Cells and Monocytes Promotes Trogocytosis Mediated by THP-1

Binding of Rituximab, Trastuzumab, Cetuximab, or mAb T101 to Cancer Cells Promotes Trogocytosis Mediated by THP-1 Cells and Monocytes This information is current as of October 1, 2021. Paul V. Beum, David A. Mack, Andrew W. Pawluczkowycz, Margaret A. Lindorfer and Ronald P. Taylor J Immunol 2008; 181:8120-8132; ; doi: 10.4049/jimmunol.181.11.8120

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Binding of Rituximab, Trastuzumab, Cetuximab, or mAb T101 to Cancer Cells Promotes Trogocytosis Mediated by THP-1 Cells and Monocytes1

Paul V. Beum,* David A. Mack,† Andrew W. Pawluczkowycz,* Margaret A. Lindorfer,* and Ronald P. Taylor2*

More than 20 years ago clinical investigations in the immunotherapy of cancer revealed that infusion of certain immunotherapeutic mAbs directed to tumor cells induced loss of targeted epitopes. This phenomenon, called antigenic modulation, can com- promise mAb-based therapies. Recently we reported that rituximab (RTX) treatment of chronic lymphocytic leukemia patients induced substantial loss of targeted CD20 on B cells found in the circulation after RTX infusion; this “shaving” of RTX-CD20 complexes from B cells is also promoted in vitro by THP-1 monocytes and by PBMC in a reaction mediated by Fc␥ receptors. The mechanism responsible for shaving appears to be trogocytosis, a process in which receptors on effector cells remove and internalize Downloaded from cognate ligands and cell membrane fragments from target cells. We now report that three therapeutic mAbs approved by the U.S. Food and Drug Administration for the treatment of cancer, RTX, cetuximab, and trastuzumab, as well as mAb T101, which has been shown to induce antigenic modulation in the clinic, promote trogocytosis in vitro upon binding to their respective target cells. Trogocytosis of the mAb-opsonized cells is mediated by THP-1 monocytes and by primary monocytes isolated from PBMC. In view of these results, it is likely that these mAbs and possibly other anticancer mAbs now used in the clinic may promote trogocytic removal of the therapeutic mAbs and their cognate Ags from tumor cells in vivo. Our ﬁndings may have important implications http://www.jimmunol.org/ with respect to the use of mAbs in cancer immunotherapy. The Journal of Immunology, 2008, 181: 8120–8132.

ecognition of ligands on target cells by cognate receptors doses (6, 7). Soon after completion of RTX infusion, substantial on acceptor cells such as B cells, T cells, or NK cells can numbers of viable malignant B cells are demonstrable in the blood- R lead to transfer of the ligand and closely associated mem- stream. These cells have markedly reduced levels of CD20 and brane fragments from the target cell to the acceptor cell (1–4). In have very little bound RTX. Moreover, these B cells are tagged this endocytic reaction, called trogocytosis or nibbling, both the covalently with C3dg, indicating that during the infusion the cells

captured ligand and its receptor on the acceptor cell are internal- must have been previously opsonized with RTX, which promotes by guest on October 1, 2021 ized. We have recently demonstrated that a similar transfer occurs complement activation and deposition of C3b activation frag- in vitro when B cells opsonized with anti-CD20 mAb rituximab ments. We have also demonstrated in several in vitro models that 3 (RTX) are reacted with either PBMC or monocytic THP-1 cells this loss of CD20 and bound RTX can occur in the complete ab- (5). In this case the RTX-CD20 immune complexes (IC) on the sence of complement, thus precluding a causative role for C3b/ ␥ target cell serve as ligands for Fc RI on the acceptor cell. C3dg in the CD20 loss (5, 8). This removal of cell-bound RTX and We established this in vitro system as a model to investigate the CD20 by acceptor cells, which we termed “shaving”, is quite sim- mechanism of loss of bound RTX and CD20 from targeted circu- ilar to trogocytosis and may be responsible for the phenomenon of lating malignant B cells, which occurs in vivo when patients with antigenic modulation seen in earlier cancer immunotherapy studies chronic lymphocytic leukemia (CLL) are treated with doses of 2 2 (9–14). For example, treatment of T cell lymphoma patients with RTX ranging from as low as 60 mg/m up to the usual 375 mg/m the anti-CD5 mAb T101 led to rapid and profound reduction in the level of CD5 on malignant T cells in the bloodstream (14). The mechanism responsible for this antigenic modulation was not *Department of Biochemistry and Molecular Genetics and †Hematology/Oncology Division, University of Virginia School of Medicine, Charlottesville, VA 22908 known, but our studies of shaving and those of other investigators Received for publication June 12, 2008. Accepted for publication October 2, 2008. on trogocytosis provide a likely mechanism for the observed loss ␥ The costs of publication of this article were defrayed in part by the payment of page of CD5, namely removal of the T101/CD5 IC by Fc R-expressing charges. This article must therefore be hereby marked advertisement in accordance acceptor cells. All of these reactions, that is, trogocytosis, shaving, with 18 U.S.C. Section 1734 solely to indicate this fact. and antigenic modulation, may share a common pathway for pro- 1 This research was supported through a grant to the University of Virginia Cancer cessing of targeted ligands removed from donor cells and taken up Center from the James and Rebecca Craig Foundation, by a grant from CLL (Chronic Lymphocytic Leukemia) Topics, and by the University of Virginia Cancer Center by acceptor cells. Support Grant. To further characterize shaving of RTX-CD20 complexes and 2 Address correspondence and reprint requests to Dr. Ronald P. Taylor, Department the relationship of this reaction to trogocytosis, we used the mem- of Biochemistry and Molecular Genetics, P.O. Box 800733, University of Virginia, Charlottesville, VA 22908. E-mail address: [email protected] brane dye PKH26 to test for the transfer of membrane fragments from RTX-opsonized donor cells to acceptor THP-1 cells. Multi- 3 Abbreviations used in this paper: RTX, rituximab; Al, Alexa; bt, biotinylated; CET, cetuximab; CLL, chronic lymphocytic leukemia; Gt, goat; IC, immune complex(es); spectral image analysis and flow cytometry experiments revealed MESF, molecules of equivalent soluble fluorochome; Ms, mouse; RA, retinoic acid; that both RTX and PKH26 are taken up by the acceptor cells, and SA, streptavidin; TRA, trastuzumab. this finding extends our previous work in this system (5). We in- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 vestigated the possible generality of these reactions with respect to www.jimmunol.org The Journal of Immunology 8121

