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 compromise 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 Fcγ receptors. The mechanism responsible for shaving appears to be trogocytosis, a process in which receptors on effector cells remove and internalize 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 with respect to the use of mAbs in cancer immunotherapy.

Recognition of ligands on target cells by cognate receptors on acceptor cells such as B cells, T cells, or NK cells can lead to transfer of the ligand and closely associated membrane fragments from the target cell to the acceptor cell (1, 2, 3, 4). In this endocytic reaction, called trogocytosis or nibbling, both the captured ligand and its receptor on the acceptor cell are internalized. We have recently demonstrated that a similar transfer occurs in vitro when B cells opsonized with anti-CD20 mAb rituximab (RTX)3 are reacted with either PBMC or monocytic THP-1 cells (5). In this case the RTX-CD20 immune complexes (IC) on the target cell serve as ligands for FcγRI on the acceptor cell.

We established this in vitro system as a model to investigate the mechanism of loss of bound RTX and CD20 from targeted circulating malignant B cells, which occurs in vivo when patients with chronic lymphocytic leukemia (CLL) are treated with doses of RTX ranging from as low as 60 mg/m2 up to the usual 375 mg/m2 doses (6, 7). Soon after completion of RTX infusion, substantial numbers of viable malignant B cells are demonstrable in the bloodstream. These cells have markedly reduced levels of CD20 and have very little bound RTX. Moreover, these B cells are tagged covalently with C3dg, indicating that during the infusion the cells must have been previously opsonized with RTX, which promotes complement activation and deposition of C3b activation fragments. We have also demonstrated in several in vitro models that this loss of CD20 and bound RTX can occur in the complete absence of complement, thus precluding a causative role for C3b/C3dg in the CD20 loss (5, 8). This removal of cell-bound RTX and CD20 by acceptor cells, which we termed “shaving”, is quite similar to trogocytosis and may be responsible for the phenomenon of antigenic modulation seen in earlier cancer immunotherapy studies (9, 10, 11, 12, 13, 14). For example, treatment of T cell lymphoma patients with 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 known, but our studies of shaving and those of other investigators on trogocytosis provide a likely mechanism for the observed loss of CD5, namely removal of the T101/CD5 IC by FcγR-expressing acceptor cells. All of these reactions, that is, trogocytosis, shaving, and antigenic modulation, may share a common pathway for processing of targeted ligands removed from donor cells and taken up by acceptor cells.

To further characterize shaving of RTX-CD20 complexes and the relationship of this reaction to trogocytosis, we used the membrane dye PKH26 to test for the transfer of membrane fragments from RTX-opsonized donor cells to acceptor THP-1 cells. Multispectral image analysis and flow cytometry experiments revealed that both RTX and PKH26 are taken up by the acceptor cells, and this finding extends our previous work in this system (5). We investigated the possible generality of these reactions with respect to the use of other immunotherapeutic mAbs in targeting malignant cells. Flow cytometry and fluorescence microscopy were employed to examine three different mAb-opsonized/donor cell pairs: mAb T-101/MOLT-4 cells; trastuzumab (TRA, used in the treatment of breast cancer (15, 16))/BT-474 cells; and cetuximab (CET, used in the treatment of colorectal and other cancers (17, 18))/SCC-25 cells. In all three cases we observe similar reactions that closely follow the tenets defined for trogocytosis: transfer of donor cell-bound mAb/target Ag IC and membrane fragments to the acceptor cells, as well as internalization of acceptor cell FcγRI.

The Her2/Neu+ BT-474 cell line and the CD5+ MOLT-4 cell line were obtained from American Type Culture Collection. The epidermal growth factor receptor-positive SCC-25 cell line was kindly provided by Dr. Christopher Thomas (University of Virginia). BT-474 cells and MOLT-4 cells were cultured in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen). SCC-25 cells were cultured in DMEM/F-12 medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM HEPES, and 500 ng/ml hydrocortisone. BT-474 and SCC-25 cells were trypsinized before their use in experiments. These three cell types, along with Daudi, Raji, and Z138 cells (5, 8, 19), were used as donor cells. THP-1 cells were used as acceptor cells in most experiments and were activated by incubation with PMA or with all-trans retinoic acid (RA) (5). In some experiments primary monocytes isolated from PBMC were used as acceptor cells without any previous activation (20, 21).

PKH26 was obtained from Sigma-Aldrich; human IgG and mouse (Ms) IgG from Lampire Biological Laboratories; RTX, TRA, and CET from the University of Virginai hospital pharmacy; mAb M22 (mouse IgG1) and mAb 10.1 (mouse IgG1), both specific for FcγRI, from Medarex and Caltag Laboratories, respectively; PE goat (Gt) anti-Ms IgG (Fc-specific) from Jackson ImmunoResearch Laboratories; biotinylated (bt) anti-CD11b from BD Pharmingen and bt anti-CD14 and tricolor (PE-Cy5) streptavidin (SA) from Caltag Laboratories. mAb T101 (mouse IgG2a) was kindly provided by Dr. Jorge Carrasquillo (National Institutes of Health). mAb HB43 (mouse IgG1), specific for human IgG Fc (22), and mAb IV.3 (mouse IgG2b, derived from the HB217 cell line), specific for FcγRII (CD32), were purified from hybridomas obtained from American Type Culture Collection. mAbs were labeled with Alexa (Al) 488 (Invitrogen), according to the manufacturer’s instructions. Al647 Gt anti-Ms IgG1 (Fc-specific) and 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 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 citric acid (pH 3.5), and the mAb and pepsin solutions were prewarmed to 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 1 N NaOH to raise the pH to 7 and the sample was dialyzed into borate saline (pH 7.8). FITC-labeled fragments were prepared and tested for complete removal of Fc regions by binding them to cognate cells followed by probing with Al647 mAb HB43, which is specific for the Fc region of human IgG (22). mAb HB43 bound well to cells opsonized with intact TRA and CET, but virtually no mAb HB43 was bound to cells opsonized with F(ab′)2 TRA or CET (not shown).

Raji or Z138 cells, MOLT-4 cells, trypsinized BT-474 or trypsinized SCC-25 cells (∼107 cells/ml) were labeled with 4 μM PKH26 for 2 min followed by quenching with RPMI 1640 containing 10% FBS and after two washes were incubated with and without 10 μg/ml Al488-labeled mAbs (RTX, T101, TRA, or CET, respectively) in RPMI 1640 media for 20 min at 37°C with gentle shaking. The opsonized cells were washed with cold BSA/PBS to remove unbound mAb and then resuspended in media. The nonopsonized (naive) or Al488 mAb-opsonized PKH26-labeled donor cells were combined with PMA-treated adherent acceptor THP-1 cells in 24-well plates, or with RA-treated THP-1 cells in tubes. Various ratios of donor cells/acceptor cells were used, but typically 2–10 × 105 donor cells were added to 1 × 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 × g for 15 s, and then incubated for various times (usually 45 min) at 37°C in 5% CO2. Zero-time control samples, consisting only of opsonized or naive donor cells in RPMI 1640 medium, were subjected to identical conditions. 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 samples were immediately quenched by addition of cold BSA/PBS. All experiments were performed in duplicate. In some experiments, THP-1 cells were preincubated for 60 min at 37°C with 2 mg/ml human IgG or with 30 μg/ml anti-FcγR mAbs, and then used in the trogocytosis reactions following the above procedures. Blocking mAbs specific for FcγRI (mAb 10.1) and FcγRII (mAb IV.3) were used at concentrations (30 μg/ml) that were previously demonstrated to lead to saturation of binding to these receptors (23, 24, 25, 26). After the trogocytosis reaction, the donor and acceptor cells were analyzed in a variety of assays, as detailed below.

