Cells a Selected Set of Surface Molecules on T Or B by Components Via Trogocytosis Is Triggered Capture of Target Cell Membrane

Capture of Target Cell Membrane Components via Trogocytosis Is Triggered by a Selected Set of Surface Molecules on T or B Cells This information is current as of October 2, 2021. Denis Hudrisier, Anne Aucher, Anne-Laure Puaux, Christine Bordier and Etienne Joly J Immunol 2007; 178:3637-3647; ; doi: 10.4049/jimmunol.178.6.3637

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Capture of Target Cell Membrane Components via Trogocytosis Is Triggered by a Selected Set of Surface Molecules on T or B Cells1

Denis Hudrisier,2*† Anne Aucher,* Anne-Laure Puaux,* Christine Bordier,* and Etienne Joly*

Key events of T and B cell biology are regulated through direct interaction with APC or target cells. Trogocytosis is a process whereby CD4؉ T, CD8؉ T, and B cells capture their specific membrane-bound Ag through the acquisition of plasma membrane fragments from their cellular targets. With the aim of investigating whether the ability to trigger trogocytosis was a selective property of Ag receptors, we set up an assay that allowed us to test the ability of many different cell surface molecules to trigger trogocytosis. On the basis of the analysis of a series of surface molecules on CD4؉ T, CD8؉ T, and B cells, we conclude that a set of cell type-specific surface determinants, including but not limited to Ag receptors, do trigger trogocytosis. On T cells, these determinants include components of the TCR/CD3 as well as that of coreceptors and of several costimulatory molecules. On B Downloaded from cells, we identified only the BCR and MHC molecules as potentials triggers of trogocytosis. Remarkably, latrunculin, which prevents actin polymerization, impaired trogocytosis by T cells, but not by B cells. This was true even when the same Abs were used to trigger trogocytosis in T or B cells. Altogether, our results indicate that although trogocytosis is performed by all hemopoietic cells tested thus far, both the receptors and the mechanisms involved can differ depending on the lineage of the cell acquiring membrane materials from other cells. This could therefore account for the different biological consequences of Ag capture via trogocytosis proposed for different types of cells. The Journal of Immunology, 2007, 178: 3637–3647. http://www.jimmunol.org/

n many situations, during their development, activation, and their targets (7). In such cases, however, it is unclear whether this effector functions, T and B cells interact with other cellular bidirectional transfer takes place within a single area of contact or I partners. For instance, development of both CD4ϩ and CD8ϩ via the formation of separate structures where membrane traffic T cells is controlled via direct interactions with epithelial cells or follows antiparallel directions. APCs in the thymus (1). Their activation requires interactions with One primary consequence of trogocytosis is that not only the Ag APC in secondary lymphoid organs and their effector functions but also other target cell surface molecules are acquired by effector depend on Ag recognition on APC or target cells. Even though B cells (8, 9). Interestingly, although a portion of the acquired mol- cells can recognize soluble Ags, they very often recognize (both ecules is internalized, a fraction also remains on the cell surface of by guest on October 2, 2021 during their development and/or activation) membrane-bound Ags the effector, in an apparently functional conformation (4, 10). Al- or soluble Ag adsorbed on cell surfaces (2). These recognition though a clear in vivo demonstration of the physiological signifi- processes largely determine the outcome of the target cell-effector cance of trogocytosis is still lacking, the biological and patholog- cell interaction (i.e., activation or tolerance). Ag recognition by ical consequences envisaged for trogocytosis are very diverse and one Ag receptor is followed by receptor as well as Ag internal- depend on the type of target and effector cells to a large extent (5, ization by the lymphocyte, even when the Ag was initially bound 6, 11). For instance, trogocytosis allows B cells to internalize to the membrane of another cell (3, 4). It is now clear that the membrane-bound ligands and to present them to CD4ϩ T cells in internalization of membrane-bound Ag starts with its acquisition in the context of MHC class II molecules (12). In the case of T cells, a membrane-associated form by the effector cell, a process we it is thought that, after internalization, the Ag-TCR interaction may termed trogocytosis (5, 6). Although trogocytosis is usually uni- be maintained to sustain T cell activation (13), whereas the Ag directional, exceptions exist whereby membrane materials are remaining on the surface of the T cells after acquisition may con- transferred in both direction, for example between NK cells and trol T-T interactions and could contribute to activation of tolerance. The decision between activation or tolerance could also depend on the acquisition of costimulatory molecules together with *Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recher- the Ag (11). Apart from CD8ϩ T, CD4ϩ T, and B cells (14), other che Scientifique Unite´Mixte de Recherche 5089, Toulouse, France; and †Universite´ Paul Sabatier, Toulouse, France types of hemopoietic cells have been shown or suggested to per- Received for publication August 16, 2006. Accepted for publication December 18, 2006. form trogocytosis. This is the case of ␥␦T cells (15), NK cells (16), ␣␣ The costs of publication of this article were defrayed in part by the payment of page CD8 intraepithelial lymphocytes (10), dendritic cells (17, 18), charges. This article must therefore be hereby marked advertisement in accordance and also very likely monocytes and macrophages. Even outside the with 18 U.S.C. Section 1734 solely to indicate this fact. immune system, transfer of membrane-bound ligands on recipient 1 This work was supported by a grant from the Agence Nationale de Recherches to the cells has been described and was shown to involve the capture of project Tumor-Antigen Associated Vaccine from Ligue Re´gionale contre le Cancer, from Re´gion Midi-Pyreńeés, and by core funding from Centre National de la Recherche plasma membrane components (19–22). A common feature of the Scientifique. active capture of membrane-bound ligands is the engagement of 2 Address correspondence and reprint requests to Dr. Denis Hudrisier, Institut de receptors on recipient cells. On T cells, the use of anti-TCR block- Pharmacologie et de Biologie Structurale, 205 route de Narbonne, 31077 Toulouse ing mAbs has strongly suggested the involvement of the TCR (4). Cedex 3, France. E-mail address: [email protected] Similarly, activatory and inhibitory receptors on NK cells (16, 23– Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 25), the BCR on B cells (3, 14), and scavenger receptors on DC www.jimmunol.org 3638 Table I. Summary of the effect of mAb against the indicated determinants on trogocytosis and functions performed by CD4ϩ , CD8ϩ , and B cells

ϩ ϩ DO11.10 CD4 T Cells OT-I CD8 T Cells MD4 B cells

Determinant Alternate name Clone Isotype Expressiona Trogocytosisb Functionsc Expressiona Trogocytosisb Functionsd Expressiona Trogocytosisb Functionse

