Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls

Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb‐based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA‐mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell‐bound cognate IgG mAbs for several different mAb‐donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP‐1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody‐drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.


INTRODUCTION mAb-Mediated Loss of CD20 on B cells Requires an Explanation
We have reported that prolonged intravenous infusion of the usual high doses of CD20 mAbs rituximab (RTX) or ofatumumab (OFA) (600 to 2000 mg over ∼8 hr), used in targeting circulating malignant B cells in the treatment of chronic lymphocytic leukemia (CLL), leads to pronounced loss of CD20 from circulating B cells in the later phase of the infusion (Beurskens et al., 2012;Kennedy et al., 2004).Initially these IgG mAbtargeted CLL B cells were cleared from the circulation and presumably phagocytosed by fixed tissue macrophages in the liver (Kupffer cells) (Atkinson & Frank, 1974;Atkinson et al., 1973;Schreiber & Frank, 1972a, 1972b).However, ∼1 hour after the start of this RTX/OFA infusion, a different pattern became evident: due to re-equilibration of more CLL B cells into the circulation from other compartments including the spleen and lymphatics, and likely saturation of Kupffer cells, the concentration of circulating CLL B cells increased from the previous RTX/OFA-induced nadir, even though additional CD20 mAb was still being infused into the bloodstream.These B cells became covalently tagged with C3 activation fragments, indicating that RTX /OFA must have bound to CD20 proteins initially present on the re-equilibrated cells, thus forming immune complexes that activated complement.However, the levels of CD20 on these isolated cells were soon substantially reduced, typically down to ∼15% or less of pre-RTX/OFA infusion levels (Beurskens et al., 2012;Kennedy et al., 2004).In the absence of CD20 mAb therapy, CD20 levels on re-equilibrated circulating B cells from the lymphatics and spleen are comparable or greater than CD20 levels of circulating cells (Pavlasova et al., 2016).The pattern of recrudescence of "low CD20" B cells covalently tagged with C3 activation fragments has now been replicated without exception in studies of more than 100 CLL patients who received either RTX or OFA therapy at four different institutions: the University of Virginia School of Medicine, the NIH, the Mayo Clinic, and the University of Rochester School of Medicine (Beurskens et al., 2012;Kennedy et al., 2004;Wallace et al., 2023;Zent et al., 2014Zent et al., , 2016)).
Based on these clinical observations, we designed an in vitro model to investigate the mechanism by which substantial amounts of CD20 could have been removed (i.e., trogocytosed) from these re-equilibrated cells in the presence of excess circulating amounts of RTX/OFA (>30 μg/ml) (Beum et al., 2006).The in vitro experiments we conducted were based on incubating Alexa Fluor 488 (Al488)-labeled RTX/OFA-opsonized B cells with acceptor cells such as THP-1 monocytes.We demonstrated that Fcγ receptor-expressing acceptor cells are capable of rapidly (10 to 45 min) removing both cell-bound Al488labeled CD20 mAb as well as CD20 from opsonized B cells in a concerted reaction in which portions of the plasma membrane of the donor B cell are also transferred to the acceptor cell.These observations, applied to the special case of cell-bound IgG immune complexes interacting with Fcγ receptor-expressing cells, recapitulate the original definition of trogocytosis (transfer of plasma membrane-associated ligands from donor cells to cognate receptors on acceptor cells) for this special case (Joly & Hudrisier, 2003).Other antigen substrates (on donor cells) that we have demonstrated to be successfully targeted for trogocytosis with specific mAbs include CD5 (MOLT-4 T cells), Her2/Neu (BT-474 breast cancer cells), and the epidermal growth factor receptor (SSC-25 squamous carcinoma cells) (Beum et al., 2008).We have found that additional donor cells are also susceptible to trogocytosis of CD20 include several B cell lines (Raji, Daudi, Z138 cells), as well as B cells isolated from normal individuals and patients with CLL.Acceptor cells (all of which express Fcγ receptors) include human monocytes, macrophages, neutrophils, and NK cells (Beum et al., 2006;Beum, Lindorfer, & Taylor, 2008;Beum et al., 2008;Beum et al., 2011;Taylor & Lindorfer, 2015).Here, we describe in detail, the general methodology we have used to examine trogocytosis mediated by THP-1 cells and monocytes.
The measurement of the transfer of fluorescent or radioactive probes from one cell to another is straightforward and its demonstration provides powerful first order evidence for trogocytosis.However, it is also necessary to include critical control experiments to clearly demonstrate and validate the reaction.These experiments include careful interrogation of unambiguously identified donor and acceptor cells.We will describe experiments that demonstrate trogocytosis based on flow cytometry or fluorescence microscopy.These experiments include trogocytosis mediated by adherent THP-1 cells as well as by THP-1 cells and monocytes in solution.Several Al488 mAb-donor cell pairs are examined.The general methods we describe allow for measurement of the transfer of the Al488 mAbs along with portions of the donor cell plasma membrane from the donor to acceptor cells.We also follow trogocytosis by making use of a secondary developing agent, Al488 mAb HB43, specific for the Fc region of human IgG.Use of this reagent reinforces the lessons learned with the Al488 mAbs because it tracks removal of unlabeled mAbs from the donor cells.NOTE: Appropriate informed consent is necessary for obtaining and use of human study material.

