Kinetics and mechanics of 2D interactions between T cell receptors and different activating ligands

Adaptive immune responses are driven by interactions between T cell antigen receptors (TCRs) and complexes of peptide antigens (p) bound to Major Histocompatibility Complex proteins (MHC) on the surface of antigen presenting cells. Many experiments support the hypothesis that T cell response is quantitatively and qualitatively dependent on the so-called strength of TCR/pMHC association. Most available data are correlations between binding parameters measured in solution (3D) and pMHC activation potency, suggesting that full lymphocyte activation required a minimal lifetime for TCR/pMHC interaction. However, recent reports suggest important discrepancies between the binding properties of ligandreceptor couples measured in solution (3D) and those measured using surface-bound molecules (2D). Other reports suggest that bond mechanical strength may be important in addition to kinetic parameters. Here, we used a laminar flow chamber to monitor at the single molecule level the 2D interaction between a recombinant human TCR and eight pMHCs with variable potency. We found that (i) 2D dissociation rates were comparable to 3D parameters previously obtained with the same molecules, (ii) no significant correlation was found between association rates and activating potency of pMHCs, (iii) bond mechanical strength was partly independent of bond lifetime, and (iv) a suitable combination of bond lifetime and bond strength displayed optimal correlation with activation efficiency. These results suggest possible refinements of current models of signal generation by T cell receptors. In conclusion, we reported for the first time the 2D binding properties of eight TCR/pMHC couples in a cell-free system with single bond resolution. INTRODUCTION. An essential component of adaptive immune responses is the capacity of T lymphocytes to detect foreign peptides bound to major histocompatibility complex molecules (pMHC) on the surface of virus-infected cells or specialized cells that have ingested and processed microbial pathogens. This recognition event is a remarkable feat since a T lymphocyte bearing typically 50,000 identical receptors (TCRs) is able to scan within a few minutes the surface of a cell TCR 2D binding properties 2/16 exposing more than 100,000 different pMHC complexes and detect a few or even a single (1,2) cognate peptide differing from self-peptides by a few or even a single amino acid. In addition to remarkable specificity and sensitivity, the recognition event may trigger a variety of responses ranging from lymphocyte unresponsiveness or anergy to full activation (3). It is therefore not surprising that intense efforts have been made to determine the criteria used by a T lymphocyte to select a particular outcome after briefly encountering a given pMHC. A number of separate reports have supported the view that the TCR response is determined by some physical-chemical property of TCR/pMHC interaction such as affinity (4-7), dissociation rate of the complex (8-11) or the association rate (11). However, while most authors found a positive correlation between the lifetime of TCR/pMHC complexes and T lymphocyte activation, some discrepancies remain (12). In addition, two points remain incompletely understood. First, since interaction lifetime is a random event that may display important fluctuations, it is difficult to understand how T lymphocytes can discriminate between quite similar ligands with exquisite accuracy. It was suggested that the involvement of a complex series of time-requiring reactions in TCR signaling might be necessary to overcome this paradox (13, 14). This was the basis of the so-called kinetic proofreading hypothesis. Secondly, the aforementioned studies of the interaction between TCR and pMHC were performed using soluble molecules (i.e. three-dimensional or 3D conditions) using techniques such as surface plasmon resonance. It has long been emphasized that measured 3D parameters could not fully account for the behaviour of surface-attached (2D conditions) molecules (15-18) because of major differences in diffusion conditions and the forces that the interacting molecules are subjected to. During the last two decades, a number of methods, including laminar flow chambers, atomic force microscopy, biomembrane force probes or optical traps have yielded very useful information on interactions between surface-attached molecules at the single bond level (19). However, it is only recently that TCR/pMHC interactions could be monitored at the single bond level. A kinetic study performed with single-molecule fluorescence resonance energy transfer (FRET) microscopy showed that the dissociation rate of bonds formed between cell-bound TCR and pMHC was 4-12 fold higher than on soluble molecules, and this was ascribed to an active participation of lymphocyte cytoskeleton (20). In other