CD2 RM2-2 Rat IgG2b.␬ ϩ 6.7 ؎ 3.23 None ϩ 5.59 ؎ 1.62 None Ϫ 1 Ϯ 0 CD3⑀ 17A2 Rat IgG2b.␬ ϩ 8.83 ؎ 5.63 CD69a; CD154a ϩ 5.60 ؎ 3.14 CD69a; CD107a; IFN-␥ Ϫ 1 Ϯ 0 CD3⑀ 500A2 Hamster IgG2␬ ϩ 10.37 ؎ 5.67 CD69a; CD154a ϩ 6.35 ؎ 4.69 CD69a; CD107a; IFN-␥ Ϫ 1 Ϯ 0 CD3⑀ 145-2C11 Hamster IgG1␬ ϩ 8.87 ؎ 4.32 CD69a; CD154a ϩ 6.92 ؎ 4.98 CD69a; CD107a; IFN-␥ Ϫ 1 Ϯ 0 CD4 RM4-4 Rat IgG2b.␬ ϩ 2.33 ؎ 1.52 CD154a Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 CD4 RM4-5 Rat IgG2a.␬ ϩ 7.2 ؎ 3.43 CD154a Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 CD4 GK1.5 Rat IgG2b.␬ ϩ 2.66 ؎ 2.08 CD154a Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 CD4 H129 Rat IgG2a.␬ ϩ 6.5 ؎ 1.91 CD154a Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 CD5 53.73 Rat IgG2a.␬ ϩ 2 ؎ 0.3 None ϩ 1.66 ؎ 0.28 None ϩ 1 Ϯ 0 None CD8␣ 53.6.7.2 Rat IgG2a.␬ Ϫ 1 Ϯ 0 ϩ 4.87 ؎ 2.23 None Ϫ 1 Ϯ 0 CD8␣ 5H10 Rat IgG2b.␭ Ϫ 1 Ϯ 0 ϩ 2.07 ؎ 0.83 None Ϫ 1 Ϯ 0 CD8␤ H35-12.2 Rat IgG2b.␬ Ϫ 1 Ϯ 0 ϩ 5.07 ؎ 1.83 None Ϫ 1 Ϯ 0 CD8␤ 53.5.8 Rat IgG1.␬ Ϫ 1 Ϯ 0 ϩ 4.93 ؎ 3.14 None Ϫ 1 Ϯ 0 CD9 KMC8 Rat IgG2a.␬ ϩ 2 ؎ 1 None ϩ 1.33 ؎ 0.25 None ϩ 1 Ϯ 0 None CD11a LFA-1 ␣-chain 2D7 Rat IgG2a.␬ ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None CD11a LFA-1 ␣-chain M17/4 Rat IgG2a.␬ ϩ 1 Ϯ 0 ϩ 1 Ϯ 0 ϩ 1 Ϯ 0 ␣ ␬ ϩ Ϯ ϩ Ϯ ϩ Ϯ CD11b Integrin M M1/70 Rat IgG2b. 1 0 1 0 1 0 CD16/32 Fc␥III/IIR 2.4G2 Rat IgG2b.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 1 Ϯ 0 None CD18 LFA-1 ␤-chain M18/2 Rat IgG2a.␬ ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None CD18 LFA-1 ␤-chain GAME-46 Rat IgG1.␬ ϩ 1.1 Ϯ 0.14 ϩ 1.05 Ϯ 0.1 ϩ 1 Ϯ 0 CD19 1D3 Rat IgG2a.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 None CR2/CR1 7G6 Rat IgG2b.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 None CD27 LG.3A10 Hamster IgG1.␬ ϩ 2.5 ؎ 0.57 CD154a ϩ 4.12 ؎ 1.32 CD107a Ϫ 1 Ϯ 0 CD28 Hamster IgG2.␬ ϩ 7.25 ؎ 5.67 CD154a ϩ 3.62 ؎ 1.40 None Ϫ 1 Ϯ 0 CD40 Rat IgG2a.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 1 Ϯ 0 CD69a; BCR2 CD43 S7 Rat IgG2a.␬ ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None CD45 M1-9-3-4 ? ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None Ϫ 1 Ϯ 0 CD62L L-Selectin Mel-14 Rat IgG2a.␬ ϩϮNone ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None CD69 H1-2F3 Hamster IgG ϩϮNone ϩ 1 Ϯ 0 None ϩ/Ϫ 1 Ϯ 0 None CD71 TfR C2F2 Rat IgG1.␬ ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None CD71 TfR R17.217 ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None CD79b Ig-␤ HM79b Hamster IgG2.␭ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 CD69a CD80 B7.1 16-10A1 Hamster IgG2.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 1 Ϯ 0 None CD80 B7.1 1G10 Rat IgG2a.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 1 Ϯ 0 None ؎ ␬ ϩ Ϫ Ϯ ϩ Ϫ Ϯ ϩ CD81 Eat-1 Hamster IgG1 / 1 0 None / 1 0 None 1.75 0.5 None TROGOCYTOSIS OF TRIGGERS MOLECULAR CD86 B7.2 GL1 Rat IgG2a.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 1.1 Ϯ 0.14 None CD86 B7.2 PO3 Rat IgG2b.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 1 Ϯ 0 None CD117 c-kitR ACK-45 Rat IgG2b.␬ ϩ/Ϫ 1.5 Ϯ 0.70 None ϩ/Ϫ 1 Ϯ 0 None ϩ/Ϫ 1 Ϯ 0 None CD117 c-kitR 2B8 Rat IgG2b.␬ ϩ/Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 CD132 Common ␥-chain 4G3 Rat IgG2a.␬ ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None ϩ 1 Ϯ 0 None CD184 CXCR-4 2B11 Rat IgG2b.␬ ϩ/Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 CD195 CCR-5 C34 Rat IgG2c ϩ/Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 TCR␤ H57 Hamster IgG2␭ ϩ 5.16 ؎ 4.19 CD69a; CD154a ϩ 3.58 ؎ 1.42 CD69a; CD107a; IFN-␥ Ϫ 1 Ϯ 0 IgM BCR 56-60.2 Rat IgG2a.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 1.4 Ϯ 0.52 CD69a; BCR2 IgM BCR II/41 Rat IgG2a.v Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 2.88 Ϯ 1.07 CD69a; BCR2 ␬-chain 187.1 Rat IgG1.␬ Ϫ 1 Ϯ 0 Ϫ 1.16 Ϯ 0.28 ϩ 10.5 Ϯ 6.86 CD69a; BCR2 2m S-19-8 Mouse IgG2b.v Ϫ 1 Ϯ 0 ϩ 3.68 ؎ 1.03 None Ϫ 1 Ϯ 0␤ MHC-I (H-2Kb) Y3 1 Ϯ 0 7.87 ؎ 2.58 None 1 Ϯ 0 MHC-I (H-2Kd) H97 2.3 ؎ 1.11 1 Ϯ 0 8.75 ؎ 4.59 MHC-II M5-114 Rat IgG2b.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 6.71 Ϯ 3.03 None TLR4/MD2 MTS510 Rat IgG2a.v Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 Notch-1 MN1A Mouse IgG1 ϩ/Ϫ 1 Ϯ 0 ϩ/Ϫ 1 Ϯ 0 None ϩ/Ϫ 1 Ϯ 0 B220 RA3-6B2 Rat IgG2a.␬ Ϫ 1 Ϯ 0 Ϫ 1 Ϯ 0 ϩ 1 Ϯ 0

a Expression was assessed by flow cytometry using a mix of anti-mouse Ig-Alexa-647 plus anti-rat IgG-Alexa-488 to reveal lymphocytes preincubated with the indicated mAb. In B cells, for which anti-mouse Ig cannot be used, we also used directly labeled mAb. ϩ, Ϫ, and ϩ/Ϫ refer, respectively, to strong, no, and very weak binding compared with the adequate isotype control. b Boldface type represents the mean fold induction of trogocytosis (median fluorescence of PKH-67 on effector cell in the presence of the indicated mAb divided by the median fluorescence intensity in the absence of any Ab added) Ϯ SD. c CD69 up-regulation and CD154 detection at the cell surface were determined by flow cytometry after a 4-h coincubation between effector cells and P815 in the presence of the indicated mAb. d CD69 up-regulation, CD107 detection at the cell surface, and intracellular IFN-␥ staining was determined by flow cytometry after a 4-h coincubation between effector cells and P815 in the presence of the indicated mAb.

e CD69 up-regulation and BCR down-modulation were measured by ﬂow cytometry after a 4-h coincubation between effector cells and P815 in the presence of the indicated mAb.