TROGOCYTOSIS OF mAb-OPSONIZED DONOR CELLS MEDIATED BY ADHERENT THP-1 CELLS
The experimental paradigm (Fig. 1) that we have employed for quantitative measurement of trogocytosis involves a 15 to 60 min incubation at 37°C of Al488-(or Al647-) mAb-opsonized donor cells with acceptor cells such as adherent THP-1 monocytic cells (see Support Protocol 1).All samples are run in duplicate or triplicate and usually three or more controls are included to validate the experimental trogocytosis condition.Opsonized donor cells are added to empty wells for a 45 min incubation at 37°C to determine if the cell-bound opsonizing mAb simply dissociates from (or is internalized by) the donor cells (Control 1).Opsonized donor cells (first held on ice for 45 min) are added to either empty wells (Control 2, no trogocytosis), or to wells with adherent THP-1 cells (Control 3, zero-time), followed by immediate quenching with cold BSA-PBS to test for the effect of simple processing of the samples (centrifugation and wash steps).In previous experiments we reported that samples that were simply held on ice and never exposed to THP-1 cells had identical signals to the zero-time samples.In this article we only report the zero-time controls.Finally, sample 4 is the experimental trogocytosis protocol in which opsonized donor cells are reacted with adherent acceptor THP-1 cells for 15 to 60 min at 37°C and are then quenched by addition of cold BSA-PBS.After quenching, the donor cells are transferred from the adhered THP-1 cells to a FACS tube by gentle pipetting, washed with BSA-PBS (see Support Protocol 2), and then the (putatively) trogocytosed donor cells, along with the control samples, are analyzed by flow cytometry.
The flow cytometry analyses include several of the following determinations: The trogocytosed donor cells are analyzed for the level of (residual) bound Al488 mAb (FL1).Alternatively, the trogocytosed donor cells are re-opsonized with Al488 mAb, washed, and then analyzed by flow cytometry.This procedure, combined with the other measurements, tests for simple dissociation of cell-bound Al488 mAb and allows for determination of the total amount of target antigen available on the trogocytosed donor cells after the trogocytosis reaction.As is illustrated below, we find that the % trogocytosis, which reflects loss of the Al488 mAb from donor cells, is in very good agreement with the % trogocytosis determined based on the % available antigen after re-opsonization.In some experiments the donor cells are also stained with the membrane dye PKH 26, immediately before or after opsonization is equally effective (see Support Protocol 3).After the trogocytosis reaction the acceptor cells are examined for uptake of PKH26 (FL2 signal), which reflects transfer of plasma membrane fragments from the donor cells that occurred in concert with removal of the opsonizing mAb.These experiments make use of either 2 or 3 different colors in the flow cytometry analyses (FL1, FL2, FL4) and in all cases compensation controls with cells stained with a single dye are first examined.The level of PKH26 dye remaining on the donor cells is considerably greater than the amount of PKH26 taken up by the acceptor cells, and this allows for additional and reliable analyses of the donor and acceptor cells.As is illustrated in selected experiments below, trogocytosis always includes transfer of membrane fragments from the donor cell to the acceptor cell.
The acceptor cells, which in the illustrated examples are either THP-1 cells or freshly isolated human monocytes, are examined by flow cytometry.In reactions in which the THP-1 cells are adhered to wells, after the trogocytosed donor cells are gently removed from the wells followed by a wash, the adherent THP-1 cells are then removed from the wells by vigorous pipetting.
We will describe additional controls in several experiments that are designed to enhance the validity of the measurements.For example, we have determined that the processes we are studying are mediated by Fcγ receptors on acceptor cells (Beum et al., 2006), and we find (see below) that pre-incubation of acceptor cells with human IgG (to block Fc receptors) also substantially blocks trogocytosis.We have also demonstrated trogocytosis based on probing donor cells with Alexa-labeled mAb HB43, which binds to the Fc region of chimeric/humanized mAbs (see Basic Protocol 3).Use of mAb HB43 allows for evaluation of unlabeled cell-bound mAb [e.g., cetuximab (CET), trastuzumab (TRA)].As already noted, it is certainly possible that the Al488 mAb simply dissociates from the opsonized donor cell, and then the released mAb may be taken up by Fcγ receptors on the acceptor cell.To test for this possibility, we incubate the opsonized donor cells in buffer alone for 45 min at 37°C (Control 1).The supernatant, possibly containing released opsonizing mAb, is then added to unreacted but adherent THP-1 cells to determine if they take up any putatively released Al488 mAb. 1. Count THP-1 cells using a hemocytometer.Suspend THP-1 cells at 1 × 10 6 cells/ml in complete RPMI 1640 medium supplemented with 1 μM PMA.Transfer 1 ml aliquots to the wells of a 24-well culture plate, cover and culture for 2 to 3 days at 37°C in 5% CO 2 in a tissue culture incubator.

