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Dynamics of macrophage trogocytosis of rituximab-coated B cells.

Pham T, Mero P, Booth JW - PLoS ONE (2011)

Bottom Line: Inhibition of Src family kinase activity both slowed acquisition and reduced the extent of trogocytosis.The effects of inhibiting these kinases are likely due to their role in efficient formation of cell-cell conjugates.Understanding the mechanistic determinants of macrophage trogocytosis will be important for optimal design of antibody therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Macrophages can remove antigen from the surface of antibody-coated cells by a process termed trogocytosis. Using live cell microscopy and flow cytometry, we investigated the dynamics of trogocytosis by RAW264.7 macrophages of Ramos B cells opsonized with the anti-CD20 monoclonal antibody rituximab. Spontaneous and reversible formation of uropods was observed on Ramos cells, and these showed a strong enrichment in rituximab binding. RAW-Ramos conjugate interfaces were highly enriched in rituximab, and transfer of rituximab to the RAW cells in submicron-sized puncta occurred shortly after cell contact. Membrane from the target cells was concomitantly transferred along with rituximab to a variable extent. We established a flow cytometry-based approach to follow the kinetics of transfer and internalization of rituximab. Disruption of actin polymerization nearly eliminated transfer, while blocking phosphatidylinositol 3-kinase activity only resulted in a delay in its acquisition. Inhibition of Src family kinase activity both slowed acquisition and reduced the extent of trogocytosis. The effects of inhibiting these kinases are likely due to their role in efficient formation of cell-cell conjugates. Selective pre-treatment of Ramos cells with phenylarsine oxide blocked uropod formation, reduced enrichment of rituximab at cell-cell interfaces, and reduced the efficiency of trogocytic transfer of rituximab. Our findings highlight that dynamic changes in target cell shape and surface distribution of antigen may significantly influence the progression and extent of trogocytosis. Understanding the mechanistic determinants of macrophage trogocytosis will be important for optimal design of antibody therapies.

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Kinetic analysis of acquisition and internalization of RTX by flow cytometry.Internalization of acquired RTX was assessed by its inaccessibility to labelling with anti-human antibody. (A) RTX-Al488 coated Ramos cells were coincubated with RAW cells at 37°C for the times indicated. After staining with anti-human antibody, RAW cells were detached and identified by gating on CD11b+ events. Bottom right plot shows a schematic of the progression of RAW cells in the kinetic analysis of trogocytosis. Indicated numbers are percentage of events in each quadrant out of the DN, DP, and SP populations. (B) The proportion of RAW cells in the DN population was fitted to a model as described in Methods. (C) Confocal slices of cells representative of trogocytic stages. Scale bar 10 µm.
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pone-0014498-g006: Kinetic analysis of acquisition and internalization of RTX by flow cytometry.Internalization of acquired RTX was assessed by its inaccessibility to labelling with anti-human antibody. (A) RTX-Al488 coated Ramos cells were coincubated with RAW cells at 37°C for the times indicated. After staining with anti-human antibody, RAW cells were detached and identified by gating on CD11b+ events. Bottom right plot shows a schematic of the progression of RAW cells in the kinetic analysis of trogocytosis. Indicated numbers are percentage of events in each quadrant out of the DN, DP, and SP populations. (B) The proportion of RAW cells in the DN population was fitted to a model as described in Methods. (C) Confocal slices of cells representative of trogocytic stages. Scale bar 10 µm.

Mentions: It is clear from microscopy that the RTX is not only transferred to the RAW cell but becomes internalized, as was also seen in earlier studies of macrophage trogocytosis [12]. We wanted to analyze this process by flow cytometry and differentiate between internalized and surface-exposed RTX over time. To this end, we incorporated labelling of surface-exposed RTX on RAW cells with anti-human antibodies (recognizing the Fc portion of this chimeric antibody). After incubation together, RAW cells were washed, detached, and then distinguished from remaining Ramos cells by staining for CD11b. Cells were then incubated with anti-human antibodies. Using this analysis, three populations of RAW cells were observed to develop over time. Initially, the RAW cells were negative for both RTX and anti-human antibody (Figure 6A; DN quadrant). Then over time a population arose that was positive for both RTX and anti-human (Figure 6A, DP quadrant). This represents trogocytic intermediates in which transfer but not internalization of RTX has occurred; these may either be free RAW cells that have detached a piece(s) of Ramos cells, or the RAW cell halves of RAW-Ramos conjugates that are dissociated in the process of detachment and resuspension of the RAW cells (few if any conjugates remained intact after RAW detachment (Figure S1A)). In either case, appearance of this population represents a first step in which RTX becomes associated with the RAW cell prior to its internalization. Finally, double-positive cells disappeared and RTX single-positive cells appeared (Figure 6A, SP quadrant), as RTX became internalized into the RAW cell and was no longer accessible to the added anti-human antibody. Thus, this analysis highlights that trogocytosis by macrophages can be thought of as involving two sequential steps: conjugate formation and antigen internalization.