Downloaded from from Downloaded http://www.jimmunol.org/ by guest on October 2, 2021 2, October on guest by The Journal of Immunology 3639 Downloaded from

FIGURE 1. Principle and proof of concept of the redirected trogocytosis assay. A, Scheme representing the principle of the redirected trogocytosis assay. http://www.jimmunol.org/ Previously labeled FcR-expressing target cells, such as P815, are bridged with effector cells (in our case T or B cells) via a mAb recognizing a cell surface molecule on the effector cells. The capacity of a given mAb to trigger trogocytosis is monitored by flow cytometric analyses, via the detection, on the effector cells, of the label initially incorporated in the plasma membrane of the P815 cell. B, Efficient redirected trogocytosis is triggered by the 145-2C11 anti-CD3 mAb on CD8ϩ OT-I (left panels)and CD4ϩ DO11.10 (right panels) T cells. Both types of activated T cells were mixed with PKH-67-labeled P815 in the presence of the 145-2C11 mAb (middle and lower panels) or its isotype control (top panels). Bottom panels, P815 cells were preincubated with the 2.4G2 anti-Fc␥RIII/II mAb. Histograms represent the PKH-67 fluorescence recorded on cells gated as CD8ϩ or CD4ϩ, and the numbers provided correspond to the median fluorescence intensities (MFI) of the cells in the upper left quadrant, C,AsinB except that P815 cells that had been prelabeled with biotin were used and that trogocytosis was detected using streptavidinPE (SA-PE) staining. D, The indicated concentrations of the 145-2C11 mAb were ϩ

used to coat either or both P815 and OT-I cells before mixing the cells and analyzing trogocytosis. The percentage of trogocytosis of CD8 cells is shown by guest on October 2, 2021 as a function of the concentration of mAb. Percentage of trogocytosis corresponds to the ratio of signal due to the capture of the PKH-67 fluorescent lipid by effector cells compared with that recorded on P815. It is calculated as 100 ϫ [(MFI of lymphocytes with mAb) Ϫ (MFI of lymphocytes without mAb)/(MFI on P815 cells)]. The results are representative of more than five independent experiments. E, Median fluorescence intensity for PKH-67 on gated effector cells. DO.11.10 CD4ϩ T cells, OT-I CD8ϩ T cells, or MD4 B cells were cocultured with P815 in the absence (f, in duplicates) or presence of isotype control Abs: mouse (E, IgG1, IgG2a, and IgG2b from left to right), rat (U, IgG1, IgG2a, and IgG2b from left to right) or hamster (F, IgG1 and IgG2 from left to right).

(18) have been proposed to be implicated in trogocytosis. With a mass spectrometry. The fluorescent lipid PKH-67, cytochalasin D, and sol- view to reach a better understanding of the type of receptors in- uble HEL were from Sigma-Aldrich. Biotinylation reagent (sulfo-N-hy- volved in triggering trogocytosis on ␣␤ T and B cells, we set up a droxysuccinimidyl ester-long chain biotin) was from Pierce. Latrunculin B was from Calbiochem. Fluorescently labeled mAbs against mouse CD4 versatile screening assay inspired from the classical redirected cy- (GK1.5 or RM4-4), CD8␣ (53.6.7.2), CD8␤ (53.5.8), the DO11.10 TCR tolysis approach (26). (KJ1.16), B220 (RA3-6B2), CD154 (MR1), CD107 (ABL-93), IFN-␥ (XMG1.2), CD69 (H1.2F3), CD3␧ (2C11), ␬ chain (187.1) as well as flu- Materials and Methods orescent streptavidin were all purchased from BD Pharmingen. Fluorescent anti-mouse and anti-rat IgG were from Molecular Probes. Unlabeled mAbs Cell lines and mice listed in Table I were either obtained from BD Pharmingen or purified in The plasmacytoma cell line P815 of the H-2d MHC haplotype was used as our laboratory from hybridoma supernatant. ϩ b target cell. The CD8 T cells specific for OVA257–264 presented by H-2K were obtained from OT-I TCR-transgenic mice (27). CD4ϩ T cells specific d Target cell staining for OVA323–339 presented by I-A were obtained from DO11.10 TCR- transgenic mice (28). MD4 mice, expressing a transgenic BCR specific for 3 For PKH-67 staining of P815 cells, 50 million cells were washed in PBS, hen egg lysozyme (HEL) served as a source of B cells (29). T cells were and the pellet was resuspended in 2.5 ml of diluent C (Sigma-Aldrich) in ␮ placed in culture in the presence of the corresponding peptide (0.1 M for a 15-ml tube. PKH-67 was diluted at a final concentration of 2–4 ␮Min2.5 ␮ OVA257–264 and 1 M for OVA323–339). MD4 B cells were used either ex ml of diluent C in a 15-ml tube, and the solution was rapidly added to cells vivo or after an overnight culture in complete culture medium. Splenic T and incubated for 5 min at room temperature with occasional agitation. and B cells were obtained from naive B6 and BALB/c animals. Five milliliters of FCS were then added, and after 1 min of incubation at room temperature, cells were washed three times in complete culture me- Reagents and Abs dium. For surface biotinylation, 50 million live cells were resuspended in Peptides were synthesized using an Applied automated synthesizer. All PBS containing 0.2 mg/ml sulfo-N-hydroxysuccinimidyl ester-long chain peptides were HPLC purified (Ͼ98%), and their identity was confirmed by biotin (Pierce). After 10 min at room temperature, cells were washed twice in culture medium, then incubated for 10 min at 4°C in PBS containing 100 mM glycine, and then washed twice again. In all cases, target cells were 3 ␤ ␤ Abbreviations used in this paper: HEL, hen egg lysozyme; 2m, 2-microglobulin. left1hat37°C and then washed once again in complete culture medium 3640 MOLECULAR TRIGGERS OF TROGOCYTOSIS

