Article Figures & Data
Figures
- Figure 1.
The role of PI3K/ERK and PLC/PKC signaling pathways in CD103-dependent T-cell clone–mediated lysis. A, surface expression of αE(CD103)β7 integrin on 2 tumor-specific T-cell clones derived from a lung cancer patient TIL (Heu171) and PBL (H32-8). Immunofluorescence analysis was done with anti-CD103 (black fill) mAb or an isotypic control (without fill). Mean fluorescence intensity values are in parentheses. B, the cytotoxic activity of the Heu171 and H32-8 clones toward the specific IGR–Heu tumor cell line was determined by a conventional 4-hour 51Cr-release assay at indicated E:T ratios. The T-cell clones were preincubated either in a medium or with indicated concentrations of PI3K inhibitor Wortmannin, ERK1/2 inhibitor U0126, PKC inhibitor piceatannol, PLCγ inhibitors U-73122 and U-73343 inactive form, or a vehicle control. Data shown are representative of 3 independent experiments.
- Figure 2.
Engagement of immobilized E-cadherin-Fc with its ligand on CTL clones results in the activation of ERK1/2 and PLCγ signaling pathways. A, Western blot analysis of ERK1/2 and PLCγ1 protein phosphorylation following the stimulation of CD103high Heu171 and CD103low H32-8 clones with plastic-coated rE-cadherin-Fc or autologous tumor cells. Top panels: total protein extracts were prepared from effector cells incubated with IGR-Heu (at 5:1 E:T ratio) or plastic-coated E-cadherin-Fc (5 μg/mL) for the indicated time-points. Actin was used as a loading control. Bottom panels: the normalization of phosphorylated proteins relative to total proteins. Data shown represent 1 of 3 independent experiments. B, the potentiation of ERK1/2 phosphorylation following the coengagement of CD103 and TCR. Top panels: the H32-8 clone was incubated in the presence of anti-CD3 mAb (1 μg/mL), immobilized E-cadherin-Fc (5 μg/mL), or a combination of both reagents for indicated times. An IgG1 isotypic control (1 μg/mL) was included. Cell extracts were analyzed by Western blot using the specified Ab. Actin was used as a loading control. Bottom panels: the normalization of phosphorylated proteins relative to total proteins. Data shown correspond to 1 representative experiment of 3. E-cadh, E-cadherin.
- Figure 3.
Minimal interaction of the αEβ7 integrin on the TIL clone with rE-cadherin–coated beads is sufficient to induce lytic granule polarization. A, conjugates formed between the Heu171 TIL clone and E-cadherin–coated beads were analyzed by confocal microscopy after 15 minutes of incubation. Granule polarization, as defined by the accumulation of granzyme B in the contact area between effector T cells and E-cadherin–coated beads, was followed up with anti-granzyme B mAb (green fluorescence). Nuclei were stained with TO-PRO-3 iodide (blue fluorescence) and polymerized actin with rhodamin–phalloidin (red fluorescence). Scale bars, 10 μm. B, time-lapse gallery (intervals of approximately 150 seconds) of lysosome polarization in the presence or absence of U-73122 (0.2 μmol/L), U-73343 (0.2 μmol/L), or a vehicle control. The Heu171 TIL clone was labeled with a LysoTracker Red probe and conjugates formed with E-cadherin-Fc–coated or -uncoated beads were followed up by confocal microscopy. Scale bars, 10 μm. Arrows point to lysosome polarization at the contact area between CTL and rE-cadherin–coated beads. C, percentages of CTL displaying lysosome relocalization during conjugate formation between the Heu171 and rE-cadherin–coated beads, after preincubation of T-cell clones with U-73122, U-73343, or a vehicle control. Data show mean ± SD of 4 different fields and are representative of 3 independent experiments. Numbers (n) of analyzed conjugates are indicated. E-cadh, E-cadherin
- Figure 4.
Ligation of CD103 on freshly isolated CD8+ TIL with rE-cadherin–coated beads induces lytic granule polarization A, phenotypic analysis of freshly isolated TIL. Immunofluorescence analysis was done with anti-CD3, anti-CD103, anti-CD8, and anti-granzyme B mAb. SSC, side scatter; FSC, forward scatter. B, freshly isolated TIL were labeled with a LysoTracker Red probe and conjugates formed with E-cadherin–coated beads were followed up by confocal microscopy. Time-lapse gallery (intervals of approximately 150 seconds) of lysosome polarization in the presence of uncoated beads (top) or E-cadherin-Fc–coated beads (bottom). Scale bars, 10 μm. Bead aggregates most likely result from the homotypic bonds between adjacent E-cadherin–coated beads. Arrows indicate the granule relocalization at the contact zone between TIL and rE-cadherin–coated beads. The data shown correspond to 1 experiment of 3. C, analysis of CD103+ TIL behavior within a fresh human lung tumor specimen. Tumor slices were stained with a combination of anti-granzyme B (green fluorescence), anti-CD103 (red fluorescence) mAb, and SYTOX Green (blue fluorescence, nuclei). Scale bar, 10 μm. Data shown correspond to 1 representative experiment of 3.
- Figure 5.
Engagement of both CD103 and TCR is required for lytic granule exocytosis. A, CD107a externalization on the surface of Heu171 CTL cocultured with the indicated concentration of immobilized E-cadherin-Fc, suboptimal concentrations of anti-CD3 mAb or a combination of both reagents. Immunofluorescence analyses were done at indicated time-points. B, the involvement of PI3K/ERK and PLC/PKC pathways in cytotoxic granule exocytosis. The Heu171 clone was incubated with indicated suboptimal concentrations of surface-bound anti-CD3 mAb or a combination of immobilized anti-CD3 mAb and E-cadherin-Fc (2.5 μg/mL), in medium alone, with vehicle control, or in the presence of indicated inhibitors. CD107a induction was monitored by immunofluorescence analysis. Data shown are representative of 3 independent experiments. E-cadh, E-cadherin.
Additional Files
Supplementary Data, Le Floc'h, et al.
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Volume 71, Issue 2