In experiments employing PMA-treated adherent THP-1 cells, after the trogocytosis reaction donor cells were separated from THP-1 cells by gentle aspiration, and then residual THP-1 cells were removed from the wells by vigorous pipetting. In experiments using RA-treated THP-1 cells, donor cells were not separated from the THP-1 cells. The amount of residual opsonizing mAb on the donor cells was determined by flow cytometry by gating on FL2+ PKH26-labeled donor cells or by multispectral image analysis. In tests for loss of targeted epitope (i.e., shaving) the Al488 mAb-opsonized or nonopsonized donor cells were reprobed with the same Al488 mAb used in the initial opsonization step (“reopsonized”) in the presence of 2 mg/ml Ms IgG to block any nonspecific binding. The cells were then washed with PBS and examined by flow cytometry. In certain experiments shaved cells and control cells were stained with either FITC annexin V or with TOPRO-3 (Invitrogen) to determine whether shaving promoted apoptosis or direct killing of the cells (5, 8).

All probing steps were performed for 1 h on ice. Following their removal from the 24-well plates, THP-1 cells were probed in the presence of Ms IgG with a cocktail of bt anti-CD11b and bt anti-CD14, washed with BSA/PBS, and then secondarily probed with PE-Cy5 (tricolor) for multispectral image analyses or with either Al594 SA or Al647 SA for fluorescence microscopy or with Al647 SA for flow cytometric analysis. To monitor FcγRI levels, THP-1 cells were probed, before the trogocytosis reaction, with Al488 mAb M22, specific for a site on FcγRI that is not blocked by bound human IgG ligand (27). After the trogocytosis reaction the THP-1 cells were analyzed for residual bound Al488 mAb M22 and probed secondarily with PE Gt anti-Ms IgG (Fc-specific) or with Al647 Gt anti-Ms IgG1 (Fc-specific) to determine the percentage of Al488 mAb M22 remaining on the surface of the THP-1 cells.

Flow cytometry was performed using a dual-laser FACSCalibur cytometer (BD Biosciences). Mean fluorescence intensities were converted to molecules of equivalent soluble fluorochrome (MESF) using standard fluorescent beads (Spherotech). Multispectral image analysis was performed on an ImageStream imaging cytometer (Amnis) as previously described (28, 29). Fluorescence microscopy was performed under oil at high magnification (×100) using a BX40 fluorescent microscope (Olympus). Images were captured with a digital camera and visualized with MagnaFire analysis software.

To distinguish mAb bound to the cell surface from internalized mAb, donor cells were opsonized with Al488 mAbs and then incubated for 5 min at pH 2.5 in RPMI 1640 medium supplemented with 2% FBS (30). This procedure has been used to identify ligands bound to the external surface of a cell, which are released due to incubation at low pH. After incubation the cells were washed twice in PBS and then subjected to flow cytometry analyses. This same procedure was used to examine both donor and acceptor cells after the shaving reaction.

Statistical significance was determined using t tests performed with SigmaStat software (Jandel).

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 = 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 lipid is removed from donor cells during the trogocytosis reaction (31). Inspection of images of individual cells (Fig. 1,E–H) clearly reveal that the THP-1 cells had taken up both Al488 RTX and PKH26 after 45 min (compare Fig. 1,G with Fig. 1,H). Some Raji cell/THP-1 cell pairs were discernable and appeared to be connected in an immunologic synapse; after 45 min, both the Al488 and PKH26 signals were visible on both cells (Fig. 1 I).

FIGURE 1.

Both Al488 RTX and PKH26 are transferred from opsonized Raji cells to THP-1 cells. A–D, Al488 RTX-opsonized, PKH26-dyed Raji cells were incubated with THP-1 cells for 45 min at 37°C. THP-1 cells were then identified as described in the Materials and Methods. The dot plots, obtained by multispectral image analysis, show the fluorescent signals for Al488 RTX (A and C) and PKH26 (B and D) at time 0 (A and B) and after 45 min (C and D) for the PE Cy5-negative population (Raji cells in red) and for the PE Cy5-positive population (THP-1 cells in yellow). E–H, Representative images of Al488 RTX-opsonized, PKH26-dyed Raji cells (E and F) and of CD11b+/CD14+ RA-activated THP-1 cells (G and H) after 0 and 45 min incubation at 37°C. I, Representative images of cell pairs after 45 min incubation at 37°C. SSC, Side scatter; BF, brightfield. Representative of three similar experiments.

FIGURE 1.

Both Al488 RTX and PKH26 are transferred from opsonized Raji cells to THP-1 cells. A–D, Al488 RTX-opsonized, PKH26-dyed Raji cells were incubated with THP-1 cells for 45 min at 37°C. THP-1 cells were then identified as described in the Materials and Methods. The dot plots, obtained by multispectral image analysis, show the fluorescent signals for Al488 RTX (A and C) and PKH26 (B and D) at time 0 (A and B) and after 45 min (C and D) for the PE Cy5-negative population (Raji cells in red) and for the PE Cy5-positive population (THP-1 cells in yellow). E–H, Representative images of Al488 RTX-opsonized, PKH26-dyed Raji cells (E and F) and of CD11b+/CD14+ RA-activated THP-1 cells (G and H) after 0 and 45 min incubation at 37°C. I, Representative images of cell pairs after 45 min incubation at 37°C. SSC, Side scatter; BF, brightfield. Representative of three similar experiments.

Close modal

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 Raji cells were not opsonized with the Al488 RTX bait (Fig. 2, C and D, open circles).

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 Methods to identify THP-1 cells and then subjected to multispectral image analysis (A and C, geometric mean fluorescence displayed) or to conventional 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.

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 Methods to identify THP-1 cells and then subjected to multispectral image analysis (A and C, geometric mean fluorescence displayed) or to conventional 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.

Close modal

We extended this in vitro assay system to examine trogocytosis of donor cells opsonized with mAbs T101, CET, or TRA, and then 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. 3,A 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 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 FcγR. Moreover, as defined by PKH26 fluorescence, membrane fragments from these donor cells were also taken up by the acceptor THP-1 cells (Fig. 3,D), 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. 3 D, 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.

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 at 37°C with THP-1 cells (1:10 donor/acceptor ratio in A and 1:1 ratio in B–E) for 0 (open bar), 45 (filled bar), or for 45 min with THP-1 cells pretreated with 2 mg/ml hu IgG for 60 min (striped bar). Al488 signals for each cell type were determined by flow cytometry and are displayed as MESF values. D, Uptake of PKH26 by THP-1 cells from Al488 mAb-opsonized cells vs uptake from nonopsonized donor cells (gray cross-hatched bars) E, Uptake of PKH26. Donor cells were not opsonized (open bars) or were opsonized with unlabeled mAbs T101, CET, or TRA (filled bars) and incubated as in B–D. Results are representative of nine similar experiments. Means and SD are reported in this figure and in the following figures. Significant differences in this figure and in those that follow are all vs the 45-min incubation and are denoted as: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

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 at 37°C with THP-1 cells (1:10 donor/acceptor ratio in A and 1:1 ratio in B–E) for 0 (open bar), 45 (filled bar), or for 45 min with THP-1 cells pretreated with 2 mg/ml hu IgG for 60 min (striped bar). Al488 signals for each cell type were determined by flow cytometry and are displayed as MESF values. D, Uptake of PKH26 by THP-1 cells from Al488 mAb-opsonized cells vs uptake from nonopsonized donor cells (gray cross-hatched bars) E, Uptake of PKH26. Donor cells were not opsonized (open bars) or were opsonized with unlabeled mAbs T101, CET, or TRA (filled bars) and incubated as in B–D. Results are representative of nine similar experiments. Means and SD are reported in this figure and in the following figures. Significant differences in this figure and in those that follow are all vs the 45-min incubation and are denoted as: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Close modal

To verify that the uptake of the PKH26 illustrated in Fig. 3,D was not simply a spectral overlap artifact caused by uptake of the Al488-labeled opsonizing mAbs, the experiment illustrated in Fig. 3,E 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 these cells were opsonized with the Al488-labeled mAbs. Once again, substantially less of the membrane dye was taken up from naive cells relative to mAb-opsonized cells (Fig. 3 E, gray hatched bars vs black bars).