experiments, mechanical studies showed that the dissociation rate of bonds involving cell-attached TCR and pMHC could be 8,300 fold higher than reported for soluble molecules. Further, quite surprisingly, the pMHC with maximal activating potency displayed higher dissociation rate, in contrast with conclusions from 3D studies (21). One possible complicating factor in these studies is the fact that cellular responses that affect the measured binding parameters, such as cytoskeletal processes, are influenced by TCR signaling. Thus the higher off-rate measured with more potent ligands may be a consequence of stimulating cytoskeletal processes. Here, we used a laminar flow chamber to probe the interaction between a recombinant TCR and 8 pMHC complexes previously shown to have varying activation potency in functional assays. As recently reviewed (22), the laminar flow chamber operating at a low shear rate is an efficient way of probing the kinetics of association and force-induced dissociation of weak molecular bonds at the single molecule level. The use of model surfaces instead of cells allowed us to probe intrinsic 2D molecular properties in the absence of potentially-confounding cellular effects. These 2D binding properties were compared with 3D binding properties and pMHC activation potency. We found: (i) that there is an essential agreement between 3D dissociation rate and 2D dissociation rate measured with the lowest disrupting force; (ii) that the force resistance is not entirely accounted for by spontaneous bond duration; and (iii) that the activation potency is positively correlated with bond duration TCR 2D binding properties 3/16 and, surprisingly, correlated negatively with bond mechanical strength. Our results may provide new insights into potential mechanisms of signal generation by TCR/pMHC interactions. MATERIALS AND METHODS. Molecules. 1G4 TCR is specific for the peptide 157-165 derived from the tumour associated protein NYESO-1 protein presented on HLA-A2 (23). Soluble forms of the 1G4 TCR and pMHC variants were produced as previously described (11). Briefly, wild type or mutant forms of HLA-A2 heavy chain (residues 1-278) with C-terminal BirA tag and β2-microglobulin were expressed as inclusion bodies in E.coli, refolded in vitro in the presence of synthesized peptides, and purified using size-exclusion chromatography. Purified pMHC was biotinylated in vitro by BirA enzyme (Avidity, Hounslow, England). The 1G4 TCR α and β subunits were expressed in E.coli as inclusion bodies, refolded in vitro, and purified using size exclusion chromatography. As suggested by their names, all pMHCs differed by a single aminoacid of the peptide (3A, 3Y, 6T, 9L or 9V) or the MHC (H70, H74, or R65) (11). Preparation of surfaces and microspheres 24×24 mm glass slides (Assistent, Sondheim Germany) were rinsed twice in ethanol, then rinsed thoroughly with water. Glass slides were then cleaned in a mix of 70% sulphuric acid (Fisher Bioblock, Illkirch France) and 30% H2O2 (50% in water, Sigma-Aldrich, St-Quentin Fallavier, France) for ten minutes, then rinsed thoroughly with deionized water and stocked in deionized water. Glass slides were coated with a poly-l-lysine solution (150000-300000 Da, 100μg/ml; Sigma-Aldrich) in 0,02 M phosphate buffer, pH 7,4 for 30 minutes, rinsed in PBS, then incubated in a glutaraldehyde solution (2,5 % in 0,1 M borate buffer, pH 9,5; SigmaAldrich) for 10 minutes, and rinsed in PBS. Glass slides were then incubated in a solution of bovine serumalbumin (BSA; 90μg/ml; Sigma-Aldrich) and biotinylated BSA (10μg/ml; Sigma-Aldrich) in PBS, for 30 minutes, then rinsed with PBS. Glass slides were incubated for 30 minutes in a blocking solution of glycin (0,2M in PBS), rinsed in PBS, then incubated in a streptavidin solution (10μg/ml in PBS; Sigma-Aldrich) for 30 minutes, then rinsed with PBS. Glass slides were incubated with biotinylated pMHC in PBS, at different concentrations, for one hour, then mounted in the flow chamber. Dynabeads M500 tosylactivated microspheres (Invitrogen, Cergy Pontoise France) were coated first with a mouse anti-histidine tag antibody (MCA1396, Serotec France, Colmar), according to the manufacturer protocol. Briefly, microspheres were rinsed in 0,1 M pH 9 borate buffer, incubated 24H at 37C in antibody solution in 0,1 M pH 9 borate buffer, then rinsed in PBS, and incubated in a blocking solution of TRIS 0,1M and BSA 0,1% for 4hours at 37°C. Microspheres were stored in this solution at 4°C with 0,1% sodium azide added, and incubated in histidine-tagged TCR molecules for one hour before experiment. It was checked that these incubation times were sufficient to ensure irreversible binding of the reagents on the timescale of experiments (not shown). Flow chamber setup and data analysis. We used a custom-made flow chamber with a machined Plexiglas top forming a 0.2×2×6 mm chamber (height×width×length), the functionalysed glass slide forming