the use of other immunotherapeutic mAbs in targeting malignant 5% CO2. Zero-time control samples, consisting only of opsonized or naive cells. Flow cytometry and fluorescence microscopy were em- donor cells in RPMI 1640 medium, were subjected to identical conditions. ployed to examine three different mAb-opsonized/donor cell pairs: After these incubations the samples were placed on ice for 10 min, and then the zero-time control donor cells were added to the THP-1 cells and all mAb T-101/MOLT-4 cells; trastuzumab (TRA, used in the treat- samples were immediately quenched by addition of cold BSA/PBS. All ment of breast cancer (15, 16))/BT-474 cells; and cetuximab (CET, experiments were performed in duplicate. In some experiments, THP-1 used in the treatment of colorectal and other cancers (17, 18))/ cells were preincubated for 60 min at 37°C with 2 mg/ml human IgG or SCC-25 cells. In all three cases we observe similar reactions that with 30 ␮g/ml anti-Fc␥R mAbs, and then used in the trogocytosis reactions following the above procedures. Blocking mAbs specific for Fc␥RI (mAb closely follow the tenets defined for trogocytosis: transfer of donor 10.1) and Fc␥RII (mAb IV.3) were used at concentrations (30 ␮g/ml) that cell-bound mAb/target Ag IC and membrane fragments to the ac- were previously demonstrated to lead to saturation of binding to these ceptor cells, as well as internalization of acceptor cell Fc␥RI. receptors (23–26). After the trogocytosis reaction, the donor and acceptor cells were analyzed in a variety of assays, as detailed below. Materials and Methods Cells Analysis of target cells for loss of opsonizing mAb and loss of targeted Ag (shaving) The Her2/Neuϩ BT-474 cell line and the CD5ϩ MOLT-4 cell line were obtained from American Type Culture Collection. The epidermal growth In experiments employing PMA-treated adherent THP-1 cells, after the factor receptor-positive SCC-25 cell line was kindly provided by Dr. Chris- trogocytosis reaction donor cells were separated from THP-1 cells by gen- topher Thomas (University of Virginia). BT-474 cells and MOLT-4 cells tle aspiration, and then residual THP-1 cells were removed from the wells were cultured in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, and by vigorous pipetting. In experiments using RA-treated THP-1 cells, donor 100 ␮g/ml streptomycin (all from Invitrogen). SCC-25 cells were cultured cells were not separated from the THP-1 cells. The amount of residual in DMEM/F-12 medium containing 10% FBS, 100 U/ml penicillin, 100 opsonizing mAb on the donor cells was determined by flow cytometry by ϩ Downloaded from ␮g/ml streptomycin, 20 mM HEPES, and 500 ng/ml hydrocortisone. BT- gating on FL2 PKH26-labeled donor cells or by multispectral image anal- 474 and SCC-25 cells were trypsinized before their use in experiments. ysis. In tests for loss of targeted epitope (i.e., shaving) the Al488 mAb- These three cell types, along with Daudi, Raji, and Z138 cells (5, 8, 19), opsonized or nonopsonized donor cells were reprobed with the same Al488 were used as donor cells. THP-1 cells were used as acceptor cells in most mAb used in the initial opsonization step (“reopsonized”) in the presence experiments and were activated by incubation with PMA or with all-trans of 2 mg/ml Ms IgG to block any nonspecific binding. The cells were then retinoic acid (RA) (5). In some experiments primary monocytes isolated washed with PBS and examined by flow cytometry. In certain experiments from PBMC were used as acceptor cells without any previous activation shaved cells and control cells were stained with either FITC annexin V or

(20, 21). with TOPRO-3 (Invitrogen) to determine whether shaving promoted http://www.jimmunol.org/ apoptosis or direct killing of the cells (5, 8). Reagents and Abs PKH26 was obtained from Sigma-Aldrich; human IgG and mouse (Ms) Analysis of THP-1 cells for uptake of opsonizing mAb, PKH26, IgG from Lampire Biological Laboratories; RTX, TRA, and CET from the and for Fc␥RI levels University of Virginai hospital pharmacy; mAb M22 (mouse IgG1) and mAb 10.1 (mouse IgG1), both specific for Fc␥RI, from Medarex and All probing steps were performed for1honice. Following their removal Caltag Laboratories, respectively; PE goat (Gt) anti-Ms IgG (Fc-specific) from the 24-well plates, THP-1 cells were probed in the presence of Ms from Jackson ImmunoResearch Laboratories; biotinylated (bt) anti-CD11b IgG with a cocktail of bt anti-CD11b and bt anti-CD14, washed with BSA/ from BD Pharmingen and bt anti-CD14 and tricolor (PE-Cy5) streptavidin PBS, and then secondarily probed with PE-Cy5 (tricolor) for multispectral image analyses or with either Al594 SA or Al647 SA for fluorescence (SA) from Caltag Laboratories. mAb T101 (mouse IgG2a) was kindly pro- by guest on October 1, 2021 vided by Dr. Jorge Carrasquillo (National Institutes of Health). mAb HB43 microscopy or with Al647 SA for flow cytometric analysis. To monitor Fc␥RI levels, THP-1 cells were probed, before the trogocytosis reaction, (mouse IgG1), specific for human IgG Fc (22), and mAb IV.3 (mouse ␥ IgG2b, derived from the HB217 cell line), specific for Fc␥RII (CD32), with Al488 mAb M22, specific for a site on Fc RI that is not blocked by were purified from hybridomas obtained from American Type Culture Col- bound human IgG ligand (27). After the trogocytosis reaction the THP-1 lection. mAbs were labeled with Alexa (Al) 488 (Invitrogen), according to cells were analyzed for residual bound Al488 mAb M22 and probed sec- the manufacturer’s instructions. Al647 Gt anti-Ms IgG1 (Fc-specific) and ondarily with PE Gt anti-Ms IgG (Fc-specific) or with Al647 Gt anti-Ms Ј IgG1 (Fc-specific) to determine the percentage of Al488 mAb M22 re- Al594- and Al647-labeled SA were from Invitrogen. F(ab )2 of TRA and CET were obtained by pepsin digestion of intact mAbs as follows. The maining on the surface of the THP-1 cells. mAbs were dialyzed into 0.1 M acetate (pH 7) and then titrated with 1.5 M citric acid, to a final pH of 3.5. Pepsin was dissolved at 2 mg/ml in 1.5 M Flow cytometry, multispectral image analysis, and fluorescence citric acid (pH 3.5), and the mAb and pepsin solutions were prewarmed to microscopy 37°C. They were then combined at a mAb/pepsin ratio of 80:1 (w/w) and incubated at 37°C for 60 min. The digestion was stopped with addition of Flow cytometry was performed using a dual-laser FACSCalibur cytometer 1 N NaOH to raise the pH to 7 and the sample was dialyzed into borate (BD Biosciences). Mean fluorescence intensities were converted to mole- saline (pH 7.8). FITC-labeled fragments were prepared and tested for com- cules of equivalent soluble fluorochrome (MESF) using standard fluores- plete removal of Fc regions by binding them to cognate cells followed by cent beads (Spherotech). Multispectral image analysis was performed on an probing with Al647 mAb HB43, which is specific for the Fc region of ImageStream imaging cytometer (Amnis) as previously described (28, 29). human IgG (22). mAb HB43 bound well to cells opsonized with intact Fluorescence microscopy was performed under oil at high magnification ϫ TRA and CET, but virtually no mAb HB43 was bound to cells opsonized ( 100) using a BX40 fluorescent microscope (Olympus). Images were with F(abЈ) TRA or CET (not shown). captured with a digital camera and visualized with MagnaFire analysis 2 software. Protocols for trogocytosis experiments Acid wash protocol Raji or Z138 cells, MOLT-4 cells, trypsinized BT-474 or trypsinized SCC-25 cells (ϳ107 cells/ml) were labeled with 4 ␮M PKH26 for 2 min To distinguish mAb bound to the cell surface from internalized mAb, donor followed by quenching with RPMI 1640 containing 10% FBS and after two cells were opsonized with Al488 mAbs and then incubated for 5 min at pH washes were incubated with and without 10 ␮g/ml Al488-labeled mAbs 2.5 in RPMI 1640 medium supplemented with 2% FBS (30). This proce- (RTX, T101, TRA, or CET, respectively) in RPMI 1640 media for 20 min dure has been used to identify ligands bound to the external surface of a at 37°C with gentle shaking. The opsonized cells were washed with cold cell, which are released due to incubation at low pH. After incubation the BSA/PBS to remove unbound mAb and then resuspended in media. The cells were washed twice in PBS and then subjected to flow cytometry nonopsonized (naive) or Al488 mAb-opsonized PKH26-labeled donor analyses. This same procedure was used to examine both donor and ac- cells were combined with PMA-treated adherent acceptor THP-1 cells in ceptor cells after the shaving reaction. 24-well plates, or with RA-treated THP-1 cells in tubes. Various ratios of 5 donor cells/acceptor cells were used, but typically 2–10 ϫ 10 donor cells Statistical analysis were added to 1 ϫ 106 THP-1 cells, giving a ratio of donor cells/acceptor THP-1 cells between 1:5 and 1:1. The plates were centrifuged at 300 ϫ g Statistical significance was determined using t tests performed with Sigma- for 15 s, and then incubated for various times (usually 45 min) at 37°C in Stat software (Jandel). 8122 MONOCYTE-MEDIATED TROGOCYTOSIS Downloaded from