To generalize our findings we also tested freshly isolated human monocytes for their potential to serve as acceptor cells and promote trogocytosis. The results in Fig. 4 in which monocytes were tested as acceptor cells are quite similar to those illustrated in Fig. 3,B–D in which THP-1 cells were examined. Indeed, all three Al488 mAbs were removed from their respective target cells and taken up by the monocytes, and substantial transfer of the PKH26 dye from donor cells to monocytes was only seen when the donor cells were opsonized with their respective mAbs (Fig. 4). These transfer reactions were inhibited when the monocytes were preincubated with human IgG, again providing evidence that FcγR play an important role in the trogocytosis reaction for these primary cells as well.

FIGURE 4.

Isolated human monocytes take up opsonizing mAb and PKH26 from opsonized donor cells, and transfer can be blocked with hu IgG. A–C, Similar to Fig. 3 B–D, except human monocytes, isolated from PBMC, were used as acceptor cells. In B and C, all differences between the 45-min samples and the other conditions were significant at p < 0.006, and asterisks were omitted for clarity. Representative of three similar experiments.

FIGURE 4.

Isolated human monocytes take up opsonizing mAb and PKH26 from opsonized donor cells, and transfer can be blocked with hu IgG. A–C, Similar to Fig. 3 B–D, except human monocytes, isolated from PBMC, were used as acceptor cells. In B and C, all differences between the 45-min samples and the other conditions were significant at p < 0.006, and asterisks were omitted for clarity. Representative of three similar experiments.

Close modal

To further investigate the role of FcγR in trogocytosis, we examined the potential of blocking mAbs, specific for FcγRI and FcγRII, to inhibit the reaction. The FcγR-specific mAbs were able to modestly block both removal of the Al488 mAbs from the donor cells and uptake of Al488 mAbs and PKH26 by the THP-1 cells in all four systems (Fig. 5), but blocking was not nearly as effective as was observed with human IgG (Figs. 3 and 4). Human IgG can contain aggregates that can bind with high affinity to FcγR (32), and considerably higher concentrations of human IgG were used, likely explaining the higher level of inhibition observed. Fig. 5 A also shows that the percentage of Al488 RTX removed from the Raji cells (80%) was greater than the percentage of the other mAbs removed from their respective donor cells (25–50%), possibly suggesting that the nature of the Ag chelated by a given mAb may influence the degree of trogocytosis (see Discussion).

FIGURE 5.

Blockade of FcγRI (CD64) and FcγRII (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 μg/ml) specific for FcγRI (mAb 10.1) or for FcγRII (mAb IV.3) for 60 min at 37°C before incubation with donor cells. Cell mixtures were analyzed by flow cytometry for residual Al488 mAb on donor cells (A) and acquired Al488 mAb and PKH26 by THP-1 cells (B and C, respectively). In most experiments, the differences between the 45-min points and the samples reacted with anti-FcγR mAbs were significant, but because the absolute differences were small, asterisks were omitted. The dashed line in B represents the background immunofluorescence of naive THP-1 cells.

FIGURE 5.

Blockade of FcγRI (CD64) and FcγRII (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 μg/ml) specific for FcγRI (mAb 10.1) or for FcγRII (mAb IV.3) for 60 min at 37°C before incubation with donor cells. Cell mixtures were analyzed by flow cytometry for residual Al488 mAb on donor cells (A) and acquired Al488 mAb and PKH26 by THP-1 cells (B and C, respectively). In most experiments, the differences between the 45-min points and the samples reacted with anti-FcγR mAbs were significant, but because the absolute differences were small, asterisks were omitted. The dashed line in B represents the background immunofluorescence of naive THP-1 cells.

Close modal

Previously we reported that F(ab′)2 of cell-bound RTX do not promote shaving (5), and we have now extended this observation to the F(ab′)2 of TRA and CET. FITC-labeled F(ab′)2 of the mAbs bound to their target epitopes on donor cells, and prior opsonization with the intact cognate IgG mAbs blocked this binding (Fig. 6, A and B). When cells were opsonized with F(ab′)2 or with intact IgG mAbs and then reacted with THP-1 cells for 45 min at 37°C, there was little if any loss of bound F(ab′)2, but substantial fractions 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 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 FcγR on the acceptor cell is a key step in the shaving reaction.

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.

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.

Close modal

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 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).

FIGURE 7.

Fluorescence microscopy analyses confirm transfer of Al488 mAbs to THP-1 cells. Donor cells were opsonized with Al488 mAbs and reacted with THP-1 cells as in Fig. 3. THP-1 cells were identified by probing with a cocktail of bt anti-CD11b and bt anti-CD14 followed by Al594 SA. A, Brightfield images. B, Merged images show some red THP-1 cells containing Al488 mAbs. C, Texas Red filter shows red THP-1 cells. D, FITC filter shows Al488 mAbs. Original magnification, ×100. Representative of two similar experiments.

FIGURE 7.

Fluorescence microscopy analyses confirm transfer of Al488 mAbs to THP-1 cells. Donor cells were opsonized with Al488 mAbs and reacted with THP-1 cells as in Fig. 3. THP-1 cells were identified by probing with a cocktail of bt anti-CD11b and bt anti-CD14 followed by Al594 SA. A, Brightfield images. B, Merged images show some red THP-1 cells containing Al488 mAbs. C, Texas Red filter shows red THP-1 cells. D, FITC filter shows Al488 mAbs. Original magnification, ×100. Representative of two similar experiments.

Close modal

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 ± 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 ± 2,000 cells. Comparable results for recovery of donor cells in the CET/SCC-25 system were 9,100 ± 700 and 7,500 ± 100 cells, respectively. These results indicate that little if any phagocytosis occurred during the shaving reaction. Finally, inspection of fields by fluorescence microscopy such as those illustrated in Fig. 7 did not reveal any obviously phagocytosed cells.

We performed control experiments to determine whether the uptake of the opsonizing mAbs and PKH26 by the THP-1 cells could have resulted from passive dissociation of the mAb and 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 donor 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 ∼15% for RTX-opsonized cells that were incubated at 37°C. However, there was <3% dissociation for the other mAb-opsonized donor cells, and loss of PKH26 from all four donor cell types was <3%. 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 (<2% of the positive controls, data not shown), thus ruling out passive dissociation and uptake as a transfer mechanism.

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 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 after opsonization, between 75% and 90% of bound mAbs could be released by acid wash for the four mAb/donor cell pairs under investigation, thus indicating that most of the mAbs were bound to the cell surfaces and not internalized during opsonization. Moreover, the acid wash did not appear to induce irreversible denaturation of the epitopes targeted by the mAbs, because after the acid wash and a reequilibration to neutral pH, the cells were again capable of binding the cognate mAbs (third bar from the left in each graph of Fig. 8). As already demonstrated, a substantial fraction of the mAbs could be shaved off the donor cells by THP-1 acceptor cells (fourth set of bars from the left of Fig. 8) and most of the material that was not removed by the THP-1 cells could still 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 mAbs that were not removed from the donor cells in a single pass of shaving were indeed still located on the cell surface. Finally, a considerably different pattern was evident when the THP-1 cells were subjected to acid wash after the shaving reaction (right sides of each graph in Fig. 8). The results indicate that most of the Al488 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 reaction.

