Dynamics of macrophage trogocytosis of rituximab-coated B cells.
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.
Department of Immunology, University of Toronto, Toronto, Ontario, Canada.
Theodore
T
Pham
Patricia
P
Mero
James W
JW
Booth
D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology*; D018951:Antigens, CD20 / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D002460:Cell Line; D048430:Cell Shape; D005434:Flow Cytometry / Q000379:methods; D007700:Kinetics; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D009895:Opsonin Proteins / Q000276:immunology*; D000069283:Rituximab
D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D009895:Opsonin Proteins; D000069283:Rituximab
J Leukoc Biol. 2004 Dec;76(6):1093-103
15466916
PLoS One. 2008 Aug 29;3(8):e3097
18769733
Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11613-8
18697944
PLoS One. 2010 Jan 14;5(1):e8716
20090930
Curr Opin Immunol. 2002 Feb;14(1):136-45
11790544
Nat Clin Pract Rheumatol. 2007 Feb;3(2):86-95
17299446
Cell Mol Life Sci. 2010 Apr;67(7):1133-45
20238479
J Immunol. 2002 Sep 15;169(6):2886-91
12218101
J Biol Chem. 1999 Jan 15;274(3):1240-7
9880492
J Biol Chem. 2003 Jan 31;278(5):3331-8
12424246
J Immunol. 2007 Dec 1;179(11):7967-74
18025245
Blood. 2008 Jun 15;111(12):5621-8
18381976
Blood. 2004 Nov 1;104(9):2635-42
15226177
EMBO J. 2002 Feb 1;21(3):251-8
11823418
Nat Rev Drug Discov. 2003 Jan;2(1):52-62
12509759
J Immunol. 2006 Nov 15;177(10):7435-43
17082663
J Immunol. 2008 Dec 1;181(11):8120-32
19018005
Science. 1999 Oct 29;286(5441):952-4
10542149
J Exp Med. 1975 Nov 1;142(5):1263-82
1194852
J Immunol. 2008 Aug 15;181(4):2271-5
18684915
J Immunol. 2007 Mar 15;178(6):3418-26
17339436
J Immunol. 2009 Nov 15;183(10):6102-13
19841164
J Immunol. 2001 Mar 15;166(6):3645-9
11238601
J Immunol. 2010 Feb 15;184(4):1897-908
20089699
J Biol Chem. 2009 Oct 30;284(44):30288-97
19740737
Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):4930-4
16549762
J Cell Biol. 2001 Jun 25;153(7):1369-80
11425868
J Exp Med. 1976 Sep 1;144(3):788-809
1085341
Nature. 2001 May 24;411(6836):489-94
11373683
Nat Immunol. 2003 Sep;4(9):815
12942076
J Biol Chem. 2006 Nov 3;281(44):33242-9
16959774
J Immunol. 2006 Feb 15;176(4):2600-9
16456022
J Immunol. 2008 Aug 15;181(4):2916-24
18684983