temperature. Plates were washed three times with PBST, and developed with 50 μl of tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories) until a color change was seen in the control wells. The reaction was stopped with 50 μl of 1 N H2SO4, and absorbance at 450 nm was measured in each well using an ELx800 universal microplate reader (Bio-Tek Instruments). Activation experiments detecting IFN-γ were performed similarly to IL-2 detection, but supernatants were analyzed by IFN-γ ELISA (eBioscience).

For tetramer staining, 58−/− cells transduced with 2C TCR mutants were incubated with various concentrations of streptavidin-PE SIYR/Kb tetramer on ice for at least 2 h in the dark. After washing, cells were resuspended in ice-cold PBS containing 1% BSA and 0.02% azide, and analyzed for bound fluorescent tetramers by flow cytometry. The parental 58−/− cell line was used as a control, and background fluorescence levels were subtracted from the TCR transfectant values at the same staining concentration.

Tetramer dissociation experiments were performed, as described previously (38, 39, 40). Briefly, 58−/− cells transfected with mutant TCR chains were stained with 293 nM (for m33, N27βA, Y49αA, S51αA, and Y26αA) or 5.85 μM (for Y48βA) streptavidin-linked SIYR/Kb tetramer, respectively, on ice for 2 h. Cells were washed and suspended in dissociation buffer containing 2% FCS, 0.1% azide, 100 μM cytochalasin D, and either with or without 200 μg/ml Kb blocking Ab (B.8.24.3) in RPMI 1640. At various times, cells were diluted in ice-cold PBS containing 1% BSA and 0.02% azide and analyzed by flow cytometry. Complete dissociation was determined to be the level of staining observed for the parental 58−/− cell line.

T cells were incubated with various stimuli at 37°C for 30 min. Cells were then fixed via addition of paraformaldehyde to a final 2% concentration, and continued incubation for at least 10 min. Cells were permeabilized in ice-cold methanol at least 30 min. Then the cells were extensively washed in ice-cold PBS containing 1% BSA and 0.02% azide before staining with anti-phospho-p44/42 Ab on ice (ERK(1/2), Thr202/Tyr204; Cell Signaling Technology). The cells were washed again and stained with a fluorescently labeled secondary Ab (goat anti-mouse IgG, F(ab′)2, labeled with Alexa 488; Molecular Probes). After this stain, the cells were washed twice and analyzed by flow cytometry.

The high-affinity 2C TCR mutant m33 was generated through directed evolution of the CDR3α, using a single-chain (Vβ-linker-Vα) form of the wild-type (WT) receptor in a yeast display system (34). To examine the affinity threshold for CD8 independence more precisely and to explore the role of binding kinetics and multimerization in T cell activation, we generated a panel of receptors with affinities for SIYR/Kb (measured by SPR) that varied between 2C WT (KD = 30 μM) and m33 (KD = 16 nM). To accomplish this, single-site alanine mutations were introduced into the high-affinity TCR m33. These mutations were guided by two previous papers in which we performed alanine scans of CDR residues in the WT 2C TCR, examining their binding to SIY/Kb (41) and QL9/Ld (42). In the 2C WT TCR, the chosen mutations yielded a range of effects on pMHC binding, and we anticipated the same might be the case if these mutations were cloned into the higher affinity m33 mutant. Seven mutant TCRs carrying single alanine mutations in either the Vα or Vβ were chosen as follows: N27βA, N30βA, Y48βA, Y26αA, Y49αA, Y50αA, and S51αA (supplemental Fig. 1).5 The single-site alanine mutant receptors were each introduced into the m33 yeast surface display vector, and the expression of properly folded scTCR on the yeast was confirmed in each case using a Vβ-specific Ab (anti-Vβ8.2 mAb F23.2; data not shown). The mutants were also assayed for ability of the yeast cells displaying them to be stained with SIYR/Kb tetramers (supplemental Fig. 2a).5 Under the conditions used, the tetramers stained yeast expressing m33 and four of the mutants (Y26αA, S51αA, Y49αA, and N27βA), but not WT 2C and three other mutants (Y50αA, N30βA, and Y48βA). The levels of tetramer staining correlated with the magnitude of the binding effects observed in the original alanine scan of the 2C TCR (supplemental Fig. 2b).5 Accordingly, we examined in more detail the binding properties of all nine TCRs and their ability to mediate T cell activity.

To measure the equilibrium-binding and kinetic (on/off-rate) constants for their reaction with SIYR/Kb, each TCR was cloned into E. coli and expressed as a soluble single-chain protein (36) for use in SPR (Fig. 1,a). The affinities of scTCR forms of 2C WT and m33 (KD values of 30 μM and 16 nM, respectively) were found to be similar to the affinities previously measured by this method for full-length 2C WT (43) and full-length m33 (34). The m33 mutants displayed affinities ranging from 2C WT at the low end, and extending to near m33 at the high end (Table I). S51αA and Y26αA displayed high affinities for SIYR/Kb similar to m33 (KD values of 15 and 17 nM, respectively); m33 bound with the fastest on-rate (1.37 × 106 M−1s−1), and S51αA with the slowest off-rate of the panel (t1/2 almost 90 s). For the weakest binding TCRs (2C and Y50αA), dissociation was extremely rapid (t1/2 ∼ 1 s). The single-site mutations yielded differences in binding affinity among the panel, but there was not a mutant in the intermediate affinity range (KD ∼ 1 μM). In hopes of generating such a mutant, we cloned the double mutation S51αA/Y48βA, with the idea that the slight increase in affinity of the S51αA mutation (compared with m33) would be additive with the Y48βA mutation. This was indeed the case because the S51αA/Y48βA mutant exhibited a KD value of 540 nM.

FIGURE 1.