FIGURE 8.

Most mAbs bound to donor cells are surface bound, and after the shaving reaction the mAbs remaining on the donor cells are not internalized, but the mAbs taken up by the THP-1 cells are internalized. A–D, Results for RTX/Daudi cells, T101/MOLT-4 cells, CET/SCC-25 cells, and TRA/BT-474 cells, respectively. Bars in panels on the left, from left to right, indicate binding of Al488 mAbs to: opsonized donor cells (OP); opsonized and acid-washed donor cells (OP, AW); opsonized and acid-washed donor cells, followed by reopsonization (OP, AW, R); opsonized donor cells subjected to trogocytosis/shaving with THP-1 cells (OP, 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 by the signal for THP-1 cells after taking up the mAb from donor cells (45′ T), followed by acid wash of the THP-1 cells (45′ T, AW). Representative of two similar experiments.

FIGURE 8.

Most mAbs bound to donor cells are surface bound, and after the shaving reaction the mAbs remaining on the donor cells are not internalized, but the mAbs taken up by the THP-1 cells are internalized. A–D, Results for RTX/Daudi cells, T101/MOLT-4 cells, CET/SCC-25 cells, and TRA/BT-474 cells, respectively. Bars in panels on the left, from left to right, indicate binding of Al488 mAbs to: opsonized donor cells (OP); opsonized and acid-washed donor cells (OP, AW); opsonized and acid-washed donor cells, followed by reopsonization (OP, AW, R); opsonized donor cells subjected to trogocytosis/shaving with THP-1 cells (OP, 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 by the signal for THP-1 cells after taking up the mAb from donor cells (45′ T), followed by acid wash of the THP-1 cells (45′ T, AW). Representative of two similar experiments.

Close modal

We previously reported that THP-1 cell-promoted shaving of CD20 from RTX-opsonized Z138 cells neither killed the Z138 cells nor rendered them apoptotic (5), and we have now generalized this observation to two of the mAb-donor cell pairs in the present system. As presented in Table I, we find that reaction of mAb-opsonized donor cells with THP-1 cells for 45 min at 37°C followed by quenching with human IgG and cold BSA/PBS does not kill the cells or render them apoptotic. Only a small percentage of these donor cells were positive for the annexin V or TOPRO-3 stains, nearly identical to the values obtained for the 0-min control samples consisting of opsonized donor cells mixed with THP-1 cells preblocked with human IgG to preclude shaving. Thus, there is negligible cell killing or induction of apoptosis as a consequence of the shaving reaction.

Table I.

Effect of the shaving reaction on donor cell viability

mAb/CellIncubation Time with THP-1 Cells (min)Annexin V+ (% ± SD)TOPRO-3+ (% ± SD)
TRA/BT-474 45 5.6 ± 0.4 4.7 ± 0.4 
 5.1 ± 0.1 5.1 ± 0.8 
CET/SCC-25 45 9.2 ± 0.1 6.6 ± 0.1 
 12.7 ± 0.2 6.8 ± 0.4 
mAb/CellIncubation Time with THP-1 Cells (min)Annexin V+ (% ± SD)TOPRO-3+ (% ± SD)
TRA/BT-474 45 5.6 ± 0.4 4.7 ± 0.4 
 5.1 ± 0.1 5.1 ± 0.8 
CET/SCC-25 45 9.2 ± 0.1 6.6 ± 0.1 
 12.7 ± 0.2 6.8 ± 0.4 

We next investigated whether the degree of mAb opsonization for one representative mAb/donor cell pair, Al488 TRA/BT-474 cells, would influence the degree of trogocytosis. The results in Fig. 9,A indicate that over a wide range of TRA concentrations used for opsonization, ∼50% of the Al488 TRA initially bound to BT-474 cells was removed during incubation with THP-1 cells. Consistent with this finding, considerably more Al488 TRA was taken up by the THP-1 cells which were reacted with the most heavily opsonized BT-474 cells (Fig. 9,B). Both the loss of Al488 by the donor cells and the gain of Al488 by the acceptor cells were inhibited in the presence of 2 mg/ml human IgG. Next, for the T101/MOLT-4 system, we held the degree of opsonization constant at 10 μg/ml Al488 mAb T101 and varied the donor cell/acceptor cell ratio (Fig. 9 C). Not surprisingly, we find that as the donor cell/acceptor cell ratio increased, the amount of Al488 mAb T101 taken up by the acceptor cells increased. This result suggests that a single acceptor cell is capable of taking up mAb ligands from more than one donor cell. Finally, similar results were respectively obtained in both dose-response studies and in experiments in which cell ratios were varied, based on using T101-opsonized MOLT-4 cells and CET-opsonized SCC-25 cells, as well as on TRA-opsonized BT-474 cells and CET-opsonized SCC-25 cells (results not shown).

FIGURE 9.

Increasing opsonization of donor cells with Al488 mAb or 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 indicated concentrations of Al488 TRA, and after two washes were incubated with THP-1 cells at 37°C for 45 min. Uptake of Al488 TRA by THP-1 cells increased with increasing opsonization in the range 0.05–10 μg/ml. The dashed line (also in C) indicates the background fluorescence of naive THP-1 cells. C, MOLT-4 cells were opsonized with 10 μg/ml Al488 mAb T101 and then incubated with THP-1 cells at the indicated ratios of donor/acceptor cells. Representative of two similar experiments.

FIGURE 9.

Increasing opsonization of donor cells with Al488 mAb or 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 indicated concentrations of Al488 TRA, and after two washes were incubated with THP-1 cells at 37°C for 45 min. Uptake of Al488 TRA by THP-1 cells increased with increasing opsonization in the range 0.05–10 μg/ml. The dashed line (also in C) indicates the background fluorescence of naive THP-1 cells. C, MOLT-4 cells were opsonized with 10 μg/ml Al488 mAb T101 and then incubated with THP-1 cells at the indicated ratios of donor/acceptor cells. Representative of two similar experiments.

Close modal

A key late step in the endocytic process of shaving or trogocytosis is the internalization by the acceptor cell of the transferred ligand and the cognate receptor of the acceptor cell. Our previous work using RTX-opsonized Z138 cells as donor cells indicated that FcγRI is the receptor principally responsible for removal of RTX (5), and therefore we first tested for FcγRI internalization during incubation of THP-1 cells with RTX-opsonized Z138 cells. THP-1 cells were first labeled with Al488 mAb M22, which binds to FcγRI at a site not blocked by bound human IgG (27), and then reacted with RTX-opsonized Z138 cells for 45 min at 37°C. To determine whether mAb M22 was still bound to FcγRI on the surface of the THP-1 cells or had been internalized, these reaction samples were probed secondarily with PE anti-Ms IgG (Fc-specific) after the trogocytosis reaction. Compared with the 0-time controls, there was a modest reduction (∼30%) in the amount of Al488 mAb M22 associated with THP-1 cells after reaction with donor cells (Fig. 10,A), likely due to passive dissociation of the mAb. However, as shown in Fig. 10,B, compared with the 0-time controls, far less mAb M22 was detectable on the THP-1 cell surface after incubation with opsonized donor cells for 45 min at 37°C (∼70% reduction in signal). These results suggest that much of the residual cell-associated mAb M22 (and presumably the FcγRI to 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. 10,C) and probed after the trogocytosis reaction with Al647 Gt anti-Ms IgG1 (Fig. 10,D). 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 (∼15%) of Al488 mAb M22 associated with the THP-1 cells (Fig. 10,C), 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. 10 D), indicating that much of the cell-bound Al488 mAb M22 had been internalized, presumably along with FcγRI. Finally, there was no internalization of cell-bound mAb M22 when the THP-1 cells were incubated with any of the nonopsonized donor cells (results not shown).