Materials
2. Inspect the wells under 10× magnification to verify that cells have adhered.Remove the medium by aspiration and incubate the adherent THP-1 cells for 1 h at 37°C with either 100 μl complete RPMI 1640 medium containing 2 mg/ml purified human IgG or complete RPMI 1640 medium alone (control).
4. Centrifuge the plate 1 min at 50 × g, 4°C, to pellet the donor cells.Cover the plate and incubate for 45 min at 37°C, 5% CO 2 .
5. Add 2 ml ice-cold BSA-PBS to each well to quench the reaction samples.In additional controls, add PKH26-labeled Al488 T101-opsonized donor cells (held on ice for 45 min) to wells containing THP-1 cells, followed by immediate quenching with 2 ml cold BSA-PBS.This is the zero-time control.
6. Transfer all quenched samples to FACS tubes, wash the samples twice (see Support Protocol 2) and reconstitute in 250 μl PBS after the last wash.
7. Examine the PKH26-labeled donor cells by flow cytometry using an FL2 gate that excludes the weakly-positive PKH26 signal on the THP-1 cells.Count 8000 events.
The testable hypothesis is that after the incubation there will be loss of the Al488 signal from the donor cells reacted with the THP-1 acceptor cells, compared to the zero-time control.We also expect that there will be little, if any, loss of signal from the donor cells when they are incubated with acceptor cells that are blocked with human IgG.
8. Isolate the THP-1 cells after the trogocytosis reaction as follows: a. Wash the wells previously used for the trogocytosis reaction 3 times with BSA-PBS.b.Add 1 ml BSA-PBS and pipet vigorously 10 times to release the THP-1 cells.c.Transfer the released THP-1 cells to FACS tubes.d.Wash once (see Support Protocol 2).e. Reconstitute to 250 μl in PBS containing 2 mg/ml mouse IgG to reduce nonspecific binding of probes.9. Probe the released THP-1 cells as follows: a. Incubate the THP-1 cells with 10 μl of a cocktail containing 0.06 μg/ul each of biotinylated antibodies to CD11b and to CD14.b.Incubate these samples for 30 min on ice, and then wash twice (see Support Protocol 2).c.Resuspend the cells to 100 μl in BSA-PBS and add 10 μl of 0.1 mg/ml Al647 streptavidin.d.After a 30 min incubation on ice, wash the samples twice in BSA-PBS and resuspend to 250 μl in PBS.e. Analyze by flow cytometry based on FL1/FL2/FL4 (collect 12,000 events).have been released into the supernatant from the PKH26-labeled Al488 T101opsonized donor cells.

Compared to the zero-time samples, the released THP-1 cells (FL4 positive) should manifest increases in the Al488 and PKH26 (FL1 and FL2) signals, due to trogocytosis. However, these signals should remain at baseline for the THP-1 cells incubated with un-opsonized donors or cells subject to dissociation. Finally, the FL1 and FL2 signals for THP-1 cells blocked with human
Based on numerous calibration studies, we expect that there will be little, if any increase in theAl488 and PKH26 background signals (compared to background) for the released THP-1 cells.

MESF calculations for flow cytometry experiments
In order to obtain a rigorous quantitative measure of trogocytosis, mean fluorescence intensities, recorded as channel numbers, are converted to molecules of equivalent soluble fluorophore (MESF units) based on using a graduated series of fluorescent beads, with defined MESF values (Spherotech).The channel number of each standard bead is determined by flow cytometry under the exact instrument settings for each experiment.The slope (m) and intercept (b) of a plot of the log defined MESF vs experimental channel number are calculated using the equation: log MESF (y) = mx + b, where x = channel number.In the example in Figure 2 the derived values are m = 0.00405 and b = 2.865.
Based on these values, it is then straight-forward to convert any given channel number into an MESF value, based on calculating anti-logs.For instance, in this example, a channel number of 600 would convert to an MESF of 197,701.
Figure 3 shows flow cytometry analyses of trogocytosis of donor PKH26-labeled MOLT 4 cells opsonized with Al488 T101 and then reacted with adherent acceptor THP-1 cells at donor to acceptor ratios of 1:10 (Fig. 3A) or 1:1 (Fig. 3B).The light scattering gating scheme identifies the MOLT 4 cells.Cells were reacted for 45 min at 37°C with THP-1 cells alone (trogocytosis, pink) or with THP-1 cells first blocked with 2 mg/ml human IgG Taylor and Lindorfer

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Current Protocols (blocked, green).Alternatively, the donor cells were first incubated in medium alone for 45 min at 37°C and then combined with THP-1 cells and immediately quenched with cold BSA-PBS (blue, zero-time).The gating scheme was set to eliminate any cells that were weakly positive for PKH26, thus only showing the donor MOLT 4 cells.Figure 3A shows MESF values for the respective 3 samples reacted at donor:acceptor ratios of 1:10 were 29,000; 54,000; and 59,000.The % trogocytosis, based on comparing the trogocytosis sample with the zero-time sample is therefore ∼50%: 100 × [(59,000-29,000)/59,000)] %, and it is evident that human IgG substantially blocked trogocytosis.In Figure 3B, a similar but separate experiment with a donor:acceptor ratio of 1:1, the MESF values for the three samples were 37,000 (pink, trogocytosis); 49,000 (green, IgG block); and 54,000 (blue, zero-time), corresponding to ∼30% trogocytosis.
Figure 4 shows the flow cytometry analyses of the uptake of both Al488 T101 (FL1) and PKH26 (FL2) by THP-1 cells for the experiment illustrated in Figure 3B.The gating scheme was set to include cells weakly positive for PKH26 (Fig. 4A).Dot plots illustrate the uptake of Al488 (top plot) and PKH26 (bottom plot) for FL4 positive THP-1 cells identified by probing with biotinylated CD11b/CD14 and secondarily developed with Al647 streptavidin.The quadrants were defined by analysis of naïve THP-1 cells (not shown).The results demonstrate that approximately 26% and 43% of the THP-1 cells acquire Al488 and PKH26, respectively (Fig. 4B).Uptake is substantially blocked by pre-incubation of the THP-1 cells with human IgG (Fig. 4D).It should be noted that in the zero-time sample (Fig. 4C), very few donor cells are demonstrable, because they were only added after the 45 min incubation and then immediately quenched and processed.In the dissociation control (Fig. 4E), the donor cells were incubated for 45 min at 37°C in the absence of THP-1 cells.The cells were pelleted, and the supernatant Current Protocols For each condition, transfer of Al488 mAb T101 (FL1, top panel) and PKH26 (FL2, bottom panel) is given.The % positive cells in each quadrant is provided.There is substantial uptake of both Al488 (26.2% vs 0.5%) and PKH26 (42.8% vs 0.3%) for the trogocytosis sample relative to zero-time.

