T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR-binding parameters

Abstract

T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self-peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide-MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN-γ production by T cells, and 1G4-pMHC dissociation rates measured in soluble phase or on surface-bound molecules, displayed six- to sevenfold variation among pMHCs. When T cells were dropped onto pMHC-coated surfaces, rapid spreading occurred after a 2-min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all-or-none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.

Keywords: Affinity; Early T-cell activation; Interference reflection microscopy; Kinetics; Spreading; TCR; pMHC.

Figure 1

Typical images of spreading Jurkat cells on pMHC-coated surfaces. 1G4-transfected Jurkat T cells were sedimented onto pMHC-coated surfaces under microscopic observation and recording (One image per second). (A to C, G) Sequential IRM images of a typical cell. (D to F, H) Computer-calculated contact areas (shown as black pixels on a gray area). (A) Initial aspect of sedimenting cells with concentric rings indicative of a fairly spherical shape. (B and E) Point-like contact (0.03 μm²) that appeared 10 s later. (C and F) Display two contact spots with a total area of 1.1 μm² (time = 26 s). (G and H) Represent a more extensive contact (4150 pixels corresponding to 5.9 μm²; time = 379 s). Bar = 2 μm. Images are representative of 24 independent experiments performed, with about 20 studied individual cells per experiment.

Figure 2

Spreading of 1G4-transfected Jurkat T cells onto pMHC-coated surfaces as plotted over time. (A–H) 1G4-transfected Jurkat T cells were sedimented onto surfaces coated with 1G4 ligand under IRM observation and recording (1 image/s). A total of 495 individual cells were followed for 10 min each and images were processed to build contact plots. (A and B) The most frequently recorded curves with rapid spreading frequently preceded with transient contacts are shown. (C) A less clear-cut spreading pattern with delayed appearance of a rapid spreading phase is shown. (D) A fairly rare case with repeated transient contacts without extensive spreading is shown. (E and F) The average spreading of cells on a surface coated with a lower amount (1 μg) of peptide bound by their TCR with (E) a lower dissociation rate (3A, 35 cells) or (F) a higher dissociation rate (3Y, 30 cells) is shown. (G, H) The average spreading of cells on a surface coated with a higher amount (10 μg) of (G) a more strongly bound peptide (3A, 50 cells) or (H) a less strongly bound peptide (3Y, 26 cells) are shown. (E to H) Data are shown as mean ± SEM of about 20 individual cells observed in 5 independent experiments for each condition (defined as a pMHC species and a concentration).

Figure 3

Frequency distribution of transient contacts. The frequency distribution of transient contacts such as exemplified in Figure 2B is represented as a survival plot revealing a typical power law with an exponent of −0.19. The curve was built out of 345 recorded values of contact duration. Data are representative of 24 independent experiments.

Figure 4

Dependence of spreading on deposited peptide-MHC. 1G4-transfected Jurkat cells were deposited on surfaces coated with varying amounts of peptide-MHC. 3A (diamond), H74 (square), 9V (crosses), 3Y (triangles), or 9L (circles) and the average spreading area was determined in the period of time ranging between 15 and 20 min after deposition. (A) Each point represents a mean of 249–1096 values. (B) Each point represents the average spreading rate of cells that displayed an active response. (A and B) A total number of 495 cells were studied. Vertical bar length = twice the SD. Data shown are representative of 24 independent experiments.

Figure 5

Spreading rate, contact area, and spreading fraction are tightly correlated. 1G4-transfected Jurkat cells were deposited on surfaces coated with varying amounts of MHC-coupled peptide 3A (diamond), H74 (square), 9V (crosses), 3Y (triangles), or 9L (circles). The fraction of cells with a spreading response (13 574 cells), the contact area of cells that displayed a spreading response (6622 cells), and the initial spreading rate of cells that displayed a spreading response were measured. (A) The correlation coefficient between spreading rate and contact area was 0.8915 and (B) the correlation coefficient between the spreading fraction and the contact area was 0.9362. Data shown are representative of 24 independent experiments, with an average of 20 samples/group.

Figure 6

Dependence of spreading fraction and maximum slope on activating surfaces. 1G4-transfected Jurkat cells were deposited on surfaces coated with varying amounts of the 3A (diamond), H74 (square), 9V (crosses), 3Y (triangles), or 9L (circles) pMHCs. (A) The fraction of cells that displayed an active spreading response was determined. (B) The maximum spreading rate was determined in the period of time ranging between 0 and 10 min after deposition. A total number of 297 cells that could be monitored for 10 min and that did not display any contact at time zero were studied. (A and B) Vertical bar length = twice the SEM. Data shown are representative of 24 independent experiments, with an average of 20 samples/group.

Figure 7

Relationship between surface coating and duration of analysis. 1G4-transfected Jurkat cells were deposited on surfaces coated with varying amounts of the 3A (diamond), H74 (square), 9V (crosses), 3Y (triangles), or 9L (circles) pMHCs and the duration of the lag between initial contact and beginning of spreading at maximal rate was determined on 297 cells that displayed a spreading response and were not in contact with the surface at the onset of the observation period. (A) Average value of the lag between initial contact and spreading at maximum rate. Vertical bar length = twice the SEM. (B) Individual values of the lag between initial contact and spreading at the maximum rate. Data shown are representative of 24 independent experiments, with an average of 20 samples/group.