FIGURE 10.

FcγRI is internalized during trogocytosis. A and B, THP-1 cells were reacted with 10 μg/ml Al488 mAb M22 (specific for FcγRI (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 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 experiments.

FIGURE 10.

FcγRI is internalized during trogocytosis. A and B, THP-1 cells were reacted with 10 μg/ml Al488 mAb M22 (specific for FcγRI (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 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 experiments.

Close modal

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 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-CD11b and bt anti-CD14, followed by Al647 SA, to identify THP-1 cells. Inspection of the bright field (Fig. 11,A) and fluorescent images reveals red Al647 THP-1 cells (Fig. 11,B) that had taken up green Al488-labeled TRA or CET (Fig. 11,C). However, these THP-1 cells were not stained with Al546 mAb HB43 (Fig. 11,D), 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. 11 E).

FIGURE 11.

Fluorescence microscopy analyses confirm internalization of opsonizing mAbs by THP-1 cells during trogocytosis. Donor cells were opsonized with Al488-labeled TRA or CET and reacted with THP-1 cells as in Fig. 3. After 45 min at 37°C, cell mixtures were probed with bt anti-CD11b and bt anti-CD14 followed by Al647 SA to identify THP-1 cells, and with Al546 HB43 (anti-human IgG, Fcγ-specific) to detect Al488 TRA or Al488 CET on the outside of cells. A, Bright field. B, The Al647 SA identifies CD11b+/CD14+ THP-1 cells. C, A merge of Al647 and Al488 reveals Al488 associated with THP-1 cells. D, Al546 mAb HB43 detects Al488 TRA or Al488 CET on the surface of donor cells, but not on the surface of THP-1 cells. E, A merge of Al488 and Al546 shows Al488-positive, Al546-negative THP-1 cells. Representative of two similar experiments.

FIGURE 11.

Fluorescence microscopy analyses confirm internalization of opsonizing mAbs by THP-1 cells during trogocytosis. Donor cells were opsonized with Al488-labeled TRA or CET and reacted with THP-1 cells as in Fig. 3. After 45 min at 37°C, cell mixtures were probed with bt anti-CD11b and bt anti-CD14 followed by Al647 SA to identify THP-1 cells, and with Al546 HB43 (anti-human IgG, Fcγ-specific) to detect Al488 TRA or Al488 CET on the outside of cells. A, Bright field. B, The Al647 SA identifies CD11b+/CD14+ THP-1 cells. C, A merge of Al647 and Al488 reveals Al488 associated with THP-1 cells. D, Al546 mAb HB43 detects Al488 TRA or Al488 CET on the surface of donor cells, but not on the surface of THP-1 cells. E, A merge of Al488 and Al546 shows Al488-positive, Al546-negative THP-1 cells. Representative of two similar experiments.

Close modal

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, 6, 7). After the usual incubation of mAb-opsonized donor cells with THP-1 cells, we used this reopsonization paradigm to test for loss of cell surface Ag for the three mAb-donor cell pairs under investigation. The results for mAb T101-opsonized MOLT-4 cells reacted with THP-1 cells provide unambiguous evidence for CD5 shaving (Fig. 12,A). In the nonopsonized control there was little, if any, loss of the target Ag (97% of the time 0 signal remained). However, MOLT-4 cells that were first opsonized with Al488 mAb T101 and then reacted with THP-1 cells had substantially reduced mAb T101 binding capacity when they were reopsonized with Al488 mAb T101 after the trogocytosis reaction (29% of the 0-time control). In agreement with our studies for the RTX-CD20 system, the mAb T101-mediated shaving reaction is blocked by human IgG (Fig. 12,A), again suggesting that the reaction is mediated by FcγR. Comparable analyses for Al488 TRA/BT-474 and Al488 CET/SCC-25 systems also demonstrated shaving, based on the reduced mAb binding capacity of cells that were first opsonized with their respective Al488 mAbs (76% and 36% of the 0-time controls, respectively, Fig. 12, B and C). Once again, nonopsonized donor cells suffered little, if any, loss of mAb binding capacity, and shaving could be blocked by preincubation of the acceptor THP-1 cells with human IgG. These results demonstrate that, as we showed for RTX (5), acceptor cells cannot only remove the opsonizing mAb, they also remove its target Ag from the donor cells in these three mAb/donor cell combinations.

FIGURE 12.

Reopsonization of donor cells after trogocytosis confirms shaving (loss of target epitope), and the reaction can be blocked with human IgG. Donor cells, nonopsonized or opsonized with Al488-labeled mAbs, were incubated with THP-1 cells as in Fig. 3. After a 45-min incubation at 37°C, the cell mixtures were reprobed with the original Al488-labeled mAb. A–C, Shaving of Al488 mAb T101-opsonized MOLT4 cells, Al488 TRA-opsonized BT-474 cells, and Al488 CET-opsonized SCC-25 cells, respectively. Representative of three similar experiments.

FIGURE 12.

Reopsonization of donor cells after trogocytosis confirms shaving (loss of target epitope), and the reaction can be blocked with human IgG. Donor cells, nonopsonized or opsonized with Al488-labeled mAbs, were incubated with THP-1 cells as in Fig. 3. After a 45-min incubation at 37°C, the cell mixtures were reprobed with the original Al488-labeled mAb. A–C, Shaving of Al488 mAb T101-opsonized MOLT4 cells, Al488 TRA-opsonized BT-474 cells, and Al488 CET-opsonized SCC-25 cells, respectively. Representative of three similar experiments.

Close modal

We have reported previously that after RTX binds to CD20 on B lymphocytes, the constituents of the IC (both RTX and CD20) can be removed (shaved) from these cells and taken up and internalized by acceptor cells that express FcγR (5). This report presents three key new findings. First, we can now further describe the shaving reaction as being quite similar, if not identical, to a previously reported phenomenon, trogocytosis (1, 2, 3, 4), as revealed by the concerted uptake of the membrane dye PKH26 when mAb/cell surface Ag IC are removed from the donor cells (Figs. 1–4). Second, we have been able to extend and generalize our observations of FcγR-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.

FcγR-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, 36, 37, 38, 39, 40, 41, 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 target 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 involves 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 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 FcγR on the acceptor macrophages were blocked; their findings could perhaps be considered the first report of trogocytosis, long before the term was coined. We also note that in the present study, based on recovery of donor cells, there was no indication of phagocytosis of donor cells promoted by THP-1 cells; rather, the transfer of PKH26- and Al488-labeled mAbs can best be explained by trogocytosis.

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 substrate cell-bound IgG. That is, recognition of bound IgG on the opsonized cells by FcγR on the acceptor cells generates what appear to be immunologic synapses (Fig. 1,I). After formation of the immunologic synapse, target cell Ag, bound mAb IgG, and fragments of target cell membrane can be removed and internalized by the acceptor cell (Figs. 1, 7, and 11). Moreover, during this process there is negligible internalization of bound mAbs by the donor cells (Fig. 8). In the present study and in our earlier reports we have postulated a role for FcγRI in this process (5, 43). Human IgG can effectively suppress transfer of both Al488 mAb and PKH26 from donor cells to acceptor cells (Figs. 3, 4, 9, and 12). Furthermore, our results based on the use of mAb M22, which binds to FcγRI at a site not blocked by human IgG, demonstrate that FcγRI is indeed internalized by the acceptor cells following the trogocytosis reaction (Fig. 10), thus providing support for its role in trogocytosis. In agreement with our previous study in the RTX/Z138 cell system (5), shaving is abrogated when donor cells are opsonized with F(ab′)2 of CET or TRA (Fig. 6), again indicating a role for FcγR on acceptor cells recognizing mAb-opsonized cells. Internalization of the F(ab′)2 by the donor cells is quite unlikely; we note that previous reports of antigenic modulation indicated that intact IgG mAbs were required to promote monocyte-mediated loss of targeted epitiopes (44). Attempted blockade of FcγRI or FcγRII with specific mAbs at concentrations of 30 μg/ml only modestly inhibited trogocytosis (Fig. 5), likely due to higher avidity binding of clustered mAb on the opsonized donor cells to FcγR on the THP-1 cells. However, substantial blockade was evident when high concentrations of human IgG were used, and, as human IgG can contain aggregates (32), these higher concentrations may have allowed for more effective interaction with and blockade of FcγRI.