FIGURE 2. Kinetics of Al488 RTX and PKH26 transfer from Raji cells to THP-1 cells. Al488 RTX-opsonized or nonopsonized, PKH26-dyed Raji cells were incubated with THP-1 cells for up to 45 min at 37°C. Aliquots of quenched samples were probed as described in the Materials and Meth- ods to identify THP-1 cells and then subjected to multispectral image analysis (A and C, geometric mean fluorescence displayed) or to conventional http://www.jimmunol.org/ flow cytometry (B and D, displayed as MESF values). A and B, Loss of Al488 signal by Raji cells (filled squares) is correlated with gain of Al488 signal by THP-1 cells (open triangles). C and D, Uptake of PKH26 signal by THP-1 cells from Al488 RTX-opsonized Raji cells is shown by the filled circles; uptake from nonopsonized Raji cells is shown by the open circles. Representative of three similar experiments.

Results by guest on October 1, 2021 Membrane fragments are transferred to acceptor cells We have previously demonstrated that THP-1 cells remove RTX- CD20 complexes from B cells (5). Since CD20 is an integral membrane protein, we investigated whether donor B cell membrane lipids are also transferred. Raji cells were stained with the membrane dye PKH26, and then the cells were opsonized with Al488 RTX and incubated with THP-1 cells at 37°C for 45 min. After the reaction, the mixtures were probed with a cocktail of bt anti- CD11b and bt anti-CD14, followed by SA-PE-Cy5 to positively identify the THP-1 cells, and then the mixtures were examined by multispectral image analysis. This technique allows simultaneous spectral and image analysis of thousands of cells per sample (28), thus allowing for detailed and representative inspections of large numbers of individual cells. The dot plots in Fig. 1, A and B, (time ϭ 0 min) show the initial distribution of the Al488 RTX and PKH26 signals for the two populations. After 45 min the THP-1 cells (yellow dots) had clearly taken up both Al488 RTX and PKH26 (Fig. 1, C and D; compare C to A and D to B). There was a decrease in the Al488 RTX signal on the Raji cells (red dots) but the signal due to the PKH26 dye did not appear to change on the Raji cells, likely because only a small percentage of membrane

and after 45 min (C and D) for the PE Cy5-negative population (Raji cells FIGURE 1. Both Al488 RTX and PKH26 are transferred from opso- in red) and for the PE Cy5-positive population (THP-1 cells in yellow). nized Raji cells to THP-1 cells. A–D, Al488 RTX-opsonized, PKH26-dyed E–H, Representative images of Al488 RTX-opsonized, PKH26-dyed Raji Raji cells were incubated with THP-1 cells for 45 min at 37°C. THP-1 cells cells (E and F) and of CD11bϩ/CD14ϩ RA-activated THP-1 cells (G and were then identified as described in the Materials and Methods. The dot H) after 0 and 45 min incubation at 37°C. I, Representative images of cell plots, obtained by multispectral image analysis, show the fluorescent sig- pairs after 45 min incubation at 37°C. SSC, Side scatter; BF, brightfield. nals for Al488 RTX (A and C) and PKH26 (B and D)attime0(A and B) Representative of three similar experiments. The Journal of Immunology 8123

lipid is removed from donor cells during the trogocytosis reaction (31). Inspection of images of individual cells (Fig. 1E–H) clearly reveal that the THP-1 cells had taken up both Al488 RTX and PKH26 after 45 min (compare Fig. 1G with Fig. 1H). Some Raji cell/THP-1 cell pairs were discernable and appeared to be con- nected in an immunologic synapse; after 45 min, both the Al488 and PKH26 signals were visible on both cells (Fig. 1I). To place these observations in a quantitative context, we measured the kinetics of transfer of Al488 RTX and PKH26 from Raji cells to THP-1 cells by either multispectral image analysis or with conventional flow cytometry (Fig. 2). The decrease in the Al488 signal on the Raji cells occurred coincidentally with the increase of the Al488 signal on the THP-1 cells (Fig. 2, A and B), and both techniques gave similar results. Moreover, the rate of uptake of the membrane dye PKH26 by the THP-1 cells (Fig. 2, C and D, filled circles) roughly paralleled the rate of increase of the Al488 signal on these cells (Fig. 2, A and B, open triangles), suggesting that the transfer of both fluorophores occurred in a concerted reaction. Control experiments indicate that there was much less transfer of PKH26-associated membrane fragments to the THP-1 cells if the Downloaded from Raji cells were not opsonized with the Al488 RTX bait (Fig. 2, C and D, open circles).

Extension of trogocytosis to three other mAb/donor cell systems We extended this in vitro assay system to examine trogocytosis of donor cells opsonized with mAbs T101, CET, or TRA, and then http://www.jimmunol.org/ reacted with THP-1 cells at a 1:10 donor cell/acceptor cell ratio. In common with our observations for RTX-opsonized cells, the results of the experiment illustrated in Fig. 3A indicate that large amounts of the bound Al488 mAbs were removed from the donor cells after reaction with THP-1 cells (ﬁlled bars), and the loss of bound mAb could be signiﬁcantly inhibited by human IgG (striped bars). In the next series of experiments the ratio of donor cells/

acceptor cells was set at 1:1 to measure both loss of Al488 mAb by guest on October 1, 2021 from donor cells and mAb uptake by the acceptor cells. Significant amounts of the Al488-labeled mAbs were removed from the target cells and taken up by the acceptor THP-1 cells (Figs. 3, B and C, filled bars); both loss and uptake were substantially inhibited by human IgG (striped bars), providing evidence that these processes are mediated by Fc␥R. Moreover, as defined by PKH26 fluorescence, membrane fragments from these donor cells were also taken up by the acceptor THP-1 cells (Fig. 3D), and this reaction was also blocked by human IgG. As observed with the RTX/Raji cell pair, much less PKH26 was taken up by the acceptor cells if the target cells were not opsonized with mAbs (Fig. 3D, gray hatched bars); this low level of PKH26 uptake was similar to the amount of PKH26 taken up by the cells when the reaction with opsonized cells was blocked by human IgG. To verify that the uptake of the PKH26 illustrated in Fig. 3D was not simply a spectral overlap artifact caused by uptake of the Al488-labeled opsonizing mAbs, the experiment illustrated in Fig. 3E was performed with PKH26-labeled cells that were opsonized with unlabeled mAbs, and quite similar results were achieved. That is, uptake of PKH26 by THP-1 cells from donor cells opsonized with unlabeled mAbs is comparable to that obtained when