the bottom, maintained by a machined aluminium plate bolted to the top. Vacuum grease was used as a gasket. Flow was established using a 5ml glass syringe mounted on a syringe pump (A-99, Razel, St Albans, VT, USA). Microspheres were suspended in PBS with 0,1% BSA in the flow chamber. TCR 2D binding properties 4/16 An inverted microscope (Olympus France, Rungis) in bright-field illumination equipped with a 20X lens and a standard video camera was used to follow the twodimensional trajectory of the beads and to measure their adhesion to the underlying substrate. Video signal was digitized at video rate (25 Hz) by a digitization card (Hauppauge France, Paris) and compressed on-the-fly by the freeware VirtualDubMod using the DivX 5.1.1 codec. Microsphere positions were determined using a custom-made software written in C++. Arrest detection and duration measurement was done using a custom-made software written with Igor environment (Wavemetrics, Lake Oswego, OR USA). Statistics of molecular bond formation and rupture were determined by counting the frequency and duration of microspheres arrest events in the laminar flow as previously described (24). Briefly, a microsphere was considered as arrested if its position did not change by more than dx = 0.5 μm in dt = 0.2 s, and if its velocity before the arrest corresponded to that of a moving sedimented bead. The adhesion frequency was defined as the number of arrests divided by the total time spent by the microspheres after sedimentation in the velocity range of moving sedimented beads. The frequency of specific binding under a given condition (i.e. wall shear rate and ligand surface density) was estimated by subtracting the binding frequency measured with non peptide-specific TCR bearing microspheres from the binding frequency measured with peptide-specific TCR bearing microspheres. An arrest was considered to continue as long as the arrest criterion was satisfied, which yields an apparent duration dapp. The true arrest duration dtrue was derived from the apparent duration dapp with the correction dtrue=dapp+(t2dx/v), where v is the most probable velocity of the beads (25). Statistical analysis. Presented conclusions are based on the determination of about 11,000,000 microsphere coordinates and recording of about 7,106 binding events. However, the intrinsic randomness of single molecule events made it useful to estimate the statistical significance of presented data. The error in the fraction of surviving bonds was derived from the number of counted arrests on the basis of binomial law (26). The significance of correlations was obtained after calculating Pearson correlation coefficient r by using the normal transform: z = (n-3)/2 ln[(1+r)/1-r)], where n is the number of pairs (26). Bootstrapping (27) was performed with a custom-made software. RESULTS. We readily observed the formation of single specific TCR/pMHC bonds that displayed multiphasic rupture kinetics. In a first series of experiments, microspheres coated with the 1G4 TCR (23) were driven along a series of surfaces coated with 8 specific pMHC ligands previously shown to interact with 1G4 with affinity constants ranging between 1.1×10 and 29×10 M, and activation potencies spanning a 7-fold range (11). The shear rate was kept at a low value of 20 s, thus generating a pulling force of 34 piconewtons on surface-attached spheres (28). The motion was monitored with a computer-assisted method allowing 20 ms time resolution and 40 nm spatial accuracy (29). Previous experiments have shown that these conditions allow single ligand-receptor bonds to generate detectable arrests of moving spheres (22,30). Fully sedimented spheres flowed along the surface with a velocity of about 25 μm/s, and they displayed binding events with a frequency between about 0.1 s and 1 s. These binding events could be ascribed to a specific TCR/pMHC interaction since their frequency exhibited a 5-10 fold decrease when 1G4 was replaced with another TCR (G10) that does not bind to these pMHCs in solution (not shown). TCR 2D binding properties 5/16 Arrest durations were recorded and a typical plot of bond survival versus time is shown in Fig. 1A. The curve displayed upward concavity, similar to results previously obtained for avidin-streptavidin (31), integrin-fibronectin (32) or cadherin-cadherin (25) interactions. We checked that this feature was due to a time-dependent strengthening of individual bonds rather than to the formation of additional bonds by changing the surface density of pMHCs: while the binding frequency displayed an up to 4 fold decrease following Figure 1. Bond rupture follows a complex kinetic behavior that can be robustly represented by the fraction of bonds surviving 1s after formation. The figure shows a typical survival plot of bonds formed between microspheres coated with 1G4 TCR and surfaces coated with ESO-9V pMHC complex. Wall shear rate was 20 s. (A) Vertical bar length is