Based on these considerations, it would seem that shaving/trogocytosis of mAb-opsonized cells might not occur in vivo, where the concentrations of IgG in the bloodstream can be ∼10 mg/ml, thus potentially blocking the action of FcγRI. However, we emphasize that shaving of RTX-opsonized cells and antigenic modulation of T101-opsonized cells were first reported based on clinical observations (6, 7, 14), not in vitro experiments. It is therefore likely that certain compartments within the body (perhaps the sinusoids of the liver) allow for close contact between mAb-opsonized cells and FcγRI-expressing acceptor cells such as Kupffer cells, thus promoting shaving. We also note that several studies in mouse models have revealed that FcγRI-expressing acceptor cells can play roles in processing of IgG-opsonized substrates (45, 46, 47), despite the high concentrations of IgG in the bloodstream.

An important question emerges from this work: How do effector cells “decide” the fate of IgG-opsonized substrate cells? A voluminous literature reveals that fixed tissue macrophages such as Kupffer cells in the liver and macrophages in the spleen clear IgG-opsonized cells (48, 49, 50, 51, 52), and our clinical investigations have demonstrated that infusion of just 30 mg of RTX promotes rapid clearance of circulating malignant B cells in patients with CLL (6, 7). However, our findings suggested that fixed tissue macrophages may also mediate shaving, because we found that CLL B cells not cleared in the first 15–30 min of RTX infusion lost substantial amounts of CD20, suggesting that clearance and shaving occur simultaneously (7).

One factor that may determine whether an opsonized target is cleared or shaved may be its size. If the target is sufficiently small (e.g., an IgG-opsonized sheep erythrocyte, bacterium, or virus), then it is likely that the entire particle will be internalized by the zipper phagocytic mechanism. However, if the target is a malignant cell opsonized with many copies of a single mAb, and the target cell is comparable in size or larger than the monocyte/macrophage, then it is likely that the acceptor cell may instead ingest only small pieces of the target cell. Indeed, Wallace et al. examined the processing of IgG-opsonized breast cancer cells by human macrophages and reported that the macrophages appeared to be “chipping away” at these substrates and then internalizing the pieces (53).

Another important factor likely affecting the relative amounts of cell clearance vs shaving is the burden of cells that are targeted by a given mAb (6, 54, 55, 56, 57). If the initial burden of circulating malignant 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 the cytolytic capacity of NK cells are likely to be exceeded. Indeed, Bowles and Weiner have reported that CD16 (FcγRIII) is substantially down-regulated on NK cells after reaction with RTX-opsonized cells (56). Berdeja et al. have also reported that soon after RTX infusion, the cytolytic capacity of NK cells is reduced considerably (57). Thus, under conditions in which effector mechanisms that clear cells are saturated or exhausted, shaving/trogocytosis may be the default reaction.

There are reports of apparent shaving of cancer cells in solid tumors. We have found, for example, based on a SCID mouse model, that infusion of RTX can lead to loss of CD20 on Z138 cells growing in a solid tumor (43). Laurent et al. have reported that mature B lymphocytes lacking CD20 were demonstrable in nodular lymphoid infiltrates in the bone marrow of patients after treatment with RTX (58). Bertram et al. found that mAb T101 promoted antigenic modulation (loss of CD5) on malignant T cells growing in tumors of patients with cutaneous T cell lymphoma (14). Taken together with the present report, these findings may have important implications for the use of mAbs such as RTX, TRA, and CET in cancer immunotherapy for solid tumors. An important question centers on the accessibility of mAb-opsonized cells in such tumors to effector cells such as NK cells, monocytes, and macrophages. It is possible that local saturation of effector cell cytotoxic capacity within the tumors can promote shaving, and analyses of fine-needle aspirates or biopsies of tumors after mAb treatment may address this issue.

Finally, the mechanism(s) of action of immunotherapeutic mAbs as well as the rate at which they kill tumor cells are also likely to influence whether they can mediate shaving, as well as the degree to which shaving can reduce killing efficacy. Although recent evidence supports a role for Ab-dependent cellular cytotoxicity in the cytotoxic action of both CET and TRA, these mAbs may also promote direct cell killing by binding to target sites on cells, thus setting off signaling cascades that can induce apoptosis, or block cell growth and proliferation (15, 16, 17, 18). If these cytotoxic processes are sufficiently effective, then the targeted cells may be killed more rapidly than they can be shaved, or any shaving that does occur may not be sufficient to prevent killing. However, shaving may reduce killing efficacy, and this possibility should be considered in the design of clinical trials and their correlative studies (7).

In summary, our experiments demonstrate that the three newly tested mAb/cell substrate pairs are all subject to trogocytosis/shaving by acceptor monocytes, as we previously reported for the RTX/B cell system. However, we did observe quantitative differences in the degree of shaving for these systems, and we cannot at this time predict whether binding of an IgG mAb to a particular epitope on a cell will in fact promote effector cell-mediated shaving/trogocytosis. The mAbs used in the present studies include one mouse IgG2a (T101) and three human IgG1 isotypes, and strong interaction with FcγRI would therefore appear to be one prerequisite. However, the nature of the epitope targeted by the mAb, the disposition of the epitope within the cell membrane, and the lifetime of the mAb when it binds to the cells are all likely to be important. If mAb binding promotes rapid cell killing or if mAb binding is followed by internalization, or shedding, then trogocytosis/shaving may be minimized. Many other factors can also influence these reactions and a more complete understanding of these issues may allow for the improved design of immunotherapeutic mAbs targeted to malignant cells.

We thank Dr. Jorge Carrasquillo (Sloan Kettering Cancer Center) who graciously provided us with mAb T101 at the National Institutes of Health. This paper is dedicated to the loving memory of P. C. Venkat for tireless advocacy on behalf of patients with chronic lymphocytic leukemia.

The authors have no financial conflicts of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This research was supported through a grant to the University of Virginia Cancer 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 Support Grant.

3

Abbreviations used in this paper: RTX, rituximab; Al, Alexa; bt, biotinylated; CET, cetuximab; CLL, chronic lymphocytic leukemia; Gt, goat; IC, immune complex(es); MESF, molecules of equivalent soluble fluorochome; Ms, mouse; RA, retinoic acid; SA, streptavidin; TRA, trastuzumab.