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Current Protocols was transferred to THP-1 cells which were incubated for 45 min at 37°C and then washed and analyzed.

APPLICATION OF FLUORESCENCE MICROSCOPY TO EXAMINE THP-1 CELL-MEDIATED TROGOCYTOSIS
This procedure provides an alternative and quite visual determination of trogocytosis.Indeed, in this case, "seeing is believing."
2. Concentrate aliquots of the isolated THP-1 cells by centrifugation for 1 min at 800 × g, 4°C, followed by reconstitution in a small volume of PBS to ∼1 × 10 7 cells/ml.
3. Apply 10 μl of the concentrated cell suspension to a microscope slide, cover with a cover slip, and seal with fingernail polish.
4. Examine in a BX40 Olympus fluorescent microscope (100×) with oil, under either white light or based on the FITC or Texas Red filters.
Figure 5 shows the results of trogocytosis experiments in which the THP-1 cells were isolated and examined by fluorescence microscopy after the trogocytosis reaction (45 min, 37°C).This experiment is similar to the experiment described in Figure 4, except the donor cells were not labeled with PKH26.In this case they were reacted with one of the three respective Al488 cognate mAbs.After the trogocytosis reaction the THP-1 cells were identified by probing with biotinylated mAbs specific for CD11b and CD14 (see Basic Protocol 1, step 9) followed by Al647 streptavidin.It is clear that the red acceptor monocytes have taken up the green Al488 mAbs in all three cases.

ALEXA LABELING OF mAbs AND DETERMINATION OF F/P RATIOS
Almost all of our trogocytosis experiments require use of well-defined fluorescent mAbs.

Materials
2. Determine antibody concentration using a spectrophotometer by measuring the absorbance at 280 nm (A280).The A280 divided by 1.5 gives the mAb concentration in mg/ml.
Optimal protein concentration for this reaction is 2 mg/ml.Dilute with BS as needed.
Higher protein concentration results in aggregation and loss of product.
3. Allow Alexa NHS vial to come to room temperature before opening the vial.

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Current Protocols 4. Raise pH of the mAb solution by adding a 10% volume of 1 M bicarbonate buffer.
5. Use pH strip to make sure the pH is >8.
8. Mix gently by inversion.Do not vortex.9. React, protected from light, 1 hr at room temperature.
11. Remove unreacted dye from Alexa-mAb by gel filtration on BioRad columns equilibrated with BS buffer.The protein-dye conjugate exits in the void volume.
12. Measure the absorbance at 280 nm and 495 nm of the isolated Al488 mAbs.
13. Calculate mg/ml mAb concentration and F/P ratio according to manufacturer's directions as follows: Protein concentration (M) = A280 − (Adye × CF280) × dilution factor 203, 000 where 203,000 is the molar extinction coefficient (ε) in cm -1 M -1 of a typical IgG at 280 nm which is suitable for IgA, IgD, and IgE as well.The value CF280 is a correction factor for the fluorophore's contribution to the absorbance at 280 nm.For Alexa 488 the CF280 is 0.11.where εdye (in cm -1 M -1 ) is the molar extinction coefficient of the specific dye.Use 71,000 as the molar extinction coefficient for Al488 at 495 nm.A similar calculation is used for Al647 mAbs, except Al647 has a different extinction coefficient and correction factor.See the ThermoFisher web site for a table of extinction coefficients and correction factors for Document Connect (thermofisher.com).

STANDARD WASHING PROCEDURE
All of our procedures require multiple wash steps to insure the reliability of the measurements and the general wash procedure is now presented.

Vortex.
3. Pellet by centrifuging 2 to 5 min at 800 × g, 4°C.Use either FACS tubes or 15-ml conical tubes, depending upon the volume of cells to be processed.

LABELING AND OPSONIZATION OF CELLS
It is important to establish uniformity in fluorescent labeling of cells and mAbs.The procedures are presented here.

TROGOCYTOSIS MEDIATED BY HUMAN MONOCYTES AS ACCEPTOR CELLS
In this protocol monocytes are isolated from fresh blood and their ability to mediate trogocytosis of PKH26-labeled Al488 T101-opsonized MOLT4 cells is examined.Trogocytosis is conducted in solution phase; the donor and acceptor cells are mixed, briefly centrifuged, and then incubated for 60 min at 37°C.Controls that were used previously are included, and in addition, putatively trogocytosed cells (manifesting loss of opsonizing Al488 mAb) are re-opsonized to provide a direct and independent measure of the loss of the target antigen from the donor cells.