FIGURE 2. Selective sets of cell surface molecules on CD4ϩT CD8ϩ T and B cells trigger trogocytosis upon engagement with mAb. Redirected trogocytosis by DO11.10 CD4ϩ T, OT-I CD8ϩ T, and MD4 B cells was determined in the presence (white histograms) or absence (gray histograms) of the indicated mAb. Histograms were obtained with cells gated as positive for CD4 (A) CD8 (B), or B220 (C). Results are representative of at least three independent experiments. Additional results are presented in Table I. FL-1H, Fluorescence. Downloaded from

before resuspending them at 5 million/ml. Stained cells were then used in cells, and B220 for B cells). Trogocytosis was measured directly by the functional assays or in trogocytosis experiments. detection of PKH-67 on the effectors. When biotinylated target cells were used, ﬂow cytometric detection of trogocytosis was performed using a Redirected trogocytosis second step involving staining with streptavidin conjugated to PE together ϩ ϩ

with CD8 , CD4 , or B cell markers. In some experiments, T and B cells http://www.jimmunol.org/ After target cell labeling, cells were placed in U-bottom 96-well plates ␮ ␮ ϫ 6 ␮ were treated with latrunculin B (2–10 g/ml) or cytochalasin D (10 M) (0.5 10 cells/well in 100 l final volume). Effector cells were placed in for 30 min at 37°C before conjugate formation, and the drug was left V-bottom 96-well plates (0.2 ϫ 106 ␮ cells/well in 100 l final volume) and throughout the assay. incubated with 5 ␮g/ml (or the indicated concentrations) of the different unlabeled mAbs for 30 min at 4°C. After two washes in culture medium, Determination of surface receptor expression cell pellets were resuspended with 100 ␮l of culture medium of which 50 ␮l were removed for FACS staining and 50 ␮l were added to target cells, The expression of receptors on T and B cells was assessed by flow cytom- centrifuged for 30 s at 900 rpm to promote conjugate formation, and then etry on a sample of T or B cells preincubated with unlabeled mAbs pre- left at 37°C for 1 h. Conjugates were then dissociated by washing cells pared for redirected trogocytosis assay. These samples were incubated with twice in PBS containing 2 mM EDTA, before staining with anti-CD8, a second layer of anti-mouse Ig coupled to Alexa-488 and anti-rat Ig cou-

anti-CD4, or anti-B220 mAb. Cells were then analyzed on a FACSCalibur pled to Alexa-647. This combination allowed the binding to mouse, rat, and by guest on October 2, 2021 (BD Biosciences). Effector cells were then gated positively according to hamster mAbs. In the case of B cells where the use of anti-mouse Ig mAbs their staining with lineage-speciﬁc markers (CD8 for CTL, CD4 for Th is not possible due to BCR expression, directly labeled mAbs were utilized.

FIGURE 3. Effect of the dose of mAb on Ag binding and trogocytosis. A, OT-I cells were incubated with the indicated concentrations of the various mAbs and either analyzed by flow cytometry for mAb binding using a mix of fluorescently labeled anti-rat plus anti-mouse IgG (F) or used in redirected trogocytosis (E). For those mAbs binding to effector cells and triggering trogocytosis, the signals recorded for mAb binding to effector cell (binding) or of PKH-67 capture in a coculture with P815 (redirected trogocytosis) were both normalized so that they could be represented on the same graphs. Normalizations were conducted using the formula 100 ϫ [(MFI exp) Ϫ (MFI neg)]/[(MFI max) Ϫ (MFI neg)] where (MFI exp) refers to the median fluorescence intensity for a given concentration of an Ab, (MFI neg) in the absence of mAb, and (MFI max) in the presence of a saturating concentration of mAb. For the anti-CD71 mAb, which binds to effector cells but does not trigger trogocytosis (lower right panel), median fluorescence intensities obtained in binding experiments (left axis values) and redirected trogocytosis (right axis values) could not be normalized and are therefore represented directly as median fluorescence intensities for both events. B,AsinA but MD4 effector B cells were used. For both A and B, similar results were obtained in two independent experiments. The Journal of Immunology 3641 Downloaded from

FIGURE 4. Costimulatory effects in redirected trogocytosis. A, OT-I cells were incubated with 0.3 ␮g/ml concentrations of the indicated mAbs, as well ␮ lower panels as with 0.5 g/ml 145.2-C11 anti-CD3 mAb ( ). After 30 min, OT-1 cells were washed and then incubated with P815 previously labeled with http://www.jimmunol.org/ PKH-67. Trogocytosis was analyzed by flow cytometry after1hofcoculture at 37°C. Capture of PKH-67 is shown on gated CD8ϩ cells. Numbers represent the median fluorescence intensity for PKH-67 on gated effector cells. B,AsinA except that OT-I cells were incubated in the presence of the indicated mAb (white histograms), as well as with 0.3 ␮g/ml concentrations of the RM2-5 anti (␣)-CD2 mAb (gray histograms). Fold inductions of signals attained over background levels are represented, i.e., the ratio of the median fluorescence intensity attained in the presence of Ab over that recorded in the absenceof any mAb added. C,AsinB except that the indicated combinations of mAb have been used. D, MD4 B cells were stimulated with the indicated mAbs (top panels), as well as with suboptimal doses of the 187.1 anti-␬ mAb (0.3 ␮g/ml; bottom panels). E and F, MD4 B cells and the indicated combinations of mAb were used (as in B and C). For all the mAbs tested, comparable results were obtained in at least two independent experiments. by guest on October 2, 2021 Redirected T and B cell activation stimulated in an Ag-specific manner (14). On the basis of previous Redirected activation of T and B cells was measured by flow cytometry knowledge that Ag recognition by the TCR triggers trogocytosis, after4hofcoculture at 37°C of either population of effector cells with we tested the stimulatory 145-2C11 anti-CD3␧ mAb or its hamster P815 cells (E:T 1:5) in the presence of the indicated mAbs. We performed IgG1 isotype control in our initial experimental system. As shown a double staining to assess CD69 up-regulation on lineage marker-positive in Fig. 1B, 145-2C11 but not its isotype control triggers the capture cells (CD4 or CD8 or B220). In addition, T cell degranulation was mea- of membrane fragments from PKH-67-labeled P815 by both OT-I sured by flow cytometry using published procedures for the detection of CD107 (30) or CD154 expression (31) at the cell surface of CD8ϩ or and DO11.10 T cells. Trogocytosis triggered by 145-2C11 was CD4ϩ T cells, respectively. For CD8ϩ T cells, intracellular IFN-␥ stain- abolished when P815 cell were pretreated with the 2.4G2 anti- ing was performed after4hofcoculture at 37°C, with brefeldin added Fc␥RIII/II mAb indicating that binding to the Fc␥R on P815 was after 2 h (14). necessary. Similar results were obtained with biotinylated P815 cells (Fig. 1C) or with other FcRϩ targets such as A20 cells (not Results shown). Capture was unidirectional given that unlabeled P815 did Redirected trogocytosis as an assay to test receptor-mediated not acquire lipids from PKH-67-labeled OT-I cells in the presence trogocytosis of anti-CD3 mAb (not shown). Preincubation of the effector cells With the aim of identifying cell surface receptors able to trigger with the mAb followed by washing or leaving the mAb in contact trogocytosis, in other words, unidirectional capture of membrane with both effector and target cells gave the same extent and dose fragments, we evaluated the possibility to use anti-receptor mAbs dependence of trogocytosis. In contrast, preincubation of the mAb in a redirected trogocytosis assay, inspired from the commonly with P815 and washing the excess Ab off before effector cell ad- used T cell-redirected lysis and/or activation assays (26). This as- dition leads to much lower levels of trogocytosis, probably due to say is based on the use of a target cell expressing FcR receptors, the weak mAb-FcR binding (Ref. 32 and Fig. 1D). The levels of which will cause the cross-linking to mAbs bound to activatory fluorescent signal recorded at the end of the redirected trogocytosis receptors at the surface of any effector cell one wishes to study. For assays usually corresponded to 2–3% of those present on the P815 our work, we chose the FcRϩ mastocytoma cell line P815, a cell target cells (Fig. 1D). These levels are comparable with those we line commonly used in redirected cytolytic assays. Occurrence of routinely obtain in Ag-driven trogocytosis (8, 14). trogocytosis was then determined by the ability of various mAbs to trigger the capture, by the effector cells, of membrane fragments Identification of surface receptors triggering trogocytosis on from P815 cells the surfaces of which had been previously labeled activated T and naive B cells either with a lipophilic dye or with biotin (see principle in Fig. 1A). Having obtained the proof of concept that trogocytosis mediated As effector T cells, we used either activated OT-I CD8ϩ T cells or by TCR recognition of its Ag can be mimicked by anti-CD3␧ DO11.10 CD4ϩ T cells, which both perform trogocytosis when mAbs in a redirected trogocytosis assay, and with the objective to 3642 MOLECULAR TRIGGERS OF TROGOCYTOSIS Downloaded from http://www.jimmunol.org/