1
Hudrisier, D., J. Riond, H. Mazarguil, J. E. Gairin, E. Joly.
2001
. CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner.
J. Immunol.
166
:
3645
-3649.
2
Joly, E., D. Hudrisier.
2003
. What is trogocytosis and what is its purpose?.
Nat. Immunol.
4
:
815
3
Hudrisier, D., J. Riond, L. Garidou, C. Duthoit, E. Joly.
2005
. T cell activation correlates with an increased proportion of antigen among the materials acquired from target cells.
Eur. J. Immunol.
35
:
2284
-2294.
4
Hudrisier, D., A. Aucher, A. L. Puaux, C. Bordier, E. Joly.
2007
. Capture of target cell membrane components via trogocytosis is triggered by a selected set of surface molecules on T or B cells.
J. Immunol.
178
:
3637
-3647.
5
Beum, P. V., A. D. Kennedy, M. E. Williams, M. A. Lindorfer, R. P. Taylor.
2006
. The shaving reaction: rituximab/CD20 complexes are removed from mantle cell lymphoma and chronic lymphocytic leukemia cells by THP-1 monocytes.
J. Immunol.
176
:
2600
-2609.
6
Kennedy, A. D., P. V. Beum, M. D. Solga, D. J. DiLillo, M. A. Lindorfer, C. E. Hess, J. J. Densmore, M. E. Williams, R. P. Taylor.
2004
. Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia.
J. Immunol.
172
:
3280
-3288.
7
Williams, M. E., J. J. Densmore, A. W. Pawluczkowycz, P. V. Beum, A. D. Kennedy, M. A. Lindorfer, S. H. Hamil, J. C. Eggleton, R. P. Taylor.
2006
. Thrice-weekly low-dose rituximab decreases CD20 loss via shaving and promotes enhanced targeting in chronic lymphocytic leukemia.
J. Immunol.
177
:
7435
-7443.
8
Beum, P. V., M. A. Lindorfer, R. P. Taylor.
2008
. Within peripheral blood mononuclear cells, antibody-dependent cellular cytotoxicity of rituximab-opsonized Daudi cells is promoted by NK cells and inhibited by monocytes due to shaving.
J. Immunol.
181
:
2916
-2924.
9
Ritz, J., J. M. Pesando, S. E. Sallan, L. A. Clavell, J. Notis-McConarty, P. Rosenthal, S. F. Scholossman.
1981
. Serotherapy of acute lymphoblastic leukemia with monoclonal antibody.
Blood
58
:
141
-152.
10
Miller, R. A., D. G. Maloney, J. McKillop, R. Levy.
1981
. In vitro effects of murine hybridoma monoclonal antibody in a patient with T-cell leukemia.
Blood
58
:
78
-86.
11
Chatenoud, L., M. F. Baudrihaye, H. Kreis, G. Goldstein, J. Schindler, J. F. Bach.
1982
. Human in vivo antigenic modulation induced by the anti-T cell OKT3 monoclonal antibody.
Eur. J. Immunol.
12
:
979
-982.
12
Rinnooy Kan, E. A., E. Platzer, K. Welte, C. Y. Wang.
1984
. Modulation induction of the T3 antigen by OKT3 antibody is monocyte dependent.
J. Immunol.
133
:
2979
-2985.
13
Pesando, J. M., P. Hoffman, M. Abed.
1986
. Antibody-induced antigenic modulation is antigen dependent: characterization of 22 proteins on a malignant human B cell line.
J. Immunol.
137
:
3689
-3695.
14
Bertram, J. H., P. S. Gill, A. M. Levine, D. Boquiren, F. M. Hoffman, P. Meyer, M. S. Mitchell.
1986
. Monoclonal antibody T101 in T cell malignancies: a clinical, pharmacokinetic, and immunologic correlation.
Blood
68
:
752
-761.
15
Musolino, A., N. Naldi, B. Bortesi, D. Pezzuolo, M. Capelletti, G. Missale, D. Laccabue, A. Zerbini, R. Camisa, G. Bisagni, et al
2008
. Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer.
J. Clin. Oncol.
26
:
1789
-1796.
16
Hudis, C. A..
2007
. Trastuzumab: mechanism of action and use in clinical practice.
N. Engl. J. Med.
357
:
39
-51.
17
Kurai, J., H. Chikumi, K. Hashimoto, K. Yamaguchi, A. Yamasaki, T. Sako, H. Touge, H. Makino, M. Takata, M. Miyata, et al
2007
. Antibody-dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines.
Clin. Cancer Res.
13
:
1552
-1561.
18
Galizia, G., D. Lieto, F. De Vita, M. Orditura, P. Castellano, T. Troiani, V. Imperatore, F. Ciardiello.
2007
. Cetuximab, a chimeric human mouse anti-epidermal growth factor receptor monoclonal antibody, in the treatment of human colorectal cancer.
Oncogene
26
:
3654
-3660.
19
Kennedy, A. D., M. D. Solga, T. A. Schuman, A. W. Chi, M. A. Lindorfer, W. M. Sutherland, P. L. Foley, R. P. Taylor.
2003
. An anti-C3b(i) mAb enhances complement activation, C3b(i) deposition, and killing of CD20+ cells by rituximab.
Blood
101
:
1071
-1079.
20
Craig, M. L., A. J. Bankovich, R. P. Taylor.
2002
. Visualization of the transfer reaction: tracking immune complexes from erythrocyte complement receptor 1 to macrophages.
Clin. Immunol.
105
:
36
-47.
21
Gyimesi, E., A. J. Bankovich, T. A. Schuman, J. B. Goldberg, M. A. Lindorfer, R. P. Taylor.
2004
. Staphylococcus aureus bound to complement receptor 1 on human erythocytes by bispecific monoclonal antibodies is phagocytosed by acceptor macrophages.
Immunol. Lett.
95
:
185
-192.
22
Edberg, J. C., L. Tosic, E. L. Wright, W. M. Sutherland, R. P. Taylor.
1988
. Quantitative analyses of the relationship between C3 consumption, C3b capture, and immune adherence of complement-fixing antibody/DNA immune complexes.
J. Immunol.
141
:
4258
-4265.
23
Wiener, E., R. A. Dellow, F. Mawas, C. H. Rodeck.
1996
. Role of FcγRIIa (CD32) in IgG anti-RhD-mediated red cell phagocytosis in vitro.
Transfus. Med.
6
:
235
-241.
24
Flesch, B. K., G. Achtert, J. Neppert.
1997
. Inhibition of monocyte and polymorphonuclear granulocyte immune phagocytosis by monoclonal antibodies specific for Fcγ RI, II, and III.
Ann. Hematol.
74
:
15
-22.
25
Tebo, A. E., P. G. Kremsner, A. J. F. Luty.
2002
. Fcγ receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro.
Clin. Exp. Immunol.
130
:
300
-306.
26
Kou, Z., M. Quinn, H. Chen, W. W. Rodrigo, R. C. Rose, J. J. Schlesinger, X. Jin.
2008
. Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells.
J. Med. Virol.
80
:
134
-146.
27
Guyre, P. M., R. F. Graziano, B. A. Vance, P. M. Morganelli, M. W. Fanger.
1989
. Monoclonal antibodies that bind to distinct epitopes on FcγRI are able to trigger receptor function.
J. Immunol.
143
:
1650
-1655.
28
George, T. C., D. A. Basiji, B. E. Hall, D. H. Lynch, W. E. Ortyn, D. J. Perry, M. J. Seo, C. A. Zimmerman, P. J. Morrissey.
2004
. Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer.
Cytometry
59A
:
237
-245.
29
Beum, P. V., M. A. Lindorfer, B. E. Hall, T. C. George, K. Frost, P. J. Morrissey, R. P. Taylor.
2006
. Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer.
J. Immunol. Methods
317
:
90
-99.
30
Beekman, J. M., C. E. van der Poel, J. A. Van Der Linden, D. L. van den Berg, P. V. van den Berghe, J. G. van de Winkel, J. H. Leusen.