FIGURE 3. THP-1 cells take up opsonizing mAb and PKH26 from opsonized donor cells, and transfer can be blocked with hu IgG. A–C, Donor cells were opsonized with the indicated Al488-labeled mAbs and incubated E, Uptake of PKH26. Donor cells were not opsonized (open bars) or at 37°C with THP-1 cells (1:10 donor/acceptor ratio in A and 1:1 ratio in were opsonized with unlabeled mAbs T101, CET, or TRA (filled bars) B–E) for 0 (open bar), 45 (filled bar), or for 45 min with THP-1 cells and incubated as in B–D. Results are representative of nine similar pretreated with 2 mg/ml hu IgG for 60 min (striped bar). Al488 signals for experiments. Means and SD are reported in this figure and in the fol- each cell type were determined by flow cytometry and are displayed as lowing figures. Significant differences in this figure and in those that ;p Ͻ 0.05 ,ء :MESF values. D, Uptake of PKH26 by THP-1 cells from Al488 mAb-opso- follow are all vs the 45-min incubation and are denoted as .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء (nized cells vs uptake from nonopsonized donor cells (gray cross-hatched bars 8124 MONOCYTE-MEDIATED TROGOCYTOSIS Downloaded from http://www.jimmunol.org/

FIGURE 5. Blockade of Fc␥RI (CD64) and Fc␥RII (CD32) with specific mAbs weakly blocks transfer. Donor cells were opsonized and reacted with THP-1 cells as in Fig. 3, except THP-1 cells were pretreated with blocking mAbs (30 ␮g/ml) specific for Fc␥RI (mAb 10.1) or for Fc␥RII by guest on October 1, 2021 (mAb IV.3) for 60 min at 37°C before incubation with donor cells. Cell FIGURE 4. Isolated human monocytes take up opsonizing mAb and mixtures were analyzed by flow cytometry for residual Al488 mAb on PKH26 from opsonized donor cells, and transfer can be blocked with hu donor cells (A) and acquired Al488 mAb and PKH26 by THP-1 cells (B IgG. A–C, Similar to Fig. 3B–D, except human monocytes, isolated from and C, respectively). In most experiments, the differences between the PBMC, were used as acceptor cells. In B and C, all differences between 45-min points and the samples reacted with anti-Fc␥R mAbs were signif- the 45-min samples and the other conditions were significant at p Ͻ icant, but because the absolute differences were small, asterisks were omit- 0.006, and asterisks were omitted for clarity. Representative of three ted. The dashed line in B represents the background immunofluorescence similar experiments. of naive THP-1 cells. these cells were opsonized with the Al488-labeled mAbs. Once again, substantially less of the membrane dye was taken up from to modestly block both removal of the Al488 mAbs from the donor naive cells relative to mAb-opsonized cells (Fig. 3E, gray hatched cells and uptake of Al488 mAbs and PKH26 by the THP-1 cells in bars vs black bars). all four systems (Fig. 5), but blocking was not nearly as effective To generalize our findings we also tested freshly isolated human as was observed with human IgG (Figs. 3 and 4). Human IgG can monocytes for their potential to serve as acceptor cells and pro- contain aggregates that can bind with high affinity to Fc␥R (32), mote trogocytosis. The results in Fig. 4 in which monocytes were and considerably higher concentrations of human IgG were used, tested as acceptor cells are quite similar to those illustrated in Fig. likely explaining the higher level of inhibition observed. Fig. 5A 3B–D in which THP-1 cells were examined. Indeed, all three also shows that the percentage of Al488 RTX removed from the Al488 mAbs were removed from their respective target cells and Raji cells (80%) was greater than the percentage of the other mAbs taken up by the monocytes, and substantial transfer of the PKH26 removed from their respective donor cells (25–50%), possibly sug- dye from donor cells to monocytes was only seen when the donor gesting that the nature of the Ag chelated by a given mAb may cells were opsonized with their respective mAbs (Fig. 4). These influence the degree of trogocytosis (see Discussion). Ј transfer reactions were inhibited when the monocytes were prein- Previously we reported that F(ab )2 of cell-bound RTX do not cubated with human IgG, again providing evidence that Fc␥R play promote shaving (5), and we have now extended this observation Ј Ј an important role in the trogocytosis reaction for these primary to the F(ab )2 of TRA and CET. FITC-labeled F(ab )2 of the mAbs cells as well. bound to their target epitopes on donor cells, and prior opsoniza- To further investigate the role of Fc␥R in trogocytosis, we ex- tion with the intact cognate IgG mAbs blocked this binding (Fig. ␥ Ј amined the potential of blocking mAbs, specific for Fc RI and 6, A and B). When cells were opsonized with F(ab )2 or with intact Fc␥RII, to inhibit the reaction. The Fc␥R-specific mAbs were able IgG mAbs and then reacted with THP-1 cells for 45 min at 37°C, The Journal of Immunology 8125

Ј FIGURE 6. FITC-labeled F(ab )2 of TRA and CET bind speciﬁcally to cognate cells, but do not promote shaving. A and B, Binding of the fragments is demonstrable, and preincubation with the intact unlabeled IgG mAbs blocks binding of the fragments. C and D, Under the usual conditions of the shaving reaction (45 min at 37°C in the presence of THP-1 cells) only the intact mAbs are removed from opsonized cells. E and F, Reopsonization experiments reveal that the target epitopes recognized by TRA and CET are removed only if the cells are opsonized with intact mAbs. Representative of two similar experiments. Downloaded from

Ј there was little if any loss of bound F(ab )2, but substantial frac- tions of the intact IgG mAbs were once again removed (Fig. 6, C and D). When the cells were subjected to conditions of reopsonization, which allows for a determination of the availability of the http://www.jimmunol.org/ targeted epitopes, again only the cells opsonized with intact mAbs were demonstrated to have undergone shaving (Fig. 6, E and F). These results provide additional evidence that recognition of cell- bound IgG by Fc␥R on the acceptor cell is a key step in the shaving reaction. We next used ﬂuorescence microscopy to visualize transfer of Al488-labeled T101, TRA, or CET from opsonized donor cells to THP-1 acceptor cells. Al488 mAb-opsonized donor cells and by guest on October 1, 2021 THP-1 cells were incubated at a 1:1 ratio for 45 min at 37°C, and then stained with bt anti-CD11b and bt anti-CD14 followed by Al594 SA to identify the THP-1 cells. For each of the three mAbs, the ﬂuorescent micrographs clearly demonstrate uptake of the green Al488 mAb by red THP-1 cells (Fig. 7).