Analysis of TCR mutants by SPR. a, Measurements were performed on a Biacore 3000. Biotinylated SIYR/Kb and OVA/Kb monomers were immobilized to a neutravidin-coated CM5 sensor chip on separate flow cells at equal response units. Soluble TCRs were flowed over sensor chip at various concentrations, and binding was measured by change in response units. Nonspecific binding to the null ligand OVA/Kb was subtracted from final measurements. Kinetic analysis was measured using BIAEvaluation 3.0 software. All measurements were done at 25°C. Two of the mutant TCRs (S51αA/Y48βA and N30βA) exhibited residual binding at the end of the dissociation curves; however, equilibrium-binding constants calculated from kinetic measurements were consistent with those determined under equilibrium conditions using Scatchard plots (Table I and b). b, Scatchard analysis was performed on the SPR data to give an equilibrium KD value for each receptor. Bound TCR on the x-axis was plotted against bound TCR divided by concentration of TCR on the y-axis and fitted by linear regression. Equilibrium affinity was calculated as the negative reciprocal slope of this line. KD values derived from kinetic parameters and equilibrium analyses were in good agreement (Table I).

FIGURE 1.

Analysis of TCR mutants by SPR. a, Measurements were performed on a Biacore 3000. Biotinylated SIYR/Kb and OVA/Kb monomers were immobilized to a neutravidin-coated CM5 sensor chip on separate flow cells at equal response units. Soluble TCRs were flowed over sensor chip at various concentrations, and binding was measured by change in response units. Nonspecific binding to the null ligand OVA/Kb was subtracted from final measurements. Kinetic analysis was measured using BIAEvaluation 3.0 software. All measurements were done at 25°C. Two of the mutant TCRs (S51αA/Y48βA and N30βA) exhibited residual binding at the end of the dissociation curves; however, equilibrium-binding constants calculated from kinetic measurements were consistent with those determined under equilibrium conditions using Scatchard plots (Table I and b). b, Scatchard analysis was performed on the SPR data to give an equilibrium KD value for each receptor. Bound TCR on the x-axis was plotted against bound TCR divided by concentration of TCR on the y-axis and fitted by linear regression. Equilibrium affinity was calculated as the negative reciprocal slope of this line. KD values derived from kinetic parameters and equilibrium analyses were in good agreement (Table I).

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Table I.

Binding properties of soluble scTCRa

TCRka (M−1s−1 × 10−3)kd (s−1)t1/2 (s)KD(kd/ka) (nM)Equilibrium KD (nM)
S51α515 ± 2 0.008 ± 0.001 86 ± 12 15 ± 2 40 ± 13 
m33 1,370 ± 670 0.015 ± 0.003 46 ± 10 16 ± 12 22 ± 20 
Y26α850 ± 420 0.013 ± 0.001 50 ± 3 17 ± 8 24 ± 12 
N27β520 ± 30 0.021 ± 0.002 33 ± 4 40 ± 7 42 ± 8 
Y49α280 ± 140 0.012 ± 0.001 58 ± 2 47 ± 23 74 ± 57 
S51αA/Y48β136 ± 74 0.076 ± 0.048 11 ± 7 543 ± 56 545 ± 70 
Y48β136 ± 35 0.35 ± 0.18 2 ± 1 2,900 ± 2,100 1,900 ± 1,100 
N30β34 ± 11 0.27 ± 0.06 3 ± 1 8,200 ± 900 6,600 ± 1,500 
Y50α241 ± 175 1.3 ± 0.3 0.5 ± 0.1 6,950 ± 3,750 5,900 ± 1,800 
2C(T7) 20.4 ± 3.4 0.72 ± 0.06 0.93 ± 0.14 35,600 ± 710 23,800 ± 6,000 
TCRka (M−1s−1 × 10−3)kd (s−1)t1/2 (s)KD(kd/ka) (nM)Equilibrium KD (nM)
S51α515 ± 2 0.008 ± 0.001 86 ± 12 15 ± 2 40 ± 13 
m33 1,370 ± 670 0.015 ± 0.003 46 ± 10 16 ± 12 22 ± 20 
Y26α850 ± 420 0.013 ± 0.001 50 ± 3 17 ± 8 24 ± 12 
N27β520 ± 30 0.021 ± 0.002 33 ± 4 40 ± 7 42 ± 8 
Y49α280 ± 140 0.012 ± 0.001 58 ± 2 47 ± 23 74 ± 57 
S51αA/Y48β136 ± 74 0.076 ± 0.048 11 ± 7 543 ± 56 545 ± 70 
Y48β136 ± 35 0.35 ± 0.18 2 ± 1 2,900 ± 2,100 1,900 ± 1,100 
N30β34 ± 11 0.27 ± 0.06 3 ± 1 8,200 ± 900 6,600 ± 1,500 
Y50α241 ± 175 1.3 ± 0.3 0.5 ± 0.1 6,950 ± 3,750 5,900 ± 1,800 
2C(T7) 20.4 ± 3.4 0.72 ± 0.06 0.93 ± 0.14 35,600 ± 710 23,800 ± 6,000 
a

All experiments were carried out by SPR at 25°C. SIYR/Kb monomer was immobilized to sensor chip, and soluble scTCR was flowed in solution. Equilibrium affinity calculated from the negative inverse slope of Scatchard analysis.

It has been proposed that, at least for some TCRs, interactions with the helices of the MHC influence predominantly the binding on-rate, whereas TCR interactions with the peptide influence the stability (off-rate constant) of the complex (44). In this context, one might have expected CDR1 and CDR2 mutants in residues that contact the Kb helices to affect predominantly the on-rate. However, these data do not support this mechanism because there was no apparent correlation between CDR mutant location and their effects on the on-rate or off-rate constants. Collectively, the TCR mutants showed changes in on-rate, off-rate, or both (Table I).

