Article Figures & Data
Figures
- FIGURE 1.
CTLs exhibit nonmonotonic activity in response to increasing Ag doses. (A) HLA-A2+ JY cells were pulsed with MART-1 peptide at various concentrations and radioactively labeled. Labeled peptide-pulsed targets cells were exposed to MART-1–specific JKF6 CTLs, and lysis was measured. Results are shown as the percentage of maximal killing observed. (B) Increase in relative inhibition was calculated with respect to optimal number of pMHC-I complexes/cell (100 complexes). The number of HLA-A2/peptide complexes deposited on the target cell surface was determined using a PE-conjugated TCR-like mAb, which specifically recognizes the HLA-A2/Mart-1 complex, and PE calibration curves, as described in Materials and Methods and Supplemental Fig. 1. Data are from 12 independent experiments (means, error bars represent SEM). *p < 0.05, paired Student t test.
- FIGURE 2.
The effect of Ag dose on intracellular calcium and RasGRP1 localization. (A) Calcium flux increases as a function of Ag dose. PKH26-stained HLA-A2+ JY cells were deposited with high (1200 complexes), optimal (100 complexes), or low (20 complexes) MART-1 pMHCs or left untreated. A baseline ratio of calcium-bound Indo-1/unbound Indo-1 was acquired, followed by coincubation with the JY APCs (left arrow). CTL:APC conjugates (double-positive events) were gated and analyzed for violet/blue ratio for 10 min. The right arrow indicates the addition of Ionomycin. (B) Calcium-positive cells were plotted against the number of encountered pMHC-I complexes on APCs. Results are representative of three independent experiments. Error bars represent SEM. (C) PD-1 expression in JKF6 T cells. The cytotoxic activity of JKF6 T cell clone was tested using an [35S]-release assay; CTLs were challenged with [35S]-labeled target cells pulsed with MART-127–35 peptide in a dose-dependent manner. Killing was normalized to the maximal killing detected. A parallel “cold” coculture was mounted, in which CD8+ T cells were gated and analyzed for PD-1 expression. Filled squares indicate relative killing in each pMHC-I dose. Bars indicate the percentage of PD-1+ cells in the CD8+ population. (D) Confocal imaging of RasGRP1 intracellular localization. Peptide-pulsed JY cells were incubated with JKF6 CTLs for 5 min, followed by fixation and staining with anti-RasGRP1. Blue, DAPI; green: anti-human GM130 (Golgi marker); red: anti-RasGRP1. Numbers indicate the dose of pMHC-I presented on target APCs; two representatives from each treatment are displayed.
- FIGURE 3.
Expression of anergy-related genes in hyporesponsive CTLs. Real-time RT-PCR of the relative expression (arbitrary units) of cbl-b, c-cbl, and Itch at 30 min (A) and 6 h (B) after coculture with target cells presenting 20, 100, or 1200 pMHC-I complexes. No dose-dependent elevation in expression was detected. (C) Gene array analysis of anergy-related genes. Gene expression in CTLs was determined and the ratio between expression upon encountering high- and optimal Ag dose was calculated. Analysis was performed 4, 16, and 36 h after encountering the target cells. Analyzed genes show no elevated expression at high pMHC-I doses. Error bars represent SEM.
- FIGURE 4.
