Figure 4
Figure 4. The differential sensitivity of T- and B-cell trogocytosis to inhibitors is not due to differences in affinities of the TCR and BCR for their respective ligands. (A) DO11.10 CD4+ T cells (left panels), OT-I CD8+ T cells (middle panels), and MD4 B cells (right panels) were used in a redirected trogocytosis assay against PKH67-labeled P815 target cells triggered by either the 2C11 anti-CD3 mAb (open histograms for T cells, and closed for B cells) or the anti-BCR κ chain mAb (open histograms for B cells, and closed for T cells). The effector cells were treated with 25 μM latrunculin B before and during coculture with the P815 target cells (bottom panels) or left untreated (top panels). Graphs show the levels of PKH67 fluorescence on gated effector cells. Similar results were obtained in 3 independent experiments. (B) The percentages of OT-I CD8+ T cells (top panel) or MD4 B cells (bottom panel) found conjugated to P815 target cells in the absence of mAb (gray) or in the presence (black) of 2C11 anti-CD3 mAb (for OT-I T cells) or anti-BCR κ chain mAb (for MD4 B cells) was calculated as in Figure 2. Bars represent standard deviations; n = 4. Levels of statistical significance were calculated using the Student t test; **P < .01 and ***P < .001. (C) MD4 B cells were exposed to the following target cells labeled with the fluorescent lipophilic dye DiI: HEK (closed histograms), HEKmHELWT (open histograms), and HEKmHELK97A (gray line histograms). Effector cells were either left untreated (top panels), or treated for 20 minutes at 37°C with 10 μM PP2 (bottom panels) or placed at 4°C (middle, horizontal panels) before coculture with target cells. After 1 hour of coculture at 37°C (top and bottom panels) or at 4°C (middle panels), we measured the capture of membrane components (DiI; left panels) and of mHEL (biotinylated F10.6.6 mAb + fluorescent streptavidin; right, vertical panels). Similar results were obtained in 4 independent experiments. (D) Splenocytes from 3.83 mice were exposed for 1 hour at 37°C to PKH67-labeled splenocytes from Balb/c (no affinity, left panel), C57/BL6 (weak affinity, middle panel), or C3H/He (high affinity, right panel) before analysis by flow cytometry. These respective affinities for the indicated H-2 antigen were reported in.25 B220+ and B220− splenocytes (donor cells) (PKH67bright) fall within the right quadrants, while the B220+ and B220− 3.83 splenocytes (recipient cells) occupy the left quadrants. Numbers represent trogocytosis indexes (TIs) calculated as indicated below. (E) As in panel D except that the indicated inhibitors were added during trogocytosis. Trogocytosis indexes were calculated as follows: mfi on recipient B220+ cell in the upper left quadrant (B cells)/mfi on recipient B220− cells in the lower left quadrant (non-B cells). Similar results were obtained in 3 independent experiments.

The differential sensitivity of T- and B-cell trogocytosis to inhibitors is not due to differences in affinities of the TCR and BCR for their respective ligands. (A) DO11.10 CD4+ T cells (left panels), OT-I CD8+ T cells (middle panels), and MD4 B cells (right panels) were used in a redirected trogocytosis assay against PKH67-labeled P815 target cells triggered by either the 2C11 anti-CD3 mAb (open histograms for T cells, and closed for B cells) or the anti-BCR κ chain mAb (open histograms for B cells, and closed for T cells). The effector cells were treated with 25 μM latrunculin B before and during coculture with the P815 target cells (bottom panels) or left untreated (top panels). Graphs show the levels of PKH67 fluorescence on gated effector cells. Similar results were obtained in 3 independent experiments. (B) The percentages of OT-I CD8+ T cells (top panel) or MD4 B cells (bottom panel) found conjugated to P815 target cells in the absence of mAb (gray) or in the presence (black) of 2C11 anti-CD3 mAb (for OT-I T cells) or anti-BCR κ chain mAb (for MD4 B cells) was calculated as in Figure 2. Bars represent standard deviations; n = 4. Levels of statistical significance were calculated using the Student t test; **P < .01 and ***P < .001. (C) MD4 B cells were exposed to the following target cells labeled with the fluorescent lipophilic dye DiI: HEK (closed histograms), HEKmHELWT (open histograms), and HEKmHELK97A (gray line histograms). Effector cells were either left untreated (top panels), or treated for 20 minutes at 37°C with 10 μM PP2 (bottom panels) or placed at 4°C (middle, horizontal panels) before coculture with target cells. After 1 hour of coculture at 37°C (top and bottom panels) or at 4°C (middle panels), we measured the capture of membrane components (DiI; left panels) and of mHEL (biotinylated F10.6.6 mAb + fluorescent streptavidin; right, vertical panels). Similar results were obtained in 4 independent experiments. (D) Splenocytes from 3.83 mice were exposed for 1 hour at 37°C to PKH67-labeled splenocytes from Balb/c (no affinity, left panel), C57/BL6 (weak affinity, middle panel), or C3H/He (high affinity, right panel) before analysis by flow cytometry. These respective affinities for the indicated H-2 antigen were reported in.25  B220+ and B220− splenocytes (donor cells) (PKH67bright) fall within the right quadrants, while the B220+ and B220− 3.83 splenocytes (recipient cells) occupy the left quadrants. Numbers represent trogocytosis indexes (TIs) calculated as indicated below. (E) As in panel D except that the indicated inhibitors were added during trogocytosis. Trogocytosis indexes were calculated as follows: mfi on recipient B220+ cell in the upper left quadrant (B cells)/mfi on recipient B220− cells in the lower left quadrant (non-B cells). Similar results were obtained in 3 independent experiments.

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