Given our unique panel of TCRs with diverse monovalent (SPR-based) binding affinities for pepMHC, it was of interest to characterize the multivalent binding of pepMHC tetramers to these TCRs on T cells. The binding of pepMHC to T cells is, however, inherently complicated by the participation of CD8 in the binding reactions (6, 7). Hence, each of the mutant TCR subunits was cloned into full-length m33α-chain or 2Cβ-chain genes in a retroviral vector and transduced into T cell hybridoma 58−/−, which lacks CD8 and its own TCR (45). In all of the transduced 58−/− hybridomas, each one expressing one of the TCRs in the panel, the levels of TCR expression were similar, as shown by staining with an anti-Cβ Ab (supplemental Fig. 3a).5 Surface TCR levels were similar after cotransduction with CD8 coreceptor, although the Y48βA mutant expressed slightly lower levels of TCR compared with the rest of the panel (supplemental Fig. 3b).5 When TCR-transduced T cells were stained with a single concentration of SIYR/Kb tetramer (supplemental Fig. 3c),5 the same clearly positive or negative staining trend was seen for the coreceptor-negative T cells as was seen for yeast displaying the same receptors, stained under the same conditions (supplemental Fig. 2a).5

We further characterized the multivalent pepMHC-binding properties of several TCR transductants (m33, S51αA, Y26αA, S51αA/Y48βA, Y48βA, 2C) with a full titration of pepMHC tetramer equilibrium binding. Selected T cell lines were incubated with various concentrations of SIYR/Kb tetramers for 2 h at 4°C, washed free of unbound tetramer, and analyzed by flow cytometry (Fig. 2,a). Even the WT 2C TCR, the lowest affinity receptor, exhibited staining that was above the background level of the 58−/− parental cell line, although the staining intensity was lower than that observed for the higher affinity TCRs. The reasons for the marked differences in the maximal levels of tetramer binding for the various TCRs are not clear, and this phenomenon warrants further investigation. For each receptor tested, an effective tetrameric equilibrium-binding constant (KD,tet) was calculated as the tetramer concentration at which 50% maximum, saturated staining was obtained (Table II).

FIGURE 2.

SIYR/Kb tetramer-binding titrations and dissociation from CD8-negative, transduced T cell lines. a, The 58−/− cells transduced with 2C TCR mutants were incubated with various concentrations of SIYR/Kb tetramer for 2 h on ice, washed, and analyzed for bound fluorescent tetramers (see Materials and Methods). Representative data are shown for 2C, Y48βA, Y26αA, S51αA, S51αA/Y48βA, and m33. b, The calculated enhancement factor (KD/KD,tet) is plotted against the SPR-determined dissociation constant (KD) for the TCRs tested. c, Transduced cells were incubated with tetramers, as described in Materials and Methods, for 2 h on ice. Cells were then washed and resuspended in dissociation buffer containing 2% FCS, 0.1% azide, 100 μM cytochalasin D, and 200 μg/ml anti-Kb blocking Ab (B.8.24.3); after incubation for various times at 24°C, the cells were rapidly diluted in ice-cold buffer and analyzed by flow cytometry. The percentage of maximum binding that remained associated with the cells after various times is shown. Normalized data points were fit using a single exponential that corresponds to off-rate constant. Experiments were repeated two to three times. d, The relationship between t1/2,tet and monovalent t1/2 (Table I) is shown.

FIGURE 2.

SIYR/Kb tetramer-binding titrations and dissociation from CD8-negative, transduced T cell lines. a, The 58−/− cells transduced with 2C TCR mutants were incubated with various concentrations of SIYR/Kb tetramer for 2 h on ice, washed, and analyzed for bound fluorescent tetramers (see Materials and Methods). Representative data are shown for 2C, Y48βA, Y26αA, S51αA, S51αA/Y48βA, and m33. b, The calculated enhancement factor (KD/KD,tet) is plotted against the SPR-determined dissociation constant (KD) for the TCRs tested. c, Transduced cells were incubated with tetramers, as described in Materials and Methods, for 2 h on ice. Cells were then washed and resuspended in dissociation buffer containing 2% FCS, 0.1% azide, 100 μM cytochalasin D, and 200 μg/ml anti-Kb blocking Ab (B.8.24.3); after incubation for various times at 24°C, the cells were rapidly diluted in ice-cold buffer and analyzed by flow cytometry. The percentage of maximum binding that remained associated with the cells after various times is shown. Normalized data points were fit using a single exponential that corresponds to off-rate constant. Experiments were repeated two to three times. d, The relationship between t1/2,tet and monovalent t1/2 (Table I) is shown.

Close modal
Table II.

Multivalent binding properties of cell surface-expressed TCRa

TCRKD,SPR (nM)KD,tet (nM)Enhancement Factor (KD,SPR/KD,tet)t1/2 (s)t1/2,tet (s)
S51α15 ± 2 0.96 15.6 86 ± 12 906 ± 198 
Y26α17 ± 8 1.4 12.1 50 ± 3 354 ± 72 
m33 16 ± 12 1.56 ± 0.43 10.2 46 ± 10 510 ± 42 
N27β40 ± 7   33 ± 4 222 ± 12 
Y49α47 ± 23   58 ± 2 816 ± 42 
S51αA/Y48β540 ± 56 14 38.6 11 ± 7 27.6 ± 2.4 
Y48β2,900 ± 2,100 26 111 2 ± 1 13.2 ± 4.2 
2C WT 23,800 ± 6,000 11 ± 1 2,160 1.3  
TCRKD,SPR (nM)KD,tet (nM)Enhancement Factor (KD,SPR/KD,tet)t1/2 (s)t1/2,tet (s)
S51α15 ± 2 0.96 15.6 86 ± 12 906 ± 198 
Y26α17 ± 8 1.4 12.1 50 ± 3 354 ± 72 
m33 16 ± 12 1.56 ± 0.43 10.2 46 ± 10 510 ± 42 
N27β40 ± 7   33 ± 4 222 ± 12 
Y49α47 ± 23   58 ± 2 816 ± 42 
S51αA/Y48β540 ± 56 14 38.6 11 ± 7 27.6 ± 2.4 
Y48β2,900 ± 2,100 26 111 2 ± 1 13.2 ± 4.2 
2C WT 23,800 ± 6,000 11 ± 1 2,160 1.3  
a

All measurements correspond to TCR interactions with SIYR/Kb. KD values were measured by SPR using scTCRs (Table I). Multimeric KD measured from titration of SIYR/Kb-PE tetramers to TCR-transduced 58−/− T cell hybridomas (KD,tet). Enhancement factor = KD,SPR/KD,tet. SIYR/Kb tetramer off-rate (t1/2,tet) values measured in presence of anti-Kb Ab B.8.24.3.

By analogy with Ab reactions with multivalent vs monovalent ligands (46, 47, 48, 49), the tetramer-binding affinity/monomer-binding affinity (KD/KD,tet, or KA,tet/KA) may be termed the enhancement factor. This factor ranged from ∼10 to 20 for the highest affinity TCRs (m33, S51αA, and Y26αA), and was considerably higher for the weakest binding TCRs (over 100 for Y48βA, and over 2000 for 2C; Table II and Fig. 2 b).