Transfer of opsonizing mAbs is not due to phagocytosis or simple dissociation It is possible that the internalization of the Al488 mAbs and/or PKH26 by the acceptor THP-1 cells might be due in part to phagocytosis of the donor cells by the acceptor THP-1 cells. We were able to rule out this reaction in our previous studies of the RTX- Z138 cell/THP-1 cell system based on the high level of recovery of donor cells under conditions that promoted shaving (5). In the present studies we quantitated recovery of donor cells by counting constant volume aliquots of the reaction mixtures soon after quenching the shaving reaction. In a representative experiment, recovery of TRA-opsonized BT-474 cells after the shaving reaction averaged 17,000 Ϯ 1,100 cells, and recovery of these cells under control conditions that precluded shaving (THP-1 cells were preblocked with human IgG) averaged 16,000 Ϯ 2,000 cells. Com- parable results for recovery of donor cells in the CET/SCC-25 system were 9,100 Ϯ 700 and 7,500 Ϯ 100 cells, respectively. These results indicate that little if any phagocytosis occurred dur- FIGURE 7. Fluorescence microscopy analyses confirm transfer of Al488 mAbs to THP-1 cells. Donor cells were opsonized with Al488 mAbs ing the shaving reaction. Finally, inspection of fields by fluores- and reacted with THP-1 cells as in Fig. 3. THP-1 cells were identified by cence microscopy such as those illustrated in Fig. 7 did not reveal probing with a cocktail of bt anti-CD11b and bt anti-CD14 followed by any obviously phagocytosed cells. Al594 SA. A, Brightfield images. B, Merged images show some red THP-1 We performed control experiments to determine whether the cells containing Al488 mAbs. C, Texas Red filter shows red THP-1 cells. uptake of the opsonizing mAbs and PKH26 by the THP-1 cells D, FITC filter shows Al488 mAbs. Original magnification, ϫ100. Repre- could have resulted from passive dissociation of the mAb and sentative of two similar experiments. 8126 MONOCYTE-MEDIATED TROGOCYTOSIS

PKH26 dye from the donor cells into the medium, rather than by the active trogocytosis mechanism we have proposed. First we examined the degree of dissociation of the Al488 mAbs from do- nor cells after 45 min at 37°C in the absence of THP-1 acceptor cells, compared with holding the cells on ice. In agreement with the findings of Teeling et al. (33), the RTX sample was modestly labile; the percentage of Al488 signal lost was ϳ15% for RTX- opsonized cells that were incubated at 37°C. However, there was Ͻ3% dissociation for the other mAb-opsonized donor cells, and loss of PKH26 from all four donor cell types was Ͻ3%. We next tested the ability of THP-1 cells to take up either Al488 mAb or PKH26 potentially released into the medium by dissociation from donor cells. PKH26-stained, Al488 mAb-opsonized donor cells were first incubated in the absence of THP-1 cells for 45 min at 37°C. The donor cells were then removed by centrifugation and the cell-free supernatant was added to THP-1 cells. After a 45-min incubation at 37°C, the THP-1 cells were quenched and analyzed by flow cytometry. Uptake of the opsonizing Al488 mAb and PKH26 by the THP-1 cells was negligible (Ͻ2% of the positive controls, data not shown), thus ruling out passive dissociation and Downloaded from uptake as a transfer mechanism. Tests for internalization of opsonizing mAbs by donor cells It is possible that a fraction of the Al488-labeled mAbs was internalized by the substrate donor cells, either during the initial opsonization reaction or as a consequence of interaction with the http://www.jimmunol.org/ THP-1 cells. To address this question, we subjected the donor cells, both after initial opsonization and after the shaving reaction, to acid wash procedures (30) to distinguish surface-bound mAbs from mAbs that had been internalized. We also examined THP-1 cells with the acid wash procedure after the shaving reaction, at which time they had taken up Al488-labeled mAbs from the donor cells. The results (first two bars on the left in Fig. 8) indicate that FIGURE 8. Most mAbs bound to donor cells are surface bound, and after opsonization, between 75% and 90% of bound mAbs could after the shaving reaction the mAbs remaining on the donor cells are not by guest on October 1, 2021 be released by acid wash for the four mAb/donor cell pairs under internalized, but the mAbs taken up by the THP-1 cells are internalized. investigation, thus indicating that most of the mAbs were bound to A–D, Results for RTX/Daudi cells, T101/MOLT-4 cells, CET/SCC-25 the cell surfaces and not internalized during opsonization. More- cells, and TRA/BT-474 cells, respectively. Bars in panels on the left, from over, the acid wash did not appear to induce irreversible denatur- left to right, indicate binding of Al488 mAbs to: opsonized donor cells ation of the epitopes targeted by the mAbs, because after the acid (OP); opsonized and acid-washed donor cells (OP, AW); opsonized and wash and a reequilibration to neutral pH, the cells were again acid-washed donor cells, followed by reopsonization (OP, AW, R); opso- capable of binding the cognate mAbs (third bar from the left in nized donor cells subjected to trogocytosis/shaving with THP-1 cells (OP, Ј Ј each graph of Fig. 8). As already demonstrated, a substantial frac- 45 T); and finally acid washes of these latter cells (OP, 45 T, AW). Panels on the right give the Al488 signal for naive THP-1 cells (Naive), followed tion of the mAbs could be shaved off the donor cells by THP-1 by the signal for THP-1 cells after taking up the mAb from donor cells (45Ј acceptor cells (fourth set of bars from the left of Fig. 8) and most T), followed by acid wash of the THP-1 cells (45Ј T, AW). Representative of the material that was not removed by the THP-1 cells could still of two similar experiments. be removed by a subsequent acid wash, indicating that reaction with THP-1 cells did not lead to any appreciable internalization of the Al488 mAbs by the donor cells; that is, most of the bound mAb-opsonized donor cells with THP-1 cells for 45 min at 37°C mAbs that were not removed from the donor cells in a single pass followed by quenching with human IgG and cold BSA/PBS does of shaving were indeed still located on the cell surface. Finally, a not kill the cells or render them apoptotic. Only a small percentage considerably different pattern was evident when the THP-1 cells of these donor cells were positive for the annexin V or TOPRO-3 were subjected to acid wash after the shaving reaction (right sides stains, nearly identical to the values obtained for the 0-min control of each graph in Fig. 8). The results indicate that most of the Al488 samples consisting of opsonized donor cells mixed with THP-1 mAbs taken up by the THP-1 cells could not be released by the acid wash, which is consistent with internalization of the mAbs by the THP-1 cells as a consequence of the trogocytosis/shaving Table I. Effect of the shaving reaction on donor cell viability reaction. Incubation Time Effects of shaving on cell viability with THP-1 Cells Annexin TOPRO-3ϩ ϩ We previously reported that THP-1 cell-promoted shaving of mAb/Cell (min) V (% Ϯ SD) (% Ϯ SD) CD20 from RTX-opsonized Z138 cells neither killed the Z138 TRA/BT-474 45 5.6 Ϯ 0.4 4.7 Ϯ 0.4 cells nor rendered them apoptotic (5), and we have now general- 0 5.1 Ϯ 0.1 5.1 Ϯ 0.8 ized this observation to two of the mAb-donor cell pairs in the CET/SCC-25 45 9.2 Ϯ 0.1 6.6 Ϯ 0.1 Ϯ Ϯ present system. As presented in Table I, we find that reaction of 0 12.7 0.2 6.8 0.4 The Journal of Immunology 8127 Downloaded from