FIGURE 5. The mAbs directed against AgRs are the only ones triggering both trogocytosis and full lymphocyte activation. OT-I cells were incubated with P815 in the presence of either 100 nM OVA peptide or 1 ␮g/ml concentrations of the indicated mAb and analyzed after 4 h for CD107 expression (A), intracellular IFN-␥ staining (B), and CD69 up-regulation (C). DO11.10 cells were incubated with P815 in the presence of 1 ␮g/ml concentrations of the indicated mAb and analyzed after 4 h for CD69 up-regulation (D) and CD154 expression (E). MD4 B cells were incubated with P815 in the presence ␮

of 100 nM HEL or of 1 g/ml concentrations of the indicated mAb and analyzed after 4 h for CD69 up-regulation (F). The various histograms correspond by guest on October 2, 2021 to events gated on cells positive for CD8 (A and B), CD4 (D and E), or B220 (F) gated events. White histograms were obtained after coating the lymphocytes with the indicated mAb before incubation with P815, and gray histograms were obtained in the absence of any mAb. Similar results were obtained in a second independent assay. G, Summary of the results obtained in redirected trogocytosis and activation experiments for OT-I CD8ϩ T cells. Only the mAbs found to bind detectably to the OT-1 cells are listed. The rectangles are used to indicate what mAbs trigger the indicated function. H,AsinG but for DO11.10 CD4ϩ T cells. I,asinG but for MD4 B cells.

