Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy

Abstract

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

FIGURE 1.

Specific recognition of isolated CD1d-GSL molecules by an iNKT TCR-coated tip. A, schematic representation of the immobilization strategy used for binding the iNKT TCR molecule to the AFM tip and CD1d-GSL monomers onto mica. The biotinylated iNKT TCR and CD1d-GSL complexes were attached to the amino-functionalized surface of the tip and mica, respectively, through the aldehyde-PEG-NHS cross-linker and streptavidin as described under “Experimental Procedures.” B, diagram of the force-distance cycle. The tip is progressively moved until contact occurs with the molecules immobilized on mica (dotted line, steps 1 and 2). As the tip is further pushed onto the surface, the cantilever bends up in a characteristic slope (upward arrow). Withdrawing the tip restores its initial shape and follows the slope backwards (downward arrow). By continuing retraction, the cantilever bends down if binding occurs (step 3), thereby stretching the PEG linker until the bond ruptures at a critical force (f_u), termed the unbinding force (step 4). C, a typical force curve showing an iNKT TCR-unbinding event from CD1d-αGalCer upon tip retraction. Inset, the specific interaction is blocked by injecting free anti-CD1d mAb into the bath solution. D, probability density function constructed from 174 unbinding events of 1000 force-distance cycles (binding probability of 17.4%) showing the distribution of unbinding force events occurring between the iNKT TCR and CD1d-αGalCer (black line). The effect of the anti-CD1d blocking antibody on the iNKT TCR/CD1d interaction is represented by the red dashed line, showing a decrease in the binding probability to 5.7%.

FIGURE 2.

Evaluation of cell-surface expression of CD1d molecules by FACS analysis. A, the red line indicates CD1d expression on THP1 cells transduced with a lentivirus encoding CD1d molecules, and the blue line indicates the CD1d level on untransduced THP1 cells. B, iNKT TCR staining on THP1-CD1d cells pulsed either with αGalCer (blue line) or with OCH12 (orange line). Unpulsed THP1 (green line) and unstained (red lines) controls are also shown. MFI, mean fluorescence intensity.

FIGURE 3.

AFM images of fixed THP1 cell. A, topographical image; B, deflection image; C, cross-section.

FIGURE 4.

A, schematic diagram of single molecule force measurements on live cells with an iNKT TCR-modified AFM tip. The THP1-CD1d cells pulsed with the iNKT agonist αGalCer or OCH12 were attached to poly-l-lysine-coated glass coverslips. B, a typical force-distance cycle on living cells during AFM tip retraction showing a receptor/lipid interaction. Inset, blocking the specific interaction by injecting free anti-CD1d mAb into the solution. C, distribution of unbinding forces of iNKT TCR/CD1d-αGalCer (black line) based on 150 unbinding events of 1000 force-distance cycles. The binding probability of 15% decreased to 3.5% upon injection of free anti-CD1d mAb into the solution (red dashed line). D, comparison of the binding probabilities of an iNKT TCR-coated tip on live CD1d-transfected THP1 cells pulsed either with αGalCer (red bar) or with OCH12 (green bar) or unpulsed (blue bar).

FIGURE 5.

Loading rate dependence of unbinding force. Shown are the unbinding force spectra of the iNKT TCR bound to CD1d-αGalCer (red stars) and CD1d-OCH12 (green triangles) on isolated molecules (A) and on living THP1 cells (B). The unbinding forces for both interactions were found to depend linearly on the logarithm of the loading rate (see “Experimental Procedures”), as consistent with the Bell-Evans model. The uncertainty in the determination of the unbinding force is represented by error bars. Also shown is the binding probability as a function of contact time for CD1d-αGalCer (C) and OCH12 (D) on living THP1 cells.