Another approach, used widely in the literature, to compare relative TCR:pepMHC binding is to measure pepMHC streptavidin tetramer off-rate from the surface of T cells (38, 39). Again, this approach is complicated by the participation of CD8 in the dissociation reaction. To directly correlate with the monovalent TCR-binding affinities and kinetics, SIYR/Kb tetramer dissociation rates were measured for various T cell lines in the absence of CD8 (Fig. 2,c). TCR-transduced cells were incubated on ice for 2 h with tetramer concentrations that yielded approximately equal levels of bound tetramers (290 nM for higher binding TCRs, including m33, S51αA, Y26αA, N27βA, and Y49αA; 1.17 μM for Y48βA and S51αA/Y48βA). After washing the cells (at 4°C) to remove unbound tetramers, cells were suspended at 24°C in dissociation buffer, which contained anti-Kb Ab to block rebinding of dissociated tetramers and cytochalasin D and sodium azide to minimize tetramer internalization. At various times, the dissociation was stopped by dilution with ice-cold buffer, and the samples were analyzed by flow cytometry. For each receptor tested, a t1/2,tet was calculated by fitting a single exponential curve to the dissociation data (Fig. 2,c and Table II). This value correlated strongly with the monovalent t1/2 measured by SPR (Fig. 2,d). A previous study of the OT-1 TCR (monovalent KD ∼ 6 μM, t1/2 ∼ 30 s (50)) attempted to minimize the effects of CD8 by using a Kb mutant with reduced CD8 binding; it showed a dissociation rate (t1/2) for the tetramer of OVA/Kb, which is a strong agonist for the OT-1 TCR, of 80 s (at 37°C (38)). By comparison, the SIYR/Kb tetramer dissociated relatively rapidly from the Y48βA TCR (t1/2 ∼ 13 s at 24°C; Table II), which binds the SIYR/Kb monomer relatively weakly (by SPR monovalent KD ∼ 2.9 μM, t1/2 ∼ 2 s; Table I).

To assess the functional capabilities of the transduced CD8-negative T cell lines, we measured their production of IL-2 in response to APCs (T2-Kb) that were incubated with various concentrations of SIYR peptide. Five of the cell lines (expressing the TCRs N27βA, Y49αA, S51αA, Y26αA, and m33) responded well, whereas cell lines that expressed four of the receptors (Y50αA, N30βA, Y48βA, and 2C) were not stimulated at any concentration of SIYR tested (Fig. 3,a). T cells expressing TCR S51αA/Y48βA showed an intermediate level of activation (Fig. 3,a). The concentrations of SIYR peptide that yielded half-maximal activity (SD50 values) were remarkably similar for the five responding cell lines, but there was roughly a two order of magnitude reduction in potency for T cells expressing the S51αA/Y48βA receptor (Fig. 3 b). Importantly, all of the cell lines displayed a similar ability to respond by IL-2 production to an immobilized (plate-bound) anti-CD3 Ab (supplemental Fig. 4a).5

FIGURE 3.

Stimulation of TCR-transduced T cell hybridomas by peptide-loaded APCs. The 58−/− CD8-negative (a and b) and CD8-positive (c and d) T cells transduced with the panel of TCRs were incubated with T2-Kb cells and varying concentrations of SIYR for 24 h, and supernatants were assayed for IL-2 production. Raw peptide titrations (a and c) and average SD50 values (b and d) are shown. e, Relationship between KD value of TCR:SIYR/Kb interactions and T cell activation. SD50 from IL-2 activity assays plotted against log KD from SPR analysis. f, Relationship between off-rate (t1/2) of TCR:SIYR/Kb interactions and T cell activation. SD50 from activity assays plotted against t1/2 from SPR analysis.

FIGURE 3.

Stimulation of TCR-transduced T cell hybridomas by peptide-loaded APCs. The 58−/− CD8-negative (a and b) and CD8-positive (c and d) T cells transduced with the panel of TCRs were incubated with T2-Kb cells and varying concentrations of SIYR for 24 h, and supernatants were assayed for IL-2 production. Raw peptide titrations (a and c) and average SD50 values (b and d) are shown. e, Relationship between KD value of TCR:SIYR/Kb interactions and T cell activation. SD50 from IL-2 activity assays plotted against log KD from SPR analysis. f, Relationship between off-rate (t1/2) of TCR:SIYR/Kb interactions and T cell activation. SD50 from activity assays plotted against t1/2 from SPR analysis.

Close modal

When the same 10 TCRs were transduced into hybridomas that also expressed CD8, every cell line was stimulated to produce IL-2 in response to both anti-CD3 Abs and SIYR/Kb (Fig. 3, c and d, and supplemental Fig. 4b).5 Accordingly, as expected, T cells expressing the four receptors (Y48βA, N30βA, Y50αA, and 2C) that were not stimulated in the absence of CD8 were active when transduced into CD8-positive cells (Fig. 3 d). The SD50 values of these lines were slightly more varied than the responsive CD8-negative lines, and some of the variability could have been due to modest differences in CD8 expression levels (data not shown) rather than differences in TCR-binding properties. The effect of the expression levels of coreceptor, which are known to vary on the surface of T cells (reviewed in Ref. 51), on the apparent sensitivity of a CD8-dependent cell line is well known (52) and emphasizes the importance of considering CD8 levels in such systems. CD8 expression in the high-affinity, CD8-independent T cells resulted in only a modest increase in sensitivity (i.e., lower SD50 values) because of a threshold determined by the minimal number of SIY/Kb complexes presented on the surface of APCs. As in our previous studies with the m33 TCR, T2-Kb cells loaded with the null OVA peptide (OVA, SIINFEKL) failed to induce activation in any of the transduced T cell lines, with or without CD8 expression (data not shown).