FIGURE 10. Fc␥RI is internalized during trogocytosis. A and B, THP-1 cells were reacted with 10 ␮g/ml Al488 mAb M22 (speciﬁc for Fc␥RI (CD64), but does not block the ligand-binding site) before incubation with RTX-opsonized Z138 cells. After incubation, cells were probed with PE Gt

anti-Ms IgG (Fc-specific) to detect surface-bound Al488 mAb M22, and http://www.jimmunol.org/ then both signals on the cells were measured by flow cytometry. C and D, In a similar experiment, THP-1 cells were first reacted with Al488 mAb M22 and then incubated with Raji, MOLT4, SSC-25, and BT-474 cells opsonized with RTX, T101, CET, and TRA, respectively. After a 45-min incubation at 37°C, cells were probed with Al647 Gt anti-Ms IgG1 to detect cell surface-bound mAb M22. Representative of two similar FIGURE 9. Increasing opsonization of donor cells with Al488 mAb or experiments. increasing donor/acceptor cell ratios promote increased uptake of Al488 mAb by THP-1 cells. A and B, BT-474 cells were opsonized with the by guest on October 1, 2021 indicated concentrations of Al488 TRA, and after two washes were incu- cell is capable of taking up mAb ligands from more than one donor bated with THP-1 cells at 37°C for 45 min. Uptake of Al488 TRA by cell. Finally, similar results were respectively obtained in both THP-1 cells increased with increasing opsonization in the range dose-response studies and in experiments in which cell ratios were 0.05–10 ␮g/ml. The dashed line (also in C) indicates the background varied, based on using T101-opsonized MOLT-4 cells and CET- fluorescence of naive THP-1 cells. C, MOLT-4 cells were opsonized opsonized SCC-25 cells, as well as on TRA-opsonized BT-474 with 10 ␮g/ml Al488 mAb T101 and then incubated with THP-1 cells cells and CET-opsonized SCC-25 cells (results not shown). at the indicated ratios of donor/acceptor cells. Representative of two similar experiments. The role of Fc␥RI in the internalization reaction A key late step in the endocytic process of shaving or trogocytosis cells preblocked with human IgG to preclude shaving. Thus, there is the internalization by the acceptor cell of the transferred ligand is negligible cell killing or induction of apoptosis as a consequence and the cognate receptor of the acceptor cell. Our previous work of the shaving reaction. using RTX-opsonized Z138 cells as donor cells indicated that Fc␥RI is the receptor principally responsible for removal of RTX Dose-response studies and the effects of variations in cell ratios (5), and therefore we first tested for Fc␥RI internalization during We next investigated whether the degree of mAb opsonization for incubation of THP-1 cells with RTX-opsonized Z138 cells. THP-1 one representative mAb/donor cell pair, Al488 TRA/BT-474 cells, cells were first labeled with Al488 mAb M22, which binds to would influence the degree of trogocytosis. The results in Fig. 9A Fc␥RI at a site not blocked by bound human IgG (27), and then indicate that over a wide range of TRA concentrations used for reacted with RTX-opsonized Z138 cells for 45 min at 37°C. To opsonization, ϳ50% of the Al488 TRA initially bound to BT-474 determine whether mAb M22 was still bound to Fc␥RI on the cells was removed during incubation with THP-1 cells. Consistent surface of the THP-1 cells or had been internalized, these reaction with this finding, considerably more Al488 TRA was taken up by samples were probed secondarily with PE anti-Ms IgG (Fc-spe- the THP-1 cells which were reacted with the most heavily opso- cific) after the trogocytosis reaction. Compared with the 0-time nized BT-474 cells (Fig. 9B). Both the loss of Al488 by the donor controls, there was a modest reduction (ϳ30%) in the amount of cells and the gain of Al488 by the acceptor cells were inhibited in Al488 mAb M22 associated with THP-1 cells after reaction with the presence of 2 mg/ml human IgG. Next, for the T101/MOLT-4 donor cells (Fig. 10A), likely due to passive dissociation of the system, we held the degree of opsonization constant at 10 ␮g/ml mAb. However, as shown in Fig. 10B, compared with the 0-time Al488 mAb T101 and varied the donor cell/acceptor cell ratio (Fig. controls, far less mAb M22 was detectable on the THP-1 cell sur- 9C). Not surprisingly, we find that as the donor cell/acceptor cell face after incubation with opsonized donor cells for 45 min at 37°C ratio increased, the amount of Al488 mAb T101 taken up by the (ϳ70% reduction in signal). These results suggest that much of the acceptor cells increased. This result suggests that a single acceptor residual cell-associated mAb M22 (and presumably the Fc␥RI to 8128 MONOCYTE-MEDIATED TROGOCYTOSIS which it was bound) had indeed been internalized as a consequence of the trogocytosis reaction. We next applied this paradigm to all four mAb/donor cell pairs, except the RTX-opsonized donor B cells were Raji cells in this instance. The acceptor THP-1 cells were again first reacted with Al488 mAb M22 (IgG1 isotype) before the trogocytosis reaction (Fig. 10C) and probed after the trogocytosis reaction with Al647 Gt anti-Ms IgG1 (Fig. 10D). Use of Al647 Gt anti-Ms IgG1 in this experiment was necessary because the PE anti-Ms IgG would have reacted with the Ms mAb T101 as well as with mAb M22, but the Al647 Gt anti-Ms IgG1 will not react with mAb T101 (isotype IgG2a) and thus recognizes only mAb M22. Similar to the results above, after the 45-min trogocytosis reaction there was modest reduction in signal (ϳ15%) of Al488 mAb M22 associated with the THP-1 cells (Fig. 10C), compared with the 0-time controls. However, when the cells were probed for surface-bound mAb M22, the signals were reduced by at least 40% compared with the 0-time controls (Fig. 10D), indicating that much of the cell-bound Al488 mAb M22 had been internalized, presumably along with Fc␥RI. Finally, there was no internalization of cell-bound mAb M22 when the THP-1 Downloaded from cells were incubated with any of the nonopsonized donor cells (results not shown). We also studied the internalization phase of trogocytosis by probing cells with mAb HB43, which is specific for the Fc region of human IgG (22), and which should bind to TRA and CET when these human IgG1 mAbs are bound to the surface of BT474 and http://www.jimmunol.org/ SCC-25 cells, respectively. However, if these two mAbs are taken up by THP-1 cells and internalized, they will not be detected on the THP-1 cells by probing with mAb HB43. To test this hypothesis we used a three-color fluorescence microscopy paradigm: BT474 and SCC-25 cells were opsonized with Al488-labeled TRA and CET, respectively, and then reacted with THP-1 cells; after reaction the cell mixtures were probed with Al546 mAb HB43 to detect surface-bound TRA or CET, and with a cocktail of bt anti- by guest on October 1, 2021 CD11b and bt anti-CD14, followed by Al647 SA, to identify THP-1 cells. Inspection of the bright field (Fig. 11A) and fluorescent images reveals red Al647 THP-1 cells (Fig. 11B) that had taken up green Al488-labeled TRA or CET (Fig. 11C). However, these THP-1 cells were not stained with Al546 mAb HB43 (Fig. 11D), indicating that the transferred Al488 mAbs had been internalized by the THP-1 cells. The mAb-opsonized donor cells with residual Al488 mAb appear yellow when stained with Al546 mAb HB43, and thus serve as positive staining controls (Fig. 11E). Direct tests for shaving of mAb-targeted epitopes Our clinical reports of shaving of patient CLL cells after RTX infusion, as well as our in vitro observations of shaving of RTX- opsonized B cells by acceptor cells, are based on a key finding: compared with 0-time controls, the shaved B cells have considerably reduced RTX binding capacity, which is revealed when the cells are reopsonized with RTX; this reduction in binding capacity indicates loss of CD20 (5–7). After the usual incubation of mAb- FIGURE 11. Fluorescence microscopy analyses confirm internalization opsonized donor cells with THP-1 cells, we used this reopsonization of opsonizing mAbs by THP-1 cells during trogocytosis. Donor cells were paradigm to test for loss of cell surface Ag for the three mAb- opsonized with Al488-labeled TRA or CET and reacted with THP-1 cells donor cell pairs under investigation. The results for mAb T101- as in Fig. 3. After 45 min at 37°C, cell mixtures were probed with bt opsonized MOLT-4 cells reacted with THP-1 cells provide anti-CD11b and bt anti-CD14 followed by Al647 SA to identify THP-1 cells, and with Al546 HB43 (anti-human IgG, Fc␥-specific) to detect unambiguous evidence for CD5 shaving (Fig. 12A). In the nonop- Al488 TRA or Al488 CET on the outside of cells. A, Bright field. B, The sonized control there was little, if any, loss of the target Ag (97% Al647 SA identifies CD11bϩ/CD14ϩ THP-1 cells. C, A merge of Al647 of the time 0 signal remained). However, MOLT-4 cells that were and Al488 reveals Al488 associated with THP-1 cells. D, Al546 mAb first opsonized with Al488 mAb T101 and then reacted with HB43 detects Al488 TRA or Al488 CET on the surface of donor cells, THP-1 cells had substantially reduced mAb T101 binding capacity but not on the surface of THP-1 cells. E, A merge of Al488 and Al546 when they were reopsonized with Al488 mAb T101 after the tro- shows Al488-positive, Al546-negative THP-1 cells. Representative of gocytosis reaction (29% of the 0-time control). In agreement with two similar experiments. our studies for the RTX-CD20 system, the mAb T101-mediated The Journal of Immunology 8129