Additional Materials (also see Basic Protocol 1 and Support Protocols 2 to 4)
mouse IgG (Lampire Biological Laboratories, cat. no.7404304) 1. Label the donor cells with PKH26 followed by opsonization with the respective cognate Al488 mAbs (see Support Protocol 3).
2. Wash the cells 3 times in BSA-PBS, and reconstitute them in complete RPMI 1640 medium to a final concentration of 1 × 10 6 cells/ml.A total of at least 1 × 10 6 donor cells (in 1 ml) (see step 4) are needed for each experiment.
3. Incubate freshly isolated human monocytes in complete RPMI 1640 medium with or without blockade with 2 mg/ml human IgG for 1 hr at 37°C, at a cell concentration of 5 × 10 6 cells/ml.
At least 5 × 10 6 acceptor cells (in 1 ml) are required for each experiment.7. Pellet the second aliquot and resuspend cells in 100 μl of BSA-PBS containing 2 mg/ml mouse IgG (added to prevent non-specific binding of probes).
8. Probe samples A, B, and C with the biotinylated CD11b/CD14 cocktail followed by Al647 streptavidin to identify monocytes as in Basic Protocol 1, step 9.This will allow for identification of monocytes based on the FL4 signal.Sample D is not probed as it has no monocytes.To test for the total amount of antigen remaining on the donor cells, also add 10 μl of 0.1 mg/ml of the opsonizing Al488 mAb to Samples A to D and incubate for 15 min, room temperature.9. Wash the samples once and reconstitute to 250 μl with PBS and examine by flow cytometry.
If trogocytosis occurs successfully, then the FL4-positive monocytes should show increased levels of both Al488 and PKH26 in the positive control trogocytosis monocytes, compared to low background levels for both zero-time monocytes or monocytes that were blocked with human IgG.
Figure 6 shows that trogocytosis mediates loss of Al488 mAb T101 signal from opsonized MOLT4 cells incubated with freshly isolated human monocytes in solution for 1 hr, 37°C.FL2-positive donor cells were analyzed for FL1 signal.In addition to the trogocytosis sample (pink, 24,000 MESF), the controls include zero-time (blue, 64,000 MESF); IgG block (green, 64,000 MESF) and simple dissociation (yellow, 65,000 MESF).The yellow and blue histograms coincide with the green histogram.
Based on these values, the % trogocytosis was ∼63%.In additional controls, the cells were re-opsonized with Al488 T101 to allow for measurement of total available antigen accessible.This second opsonization had only marginal effects on the Al488 signals associated with the cells (results not shown).The results for the trogocytosed  sample before (pink, 24,000 MESF) and after re-opsonization (29,000 MESF) are quite similar.Moreover, the values for the zero-time samples before (64,000) and after re-opsonization (61,000) are quite similar as well.
Figure 7 shows analyses of the uptake by monocytes (identified as FL4-positive) of Al488 mAb T101 and PKH26 for the experiment illustrated in Figure 6.Under conditions that allowed for trogocytosis (Fig. 7B), approximately 37% and 33% of the monocytes took up Al488 (top plot) and PKH26 (bottom plot), respectively.However, when the reaction was blocked with human IgG (Fig. 7C), the % uptake was reduced to 1% and 3%.respectively.There is little, if any, uptake of either Al488 or PKH26 for the zero-time sample (Fig. 7D).

ISOLATION OF HUMAN MONOCYTES
Demonstration of an immunologic phenomenon such as trogocytosis with a cell line should always, if possible, be recapitulated with primary cells.In this case we show that freshly isolated human monocytes are also proficient at mediating trogocytosis.

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Current Protocols 2. Mix with an equal volume of PBS and carefully layer multiple 35 ml aliquots of diluted whole blood on 11 ml Ficoll Paque Plus in 50-ml centrifuge tubes.
4. Transfer the white PBMC layer to 50-ml tubes, dilute with complete RPMI 1640 medium and perform 2 washes, centrifuging 2 min at 1600 × g, room temperature.
5. Reconstitute pooled cells to 30 ml in complete RPMI 1640 medium and isolate monocytes based on the Current Protocols in Immunology method (Fuss et al., 2009) as follows: a. Transfer 30 ml of the PBMC suspension to a T75 flask and incubate 1.5 hr at 37°C in 5% CO 2.

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Current Protocols b.Carefully remove the non-adherent cells by aspiration followed by three gentle washes of the adherent cells with 10 ml each of complete RPMI 1640 medium.c.Add 10 ml complete RPMI 1640 medium to the flask and use a cell scraper to remove the adhered monocytes.d.Repeat this step 3 times and consolidate all the washes.e. Centrifuge 2 min at 1200 × g, 4°C.f.Resuspend the pelleted cells in 2 ml complete RPMI 1640 medium.