In light of the apparent correlation between the intensity of SIYR/Kb tetramer staining of the TCR-transduced CD8-negative T cells (supplemental Fig. 3c)5 and the ability of these cells to respond to SIYR/Kb on target cells by secreting IL-2 (Fig. 3,a), we examined in more detail the relationships between the binding parameters and T cell activity. The equilibrium constant (KD) and the t1/2 measured by SPR for each TCR-SIYR/Kb interaction were plotted vs the concentration of SIYR peptide required to elicit a half-maximal IL-2 response (SD50) of the transduced T cells (Fig. 3, e and f). This analysis shows that both the equilibrium constant (KD) and the off-rate (kd or t1/2) were related to the responsiveness to peptide-pulsed APCs. As to be expected from the correlations between equilibrium affinity and kinetic constants, no distinction could be made with this panel of receptors as to the more relevant parameter for TCR triggering. Notably, however, the results further support the view that even TCRs with slow dissociation rates (e.g., S51αA) can stimulate robust T cell activity. Conversely, in the presence of coreceptor, even TCR:pepMHC interactions with fast dissociation rates can trigger potent responses.

To further explore the signaling competency of TCRs on the CD8-negative cells, we stimulated them with either soluble or immobilized (plate-bound) recombinant SIYR/Kb MHC monomers and tetramers. In the initial experiments, SIYR/Kb streptavidin-linked tetramers were adsorbed on the surface of plastic tissue culture wells before the addition of T cells, and the subsequent IL-2 response was measured. Unexpectedly, this stimulus was able to specifically trigger all of the TCR transductants in the panel, including those for which no stimulation was seen with peptide-pulsed APCs (Fig. 4, a and b). Although the CD8-negative T cell responses to peptide-pulsed APCs largely showed SD50 values that amounted simply to positive or negative results without much differentiation between them (Fig. 3,a), the SD50 values for the cells’ responses to immobilized SIYR/Kb were more widely dispersed (Fig. 4, a and b); nevertheless, they again correlated strongly with both KD and off-rate (t1/2) values measured for the receptors by SPR (Fig. 4, c and d, compared with Fig. 3, e and f). IL-2 release was not observed when the cells were stimulated with the null pepMHC complex OVA/Kb immobilized on tissue culture plates (Fig. 4,a), or when 58−/− cells expressing a TCR with a different specificity (m6 (35)) were stimulated with immobilized SIYR/Kb tetramer (Fig. 4 a).

FIGURE 4.

Stimulation of TCR-transduced T cell hybridomas by immobilized SIYR/Kb tetramers. a, The 58−/− cells transduced with 2C TCR mutants were stimulated with various concentrations (10 pM-0.25 μM) of SIYR/Kb tetramer or the null complex OVA/Kb tetramer immobilized on plastic and analyzed for IL-2 secretion (see Materials and Methods). Representative data are shown. b, Average SD50 values from immobilized SIYR/Kb stimulation are plotted for each TCR transductant. c, Relationship between KD value of TCR:SIYR/Kb interactions and T cell activation. SD50 from activity assays plotted against log KD from SPR analysis. d, Relationship between off-rates (t1/2) of TCR:SIYR/Kb interactions and T cell activation. SD50 from activity assays plotted against t1/2 from SPR analysis.

FIGURE 4.

Stimulation of TCR-transduced T cell hybridomas by immobilized SIYR/Kb tetramers. a, The 58−/− cells transduced with 2C TCR mutants were stimulated with various concentrations (10 pM-0.25 μM) of SIYR/Kb tetramer or the null complex OVA/Kb tetramer immobilized on plastic and analyzed for IL-2 secretion (see Materials and Methods). Representative data are shown. b, Average SD50 values from immobilized SIYR/Kb stimulation are plotted for each TCR transductant. c, Relationship between KD value of TCR:SIYR/Kb interactions and T cell activation. SD50 from activity assays plotted against log KD from SPR analysis. d, Relationship between off-rates (t1/2) of TCR:SIYR/Kb interactions and T cell activation. SD50 from activity assays plotted against t1/2 from SPR analysis.

Close modal

When equivalent mass concentrations of SIYR/Kb monomers (1 μM) and tetramers (0.25 μM) were used to coat plastic wells, the plates were equally stimulatory for hybridomas that expressed high-affinity TCR m33, whether or not the CD8 coreceptor was also expressed (Fig. 5, a and b). Although there was some decrease in stimulation when SIYR/Kb was immobilized as a monomer rather than a tetramer for the 2C WT TCR (Fig. 5, c and d), both formats were able to stimulate the cells. These results suggest that it is the multivalent arraying of immobilized, closely packed ligands, rather than their special close proximity in tetramers, that was responsible for the cell response.

FIGURE 5.

Stimulation of T cells with soluble and immobilized pepMHC monomers and tetramers. a and b, IL-2 secretion by m33 T cells responding to immobilized and soluble SIYR/Kb stimulus. Tetramers were in solution or coated on plastic wells at 0.25 μM; equal mass concentrations for SIYR/Kb monomer were likewise in solution or surface immobilized (at 1 μM). Response is shown without (a) or with (b) coexpression of CD8. c and d, IL-2 secretion by 2C T cells responding to immobilized and soluble SIYR/Kb stimulus. Tetramers were in solution or coated on plastic wells at 1 μM; equal mass concentrations for SIYR/Kb monomer were also in solution or surface immobilized (at 4 μM). Response is shown without (c) or with (d) coexpression of CD8. The anti-CD3 Ab was plate bound (at 5 μg/ml).

FIGURE 5.

Stimulation of T cells with soluble and immobilized pepMHC monomers and tetramers. a and b, IL-2 secretion by m33 T cells responding to immobilized and soluble SIYR/Kb stimulus. Tetramers were in solution or coated on plastic wells at 0.25 μM; equal mass concentrations for SIYR/Kb monomer were likewise in solution or surface immobilized (at 1 μM). Response is shown without (a) or with (b) coexpression of CD8. c and d, IL-2 secretion by 2C T cells responding to immobilized and soluble SIYR/Kb stimulus. Tetramers were in solution or coated on plastic wells at 1 μM; equal mass concentrations for SIYR/Kb monomer were also in solution or surface immobilized (at 4 μM). Response is shown without (c) or with (d) coexpression of CD8. The anti-CD3 Ab was plate bound (at 5 μg/ml).