surface Ag IC are removed from the donor cells (Figs. 1–4). Sec- ond, we have been able to extend and generalize our observations of Fc␥R-mediated trogocytosis/shaving beyond the RTX/B cell system to include several other therapeutic mAb/donor cell pairs and cell types: T101/MOLT-4 (T lymphoblasts, models for T cell lymphoma); TRA/BT-474 (epithelial cells, models for breast cancer); and CET/SCC-25 (keratinocytes, models for head and neck cancer). Third, cell types that have been reported to mediate trogocytosis have included B cells, T cells, NK cells, and dendritic cells (1, 3, 4, 34), and thus monocytes would not necessarily be expected to mediate trogocytosis. We have now shown that both THP-1 cells and freshly isolated human monocytes indeed execute trogocytosis. Fc␥R-promoted recognition and binding of both soluble and particulate IgG-containing IC by acceptor cell monocyte/macrophages gives rise to a complex series of events, including internalization of the entire IC by the acceptor cells (35–42). If the internalized IC substrates are large (e.g., IgG-opsonized cells), this process is called phagocytosis and is mediated by the zipper mechanism in which the phagocytic cell surrounds and engulfs the tar- Downloaded from get cell by making continuous contact with IgG ligands distributed across the target cell. If the substrates are smaller, the reaction has been described as endocytosis or macropinocytosis, which in- volves less dramatic changes in the acceptor cell membrane. In all of these reactions, the entire particulate or soluble IC is internalized by the acceptor cells. However, more than 30 years ago Griffin http://www.jimmunol.org/ et al. reported that crosslinked and capped polyclonal IgG complexes bound to surface Ags on lymphocytes were processed in a very different fashion: the macrophages “removed the immune complex caps from the lymphocyte without destroying the lymphocyte” (36). Moreover, they found that this reaction was abrogated if Fc␥R on the acceptor macrophages were blocked; their FIGURE 12. Reopsonization of donor cells after trogocytosis confirms findings could perhaps be considered the first report of trogocyto- shaving (loss of target epitope), and the reaction can be blocked with hu- sis, long before the term was coined. We also note that in the man IgG. Donor cells, nonopsonized or opsonized with Al488-labeled by guest on October 1, 2021 mAbs, were incubated with THP-1 cells as in Fig. 3. After a 45-min in- present study, based on recovery of donor cells, there was no incubation at 37°C, the cell mixtures were reprobed with the original Al488- dication of phagocytosis of donor cells promoted by THP-1 cells; labeled mAb. A–C, Shaving of Al488 mAb T101-opsonized MOLT4 cells, rather, the transfer of PKH26- and Al488-labeled mAbs can best Al488 TRA-opsonized BT-474 cells, and Al488 CET-opsonized SCC-25 be explained by trogocytosis. cells, respectively. Representative of three similar experiments. Our present results suggest that when cells are opsonized with mAbs, the reaction described by Griffin et al. can occur even if secondary agents are not used to crosslink and cap the shaving reaction is blocked by human IgG (Fig. 12A), again sug- substrate cell-bound IgG. That is, recognition of bound IgG on gesting that the reaction is mediated by Fc␥R. Comparable anal- the opsonized cells by Fc␥R on the acceptor cells generates yses for Al488 TRA/BT-474 and Al488 CET/SCC-25 systems also what appear to be immunologic synapses (Fig. 1I). After for- demonstrated shaving, based on the reduced mAb binding capacity mation of the immunologic synapse, target cell Ag, bound mAb of cells that were first opsonized with their respective Al488 mAbs IgG, and fragments of target cell membrane can be removed and (76% and 36% of the 0-time controls, respectively, Fig. 12, B and internalized by the acceptor cell (Figs. 1, 7, and 11). Moreover, C). Once again, nonopsonized donor cells suffered little, if any, during this process there is negligible internalization of bound loss of mAb binding capacity, and shaving could be blocked by mAbs by the donor cells (Fig. 8). In the present study and in our preincubation of the acceptor THP-1 cells with human IgG. These earlier reports we have postulated a role for Fc␥RI in this pro- results demonstrate that, as we showed for RTX (5), acceptor cells cess (5, 43). Human IgG can effectively suppress transfer of cannot only remove the opsonizing mAb, they also remove its both Al488 mAb and PKH26 from donor cells to acceptor cells target Ag from the donor cells in these three mAb/donor cell (Figs. 3, 4, 9, and 12). Furthermore, our results based on the use combinations. of mAb M22, which binds to Fc␥RI at a site not blocked by human IgG, demonstrate that Fc␥RI is indeed internalized by Discussion the acceptor cells following the trogocytosis reaction (Fig. 10), We have reported previously that after RTX binds to CD20 on B thus providing support for its role in trogocytosis. In agreement lymphocytes, the constituents of the IC (both RTX and CD20) can with our previous study in the RTX/Z138 cell system (5), shav- Ј be removed (shaved) from these cells and taken up and internal- ing is abrogated when donor cells are opsonized with F(ab )2 of ized by acceptor cells that express Fc␥R (5). This report presents CET or TRA (Fig. 6), again indicating a role for Fc␥Ronac- three key new findings. First, we can now further describe the ceptor cells recognizing mAb-opsonized cells. Internalization Ј shaving reaction as being quite similar, if not identical, to a pre- of the F(ab )2 by the donor cells is quite unlikely; we note that viously reported phenomenon, trogocytosis (1–4), as revealed by previous reports of antigenic modulation indicated that intact the concerted uptake of the membrane dye PKH26 when mAb/cell IgG mAbs were required to promote monocyte-mediated loss of 8130 MONOCYTE-MEDIATED TROGOCYTOSIS targeted epitiopes (44). Attempted blockade of Fc␥RI or Fc␥RII There are reports of apparent shaving of cancer cells in solid with specific mAbs at concentrations of 30 ␮g/ml only modestly tumors. We have found, for example, based on a SCID mouse inhibited trogocytosis (Fig. 5), likely due to higher avidity bind- model, that infusion of RTX can lead to loss of CD20 on Z138 ing of clustered mAb on the opsonized donor cells to Fc␥Ron cells growing in a solid tumor (43). Laurent et al. have reported the THP-1 cells. However, substantial blockade was evident that mature B lymphocytes lacking CD20 were demonstrable in when high concentrations of human IgG were used, and, as nodular lymphoid infiltrates in the bone marrow of patients after human IgG can contain aggregates (32), these higher concen- treatment with RTX (58). Bertram et al. found that mAb T101 trations may have allowed for more effective interaction with promoted antigenic modulation (loss of CD5) on malignant T cells and blockade of Fc␥RI. growing in tumors of patients with cutaneous T cell lymphoma Based on these considerations, it would seem that shaving/ (14). Taken together with the present report, these findings may trogocytosis of mAb-opsonized cells might not occur in vivo, have important implications for the use of mAbs such as RTX, where the concentrations of IgG in the bloodstream can be ϳ10 TRA, and CET in cancer immunotherapy for solid tumors. An mg/ml, thus potentially blocking the action of Fc␥RI. However, important question centers on the accessibility of mAb-opsonized we emphasize that shaving of RTX-opsonized cells and anti- cells in such tumors to effector cells such as NK cells, monocytes, genic modulation of T101-opsonized cells were first reported and macrophages. It is possible that local saturation of effector cell based on clinical observations (6, 7, 14), not in vitro experi- cytotoxic capacity within the tumors can promote shaving, and ments. It is therefore likely that certain compartments within analyses of fine-needle aspirates or biopsies of tumors after mAb the body (perhaps the sinusoids of the liver) allow for close treatment may address this issue. ␥ Finally, the mechanism(s) of action of immunotherapeutic contact between mAb-opsonized cells and Fc RI-expressing Downloaded from acceptor cells such as Kupffer cells, thus promoting shaving. mAbs as well as the rate at which they kill tumor cells are also We also note that several studies in mouse models have re- likely to influence whether they can mediate shaving, as well as vealed that Fc␥RI-expressing acceptor cells can play roles in the degree to which shaving can reduce killing efficacy. Al- processing of IgG-opsonized substrates (45–47), despite the though recent evidence supports a role for Ab-dependent cel- high concentrations of IgG in the bloodstream. lular cytotoxicity in the cytotoxic action of both CET and TRA, these mAbs may also promote direct cell killing by binding to