twice the standard error calculated on the basis of binomial law (162 binding events at time zero). The straight line is the least square linear fit calculated on [0s,1s] time interval. (B) Enlarged initial part of the curve. The slope of the linear fit (straight line), fraction of surviving bonds as 1s (B1s) and median bond lifetime (t1/2) are shown. pMHC dilution , the distribution of arrest durations was not changed (Fig. 2). The stopping events could thus be considered to be mediated by single molecular bonds (32). The bond-strengthening phenomenon that we describe has been reported by several different laboratories and termed the history dependence of bond dissociation (34,35). This is indicative of the existence of a number of different bound states potentially formed by a given ligand-receptor couple (30,31,36). As a consequence, it is not straightforward to account for the dissociation behaviour of a well defined ligand-receptor couple with a single parameter (Fig. 1 A&B). TCR 2D binding properties 6/16 Figure 2. Independence of rupture kinetics on pMHC density supports the assumption that single bonds are observed. For each pMHC, binding frequency and detachment curves were determined for at least two pMHC coating densities. The assumption that binding events were mediated by single bonds was based on the finding that two-fold decrease of coating density resulted in about two-fold decrease of adhesion frequency while survival curves were unaltered. A typical example is shown: ESO-3A peptide studied at 20 (red), 40 (purple) and 80 (blue) s shear rate with coating densities of 0.5 μg/ml (crosses) or 1 μg/ml (circles). The mean number of binding events used to build each curve is 262 (range: 79-548). The fraction of bonds surviving at least 1 second gives a robust account of bond lifetime during the first 10-20s following ligand-receptor association. The statistics of bond duration were determined for the eight pMHC variants and three different values of the shear rate (20, 40 and 80 s). Typical detachment curves are shown on Fig. 3. A natural way of translating experimental data into quantitative lifetime parameters is to fit survival curves to mathematical models. The simplest model involves a single fitted parameter and would be applicable if bond rupture followed monophasic kinetics with a timeindependent rupture frequency koff, yielding : B(t) = exp(-kofft) [1] where B(t) is the fraction of bonds surviving at time t after formation. However, experimental curves were not consistent with this assumption since they did not appeared as straight lines on semi-log plots. Another possibility would be to assume a single first-order Markovian unbinding process : TCR 2D binding properties 7/16 B(t) = A0 + A1 exp(-k'offt) [2] Where A0, A1 and koff are three fitted parameters, where k'off would be equal to the off-rate if bond rupture followed monophasic kinetics. This model allowed accurate fitting of all survival curves (not shown). However, a single parameter (among A0, A1 and k'off) could not give an adequate account of bond lifetime. Thus, while k'off appeared an attractive candidate, the fitted k'off values for pMHC variants R65 and 6T were respectively 1.44 and 1.45 at 20 s -1 shear rate, while the survival curves were markedly different (Fig. 3). Figure 3. Survival statistics of the rupture of bonds formed with 8 pMHCs display minimal crossing between curves, supporting the feasibility of accounting for these curves with a single parameter such as B1s. The rupture kinetics of bonds formed with 8 pMHCs and TCR 1G4 was studied at 20 s wall shear rate. The survival during the first 5 seconds is shown. Color codes are indicated in insert. These plots are based on 1,827 arrest durations (between 110 and 356 events per curve). A simple way of accounting for a full detachment curve with a single parameter was suggested by the observation that crossings between survival curves were rare (e.g. ESO-6T and ESO-3Y at 20 s shear rate) and the rank of bond survival yielded by tested pMHCs was nearly unchanged during the first 10 s following attachment. Thus, bond survival at a given time tref after association can provide a satisfactory index of bond lifetime. Hopefully, B(tref) might account for the information drawn by a T lymphocyte from a cycle of TCR/ligand association/dissociation, whatever the underlying mechanism. In order to choose the reference time tref for bond survival determination, we used the binomial law to estimate the statistical accuracy of survival information (26) : As shown on Fig. 1A, the relative accuracy of survival determination decreased with time, due to a TCR 2D binding properties 8/16 concomitant decrease of the number of remaining bonds. The fraction of bonds surviving 1s after association (B1s) was therefore tentatively used as a single robust parameter to account for bond lifetime. We used bootstrapping (27) to assess the robustness of the B1s