Close modal

There has been some controversy in the literature about the ability of soluble pepMHC monomers, or even tetramers, to stimulate T cell activity. Having a collection of high-affinity TCRs, we were in a position to test the ability of these reagents to stimulate a late, downstream response (IL-2 secretion) under conditions in which a high proportion of the surface TCRs bound the pepMHC ligand. This system is also advantageous in that the transduced T cell lines do not express the cognate MHC (Kb) that would enable re-presentation of dissociated peptide (SIYRYYGL) from the soluble pepMHC (32, 33). Soluble MHC monomers at the same concentration or higher (>1 μM) than was used to prepare the plate-bound (immobilized) monomers provided no stimulation, even for cells with the high-affinity TCR m33, in which SIYR/Kb monomer would be at saturating levels (Fig. 5, a and b). Perhaps more surprisingly, little or no IL-2 release was observed in response to soluble SIYR/Kb tetramers even for cells that expressed CD8 coreceptor (Fig. 5, a and b). The same lack of activity was observed at various concentrations of the soluble tetramer (data not shown).

We further explored this phenomenon with soluble IgG1 fusion MHC dimers to ensure that the lack of stimulation with the streptavidin reagent was not merely a limitation of the tetramer geometry. In this system, we used both SIYR/Kb and the well-known alloantigen QL9/Ld that stimulates both the WT 2C TCR (KD = 1.5 μM) (53, 54) and the high-affinity m6 TCR engineered against this complex (KD = 14 nM) (35, 54). The 2C and m33 T cell hybridomas that did or did not coexpress CD8αβ were incubated with 10 nM (1.25 μg/ml) SIYR/Kb IgG1 fusion dimer either preimmobilized on the surface of plastic tissue culture wells, or maintained as a soluble stimulus for 24 h at 37°C. Supernatants were analyzed for IL-2 secretion. As was seen for the peptide-MHC tetramer, immobilized dimeric ligand was quite potent at stimulating a cytokine response, whereas soluble dimeric ligand was unable to stimulate IL-2 release (Fig. 6,a). This behavior was also observed when QL9/Ld-Ig fusion dimer was used to stimulate T cell hybridomas carrying either the WT 2C TCR or the high-affinity m6 TCR, with or without CD8αβ (Fig. 6 b). As with soluble pepMHC tetramer, a titration of either soluble SIYR/Kb-Ig fusion or soluble QL9/Ld-Ig fusion did not stimulate T cells to release IL-2 (data not shown). Studies to address the minimum size of an oligomeric complex that would stimulate these T cells are currently ongoing.

FIGURE 6.

Stimulation of T cells with soluble and immobilized pepMHC IgG1 fusion dimers. a, IL-2 secretion by T cell hybridomas responding to 10 nM immobilized or soluble SIYR/Kb IgG1 dimers. Immobilized anti-CD3 Ab was used as a positive control stimulus. Response is shown for 2C (top panel), 2C + CD8αβ (second panel), m33 (third panel), and m33 + CD8αβ (bottom panel). b, IL-2 secretion by T cell hybridomas responding to 10 nM immobilized or soluble QL9/Ld IgG1 dimers. Response is shown for 2C (top panel), 2C + CD8αβ (second panel), m6 (third panel), and m6 + CD8αβ (bottom panel). c, IFN-γ secretion by effector T cells from a 2C tg mouse (ex vivo, CD8+) stimulated with 10 nM immobilized or soluble QL9/Ld IgG1 dimers.

FIGURE 6.

Stimulation of T cells with soluble and immobilized pepMHC IgG1 fusion dimers. a, IL-2 secretion by T cell hybridomas responding to 10 nM immobilized or soluble SIYR/Kb IgG1 dimers. Immobilized anti-CD3 Ab was used as a positive control stimulus. Response is shown for 2C (top panel), 2C + CD8αβ (second panel), m33 (third panel), and m33 + CD8αβ (bottom panel). b, IL-2 secretion by T cell hybridomas responding to 10 nM immobilized or soluble QL9/Ld IgG1 dimers. Response is shown for 2C (top panel), 2C + CD8αβ (second panel), m6 (third panel), and m6 + CD8αβ (bottom panel). c, IFN-γ secretion by effector T cells from a 2C tg mouse (ex vivo, CD8+) stimulated with 10 nM immobilized or soluble QL9/Ld IgG1 dimers.

Close modal

Finally, to assess whether normal effector T cells from a 2C tg mouse responded similarly, the QL9/Ld IgG1 dimers were used. T cells from the 2C tg mice are H2-Kb positive, but negative for H-2Ld, so there is no possibility of ambiguous results based on re-presentation of the peptide (32, 33). The 2C tg T cells were stimulated with the alloantigen QL9/Ld (KD = 1.5 μM) by immobilized or soluble Ig dimer. Consistent with the results from the transduced T cell hybridomas, immobilized QL9/Ld Ig dimer was able to stimulate IFN-γ release from the 2C tg T cells, whereas soluble dimer was ineffective (Fig. 6 c). Results were identical if a 10-fold higher concentration of QL9/Ld Ig dimer was used in the assay (data not shown).

It was of some interest to note that the magnitude of the maximum tetramer staining (Fig. 2,a) varied with the TCR-binding affinity in the same way that the efficiency of IL-2 release varied upon stimulation with immobilized SIYR/Kb tetramers (Fig. 4 a). In particular, the maximal tetramer-staining levels and the levels of IL-2 release were consistently lower for the CD8-dependent receptors than for the CD8-independent receptors.

In the tetramer titration experiments, the histograms of the fluorescently labeled cells followed a normal (Gaussian) distribution at each ligand concentration (data not shown). This homogeneity contrasts with activation of intracellular phosphorylation cascades that seem to follow a more bimodal on or off distribution when measured on CD8+ T cells (55). We asked therefore whether the reduced responsiveness of our CD8-negative hybridomas that expressed the lower affinity TCRs, which required high levels of plate-bound SIYR/Kb, resulted from the response of only a small fraction of the cells or from a weaker response by all cells in the entire population. To investigate this issue, we monitored phosphorylation of ERK in response to immobilized stimuli by flow cytometry, allowing us to look at the responses of individual cells. We incubated T cells with either plate-bound anti-CD3 or SIYR/Kb for 30 min and then measured the level of phosphorylated ERK (pERK) (56). The pERK response under these conditions clearly showed the response was indeed bimodal; only a relatively small fraction of cells responded in cases where IL-2 secretion was low (Fig. 7). The fraction of pERK-positive 2C T cells stimulated by immobilized SIYR/Kb was ∼40% of the fraction responding to anti-CD3, consistent with the relative levels of total IL-2 detected in supernatants of the same cells (Fig. 4 a). In general, the percentage of pERK-positive cells corresponded to previous IL-2 secretion experiments: responses to SIYR/Kb were improved by addition of CD8 coreceptor, expression of a higher affinity TCR (m33), or stimulation with immobilized anti-CD3 Ab.

