Figure 1
Figure 1. The differential effect of inhibitors and temperature on T-cell and B-cell trogocytosis. (A) DO11.10 CD4+ T cells (left panels), OT-I CD8+ T cells (middle panels), and MD4 B cells (right panels) were exposed in the presence or absence of 25 μM latrunculin B (Lat. B) to their respective biotinylated target cells that either expressed, on their surface, the appropriate antigen (open histograms) or not (closed histograms). After 1 hour at 37°C, conjugates were dissociated and lymphocytes were stained with fluorescent streptavidin to detect capture of biotinylated components, and with lineage-specific mAbs. Graphs show the level of biotin staining on gated effector cells. (B) Quantification of trogocytosis efficiency performed by lymphocytes in the presence or absence of 25 μM latrunculin B, 10 μM cytochalasin D (Cyt. D), 100 nM nocodazole (Noco.), 10 μM PP2, 100 μM piceatannol (Pice.), and 100 nM wortmannin (Wort.). Residual trogocytosis in the presence of the indicated inhibitors was calculated using the formula described in Document S1. DO11.10 CD4+ T cells were not tested in presence of cytochalasin D and nocodazole. Bars represent the standard deviations; n = 5 for T CD4+ cells and n > 6 for T CD8+ cells and B cells. Levels of statistical significance were calculated using the Student t test; **P < .01. (C) As in panel A, except that 10 μM PP2 was used instead of latrunculin B. The data are from experiments in which all cells were treated in parallel with the same aliquots of the indicated inhibitors. (D) OT-I CD8+ T cells (squares) and MD4 B cells (diamonds) were analyzed as in panel A except that a range of concentrations of latrunculin B (top panel) or PP2 (bottom panel) was used. Residual trogocytosis was calculated as in panel B. Similar results were obtained in 4 independent experiments. (E) As in panel A except that cocultures were incubated at 37°C or at 4°C rather than with or without inhibitor. Note that for experiments performed at 4°C, we used an effector to target ratio of 15:1 to ensure that all B cells were in contact with a target cell.

The differential effect of inhibitors and temperature on T-cell and B-cell trogocytosis. (A) DO11.10 CD4+ T cells (left panels), OT-I CD8+ T cells (middle panels), and MD4 B cells (right panels) were exposed in the presence or absence of 25 μM latrunculin B (Lat. B) to their respective biotinylated target cells that either expressed, on their surface, the appropriate antigen (open histograms) or not (closed histograms). After 1 hour at 37°C, conjugates were dissociated and lymphocytes were stained with fluorescent streptavidin to detect capture of biotinylated components, and with lineage-specific mAbs. Graphs show the level of biotin staining on gated effector cells. (B) Quantification of trogocytosis efficiency performed by lymphocytes in the presence or absence of 25 μM latrunculin B, 10 μM cytochalasin D (Cyt. D), 100 nM nocodazole (Noco.), 10 μM PP2, 100 μM piceatannol (Pice.), and 100 nM wortmannin (Wort.). Residual trogocytosis in the presence of the indicated inhibitors was calculated using the formula described in Document S1. DO11.10 CD4+ T cells were not tested in presence of cytochalasin D and nocodazole. Bars represent the standard deviations; n = 5 for T CD4+ cells and n > 6 for T CD8+ cells and B cells. Levels of statistical significance were calculated using the Student t test; **P < .01. (C) As in panel A, except that 10 μM PP2 was used instead of latrunculin B. The data are from experiments in which all cells were treated in parallel with the same aliquots of the indicated inhibitors. (D) OT-I CD8+ T cells (squares) and MD4 B cells (diamonds) were analyzed as in panel A except that a range of concentrations of latrunculin B (top panel) or PP2 (bottom panel) was used. Residual trogocytosis was calculated as in panel B. Similar results were obtained in 4 independent experiments. (E) As in panel A except that cocultures were incubated at 37°C or at 4°C rather than with or without inhibitor. Note that for experiments performed at 4°C, we used an effector to target ratio of 15:1 to ensure that all B cells were in contact with a target cell.

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