understand whether other surface molecules on effector cells could molecules triggering trogocytosis included the TCR and CD3␧ as trigger trogocytosis, we tested a series of surface molecules for well as CD28. These observations are in good agreement with which mAbs of the IgG isotype were available (Table I). The ex- previous reports documenting the capture of peptide-MHC com- pression of all these receptors on T and B cells was determined by plexes triggered via the TCR and of CD80/86 via CD28 (33). In flow cytometry (see Table I). When used in redirected trogocytosis addition, we found that engagement of lineage-specific coreceptors assay, the isotype controls for each of the anti-receptor mAbs led (CD8␣ or ␤ for OT-I cells and CD4 for DO11.10 cells) and co- to levels of PKH-67 staining that were not higher than those re- stimulatory molecules such as CD2 and CD27 also triggered tro- corded in the absence of any mAb added (Fig. 1E). Initially, to gocytosis. Low but significant levels of trogocytosis were triggered manage large numbers of samples, the mAbs were added to wells by anti-CD9 or anti-CD5 mAbs on both types of T cells (Table I). containing both P815 and effector cells and were not washed away The intensities of the signals recorded with all these Abs are pro- during the assay. Because P815 were labeled with PKH-67 in vided in Table I for information, expressed as the fold induction in batches for the whole experiment, the fluorescence intensity of median fluorescence intensity compared with those obtained in the PKH-67 on the P815 in all samples was identical, which was con- absence of any mAb added. None of the other mAbs against ac- venient for comparing the extent of trogocytosis triggered by the tivation molecules, adhesion molecules (see CD18 in Fig. 2), or various mAbs. Those were tested in redirected trogocytosis assay various chemokine, cytokine, or other types of receptor (see CD71 against activated CD4ϩ DO11.10, activated CD8ϩ OT-I, and na- in Fig. 2) triggered trogocytosis. On B cells, trogocytosis was very ive MD4 B cells, which can all exhibit strong trogocytosis activity strongly triggered by anti-BCR ␬ chain and anti-MHC class II when activated via their respective cognate Ags (14). As shown in mAbs and by one of two anti-BCR ␮ chain mAbs. An anti- Fig. 2 and Table I, on each type of effector cells, we identified a CD81 mAb also triggered trogocytosis, albeit at low levels. selected set of surface receptors capable of triggering trogocytosis Interestingly, mAbs neither against other components function- upon engagement. Whereas on T cells a relatively large number of ally associated with the BCR such as CD19, CD21/35 or CD79b cell surface molecules triggered trogocytosis, a very limited set (Ig␤) nor against molecules known to transduce important was identified for B cells. On CD4ϩ and CD8ϩ T cells, cell surface signals on B cells such as CD80, CD86, or CD40 triggered The Journal of Immunology 3643 trogocytosis. At least one mAb of each isotype was found to be able to trigger trogocytosis on at least one type of effector cell, indicating that the identification of surface receptors triggering trogocytosis was not biased by limitations in mAb binding to the FcR on P815 target. Altogether, our results show that a selected set of cell-type specific surface molecules trigger trogocytosis upon engagement. The extent of trogocytosis triggered by a given mAb and the extent of its binding to Ag are strictly correlated Next, we evaluated the ability of increasing concentrations of mAbs working in redirected trogocytosis to bind to surface Ag and to trigger trogocytosis. For that, effector cells were preincubated with increasing concentrations of the indicated mAbs, washed, and then split into two halves: one was used in subsequent staining with anti-mouse and anti-rat IgG and flow cytometry analysis; and the other was tested in our redirected trogocytosis assay. There is no correlation between the expression level of a given cell surface molecule and its ability to trigger trogocytosis. Indeed, certain strongly expressed molecules do not trigger trogocytosis (see the Downloaded from examples of CD71 on OT-I and MD4 cells in Fig. 3, A and B, respectively). When a receptor mediates trogocytosis, again there is no strict correlation between the extent of trogocytosis triggered and the density of receptor expression. In contrast, the only strict rule is that there is no case in which trogocytosis is triggered without detectable staining of the lymphocyte with the corresponding http://www.jimmunol.org/ ␤ mAb. In line with this, the S19 mAb against 2-microglobulin, which stains only OT-I cells (H-2b), triggered strong trogocytosis only on these cells. The mAb concentration giving half-maximal binding was always closely correlated with that giving half-max- FIGURE 6. Receptors triggering trogocytosis on naive B6 and BALB/c imal trogocytosis, independently of what mAb or effector cell was lymphocytes. Redirected trogocytosis using the indicated mAbs was per- used (Fig. 3). formed with splenic CD8ϩ T(A), CD4ϩ T(B), and B (C) cells from C57BL/6 (H-2b) mice (black histograms) or BALB/c (H-2d) mice (white Costimulatory effects in redirected trogocytosis histograms). Ordinates represent fold induction calculated by the formula by guest on October 2, 2021 When used concomitantly with submaximal concentrations of anti- median fluorescence intensity of trogocytosis triggered by the indicated CD3 mAb on OT-I cells, mAbs triggering trogocytosis on their mAbs divided by median fluorescence intensity obtained in the absence of b own led to an increase of the extent of trogocytosis caused by the any mAb added. Anti-MHC class I mAbs were Y3 for H-2 cells and H97 for H-2d cells. For technical reasons, the H57 mAb against TCR␤ was not anti-CD3, whereas adding mAbs that did not trigger trogocytosis included in this experiment but was found to trigger trogocytosis on splenic on their own did not affect anti-CD3-mediated trogocytosis (Fig. CD4 and CD8 T cells in other experiments with efficiencies comparable 4A). Similar results were obtained when anti-CD3 was replaced by with those of the anti-CD3 mAbs. suboptimal concentrations of anti-CD2 (Fig. 4B) or anti-CD27, -CD28, -CD8 or -H-2Kb (not shown). Finally, with mAbs that could not trigger trogocytosis on their own, we could not find any weakly (not shown). In marked contrast, and with the exception of combination of two such mAbs that would trigger trogocytosis the anti-CD27 mAb which triggered a weak but reproducible (Fig. 4C). On MD4 B cells, similar results were obtained with CD107 up-regulation, none of the other mAbs tested triggered submaximal concentrations of anti-␬ mAb (Fig. 4D) or anti-MHC signs of OT-I cell activation, independent of the fact that they class II mAb (Fig. 4, E and F). could trigger trogocytosis (CD2 and CD28) or not (CD18). On In summary, when two mAbs are combined, we find an additive DO11.10 T cells, the anti-CD3 and anti-TCR mAbs but none of the effect on the levels of trogocytosis recorded. other mAbs tested triggered CD69 up-regulation (Fig. 5D and data not shown). Surface expression of CD154, a recently described test Trogocytosis triggered by surface determinants other than Ag for CD4ϩ T cell activation (31), was also triggered very efficiently receptors did not lead to full T or B cell activation by the anti-CD3␧ and anti-TCR mAbs but was also triggered, al- AgR-mediated activation of T or B cells can be mimicked by ag- though more weakly, by several other mAbs such as anti-CD27 gregating some cell surface molecules (34) or by aggregating and anti-CD28 (Fig. 5E). Other mAbs (for instance the anti-CD2 membrane microdomains (35). We therefore investigated how tro- mAb) did not trigger CD154 expression (Fig. 5E). Finally, when B gocytosis triggered by receptors other than the TCR and the BCR cells were used, soluble HEL and the anti-BCR mAbs (markedly in our redirected assay correlated with their ability to mimic some for the anti-␬ and modestly for the anti-␮) but not the anti-MHC aspects of AgR-mediated activation of T or B cell. As shown in class II mAb triggered CD69 up-regulation (Fig. 5F) or BCR in- Fig. 5, the 145-2C11 anti-CD3 or the OVA peptide recognized by ternalization (not shown). Remarkably, the mAb we used against the OT-I TCR triggered OT-I cell degranulation (as measured by CD40 did not trigger trogocytosis or BCR internalization but did CD107 surface expression; Fig. 5A), IFN-␥ production (Fig. 5B), up-regulate CD69 expression (not shown). Therefore, our results, as well as CD69 up-regulation (Fig. 5C) in redirected activation summarized in Fig. 5, G–I, show that engagement of AgRs trig- assays. Similar results were obtained with the 17A2 and 500A2 gered T or B cell activation, as well as trogocytosis, but that tar- mAb to CD3␧ and by the H57 anti-TCR mAb, although more geting molecules other than the AgRs can lead to a variety of 3644 MOLECULAR TRIGGERS OF TROGOCYTOSIS Downloaded from http://www.jimmunol.org/

FIGURE 7. The cell type rather than the nature of the receptor deﬁnes the need for an active actin cytoskeleton for trogocytosis. A, OT-I cells (top panel) or DO11.10 (bottom panel) were used in redirected trogocytosis with the indicated mAb in the presence (Ⅺ) or the absence (f)of10␮g/ml latrunculin B. Ordinates represent percentage of trogocytosis for CD8ϩ or CD4ϩ cells, calculated as in the legend to Fig. 1. B, MD4 B cells were used in redirected trogocytosis with the indicated concentrations of mAb to ␬-chain (left panel) or MHC class II (right panel) in the presence (E) or the absence (F)of10 ␮g/ml latrunculin B. Ordinates represent the percentage of trogocytosis performed by B220ϩ cells. C,AsinA except that naive B6 splenocytes were used as responder cells and gated positively for CD4 (left column), CD8 (middle column), and B cells (right column). White histograms represent ﬂuorescent

intensities on untreated cells at the end of the redirected assay with the indicated mAb, and gray histograms correspond to the same cells treated with 10 by guest on October 2, 2021 ␮g/ml latrunculin B during the assay. The numbers provided represent the percentage of reduction of trogocytosis in the presence of latrunculin B calculated as 100 Ϫ 100 ϫ [(MFI ϩ mAb in the presence of latrunculin B) Ϫ (MFI Ϫ mAb in the presence of latrunculin B)]/[(MFI ϩ mAb without latrunculin B) Ϫ (MFI Ϫ mAb without latrunculin B)] where (MFI ϩ mAb) represents the median ﬂuorescence intensity obtained in the presence of the indicated mAb and (MFI Ϫ mAb) that obtained without mAb added. D, Naive B6 splenocytes that were pretreated for 30 min with 10 ␮M cytochalasin D (bottom panels) or not (top panels) were used in redirected trogocytosis with PKH-67-labeled P815 cells. Triggering was done using the 145.2-C11 mAb for T cells or the anti-␬ for B cells (white histograms). Negative controls were performed in the absence of mAbs (black histograms). Analysis was done by gating on cells that were CD4ϩ (left panels), CD8ϩ (middle panels), or B220ϩ (right panel). Results were all conﬁrmed in at least two additional independent experiments.