2008
. Filamin A stabilizes FcγRI surface expression and prevents its lysosomal routing.
J. Immunol.
180
:
3938
-3945.
31
Daubeuf, S., A.-L. Puaux, E. Joly, D. Hudrisier.
2007
. A simple trogocytosis-based method to detect, quantify, characterize and purify antigen-specific live lymphocytes by flow cytometry, via their capture of membrane fragments from antigen-presenting cells.
Nat. Protoc.
1
:
2536
-2542.
32
Teeling, J. L., T. Jansen-Hendriks, T. W. Kuijpers, M. de Haas, J. G. J. van de Winkel, C. E. Hack, W. K. Bleeker.
2001
. Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia.
Blood
98
:
1095
-1099.
33
Teeling, J., R. R. French, M. S. Cragg, J. van den Brakel, M. Pluyter, H. Huang, C. Chan, P. W. Parren, C. E. Hack, M. Dechant, et al
2004
. Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin’s lymphomas.
Blood
104
:
1793
-1800.
34
Janssen, E., K. Tabeta, M. J. Barnes, S. Rutschmann, S. McBride, K. S. Bahjat, S. P. Schoenberger, A. N. Theofilopoulos, B. Beutler, K. Hoebe.
2006
. Efficient T cell activation via a Toll-interleukin 1 receptor-independent pathway.
Immunity
24
:
787
-799.
35
Griffin, F. M., J. A. Griffin, J. E. Leider, S. C. Silverstein.
1975
. Studies on the mechanism of phagocytosis: I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane.
J. Exp. Med.
142
:
1263
-1282.
36
Griffin, F. M., J. A. Griffin, S. C. Silverstein.
1976
. Studies on the mechanism of phagocytosis: II. The interaction of macrophages with anti-immunoglobulin IgG-coated bone marrow-derived lymphocytes.
J. Exp. Med.
144
:
788
-809.
37
Swanson, J. A., C. Watts.
1995
. Macropinocytosis.
Trends Cell Biol.
5
:
424
-427.
38
Davis, W., P. T. Harrison, M. J. Hutchinson, J. M. Allen.
1995
. Two distinct regions of FcγRI initiate separate signalling pathways involved in endocytosis and phagocytosis.
EMBO J.
14
:
432
-441.
39
Koval, M., K. Preiter, C. Adles, P. D. Stahl, T. H. Steinberg.
1998
. Size of IgG-opsonized particles determines macrophage response during internalization.
Exp. Cell Res.
242
:
265
-273.
40
Lovdal, T., E. Andersen, A. B. T. Brech.
2000
. Fc receptor mediated endocytosis of small soluble immunoglobulin G immune complexes in Kupffer and endothelial cells from rat liver.
J. Cell Sci.
113
:
3255
-3266.
41
Guyre, C., T. Keler, S. Swink, L. Vitale, R. Graziano, M. Fanger.
2001
. Receptor modulation by FcγRI-specific fusion proteins is dependent on receptor number and modified by IgG.
J. Immunol.
167
:
6303
-6311.
42
Swanson, J. A., A. D. Hoppe.
2004
. The coordination of signaling during Fc receptor-mediated phagocytosis.
J. Leukocyte Biol.
76
:
1093
-1103.
43
Li, Y., M. E. Williams, J. B. Cousar, A. W. Pawluczkowycz, M. A. Lindorfer, R. P. Taylor.
2007
. Rituximab/CD20 complexes are shaved from Z138 mantle cell lymphoma cells in intravenous and subcutaneous SCID mouse models.
J. Immunol.
179
:
4263
-4271.
44
Schroff, R. W., R. A. Klein, M. M. Farrell, H. C. Stevenson.
1984
. Enhancing effects of monocytes on modulation of a lymphocyte membrane antigen.
J. Immunol.
133
:
2270
-2277.
45
Ioan-Facsinay, A., S. J. de Kimpe, S. M. M. Hellwig, P. L. van Lent, F. M. A. Hofhuis, H. H. van Ojik, C. Sedlik, S. A. de Silveira, J. Gerber, Y. F. de Jong, et al
2002
. FcγRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection.
Immunity
16
:
391
-402.
46
Bevaart, L., M. J. H. Jansen, M. J. van Vugt, J. S. Verbeek, J. G. J. van de Winkel, J. H. W. Leusen.
2006
. The high-affinity IgG receptor, Fc-γRI, plays a central role in antibody therapy of experimental melanoma.
Cancer Res.
66
:
1261
-1264.
47
Baudino, L., F. Nimmerjahn, S. A. da Silveira, E. Martinez-Soria, T. Saito, M. Carroll, J. V. Ravetch, J. S. Verbeek, S. Izui.
2008
. Differential contribution of three activating IgG Fc receptors (FcγRI, FcγRIII, and FcγRIV) to IgG2a- and IgG2b-induced autoimmune hemolytic anemia in mice.
J. Immunol.
180
:
1948
-1953.
48
Schreiber, A. D., M. M. Frank.
1972
. Role of antibody and complement in the immune clearance and destruction of erythrocytes: in vivo effects of IgG and IgM complement fixing sites.
J. Clin. Invest.
51
:
575
-582.
49
Frank, M. M..
1989
. The role of macrophages in blood stream clearance. M. Zembala, and G. L. Asherson, eds.
Human Monocytes
337
-344. Academic Press, New York.
50
Kimberly, R. P., J. E. Salmon, J. C. Edberg, A. Gibofsky.
1989
. The role of Fcγ receptors in mononuclear phagocyte system function.
Clin. Exp. Rheumatol.
7
:
103
-108.
51
Gong, Q., Q. Ou, S. Ye, W. P. Lee, J. Cornelius, L. Diehl, W. Y. Lin, Z. Hu, Y. Lu, Y. Chen, et al
2005
. Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy.
J. Immunol.
174
:
817
-826.
52
Kavai, M., G. Szegedi.
2007
. Immune complex clearance by monocytes and macrophages in systemic lupus erythematosus.
Autoimmun. Rev.
6
:
497
-502.
53
Wallace, P. K., P. Kaufman, L. Lewis, T. Keler, A. Givan, J. Fisher, M. Waugh, A. Wahner, P. Guyre, M. Fanger, M. Ernstoff.
2001
. Bispecific antibody-targeted phagocytosis of HER-2/neu expressing tumor cells by myeloid cells activated in vivo.
J. Immunol. Methods
248
:
167
-182.
54
Golay, J., M. Manganini, V. Facchinetti, R. Gramigna, R. Broady, G. Borleri, A. Rambaldi, M. Introna.
2003
. Rituximab-mediated antibody-dependent cellular cytotoxicity against neoplastic B cells is stimulated strongly by interleukin-2.
Haematologica
88
:
1002
-1012.
55
Dall'Ozzo, S., S. Tartas, G. Paintaud, G. Cartron, P. Colombat, P. Bardos, H. Watier, G. Thibault.
2004
. Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship.
Cancer Res.
64
:
4664
-4669.
56
Bowles, J. A., G. J. Weiner.
2005
. CD16 polymorphisms and NK activation induced by monoclonal antibody-coated target cells.
J. Immunol. Methods
304
:
88
-99.
57
Berdeja, J. G., A. Hess, D. M. Lucas, P. O'Donnell, R. F. Ambinder, L. F. Diehl, D. Carter-Brookins, S. Newton, I. W. Flinn.
2007
. Systemic interleukin-2 and adoptive transfer of lymphokine-activated killer cells improves antibody-dependent cellular cytotoxicity in patients with relapsed B-cell lymphoma treated with rituximab.
Clin. Cancer Res.
13
:
2392
-2399.
58
Laurent, C., G. R. de Paiva, L. Ysebaert, G. Laurent, M. March, G. Delsol, P. Brousset.
2007
. Characterization of bone marrow lymphoid infiltrates after immunochemotherapy for follicular lymphoma.
Am. J. Pathol.
128
:
974
-980.