Force-Regulated In Situ TCR-Peptide-Bound MHC Class II Kinetics Determine Functions of CD4+ T Cells

Abstract

We have recently shown that two-dimensional (2D) and force-regulated kinetics of TCR-peptide-bound MHC class I (pMHC-I) interactions predict responses of CD8(+) T cells. To test whether these findings are applicable to CD4(+) T cells, we analyzed the in situ 3.L2 TCR-pMHC-II interactions for a well-characterized panel of altered peptide ligands on the T cell surface using the adhesion frequency assay with a micropipette and the thermal fluctuation and force-clamp assays with a biomembrane force probe. We found that the 2D effective TCR-pMHC-II affinity and off-rate correlate with, but better predict the T cell response than, the corresponding measurements with the surface plasmon resonance in three dimensions. The 2D affinity of the CD4 for MHC-II was very low, approaching the detection limit, making it one to two orders of magnitude lower than the affinity of CD8 for MHC-I. In addition, the signal-dependent cooperation between TCR and coreceptor for pMHC binding previously observed for CD8 was not observed for CD4. Interestingly, force elicited TCR-pMHC-II catch-slip bonds for agonists but slip-only bonds for antagonists, thereby amplifying the power of discrimination between altered peptide ligands. These results show that the force-regulated 2D binding kinetics of the 3.L2 TCR for pMHC-II determine functions of CD4(+) T cells.

FIGURE 1. Micropipette adhesion frequency assay and thermal fluctuation assay

(A) Schematic of micropipette apparatus. A T-cell and a pMHC-coated RBC are held by two apposing micropipettes before the contact. Below the left photomicrograph, the TCR complex and CD4 are drawn on the T-cell surface, whereas the pMHC coupled via biotin–streptavidin interaction are drawn on the RBC surface. The RBC was brought to contact the T-cell for certain contact duration and retract to observe the presence, signified by the elongation (upper right), or absence, signified by the lack of elongation (lower right), of the soft RBC membrane at the end of the contact. (B) The adhesion frequency assay was conducted for a panel of cognate peptides (Hb, T72, I72, A72) and an irrelevant peptide (MCC) with representative data (from three independent experiments) shown along with the following molecule densities (TCR:pMHC in molecules/μm²): Hb (185:33), T72 (179:67), I72 (171:114), A72 (158:186), and MCC (136: 4483, 315 for CD4). Each point represents mean ± SEM (n=3-5 cell pairs each contacted 50 times to estimate an adhesion frequency) (C) Controls for nonspecific adhesion were performed at 5s contact duration between 3.L2 T cell and unmodified RBCs, biotinylated RBCs without further coupling to streptavidin, biotinylated RBCs linked with streptavidin without further coating of pMHC, biotinylated RBCs linked with streptavidin coated with pMHC-I (OVA:H-2K^b) or noncognate pMHC-II (MCC:I-E^k), which were compared to biotinylated RBCs linked to streptavidin coated with cognate pMHC-II (Hb:I-E^k). Each bar is presented as mean ± SEM (n=5 cell pairs each contacted 50 times to estimate an adhesion frequency). (D) The thermal fluctuation assay was performed as described in Methods. Ranked bond lifetime distributions for each ligand were linearized by ln(# of events with a lifetime > t_b)/ln(total # of events) versus t_b plots to allow us to estimate k_off from the negative slope of the line. The numbers of bond lifetimes are 22 (Hb), 66 (T72), 65 (I72), and 46 (A72). The high levels of goodness-of-fit as assessed by R² (> 0.97 for all ligands) support the use of the first order kinetics model.