responses, and that triggering of trogocytosis is not necessarily cells from B6 but not BALB/c mice (Fig. 6). Similar results were linked to the induction of activation markers. obtained with the Y3 anti-H-2Kb mAb. Finally, the H97 mAb which reacts with H-2Kd and H-Dd, and not with H-2b molecules, Identification of surface receptors triggering trogocytosis on triggered trogocytosis by CD4ϩ, CD8ϩ, and B cells of the H-2d heterogeneous populations of naive C57BL/6 (B6) and BALB/c haplotype only. splenocytes Next, we tested freshly isolated splenocytes from B6 or BALB/c Trogocytosis performed by T cells, but not by B cells, requires mice in redirected experiments. As shown in Fig. 6, we found that actin cytoskeleton essentially the same receptors triggered trogocytosis in naive T and Upon Ag recognition on APC, T cells perform trogocytosis in a B cells as compared with Ag-specific activated T and naive B cells manner that requires signaling as shown by its blocking or inhi- (see the effect of anti-CD2, anti-CD3, anti-CD4/8, anti-CD27, anti- bition with various mAbs and/or pharmacological inhibitors such CD28, and anti-MHC I on B6 and BALB/c T cells and of the as PP2, an src kinase inhibitor, or latrunculin B (8, 14, 36). To anti-␬ and of anti-MHC class I and class II on B6 and BALB/c B document further the nature of the mechanisms triggered during cells). Several different anti-CD4 and anti-CD8 mAb of the IgG redirected trogocytosis by T or B cells, we next used latrunculin B, isotype but not of the IgM isotype triggered trogocytosis on CD4ϩ an inhibitor of the actin cytoskeleton. As shown in Fig. 7A, tro- T and CD8ϩ T cells (data not shown). This experimental system gocytosis performed by OT-I (upper panel) or DO11.10 (lower was better suited to illustrate the specific effect of anti-MHC class panel) was substantially decreased in the presence of latrunculin I mAb than the transgenic lines used in the previous experiments. B; this occurred for all the mAbs tested. In marked contrast, tro- ␤ ␤ ␬ For example, the anti- 2-microglobulin ( 2m), which reacts only gocytosis triggered by mAbs to IgM (not shown), -chain, or with cells of the H-2b haplotype and triggered trogocytosis only on MHC-II on MD4 B cells was insensitive to latrunculin B (Fig. 7B). OT-I cells, also triggered trogocytosis by CD4ϩ, CD8ϩ, and B On naive CD4ϩ T cells, trogocytosis triggered by anti-CD3 or The Journal of Immunology 3645 anti-CD4 was almost completely inhibited by latrunculin B (Fig. (four different mAbs), to costimulatory molecules such as CD2, 7C). Similar results were obtained with anti-CD3 or anti-CD8 CD27, and CD28 and to MHC class I molecules all triggered tro- mAb on naive CD8ϩ T cells (Fig. 7C). For MD4 B cells, we found gocytosis. The same set of mAbs triggered trogocytosis on CD8ϩ no inhibitory effect of latrunculin B on trogocytosis triggered by T cells except that anti-CD8 mAbs (four different ones, two to anti-␬ or anti-MHC class II mAb on B6 B cells (Fig. 7C). Because CD8␣ and two to CD8␤) instead of anti-CD4 mAbs worked on anti-MHC class I mAbs are the only ones we identified that can this cell type. On both types of transgenic T lymphocytes and on trigger trogocytosis on both T and B cells, we compared the effect primary spleen lymphocytes, weak trogocytosis was triggered by of latrunculin B in redirected trogocytosis assays in T and B cells an anti-CD5 mAb, whereas an anti-CD9 mAb worked only with from B6 splenocytes (Fig. 7C). Trogocytosis mediated by anti- the T cells from the transgenic lines. This difference is probably ␤ b 2m or anti-H-2K mAb was blocked by latrunculin B on both due to the activated state of the latter, possibly through elevated CD4ϩ and CD8ϩ T cells but not on B cells, although the nature levels of surface expression of the CD9 molecule. and affinity of the receptor-ligand interaction was strictly identical On B cells, only mAbs to the BCR, to MHC class II, and to in the two experimental setups. When cytochalasin D was used MHC class I triggered trogocytosis efficiently. In addition, the anti- instead of latrunculin B to inhibit the actin cytoskeleton, the same CD79b and anti-CD81 mAbs, which are molecules that participate differential effect on trogocytosis performed by T cell and B cells in the BCR complex also triggered trogocytosis, albeit weakly and, was obtained (Fig. 7D for primary splenocytes; data not shown for in the case of CD79b, was detected with B6 and BALB/c splenic OT-I T cells and MD4 B cells). By performing time course ex- B cells but not with MD4 B cells. This last observation could be periments, we have found that, under the conditions used for this due to the fact that expression of CD79b is almost undetectable on ϳ study, the t1/2 for redirected trogocytosis was 30 min for both B MD4 B cells (not shown). Similarly, the fact that anti-CD81 does and T cells (not shown), which is very similar to the rates recorded not trigger trogocytosis on T lymphocytes could be due to the fact Downloaded from for Ag-driven trogocytosis in T cells (8). This result rules out the that T cells express lower levels of this molecule than do B cells. possibility that the different requirements of trogocytosis in T and For CD5, however, the levels found on B cells are comparable B cells for an active actin cytoskeleton could be explained by a with those seen on T cells, and, unlike for T cells, we found ab- difference in the kinetics of acquisition of material. Altogether, our solutely no trogocytosis being triggered in B cells by mAbs against results strongly suggest that trogocytosis by B and T cells relies, at this molecule. least partly, on distinct cellular mechanisms. In marked contrast, a large number of mAbs to surface mole- http://www.jimmunol.org/ cules expressed at high levels never triggered trogocytosis in any Discussion of the lymphocyte types tested: those to different chemokine Using a method based on bridging mAb-coated lymphocytes to receptors (CCR5 and CXCR4), cytokine receptors (common ␥ their targets via the FcR of the target, we have been able to explore chain), FcR (CD16/32), and CR (CD21/35), adhesion molecules the ability of a large panel of proteins found at the surface of T (CD62L, LFA-1, CD11a, CD11b), activation molecules (CD69, and/or B cells to trigger trogocytosis. We report that trogocytosis CD43) or other receptors (Notch, CD117, TLR4/MD2) or surface can be triggered by the engagement of a set of lymphocyte-specific molecules (CD45). Interestingly, mAbs to costimulatory mole- surface receptors either individually or in combination. In addition, cules expressed by B cells (CD40, CD80, CD86) did not trigger by guest on October 2, 2021 based on their sensitivity to the presence of a functional actin cy- trogocytosis. toskeleton, we have found that trogocytosis can have different Regarding those surface molecules not triggering trogocytosis in functional requirements depending on the cell type used as effector our assay, the situation is somewhat difficult to interpret because, cell rather than on what receptor is involved. when no effect is obtained, one does not know whether it is be- Trogocytosis was previously reported as a process whereby cause that cell surface molecule is not a potential trigger of tro- CD4ϩ T, CD8ϩ T, and B cells capture their Ag anchored in the gocytosis (and never will be), or whether it is due to the inability membrane of APC via the transfer of membrane fragments (5). of the Ab used in the assay to trigger the right conformation. In Because this process requires the presence of Ag, it was assumed support of this view, some mAbs do trigger trogocytosis effi- that AgR were central in this process. This assumption received ciently, whereas others reacting with the same molecule trigger it more direct experimental support when blocking mAbs to the AgR much less or not at all. For example, among the three anti-BCR or coreceptors expressed by T cells were reported to inhibit Ag mAbs, one of the anti-IgMs does not trigger trogocytosis, the other capture (4, 8, 37) or when thymocytes from TCR␣-deficient mice triggers a very modest capture, and the anti-␬ triggers a very strong exhibited substantially decreased MHC capture as compared with one. Similarly, on CD8ϩ T cells, the 5H10 anti-CD8 mAb is also wild-type thymocytes (38). In addition, it was reported that CD28, much less efficient than the other three. We had, however, more a costimulatory receptor expressed by T cells, mediated capture of than one Ab for a good number of the receptors triggering trogo- CD80 by T cells, suggesting that non-AgRs could participate in cytosis (TCR/CD3, CD4, CD8, BCR) as well as for several recep- trogocytosis (33, 36). Using a large panel of anti-receptor mAbs in tors not triggering trogocytosis in our experimental conditions a redirected trogocytosis assay, i.e., trogocytosis initiated by FcR- (LFA-1, four mAbs; CD117, two mAbs; CD80, two mAbs; CD86, expressing target cells, we tested the ability of individual cell sur- two mAbs; CD71, two mAbs). In addition, we have recently un- face molecules to trigger trogocytosis upon engagement by mAbs. dertaken a similar study on human lymphocytes and have obtained We have found that this assay indeed allows identification of cap- extremely comparable results, both for the molecules with a ca- ture-promoting receptors because mAbs to the TCR/CD3 complex pacity to trigger trogocytosis and for those that do not (S. Daubeuf, mimicked Ag in their ability to trigger trogocytosis. The assay manuscript in preparation). At this stage, we therefore believe that works as expected because trogocytosis was abolished by addition the situations in which a surface molecule can trigger trogocytosis of a blocking mAb to the FcR and was not triggered by mAbs of with certain Abs and not with others are more unusual than the IgM isotype. Using either Ag-specific or nonspecific, naive or the norm. activated CD4ϩ T, CD8ϩ T, or B cells from different haplotypes, Altogether, our results yield a picture that is different for T and we have thus identified a set of cell type-specific surface molecules for B cells. For T cells, it appears that a coordinated set of recep- involved in trogocytosis. On CD4ϩ T cells, we found that mAbs to tors known to play a critical role in T cell activation (39, 40) have the TCR itself, to the CD3 complex (three different mAbs), to CD4 the ability to trigger trogocytosis and might therefore cooperate in 3646 MOLECULAR TRIGGERS OF TROGOCYTOSIS promoting the acquisition of various materials by T cells. Given The assay described here could conceptually be used to identify the varied roles proposed for trogocytosis in T cell activation, it is other receptors involved in trogocytosis in B and T cells, but also therefore possible that the presence or absence of these molecules by other types of immune cells. Regarding the relevance of our on T cells and of their ligands on APC could determine the positive method for the study of human lymphocytes, preliminary data in- (activation) or negative (induction of anergy) roles the captured dicate that the same surface molecules also trigger trogocytosis by material will play in subsequent T-T interactions (11). For B cells, circulating human CD4 and CD8 T cells obtained from healthy capture is mainly due to the BCR and poorly or not at all to the blood donors (not shown). BCR-associated costimulatory molecules, suggesting that BCR Furthermore, this method may also prove to be useful to study signaling is not important for Ag capture. Interestingly, our results the mechanisms of trogocytosis and cellular interactions within that trogocytosis by T cells is very sensitive to latrunculin B or and outside of the immune system. One such system is that of cytochalasin D but remains insensitive to high doses of these drugs reproduction where trogocytosis between the egg and the sperm is on B cells (this study) and to other inhibitors (A. Aucher, submit- proposed as a preliminary step before fusion (49), and the use of ted for publication) also goes in this direction. Furthermore, it has an experimental setup based redirected trogocytosis could provide recently been shown that BCR signaling and internalization are the means to identify the molecular partners involved in this pro- mutually exclusive events (41). In addition it was previously re- cess. Concerning immune cells, although the in vitro identification ported that several initial steps of Ag recognition by B cells do not of molecular triggers of trogocytosis does not per se provide a require signaling (42). The fact that mAbs to MHC class I and proof of the in vivo physiological relevance of this process, the MHC class II can trigger trogocytosis on T and B cells was some- results reported here may provide the experimental means to trigger trogocytosis independently of T or B cells activation. In the what unexpected but could be reminiscent of previously published Downloaded from observations that these molecules participate in cell adhesion to- newly and rapidly emerging field of cell-cell communication based gether with their respective counterreceptors, CD8 and CD4 (43, on membrane exchange, the identification of receptors involved in 44) and that the engagement of MHC molecules either via mAbs trogocytosis processes will be central to the understanding of how or with physiological partners impact on both effector and target these events are triggered and how they may impact on the ensuing cells of the immune system (45, 46). Regarding the capacity of biological processes.