An important question emerges from this work: How do effector http://www.jimmunol.org/ target sites on cells, thus setting off signaling cascades that can cells “decide” the fate of IgG-opsonized substrate cells? A volu- induce apoptosis, or block cell growth and proliferation (15– minous literature reveals that fixed tissue macrophages such as 18). If these cytotoxic processes are sufficiently effective, then Kupffer cells in the liver and macrophages in the spleen clear IgG- the targeted cells may be killed more rapidly than they can be opsonized cells (48–52), and our clinical investigations have dem- shaved, or any shaving that does occur may not be sufficient to onstrated that infusion of just 30 mg of RTX promotes rapid clear- prevent killing. However, shaving may reduce killing efficacy, ance of circulating malignant B cells in patients with CLL (6, 7). and this possibility should be considered in the design of clin- However, our findings suggested that fixed tissue macrophages ical trials and their correlative studies (7). may also mediate shaving, because we found that CLL B cells not In summary, our experiments demonstrate that the three cleared in the first 15–30 min of RTX infusion lost substantial by guest on October 1, 2021 newly tested mAb/cell substrate pairs are all subject to trogo- amounts of CD20, suggesting that clearance and shaving occur cytosis/shaving by acceptor monocytes, as we previously re- simultaneously (7). ported for the RTX/B cell system. However, we did observe One factor that may determine whether an opsonized target is quantitative differences in the degree of shaving for these sys- cleared or shaved may be its size. If the target is sufficiently small tems, and we cannot at this time predict whether binding of an (e.g., an IgG-opsonized sheep erythrocyte, bacterium, or virus), IgG mAb to a particular epitope on a cell will in fact promote then it is likely that the entire particle will be internalized by the effector cell-mediated shaving/trogocytosis. The mAbs used in zipper phagocytic mechanism. However, if the target is a malig- the present studies include one mouse IgG2a (T101) and three nant cell opsonized with many copies of a single mAb, and the human IgG1 isotypes, and strong interaction with Fc␥RI would target cell is comparable in size or larger than the monocyte/mac- therefore appear to be one prerequisite. However, the nature of rophage, then it is likely that the acceptor cell may instead ingest the epitope targeted by the mAb, the disposition of the epitope only small pieces of the target cell. Indeed, Wallace et al. exam- within the cell membrane, and the lifetime of the mAb when it ined the processing of IgG-opsonized breast cancer cells by human binds to the cells are all likely to be important. If mAb binding macrophages and reported that the macrophages appeared to be promotes rapid cell killing or if mAb binding is followed by “chipping away” at these substrates and then internalizing the internalization, or shedding, then trogocytosis/shaving may be pieces (53). minimized. Many other factors can also influence these reac- Another important factor likely affecting the relative amounts of tions and a more complete understanding of these issues may cell clearance vs shaving is the burden of cells that are targeted by allow for the improved design of immunotherapeutic mAbs tar- a given mAb (6, 54–57). If the initial burden of circulating ma- geted to malignant cells. lignant cells is high (e.g., in CLL) and is restored by reequilibration after first-pass clearance mediated by a mAb such as RTX (6, 7), then the phagocytic capacity of the liver and spleen as well as Acknowledgments the cytolytic capacity of NK cells are likely to be exceeded. In- We thank Dr. Jorge Carrasquillo (Sloan Kettering Cancer Center) who deed, Bowles and Weiner have reported that CD16 (Fc␥RIII) is graciously provided us with mAb T101 at the National Institutes of Health. substantially down-regulated on NK cells after reaction with RTX- This paper is dedicated to the loving memory of P. C. Venkat for tireless opsonized cells (56). Berdeja et al. have also reported that soon advocacy on behalf of patients with chronic lymphocytic leukemia. after RTX infusion, the cytolytic capacity of NK cells is reduced considerably (57). Thus, under conditions in which effector mech- anisms that clear cells are saturated or exhausted, shaving/trogo- Disclosures cytosis may be the default reaction. The authors have no financial conflicts of interest. The Journal of Immunology 8131

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