TROGOCYTOSIS MEDIATED BY THP-1 CELLS IN SOLUTION
Although it is technically easier to study trogocytosis with adhered acceptor cells, it is important to demonstrate that this is a general phenomenon, which also can occur if cells are in solution, as illustrated in this experiment.
4. Wash the opsonized samples 3 times with BSA-PBS and reconstitute in complete RPMI 1640 medium to starting volume.
6.After the incubation, add 100 μl of retinoic acid-treated THP-1 cells to the mediumonly samples from step 5 and then immediately quench all samples with 4 ml of cold BSA-PBS and pellet all the samples in the standard wash.
Based on experiments illustrating little dissociation of these mAbs from cells for 45 min, this procedure was used as the zero-time control.
7. Reconstitute all samples to 500 μl with PBS.Combine 100 μl of each duplicate sample with 10 μl of 0.1 mg/ml Al488 HB43, specific for human IgG Fc, and incubate for 15 min at room temperature.
8. Meanwhile, add 20 μl of a 1:500 dilution of TO-PRO-3 in PBS to duplicate 100 μl sample aliquots.Incubate at least 5 min at room temperature.
9. Wash all samples twice with PBS, aspirate to leave 250 μl, and then analyze by flow cytometry for FL1/FL2/FL4 (10,000 events per sample) where the level of binding of Al488 HB43 (FL1) reflects how much opsonizing mAb is cell-bound, and/or is lost due to trogocytosis, and the TO-PRO-3 reports on cell viability.
10. Prepare positive controls for killing of cells.React separate 100 μl aliquots of untreated donor cells with 500 μl of 70% ethanol for 15 s.Wash all samples twice with PBS and resuspend to 100 μl with PBS.Probe with 20 μl a 1:500 dilution of TO-PRO-3 and analyze cells for killing, as described above.
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Current Protocols  Based on the FL4 analyses of uptake of TO-PRO-3 it is evident that the cells were not killed during the procedures.

RETINOIC ACID TREATMENT OF THP-1 CELLS
After retinoic acid treatment, THP-1 cells are not adherent, but they are able to promote trogocytosis in solution.

Materials
See Basic Protocols 1 and 3
Generally, a 1/10 split will grow up in 5 to 7 days.
3. Resuspend pelleted cells in 50 ml fresh complete RPMI 1640 medium.4. Add 50 μl retinoic acid stock solution (1 mM in absolute ethanol) to the resuspended cells.Mix by shaking.Discard the retinoic acid aliquot.
It is important to maintain this 1/1000 dilution to keep final ethanol concentration low.
The cell density does not increase significantly after retinoic acid treatment.

CULTURING OF SCC-25, BT-474, MOLT-4 AND THP-1 CELL LINES
All the donor cells and the acceptor THP-1 cells require special and variable culture conditions, as presented below.

Cells and specific culture conditions SCC-25 cells
Grown in DMEM/F12 medium.These cells express high levels of EGFR and are a good model cell line for studies involving erbitux.They grow very rapidly as an attached monolayer.They should be split 1:40 once every week.

BT-474 cells
Grown in complete RPMI 1640 medium.These cells express high levels of Her-2/Neu and are a good model cell line for studies involving herceptin.They grow slowly (∼1/10 as fast as SCC-25) as an attached monolayer.They should be split 1:10 once every two weeks.

SCC-25 and BT-474 cells
Both SSC-25 cells and BT-474 cells are grown as monolayers in T75 flasks and are released as follows: the medium is withdrawn; and the adherent cells are treated with 5 ml trypsin-EDTA for 15 min followed by addition of 10 ml complete RPMI 1640 medium; the released cells are washed and reconstituted in complete RPMI 1640 medium.

MOLT-4 cells
Grown in complete RPMI 1640 medium.MOLT-4 cells grow rapidly in suspension and will attach to plastic to some degree.They should be split at least 1:20 once a week.The cells express CD5 and are useful for studies involving the anti-CD5 mAb T101.