parameter : starting from the 162 arrest durations exemplified on Fig. 1, we generated 25 survival curves by random sampling with repetition of 80 arrest durations from the 162 experimental values. The slope of the regression line on the [0,1s] time interval, parameter B1s and the median duration of arrests were calculated, yielding mean values of -0.588 (standard deviation SD=0.127, coefficient of variation CV = SD/mean = 0.22), 0.62 (SD=0.051, CV=0.083), and 2.59s (SD=1.35, CV=0.52). Thus B1s might be considered to provide a reproducible account of bond rupture during the first few seconds after attachment. Interestingly, the estimate of B1s CV matched the estimate obtained with binomial distribution (26), which yielded 0.052. Note that parameter B1s was strongly correlated with the fraction of bonds surviving 5s (B5s) and 20s (B20s), since Pearson correlation coefficients between B1s and B5s, and between B1s and B20s were 0.9826 and 0.9525, respectively. This suggested that our conclusions should not be strongly dependent on the choice of 1s as a reference time. 2D Bond lifetimes obtained with the flow chamber are consistent with previously reported 3D values We next examined whether 3D lifetime measurements could yield a satisfactory account of the lifetime of 2D interactions. Under 3D conditions, the expected fraction of bonds surviving at time t after attachment is: B3D(t) = exp(-koff t). [3] Where B3D(d) is the fraction of bonds surviving at time t after formation under 3D conditions and koff is the conventional dissociation rate. Thus, we compared the experimental survival fractions obtained with the flow chamber and the values of B3D(t) obtained with the dissociation rates koff determined with BIAcore. First, we compared bond survival at time 1s and zero force (BIAcore) or when bonds were subjected to hydrodynamic forces (flow chamber). Using an estimate of 32 nm for the total length of the bond maintaining a microsphere at rest (pMHC + TCR + anti-Histag, represented about 8 immunoglobulin domains of 4 nm each), the force on the bond was calculated to be 34, 68 and 136 pN at wall shear rates of 20, 40, and 80 s, respectively (30). Note that this estimate is only weakly dependent on bond length since it is inversely proportional to the square root of the bond length (30). Experimental data are shown on Fig. 4A, suggesting a qualitative agreement between 2D and 3D parameters, with two groups of more transient and more durable bonds appearing with both methods. An interesting outlier was peptide ESO-6T that exhibited highest resistance to stronger forces and rather low bond lifetime in presence of low forces. We tried to make the comparison of 2D and 3D data more quantitative and less dependent on the threshold time by calculating correlation coefficients with survival fractions measured at times 0.5, 1, 3, 5 and 10 s under wall shear rates of 20, 40 and 80 s . Results are shown on Fig. 4B, yielding the following conclusions : With the lowest two flow rates, the correlation between 2D and 3D survival at time t was fairly constant when t was lower than 10 seconds. The correlation coefficients r measured at 1s were respectively 0.8271 (P=0.0084) and 0.9332 (P=000168) at 20 and 40 s shear rate. These values were not significantly different since the normal transforms of r differed by only 1.13 standard deviations (P = 0.26) TCR 2D binding properties 9/16 With the highest flow rate, the correlation between 2D and 3D was low (ranging between 0.572 and 0.144) and not significant (P ranging between 0.15 and 0.75). Thus, 3D measurements were found to give a satisfactory account of the lifetime of surfaceattached bonds subjected to low forces. Figure 4. The correlation between 3D dissociation rates and bond survival at time tref in the flow chamber depends on the flow rate, not on tref. (A) Bond survival at time 1s is shown for 8 pMHC/TCR pairs under 3D conditions, corresponding to F=0pN, and under 2D conditions with 20, 40 and 80 s -1 flow rate, corresponding to estimated forces of 34, 68 and 136 pN on bonds. (B) The Pearson correlation coefficient was calculated between the experimental survival fraction measured in the flow chamber at different times under a wall shear rate of 20 (squares), 40 (crosses) or 80 s (circles) and the value expected on the basis of 3D measurements, i.e. exp(-koff t). The curves shows that 3D (force-free) dissociation is strongly correlated to 2D survival in presence of low forces, but not higher forces, for a wide range of time values, supporting the robustness of this conclusion. The flow chamber yielded new information on the mechanics of bond rupture as compared to 3D studies. The simplest interpretation of our results would be that the forces exerted by flow might decrease the survival of stressed bonds in proportion to a parameter unrelated to koff. Indeed, TCR 2D binding properties 10/16 TABLE 1 Summary of 2D and 3D properties of TCR/pMHC pairs