Dynamics of macrophage trogocytosis of rituximab-coated B cells.

Pham T, Mero P, Booth JW - PLoS ONE (2011)

Kinetic analysis of acquisition and internalization of RTX by flow cytometry.Internalization of acquired RTX was assessed by its inaccessibility to labelling with anti-human antibody. (A) RTX-Al488 coated Ramos cells were coincubated with RAW cells at 37°C for the times indicated. After staining with anti-human antibody, RAW cells were detached and identified by gating on CD11b+ events. Bottom right plot shows a schematic of the progression of RAW cells in the kinetic analysis of trogocytosis. Indicated numbers are percentage of events in each quadrant out of the DN, DP, and SP populations. (B) The proportion of RAW cells in the DN population was fitted to a model as described in Methods. (C) Confocal slices of cells representative of trogocytic stages. Scale bar 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3022012&req=5

pone-0014498-g006: Kinetic analysis of acquisition and internalization of RTX by flow cytometry.Internalization of acquired RTX was assessed by its inaccessibility to labelling with anti-human antibody. (A) RTX-Al488 coated Ramos cells were coincubated with RAW cells at 37°C for the times indicated. After staining with anti-human antibody, RAW cells were detached and identified by gating on CD11b+ events. Bottom right plot shows a schematic of the progression of RAW cells in the kinetic analysis of trogocytosis. Indicated numbers are percentage of events in each quadrant out of the DN, DP, and SP populations. (B) The proportion of RAW cells in the DN population was fitted to a model as described in Methods. (C) Confocal slices of cells representative of trogocytic stages. Scale bar 10 µm.
Mentions: It is clear from microscopy that the RTX is not only transferred to the RAW cell but becomes internalized, as was also seen in earlier studies of macrophage trogocytosis [12]. We wanted to analyze this process by flow cytometry and differentiate between internalized and surface-exposed RTX over time. To this end, we incorporated labelling of surface-exposed RTX on RAW cells with anti-human antibodies (recognizing the Fc portion of this chimeric antibody). After incubation together, RAW cells were washed, detached, and then distinguished from remaining Ramos cells by staining for CD11b. Cells were then incubated with anti-human antibodies. Using this analysis, three populations of RAW cells were observed to develop over time. Initially, the RAW cells were negative for both RTX and anti-human antibody (Figure 6A; DN quadrant). Then over time a population arose that was positive for both RTX and anti-human (Figure 6A, DP quadrant). This represents trogocytic intermediates in which transfer but not internalization of RTX has occurred; these may either be free RAW cells that have detached a piece(s) of Ramos cells, or the RAW cell halves of RAW-Ramos conjugates that are dissociated in the process of detachment and resuspension of the RAW cells (few if any conjugates remained intact after RAW detachment (Figure S1A)). In either case, appearance of this population represents a first step in which RTX becomes associated with the RAW cell prior to its internalization. Finally, double-positive cells disappeared and RTX single-positive cells appeared (Figure 6A, SP quadrant), as RTX became internalized into the RAW cell and was no longer accessible to the added anti-human antibody. Thus, this analysis highlights that trogocytosis by macrophages can be thought of as involving two sequential steps: conjugate formation and antigen internalization.

Bottom Line: Inhibition of Src family kinase activity both slowed acquisition and reduced the extent of trogocytosis.The effects of inhibiting these kinases are likely due to their role in efficient formation of cell-cell conjugates.Understanding the mechanistic determinants of macrophage trogocytosis will be important for optimal design of antibody therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Macrophages can remove antigen from the surface of antibody-coated cells by a process termed trogocytosis. Using live cell microscopy and flow cytometry, we investigated the dynamics of trogocytosis by RAW264.7 macrophages of Ramos B cells opsonized with the anti-CD20 monoclonal antibody rituximab. Spontaneous and reversible formation of uropods was observed on Ramos cells, and these showed a strong enrichment in rituximab binding. RAW-Ramos conjugate interfaces were highly enriched in rituximab, and transfer of rituximab to the RAW cells in submicron-sized puncta occurred shortly after cell contact. Membrane from the target cells was concomitantly transferred along with rituximab to a variable extent. We established a flow cytometry-based approach to follow the kinetics of transfer and internalization of rituximab. Disruption of actin polymerization nearly eliminated transfer, while blocking phosphatidylinositol 3-kinase activity only resulted in a delay in its acquisition. Inhibition of Src family kinase activity both slowed acquisition and reduced the extent of trogocytosis. The effects of inhibiting these kinases are likely due to their role in efficient formation of cell-cell conjugates. Selective pre-treatment of Ramos cells with phenylarsine oxide blocked uropod formation, reduced enrichment of rituximab at cell-cell interfaces, and reduced the efficiency of trogocytic transfer of rituximab. Our findings highlight that dynamic changes in target cell shape and surface distribution of antigen may significantly influence the progression and extent of trogocytosis. Understanding the mechanistic determinants of macrophage trogocytosis will be important for optimal design of antibody therapies.

Show MeSH
Related in: MedlinePlus