THP-1 cells
Grown in complete RPMI 1640 medium supplemented with 50 μM mercaptoethanol.THP-1 cells are routinely cultured in suspension.This is a monocytic cell line that is differentiated to a macrophage state by retinoic acid or PMA, and under these conditions they are particularly useful for trogocytosis experiments.They grow quickly and should be split 1:10 once a week.THP-1 cells become adherent after 2 to 4 days in culture with 1 μM PMA.Alternatively, THP-1 cells are held in suspension for 2 to 4 days after combination with 1 to 2 μM retinoic acid.
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Current Protocols et al., 1997;Taylor, Martin, et al., 1997;Taylor et al., 1992;Taylor, Sutherland, et al., 1997); 2) murine B cells or human cell lines via complement receptor 2 (CR2) (Lindorfer et al., 2003;Whipple et al., 2007;Whipple et al., 2004); and 3) platelets in rabbits and mice via the immune adherence complement receptor (Edberg et al., 1989;Taylor et al., 1985Taylor et al., , 1986)).Immune complexes were transferred to acceptor cells which included monocytes, macrophages and follicular dendritic cells and mouse and human macrophage-like cell lines.Not only were the immune-complexed substrates transferred to the acceptor cells, but almost all the CR1, CR2 and the platelet immune adherence receptors were removed from the respective donor cells.In all cases we found that transfer required recognition of the Fc regions of the immune complexes by Fcγ receptors on the acceptor cells.
We were able to document these reactions in vitro as well as in rabbit and mouse models and in non-human primate models in which mAbs specific for CR1 were used as surrogates for C3b, and the immunological significance of these processes have proven to be considerable.In agreement with the initial reports of Nelson and later Hebert and colleagues (Cornacoff et al., 1983;Hebert & Cosio, 1987;Nelson, 1953Nelson, , 1955)), we found that erythrocyte CR1 can serve as an anchor site to promote the safe and effective binding and subsequent clearance from the circulation of complement-opsonized immune complexes, both soluble and particulate (e.g., bacteria and viruses with bound IgG).The erythrocytes are not cleared and remain in the circulation, but as noted above, during this processing CR1 is removed from the erythrocytes, thereby providing a vital clue for our later mechanistic investigations.We also made use of a mAb specific for CR2, which analogously serves as a surrogate for its ligand C3d, to attach antigens to B cells in bispecific constructs (Whipple et al., 2007).We found that transfer of these prototype immune complexes from B cell CR2 to follicular dendritic cells (FDC) provoked an immune response to the transferred substrate antigens.In this case the transfer reaction was accompanied by substantial loss of CR2 from the B cells.This immunization protocol recapitulates Fearon's use of C3d opsonization to enhance immunity to selected antigens (Dempsey et al., 1996;Fearon & Carroll, 2000).
The detailed mechanisms for these transfer reactions were not altogether clear; our stud-ies suggested that one or more proteases might be involved, but definitive proof for this was never established.It was only after we carefully examined and thoroughly characterized the processing of immune complexes composed of IgG anti-CD20 mAbs RTX or OFA, bound to CD20-positive B cells, that we were able to establish and define a general pattern for all of these reactions: antigens on target cells that are opsonized with IgG are rapidly and efficiently removed from the donor cell by Fcγ-expressing acceptor cells in a process previously defined as trogocytosis by Joly and Hudrisier (2003).Most generally, trogocytosis is a process in which receptors on acceptor cells, specific for cognate plasma-membrane expressed ligands on donor cells, promote formation of immunological synapses with the donor cells which include local merging of the respective plasma membranes of the cells.Then, the cognate ligands, as well as closely associated portions of the plasma membrane of the donor cells, are pinched off and transferred to and internalized by the acceptor cells.Application of this definition to the present case is as follows: immune complexes consisting of multimers of IgG antibodies bound to target antigens on the donor cell are taken up after chelation by Fc receptors on the acceptor cell, and the antigen on the donor cell, still bound to IgG, is transferred to the acceptor cell, along with small portions of the associated plasma membrane of the donor cell (Lindorfer & Taylor, 2022;Taylor & Lindorfer, 2015).
In 1985 Ross and colleagues first reported that CR1 on erythrocytes of patients with systemic lupus erythematosus is reduced considerably compared to its levels in normal individuals (Ross et al., 1985).This phenomenon can clearly be attributed to trogocytosis in which C3b-opsonized IgG containing complexes (most likely antibody/dsDNA immune complexes) are removed from the erythrocytes (along with CR1) and are taken up by Fcγ receptor expressing acceptor cells.It is also noteworthy that in lupus mouse models, the immune adherence receptor on mouse platelets is also reduced (Alexander et al., 2007;Miller et al., 1975), and this observation recapitulates analogous findings for CR1 loss from erythrocytes in SLE.
The methods to measure trogocytosis that are documented in this contribution should provide a template for analyses of other immunological systems that are suspected to be characterized by trogocytosis.For   (Krejcik et al., 2017).We suggest that as other immunotherapeutic mAbs are developed, these patterns will be repeated.Another particularly relevant observation that is most likely related to trogocytosis concerns the severe side effects associated with the use of antibody-drug conjugates (ADC) in cancer immunotherapy.Several lines of evidence strongly suggest that these agents can induce severe liver toxicity due to uptake of the toxic conjugates by liver sinusoidal endothelial cells that express FcγRIIB (Lindorfer & Taylor, 2022).
Based on these considerations, an important question for research on trogocytosis should focus on developing specific strategies to block this reaction when it negatively impacts on immunotherapies, which we have described for CD20 mAb-directed therapies.In addition to low dose, high frequency RTX/OFA treatments (Wallace et al., 2023;Williams et al., 2006;Zent et al., 2014;Zent et al., 2016), other approaches, including specific blocking of Fcγ receptors on acceptor cells, especially FcγRIIb appear to be particularly promising.We hope that the experimental protocols that we have described herein will be of use in future investigations in this area.

Critical Parameters and Troubleshooting
See Table 1 for a list of common problems, possible causes, and solutions.

Understanding Results
The experiments illustrated in Figures 3  and 4 clearly demonstrate that the acceptor cells can remove both the Al488 mAbs as well as PKH26 from the donor cells in a brief period (45 min).The controls reveal that simple dissociation of the mAbs from the donor cells followed by uptake by the acceptor cells does not occur to any measurable extent.The cells must be in contact.It is important to provide an alternative measure of trogocytosis, and this is illustrated in Figure 5, in which substantial amounts of Al488 mAbs are easily demonstrable on the acceptor T101 cells after incubating them with three different types of donor cells opsonized with their cognate mAbs.This experiment also additionally and independently illustrates the general nature of trogocytosis.
The experiments illustrated in Figures 6 and 7 demonstrate that human monocytes are fully capable of promoting trogocytosis of cells opsonized with mAb/donor cell pairs.The gating schemes clearly allow for isolation and examination of both the donor cells and acceptor monocytes.Although the total MESF signal on cells subjected to reopsonization tends to be modestly greater than after a single opsonization, we do find that the apparent degree of trogocytosis is quite

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Current Protocols similar for the initial determination and after re-opsonization, thus again validating the trogocytosis paradigm.We also found that under conditions that precluded or should have blocked trogocytosis (zero-time or preincubation of acceptor monocytes with human IgG), uptake of the Al488 mAbs or PKH26 by the THP-1 cells was considerably less than under the condition that allowed for trogocytosis.

Time Considerations
Basic Protocol 1 can be completed in 1 day.Basic Protocol 2, that makes use of monocytes as acceptor cells, can be completed in one long day, 9 to 10 hr.Basic Protocol 3 can be completed in 1 day.Alternate Protocol can be completed in 1 day.Support Protocol 1 can be completed in 3 hr.Support Protocol 2 can be completed in 4 hr.Support Protocol 3: opsonization of cells can be completed in 1 hr; PKH26 labeling of cells can be completed in 2 hr.Support Protocol 4 requires 5 hr.Support Protocol 5 is conducted over 3 to 5 days.