FIGURE 7.

pERK(1/2) responses to efficient and inefficient stimuli. Flow cytometry analysis of pERK levels in hybridomas that expressed the 2C TCR, or the 2C TCR + CD8αβ, or the m33 TCR each in response (30 min) to no stimulus or immobilized SIYR/Kb monomer or immobilized anti-CD3.

FIGURE 7.

pERK(1/2) responses to efficient and inefficient stimuli. Flow cytometry analysis of pERK levels in hybridomas that expressed the 2C TCR, or the 2C TCR + CD8αβ, or the m33 TCR each in response (30 min) to no stimulus or immobilized SIYR/Kb monomer or immobilized anti-CD3.

Close modal

By creating a panel of TCR molecules that bind the same pepMHC ligand with a broad range of affinities and off-rates (t1/2), we have been able to examine in unprecedented detail the influence of t1/2 and affinity of the TCR:pepMHC interaction on a T cell activation response (IL-2 production). For each member of the TCR panel, we characterized its binding to the pepMHC ligand as the following: 1) a monovalent reaction, using a single-chain form of the TCR and SPR; and 2) a multivalent reaction, by analyzing the binding of pepMHC tetramers to the TCRs expressed on T cell hybridomas. The binding parameters of these interactions were then correlated with the IL-2 secretion response of these hybridomas, as either CD8 or CD8+ cells, to the pepMHC on APCs (T2-Kb). The results of the monovalent (SPR) reactions are summarized in Table I, and the multivalent (tetramer) reactions in Table II.

The most illuminating results were obtained with the T cell hybridomas that lack CD8. Those cells whose TCRs had an affinity for the pepMHC above a threshold value (delineated by the affinity of the double-mutant TCR S51αA/Y48βA, KD < 550 nM (Figs. 1–3)) made robust responses to the pepMHC on the APCs, whereas those whose TCR affinity was below this threshold did not respond. This abrupt affinity threshold of T cell activity resembles the sharp TCR:pepMHC affinity threshold recently described for thymic negative selection by Daniels et al. (38). Although the reasons differ, these all-or-none (digital) responses represent a marked departure from the graded (analog) responses that generally characterize T cell responses as well as Ab-Ag reactions. It should be noted that the threshold characterized in this paper is for the secretion of IL-2, a relatively late event in the effector T cell response (57). Other activities, such as increased intracellular calcium ion concentration, cell surface CD69 up-regulation, cytotoxicity, or T cell proliferation, could be governed by different affinity thresholds (58). Ultimately, in vivo T cell expansion and effector responses will also presumably be determined by some optimal affinity of the TCR:pepMHC interaction (59), perhaps influenced by additional innate factors (60).

The monovalent and multivalent binding of SIYR/Kb by the double-mutant S51αA/Y48βA were only slightly better than by the single-site mutant Y48βA (Figs. 1 and 2; Table I), but strikingly, responsiveness to pepMHC on APCs was observed for the double mutant but not for Y48βA, even when the peptide concentrations were orders of magnitude higher (Fig. 3 a). Hence, we conclude that the binding properties for S51αA/Y48βA lie very close to the threshold for coreceptor-independent stimulation by APCs, and may be used to define a tipping point between activating and nonactivating TCRs in the absence of CD8.

There has been much debate as to which parameter of the TCR:pepMHC-binding reaction (KD vs kd or t1/2) is the most important determinant of T cell responses. In our system, because both KD and t1/2 values were directly related to each other for every TCR, we were unable to decisively address the most relevant parameter. A less than consistent correlation between T cell responsiveness and TCR-pepMHC t1/2 has often been noted before (reviewed in Ref. 61). In some cases, TCR-pepMHC interactions can elicit T cell responses despite their having short t1/2 values, and these cases were associated with relatively large changes in heat capacity on forming TCR:pepMHC pairs (25). To account for the findings, it has been proposed (26) that constraints on the molecular flexibility of TCR and pepMHC when anchored in their respective cell membranes could result in interactions having significantly longer t1/2 than when the same TCR and pepMHC interact in SPR, where they behave essentially as though in solution, under conditions in which most TCR-pepMHC koff values are measured. Along with increased valency effects, these flexibility-related constraints could contribute to the slow dissociation of MHC tetramers from the surface of CD8-negative T cells as opposed to the rapid dissociation measured by SPR (Fig. 2, c and d, and Table II).

For the T cells that expressed the same TCR, but also CD8, the sharp difference between the high-affinity responders and the low-affinity nonresponders was obscured because in the presence of CD8 the latter also responded (Fig. 2, a and c). This effect of CD8 is in accordance with the general rule that for TCR-mediated T cell responses, the enhancing effect of the coreceptor is much more apparent for weak than strong TCR:pepMHC reactions (6, 17). Although the affinity threshold for activation of CD8+ cells was not addressed in this study, CD8 could decrease the threshold considerably, because CD8+ 2C T cells have been shown to specifically lyse target cells that present p2Ca/Kb, whose interaction with the 2C TCR has a KD = 0.3–1 mM (i.e., 10- to 30-fold lower than the 2C TCR affinity for SIY/Kb) (53, 62). Although CD8 can contribute binding energy to TCR:pepMHC complex formation, it has been suggested that it does not significantly affect the overall TCR:pepMHC dissociation rate (11, 63). Although this may be the case, the contribution of CD8 is likely to be more complicated, and possibly varies depending on the TCR or the pepMHC. A recent study explored the relative contribution of CD8 to binding of different pep-Kb ligands by comparing the binding of WT Kb tetramers vs the D227K Kb mutant tetramers that have a reduced ability to bind CD8 (38). A ∼10-fold contribution of CD8 to the equilibrium KD of multivalent binding and a 3- to 5-fold contribution to the t1/2 of tetramer dissociation were observed. (Their study was performed at 37°C, whereas ours were at 24°C, so that we could compare directly the tetramer binding with SPR results).

