Supplementary Figure 3: 2D binding at zero force does not provide clear criteria for thymocyte negative selection.
From: A TCR mechanotransduction signaling loop induces negative selection in the thymus
Related to Fig. 5. a, Lifetimes of OT1 TCR bimolecular bonds with the indicated peptides bound to H2-Kbα3A2 or of the MHC–CD8 bond (wVSV) were measured by the BFP thermal fluctuation assay24 at zero-force. Their survival probabilities were plotted as fraction of events with a lifetime ≥ tb vs. lifetime tb. The number of bond lifetime measurements are: N = 52 (OVA), 39 (Q4), 49 (Q4R7), 26 (T4), 31 (Q4H7), 39 (Q7), 52 (G4), and 20 (VSV). b, Micrographs of the micropipette adhesion frequency assay25. A DP thymocyte (right) aspirated by a pipette was aligned with a RBC held by another pipette (left). The two cells were brought into a controlled contact for a given area (Ac) and duration (tc) and then retracted to detect binding, which was observed by the absence (top, no adhesion) or presence (bottom, adhesion) of RBC elongation. Scale bar = 5 µm. c, Specificity control. At 5 s contact, thymocytes adhered to RBCs coated with wOVA (13 μm-2) at higher frequency than those with mOVA (13 μm-2), showing the contribution of CD8 binding, but did not adhere to unmodified RBCs, biotinylated RBCs, and RBCs coated with biotin–SA (4335 μm-2) or mVSV (604 μm-2). Data are presented as mean ± s.e.m. of cell pairs each contacted 50 times to estimate an adhesion frequency. The number of cell pairs are: N = 3 (unmodified), 3 (biotin), 4 (biotin-SA), 3 (mVSV), 4 (mOVA), and 5 (wOVA). d, Adhesion frequency Pa vs. contact duration tc plots for indicated peptides bound to H2-Kbα3A2 (brown circle) or H2-Kb in the absence (blue square) or presence of anti-CD8 CT-CD8a (green triangle) or anti-TCR B20.1 (red diamond). Except for OVA whose measurements required a lower pMHC density (14-46 μm-2), a narrow range of pMHC densities (84-240 μm-2) were used to compare DP thymocyte adhesions in indicated conditions for each ligand. MHC–CD8 interaction (measured by using B20.1 to block TCR binding) mediated lower but clearly measureable adhesion frequencies than the total pMHC interactions with TCR and/or CD8. The much lower adhesions mediated by TCR than CD8 result from the 10 times lower expression of TCR than CD8 on DP thymocytes; but TCR still has higher affinities for pMHC than CD8 (cf. panel e). Since TCR–pMHC interactions (measured by using H2-Kbα3A2 or CT-CD8a blocking to abolish CD8 binding) were nearly undetectable for these peptides with such low pMHC densities, higher ligand densities (840-3739 μm-2) were used to measure their effective 2D affinities, as shown in panel e. Data are presented as mean ± s.e.m. of N≥3 cell pairs for 50 touches each pair. The data are representatives from at least two independent experiments per curve. e, No significant differences (p>0.5, one-way ANOVA) were observed among effective 2D affinities AcKa of TCR for pMHCs of distinctive selection outcomes (left ordinate, open bar, mean ± s.e.m., N≥5). MHC–CD8 AcKa (left ordinate, hatched bar, mean ± s.e.m., N=5) and available 3D tetramer values (right ordinate, black bar)4 are shown for comparison. f, To directly compare different ligands, the adhesion frequency data were converted to average number of bonds by <n> = – ln(1 – Pa), normalized by dividing them by the corresponding pMHC density mpMHC, and plotted versus contact duration tc. Except for OVA whose curve (red circle) is one log higher, the collapsed <ntotal>/mpMHC curves of the other ligands show no significant difference. The <nCD8>/mpMHC curve (black filled circle) is lower. g, Normalized total adhesion bonds, <ntotal>/mpMHC = – ln(1 – Pa)/mpMHC (open bar) for the indicated pMHCs (mean ± s.e.m., N≥3) and available 3D tetramer values (black bar)4 are shown for comparison. No significant differences were found among the middle 6 ligands (p>0.5, one-way ANOVA). The 2D kinetic parameters measured at zero force by adhesion frequency assay and thermal fluctuation assay are summarized in Supplementary Table 6