Figure 1
Figure 1 Schematic procedure for measuring trogocytosis mediated by adherent THP-1 acceptor cells.Controls for dissociation, zero-time, and no acceptor cells are important for validation of trogocytosis.

Figure 2
Figure 2 Example of the calculation of MESF values from channel numbers.

Figure 3
Figure 3 Flow cytometry analyses of trogocytosis of donor PKH26-labeled Al488 T101-opsonized MOLT-4 cells reacted with adherent THP-1 cells.The light scattering profile of highly positive PKH26-donor MOLT-4 cells is shown in the top panels, and histograms representing trogocytosis (pink), zero-time (blue) and blockade by human IgG (green) are shown in the bottom panels.The % trogocytosis is calculated by comparing the zero-time sample to the trogocytosis sample (see text).
Figure4shows the flow cytometry analyses of the uptake of both Al488 T101 (FL1) and PKH26 (FL2) by THP-1 cells for the experiment illustrated in Figure3B.The gating scheme was set to include cells weakly positive for PKH26 (Fig.4A).Dot plots illustrate the uptake of Al488 (top plot) and PKH26 (bottom plot) for FL4 positive THP-1 cells identified by probing with biotinylated CD11b/CD14 and secondarily developed with Al647 streptavidin.The quadrants were defined by analysis of naïve THP-1 cells (not shown).Fig. 4B, trogocytosis; Fig. 4C, zero-time; Fig. 4D, blocked with human IgG; Fig. 4E, dissociation (see Step 10); Fig. 4F, THP-1 cells incubated with un-opsonized PKH26labeled MOLT-4 cells.The results demonstrate that approximately 26% and 43% of the THP-1 cells acquire Al488 and PKH26, respectively (Fig.4B).Uptake is substantially blocked by pre-incubation of the THP-1 cells with human IgG (Fig.4D).It should be noted that in the zero-time sample (Fig.4C), very few donor cells are demonstrable, because they were only added after the 45 min incubation and then immediately quenched and processed.In the dissociation control (Fig.4E), the donor cells were incubated for 45 min at 37°C in the absence of THP-1 cells.The cells were pelleted, and the supernatant

Figure 4
Figure 4Flow cytometry analyses of uptake of Al488 (FL1) and PKH-26 (FL2) by THP-1 cells in the experiment from Figure3B.THP-1 cells were identified by probing with a cocktail of btCD11b and btCD14 followed by secondary development with Al647 streptavidin.Gating on all cells based on light scattering is shown in (A).For each condition, transfer of Al488 mAb T101 (FL1, top panel) and PKH26 (FL2, bottom panel) is given.The % positive cells in each quadrant is provided.There is substantial uptake of both Al488 (26.2% vs 0.5%) and PKH26 (42.8% vs 0.3%) for the trogocytosis sample relative to zero-time.

Figure 6
Figure 6 Trogocytosis of Al488 mAb T101 from MOLT-4 cells mediated by monocytes.The light scattering profile of highly positive PKH26-donor MOLT-4 cells is shown in the top panel and the histogram shows the FL1 signal on samples that were subjected to trogocytosis (pink), zero-time incubation (blue), blockade with huIgG (green), and simple dissociation (yellow).The blue, green, and yellow traces were superimposable.

Figure 7
Figure 7 Uptake of Al488 and PKH-26 by monocytes for the experiment illustrated in Figure 6.(A) Light scattering profile.(B-D) The % cells in each quadrant is noted for each condition.Monocytes are identified as FL4 positive as in Figure 3.The upper right quadrant of each top dot plot represents uptake of Al488 (FL1) and the upper right quadrant of each bottom dot plot represents uptake of PKH26 (FL2).In the trogocytosed sample (B) ∼2/3 of the cells had taken up both Al488 and PKH26.There was almost no uptake in the two control conditions (C and D).

Figure 8
Figure8shows the results of trogocytosis experiments in which unlabeled CET was used to opsonize cognate SCC-25 donor cells, which were then reacted with retinoic acidactivated THP-1 cells in solution.Analysis of trogocytosis is based on development with Al488 mAb HB43, specific for IgG Fc, which detects CET bound to cells.Based on the MESF values of the reacted (pink) versus the zero-time controls (green) shown in the FL1 histogram, the % trogocytosis was ∼65%.Based on the FL4 analyses of uptake of TO-PRO-3 it is evident that the cells were not killed during the procedures.

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Pellet and wash the samples once.e.Resuspend the cells in 300 μl PBS and split each sample into two equal aliquots.f.Hold on ice.6.Examine the donor cells in one aliquot by flow cytometry.Collect 7000 events per sample.
The FL2 gate is set to exclude the weakly positive monocytes.Examination of the highlypositive PKH26 donor cells for the Al488 signal provides a quantitative evaluation of trogocytosis.In particular, we anticipate that in the positive trogocytosis sample, there will be a substantial decrease in the Al488 signal compared to the zero-time control.Moreover, under conditions that allow for simple dissociation (no acceptor cells present) or when the acceptor cells are blocked with human IgG, we expect to observe Al488 signals close to those of the zero-time controls.

Table 1
Troubleshooting Measurement of Trogocytosis