By eliminating the contribution of CD8 to pepMHC binding, we are able to show explicitly that the enhancement achieved by multivalency varies depending on the monovalent affinity. The enhancement is greatest at lower affinities, with 2C exhibiting a binding enhancement over 2000-fold with tetramer (Table II). This relationship of monovalent affinity to multivalent binding enhancement is analogous to binding studies in which monovalent and bivalent Ab fragments bound to ligands that were plate bound at high or low densities: the lower affinity Abs exhibited greater binding enhancement with the increase in ligand density compared with the higher affinity Abs (46, 48, 49). We think that a likely explanation involves the approximate 5-min washing step, wherein tetramers bound univalently will tend to be lost in proportion to their dissociation rate (t1/2): for weak binders (t1/2 ∼ 1 s) the only tetramers that survive the washing step are bound trivalently or possibly bivalently, whereas for strong binders (t1/2 > 50 s) more univalent and bivalently bound tetramers remain bound. Hence, tetramer binding and SPR binding are less divergent for the strong binders than the weak binders. This finding of varying multivalent enhancement factors points to the usefulness of multivalent binding assays to compare different TCR:pepMHC interactions, but it also suggests that quite small changes in intrinsic binding affinities could yield larger changes in multivalent binding.

We were surprised by the finding that CD8-negative T cells that expressed the very low-affinity TCRs were able to respond to the pepMHC complex immobilized on the surface of a plastic well, although they were completely unresponsive to SIYR/Kb on the surface of APCs. Preliminary efforts to quantitate the total densities of SIYR/Kb in each format suggested that they do not differ substantially (e.g., by orders of magnitude). Recent evidence indicates that class I pepMHC complexes on the surface of cells, although mobile, tend to linger in relatively immobile clusters around ICAM molecules, increasing the local concentration dramatically and improving the ability of T cells to scan those complexes (64). Thus, if anything, we might have expected our APCs to serve as a more efficient mechanism for the clustering of multiple pepMHC. It is possible that localized patches of more highly clustered pepMHC are achieved when immobilized on plastic surfaces than on the surface of an APC, but this remains to be seen. A similar observation about the potency of immobilized ligands, using CD8-positive T cells, led Ma et al. (65) to propose a deformation model of TCR triggering. Variability in immobilized surface density could, in part, explain the lower fraction of stimulated cells seen for lower affinity receptors (Fig. 7) (e.g., depending on where a cell settles, the local ligand density could either be above or below a critical threshold for stimulating the cell). Another difference between cell surface and plate-immobilized Ag presentation that may impact the cell response is the duration of the pepMHC’s presentation. The dynamics of pepMHC complex turnover expected on the surface of APC, which may result in changing Ag densities over the course of the 24-h assay (66), are absent when immobilized pepMHC are used as a stimulus.

It might reasonably have been predicted that T cells transduced with the highest affinity TCR m33, together with the coreceptor CD8αβ, would be efficiently stimulated with SIYR/Kb tetramers because this ligand should bring together multiple TCR and CD8 molecules into a cluster. Indeed, early studies showed that T cell hybridomas with lower affinity WT TCRs were stimulated by pepMHC multimerized on soluble dextran or agarose beads (67). Their study showed that multivalent interactions were important, but they were unable to determine whether this was because of the low affinity of normal TCR:pepMHC interactions, or a true requirement for extensive cross-linking. In the present study, despite highly efficient activation by immobilized SIYR/Kb, there was little stimulation of IL-2 secretion induced by SIYR/Kb dimers or tetramers in solution (Figs. 5 and 6). This result was especially unexpected given that both the t1/2,tet and the equilibrium tetrameric binding enhancement factor (Fig. 2) indicated that long-lived, multivalent binding takes place under these conditions, and it has been shown that soluble pepMHC oligomers with dimeric valency or higher can initiate early TCR recognition processes and early signaling events such as calcium influx even with WT relatively low-affinity receptors (28, 31, 68), in a mechanism that may involve the recognition of endogenous pepMHC complexes through CD8 engagement (5, 69). From our results, it seems likely that larger order clustering of TCR complexes by agonist pepMHC, beyond that obtained with a streptavidin-linked tetramer, is needed to drive T cell signaling all the way to late effector functions, such as IL-2 secretion. The specific size and nature of a fully stimulatory pepMHC complex are unclear, but most likely lies between a tetramer and the larger order organization present when pepMHC complexes are immobilized at high density on a plate. These structures may need to be large enough to recapitulate some of the functions of the immunological synapse, such as providing a directionality for secretion (reviewed in Ref. 70).

Finally, the results presented in this study provide a guide toward the use of genetically engineered TCRs in adoptive T cell therapies (71, 72, 73, 74, 75), because one of the major goals of such studies is to engineer CD8-independent, class I MHC-restricted TCRs for introduction into CD4+ Th cells. Our previous studies with the m33 TCR have shown that by raising the affinity of the TCR for SIYR/Kb to 30 nM, transduced T cells showed self-reactivity (34). It is clear from the present study that efficient CD8-independent T cell targeting can be achieved with TCRs that have considerably lower affinity, possibly avoiding a degree of self-reactivity that is the result of cross-reactivity with structurally related self peptides (such as dEV8 in the 2C system).

We thank Hans Schreiber, Max Artyomov, and Arup Chakraborty for their many helpful comments on the work and on the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants GM55767 and CA097296 (to D.M.K.), a grant from the James S. McDonnell Foundation (to D.M.K.), and National Institutes of Health Grants CA100875 and AI50631 (to J.C.). A.B. was supported in part by the Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship and postdoctoral fellowships from the Sorono Foundation and National Institutes of Health. J.D.S. was supported by the Samuel and Ruth Engelberg/Irvington Institute Fellowship of the Cancer Research Institute.

4

Abbreviations used in this paper: pepMHC, peptide-MHC; PBST, PBS containing 0.05% Tween 20; pERK, phosphorylated ERK; scTCR, single-chain TCR; SPR, surface plasmon resonance; tg, transgenic; WT, wild type; MSCV, murine stem cell virus.

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The online version of this article contains supplemental material.

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