certain molecules that are not part of the Ag receptor complex to http://www.jimmunol.org/ trigger trogocytosis by lymphocytes, our observation that engage- Acknowledgments ment of either CD8 or CD28 can trigger trogocytosis in our non- We thank Florent Navarro and the staff of Becton Dickinson Europe for their help with mAbs. We thank the staff of our animal facility for the maintenance physiological assay fits well with the reports of Pardigon et al. (10) of the various strains of mice. We thank Isabelle Maridonneau-Parini for the that intraepithelial lymphocytes acquire TL, a nonclassical MHC gift of cytochalasin D. class I molecule in a CD8␣␣-mediated manner both in vitro and in vivo and that CD28 mediates the capture of CD80 (33). The events Disclosures recorded in the somewhat artificial setup of a redirected trogocy- The authors have no financial conflict of interest. tosis assay are therefore apparently in good agreement with physiological trogocytosis processes. References by guest on October 2, 2021 An important future question will be to understand why it is 1. Anderson, G., and E. J. Jenkinson. 2001. Lymphostromal interactions in thymic only certain receptors that trigger trogocytosis, and not others, be- development and function. Nat. Rev. Immunol. 1: 31–40. 2. Carrasco, Y. R., and F. D. Batista. 2006. B cell recognition of membrane-bound cause our study has not allowed us to define a clear-cut criterion. antigen: an exquisite way of sensing ligands. Curr. Opin. Immunol. 18: 286–291. The fact that mAbs of different isotypes can trigger trogocytosis 3. Batista, F. D., D. Iber, and M. S. Neuberger. 2001. B cells acquire antigen from rules out a restrictive role of a given isotype in triggering trogo- target cells after synapse formation. Nature 411: 489–494. 4. Huang, J. F., Y. Yang, H. Sepulveda, W. Shi, I. Hwang, P. A. Peterson, cytosis. Nevertheless, the relative affinities of the different mouse, M. R. Jackson, J. Sprent, and Z. 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