Ag dose–dependent phosphorylation of major T cell–signaling components. HLA-A2+ JY cells were pulsed with increasing doses of MART peptide, washed, and cultured with MART-specific CTLs for 5 min (Materials and Methods). (A) Graphs show the gradually increasing phosphorylation of key TCR components in response to increasing Ag doses. Yellow and blue indicate increased and decreased phosphorylation, respectively. Numbers indicate the amount of pMHC-I complexes on target cells. Changes in phosphorylation of signaling proteins following activation of T cells were calculated using the inverse hyperbolic sine (arcsinh) of the MFI, with the first row of each lane used as phosphorylation reference, as previously described (50). (B) Relative phosphorylation was defined as the measured phosphorylation level of a specific protein divided by its maximal phosphorylation level under Ag saturation (see Materials and Methods). Relative phosphorylation was calculated after 5 min, and the average of eight independent experiments is shown. At 100 pMHC-I complexes, a significant elevation in relative phosphorylation is exhibited in ZAP-70, SLP-76, and LAT compared with CrkL, SHP-2, and c-cbl. Data are means; error bars represent SE. (C) The relative phosphorylation among activation molecules is significantly higher than that of inhibitory molecules at 100 pMHC complexes/cell. The activation-related molecules CD3, LAT, and SLP-76 show a significant increase in their relative phosphorylation compared with the inhibitory-related molecules SHP-2, c-cbl, and CrkL. Data are means; error bars represent SEM. (D) Dose-dependent incremental change in phosphorylation. pMHC dose-dependent incremental fold change in phosphorylation was determined by measuring the relative phosphorylation MFI of the indicated pMHC dose and dividing it by the MFI measured at previous lower dose of pMHC-I complexes. The transition from 50 pMHC-I complexes to 100 complexes generates a sharp increase in the phosphorylation of LAT, Lck, CD3, and ZAP-70. *p < 0.05 **p < 0.01, unpaired Student t test.
- FIGURE 5.
High-throughput analysis of T cell-signaling profiles by Mass-Cytometry. HLA-A2+ JY cells were pulsed with increasing number of pMHC-I complexes, and cultured with JKF6 CTLs for 2, 5 and 10 min. Subsequently, the cells were fixed, permeabilized and stained with metal-conjugated Abs (see Materials and Methods). (A), CD8+ cells were gated, and fold change increase in phosphorylation was calculated in relation to level of phosphorylation when encountered with APCs bearing no specific peptide. Note the change in the Y axis scale indicating the extent of relative fold changes across time points. (B), Mass cytometry results depicted as a heat map. Red colors indicate an increase in phosphorylation, whereas green indicate a decrease in signal. For intensities, see scale bars on the right. All values in a given row are relative to the value in the first column of that row. The data are of four individuals experiments combined. (C), Hierarchical unsupervised clustering for the fold-change phosphorylation across all doses and time points. Columns and rows correspond to analyzed molecules. Colors indicate the level of Pearson’s correlation in phosphorylation in relation to pMHC-I across all time points, where red indicates high positive correlation and green high negative correlation.
- FIGURE 6.
Integration of activatory and inhibitory signals. (A) An average of the relative phosphorylation of the activatory molecules (ZAP70, Lck, LAT, SLP-76) and the inhibitory molecules (SHP-2, c-cbl, CrkL) was calculated based on the experimental phospho-flow data obtained in 10 independent experiments. (B) The δ of the relative phosphorylation calculated for the activatory and inhibitory molecules, shown in arbitrary units. (C) SHP1/2 inhibition in CTLs exposed to high numbers of presented pMHC-I complexes abrogates Ag dose–induced unresponsiveness/anergy. HLA-A2+ JY cells were radioactively labeled, pulsed with various concentrations of MART-1, and cultured with SHP-1/2–inhibited JKF6 CTLs for 5 h. Cytotoxicity was assessed as described in Materials and Methods. Error bars represent SEM. *p < 0.05, paired Student t test.
- FIGURE 7.
Model for activatory and inhibitory signal summation by effector T cells. Upon interaction between a CTL and a target cell, Ag recognition by TCRs induces the phosphorylation of the ITAM of CD3 molecules, particularly the CD3ζ chains, by Lck kinase, leading to recruitment of ZAP70 and the subsequent activation of LAT and SLP-76. Simultaneously, inhibitory molecules are activated via phosphorylation. Below a threshold of 100 presented pMHC-I complexes, a relative activation of activatory molecules “outweigh” the net effect of the inhibitory molecules, and the intracellular balance and signal integration between these opposing signaling cascades is translated to an effector phenotype. When the number of presented pMHC-I complexes increases beyond this threshold, the relative phosphorylation of the inhibitory molecules increase, and the integration of signals generates an anergic phenotype.
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