FIGURE 2. Comparison of 2D binding parameters with 3D counterparts

(A) Lack of CD4 binding to I-E^k. Treatment with the TCR blocking antibody CAb abolished the adhesion frequency observed without blocking, but treatment with the CD4 blocking antibody (GK1.5) did not affect the binding curve. The noncognate ligand MCC:I-E^k had some binding at a longer contact duration (5s) with a very high site density of >4,000 molecules/μm², but overall binding was very low (<5%). The molecule densities used to generate these representative data are (TCR:pMHC molecules/μm²): Hb (116:34), Hb + GK1.5 (116:34), Hb + CAb (163:28), and MCC (136:4483, 315 for CD4). Each point represents mean ± SEM (n≥3 cell pairs each contacted 50 times to estimate an adhesion frequency). (B-D) Comparison between 2D vs. 3D affinity (B), off-rate (C), and on-rate (D). 2D measurements were from adhesion frequency assay and thermal fluctuation assay whereas 3D measurements were from SPR. Each 2D kinetic parameter is presented as mean ± SEM (n≥3 sets of adhesion frequency vs. contact time curve).

FIGURE 3. Correlating 2D kinetics with 3D counterparts and with T-cell response

(A-C) Correlation between 2D vs. 3D kinetics. The effective 2D affinity correlates highly (R²=0.9, p<0.05) to the 3D affinity due solely to the much higher values of the agonist pMHC-II than the other three APLs (A). The off-rates correlate well (R²=0.87, p<0.1) between 2D and 3D (B). The effective 2D on-rates poorly correlate (R²=0.14, p>0.5) between 2D and 3D (C). The mean values from both 2D (Table I) and 3D (Table II) measurements were compared. (D-F) Correlation of 2D kinetics with T-cell response. The effective 2D affinity correlates well (R²=0.84) with the reciprocal of the ligand concentration required to produce 40% B cell apoptosis (1/EC₄₀) (D). The 2D off-rate highly (R²=0.92), but negatively correlates with 1/EC₄₀ (E). The effective 2D on-rate shows a poor correlation (R²=0.36) (F). The parameters used for the 2D kinetics are the mean values from the adhesion frequency and thermal fluctuation assays. The EC₄₀ values are from Ref. (36).

FIGURE 4. Comparison between CD4⁺ and CD8⁺ 3.L2 T cells for TCR–pMHC-II binding

(A) Flow cytometry scatter plot of 3.L2 T-cells. CD4⁺ and CD8⁺ cells were purified with respective CD4 and CD8 negative purification protocols then analyzed for TCR, CD4, and CD8 expressions by flow cytometry. (B) Comparison of effective 2D affinity between CD4⁺ and CD8⁺ 3.L2 T cells. No significant difference were observed between most of the two subpopulation. Student's t-test was used for statistical analysis. Each bar represents mean ± SEM (n≥3).

FIGURE 5. Ligand dependent bond lifetime under force

(A) Mean ± SEM bond lifetimes measured by force-clamp assay are plotted vs. force. The agonist ligands, Hb and T72, show catch-slip bonds, whereas the antagonist ligands, I72 and A72, show slip-only bonds. The number of lifetime measurements for each curve is: Hb (298), T72 (300), I72 (448), and A72 (343). (B-E) Normalized lifetime distributions of bimolecular TCR–pMHC-II bonds with the indicated peptides measured by the force-clamp assay in the indicated force regimes. The shallower the curve, the slower the dissociation, and the greater the number of bonds surviving a given time. As force increases, the separation between agonist and antagonist ligands increases, reaches a maximum at 5-15pN (C and D), and then decreases.

FIGURE 6. Force amplifies ligand discrimination

(A) The ligand concentration required to generate 40% B cell apoptosis (EC₄₀) from Ref. (36) was plotted vs. mean bond lifetime for Hb (circle), T72 (square), I72 (diamond), and A72 (triangle) and fitted by a straight line, one for 0pN (blue) and another for 10pN (brown). Force tilts the EC₄₀ vs. bond lifetime curve to increase the dynamic range in the x-axis by more than one fold. (B) The ratio of bond lifetime of Hb to another peptide was plotted vs. force to show increased power of ligand discrimination around optimal force where bond lifetime for Hb reaches maximum. The increase in the ratios for Hb/I72 (green) and Hb/A72 (blue) but not for Hb/T72 (brown) further indicates separation between agonist and antagonist ligands. The data from force vs. bond lifetime were used to calculate the ratio.