Figure 6
Figure 6. ALM interferes with down-regulation of CD20 by phagocytic cells through depletion of their trogocytic capacity. (A) When incubated with RTX-FITC (top) or ALM-FITC (bottom) coated ALL-CM cells, monocytes labeled with the red membrane dye PKH-26 rapidly internalized antibodies from the leukemic cells as illustrated by the transfer of green signal into the monocytes. (B) When CD14 FITC-labeled monocytes were incubated with PKH-labeled ALL-CM cells coated with unconjugated RTX, the monocytes rapidly internalized red signal indicating membrane transfer, or trogocytosis (top). In contrast, trogocytosis did not occur when monocytes were incubated with ALL-CM cells coated with unconjugated ALM (bottom). (C) Uncoated, RTX-FITC–coated, or ALM-FITC–coated ALL-CM cells were incubated in the absence or presence of excess (200 μg/mL) RTX-FITC or ALM-FITC, and in the absence or presence of monocytes as indicated. After 45 minutes, cells were analyzed by flow cytometry. In the absence of excess RTX and the presence of monocytes, fluorescence of initially RTX-coated ALL cells was decreased compared with controls, confirming that RTX-FITC had been removed from the cell surface by monocytes, and fluorescence remained low in the presence of excess RTX-FITC, confirming that CD20 had simultaneously been removed (top). Incubation with monocytes alone also reduced fluorescence of initially ALM-FITC–coated cells, confirming that ALM-FITC, like RTX-FITC, was removed from the surface by monocytes. However, in the presence of excess ALM-FITC, fluorescence remained at normal levels, indicating that CD52 itself remained present (bottom). (D) Monocytes were first incubated with ALL cells in the absence or presence of ALM for 45 minutes, after which RTX-FITC–labeled ALL cells were added to assess remaining trogocytic capacity. Preincubation with ALL cells only did not affect the trogocytic capacity of monocytes, as internalization of RTX-FITC occurred as seen before (top). In contrast, preincubation with ALL cells and excess ALM-depleted the trogocytic capacity as no internalization of RTX-FITC was detected (bottom).

ALM interferes with down-regulation of CD20 by phagocytic cells through depletion of their trogocytic capacity. (A) When incubated with RTX-FITC (top) or ALM-FITC (bottom) coated ALL-CM cells, monocytes labeled with the red membrane dye PKH-26 rapidly internalized antibodies from the leukemic cells as illustrated by the transfer of green signal into the monocytes. (B) When CD14 FITC-labeled monocytes were incubated with PKH-labeled ALL-CM cells coated with unconjugated RTX, the monocytes rapidly internalized red signal indicating membrane transfer, or trogocytosis (top). In contrast, trogocytosis did not occur when monocytes were incubated with ALL-CM cells coated with unconjugated ALM (bottom). (C) Uncoated, RTX-FITC–coated, or ALM-FITC–coated ALL-CM cells were incubated in the absence or presence of excess (200 μg/mL) RTX-FITC or ALM-FITC, and in the absence or presence of monocytes as indicated. After 45 minutes, cells were analyzed by flow cytometry. In the absence of excess RTX and the presence of monocytes, fluorescence of initially RTX-coated ALL cells was decreased compared with controls, confirming that RTX-FITC had been removed from the cell surface by monocytes, and fluorescence remained low in the presence of excess RTX-FITC, confirming that CD20 had simultaneously been removed (top). Incubation with monocytes alone also reduced fluorescence of initially ALM-FITC–coated cells, confirming that ALM-FITC, like RTX-FITC, was removed from the surface by monocytes. However, in the presence of excess ALM-FITC, fluorescence remained at normal levels, indicating that CD52 itself remained present (bottom). (D) Monocytes were first incubated with ALL cells in the absence or presence of ALM for 45 minutes, after which RTX-FITC–labeled ALL cells were added to assess remaining trogocytic capacity. Preincubation with ALL cells only did not affect the trogocytic capacity of monocytes, as internalization of RTX-FITC occurred as seen before (top). In contrast, preincubation with ALL cells and excess ALM-depleted the trogocytic capacity as no internalization of RTX-FITC was detected (bottom).

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