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Cell Transfer of Sensitivity−after Trogocytosis Allows a Functional Cell

Proper Regrafting of Ig-Like Transcript 2

Joel LeMaoultMarina Daouya, Layale Yaghi, Edgardo D. Carosella and Kiave-Yune HoWangYin, Julien Caumartin, Benoit Favier,

http://www.jimmunol.org/content/186/4/2210doi: 10.4049/jimmunol.1000547January 2011;

2011; 186:2210-2218; Prepublished online 17J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2011 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Proper Regrafting of Ig-Like Transcript 2 after TrogocytosisAllows a Functional Cell–Cell Transfer of Sensitivity

Kiave-Yune HoWangYin,1 Julien Caumartin,1 Benoit Favier, Marina Daouya,

Layale Yaghi, Edgardo D. Carosella, and Joel LeMaoult

The acquisition by T cells of exogenous ligands originally expressed by APC has been already described. However, reports essentially

focused on the outward signaling of acquired ligands and their effects on surroundings cells. We investigated the function of trans-

ferred receptors (not ligands) on the T cells that acquired them (not on cells they interact with). We show that inhibitory Ig-like

transcript 2 receptors efficiently transfer from monocytes to autologous T cells by trogocytosis and integrate within the plasma

membrane of the acquirer T cells. Furthermore, the acquired receptors can access compatible signaling machinery within acquirer

T cells and use it to signal and alter the functions of their new host cells. These data are a formal demonstration that a transferred

moleculemay send signals to its new host cell.We also provide evidence that sensitivity tomodulatorymolecules can be acquired from

other cells and introduce the notion of intercellular transfer of sensitivities. The Journal of Immunology, 2011, 186: 2210–2218.

Trogocytosis is a mechanism of fast cell-to-cell contact-dependent uptake of membranes and associated mole-cules from one cell by another (1). Intercellular transfers

of membrane patches are observed during interactions betweenimmune cells (reviewed in Ref. 2). Trogocytosis of ligands is wellestablished, in vitro in humans and also in vivo in mice (3–8), inwhich MHC class I, MHC class II, and costimulatory moleculescan transfer from APC to T cells. The ligands acquired throughtrogocytosis may temporarily transfer functions of the donor cellsto the acceptor cells. For example, 1) CD8+ T cells that acquiredtheir MHC class I ligands became the targets of fratricide Ag-specific cytolysis (3); 2) T cells that acquired HLA-DR andCD80 could stimulate resting T cells (2); and 3) CD4+ T cells thatacquired HLA-G behaved as suppressor cells (9). Hence, acqui-sition of exogenous ligands by T cells endows them with newoutward signaling properties and enables them to influence thefunctions of surrounding immune cells.Transfers of receptors and inward signaling through acquired

receptors have been much less studied, even though work hasstarted in this direction. For instance, it has been shown thatconstitutively activated molecules that were acquired by inter-cellular transfers may signal to their acquirer cells. However, inthese reports involving cell lines, receptor–ligand interactions

played no role because of the constitutive activation of thetransferred molecules (10, 11). By contrast, a recent report in-vestigated the functional consequences of the trogocytic transferof FcRs from murine APC to murine T cells. In this report, it wasshown that FcRs could bind with their ligands after transfer, butcould not transduce signals to their new host cells (12). Hence, thecapability of a transferred receptor to bind to its ligand, transducea signal, and function remains to be demonstrated, especially fornontransformed human cells. This requires the demonstration of:1) the proper insertion of the acquired receptor within the plasmamembrane of the new host cell; 2) the capability of the transferredreceptor to access the intracellular machinery of the new host cell;and 3) the functional consequences of its binding to a ligand.In this study, we investigated these issues using the transfer of

the Ig-like transcript 2 (ILT2) inhibitory receptor (LILRB1/CD85j) from monocytes to ILT2-negative autologous T cells.The T cells we used were ILT2-negative, but nevertheless, it hasbeen shown that in cases in which ILT2 is expressed by T cells,ILT2 engagement inhibits their proliferation and cytolytic func-tions (13). In these cases, ILT2 inhibitory functions are mediatedby tyrosine phosphorylation of ITIM-like sequences in its cyto-plasmic tail and involve the p56lck kinase and recruitment of Srchomology region 2 domain-containing phosphatase-1 and -2phosphatases (14). In ILT2-transfected cells, ILT2 phosphoryla-tion could be induced by cross-linking or by the protein tyrosinephosphatase inhibitor pervanadate (PV). ILT2 signaling dependson binding to HLA class I molecules, and ILT2’s highest affinityligand is HLA-G (for a review on HLA-G, see Ref. 15).In this study, we demonstrate the ILT2 transfers from mono-

cytes to autologous activated T cells by trogocytosis and theproper integration of transferred ILT2 within the plasma mem-brane of a new host cell. Furthermore, we show that transferredILT2 behaved as an endogenously produced molecule, capableof using the intracellular biochemical machinery of its new celland send inward-oriented signals, which are then responsible forthe acquirer T cell functional inhibition. These data are a formaldemonstration that a transferred molecule may send signalsto its new host cell after ligand engagement. They imply thatsensitivity to modulatory molecules can be acquired from othercells.

Commissariat a l’Energie Atomique, Institut d’Imagerie BioMedicale, Service deRecherches en Hemato-Immunologie, 75475 Paris, France; and Institut Universitaired’Hematologie, Hopital Saint-Louis, 75475 Paris, France

1K.-Y.H. and J.C. contributed equally to this work.

Received for publication February 16, 2010. Accepted for publication December 13,2010.

This work was supported by the Commissariat a l’Energie Atomique, the Fondationpour la Recherche Medicale, and l’Association pour la Recherche sur le Cancer.

Address correspondence and reprint requests to Dr. Joel LeMaoult, Commissariata l’Energie Atomique-Division des Sciences du Vivant-Service de Recherches enHemato-Immunologie, Institut Universitaire d’Hematologie, Hopital Saint-Louis, 1Avenue Claude Vellefaux, Paris 75475, France. E-mail address: Joel.LeMaoult@cea.fr

The online version of this article contains supplemental material.

Abbreviations used in this article: GAM, goat anti-mouse; ILT2, Ig-like transcript 2;LCL, lymphoblastoid cell line; PV, pervanadate.

Copyright� 2011 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/11/$16.00

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Materials and MethodsCells and cell lines

Blood was obtained from healthy volunteers from French Blood Bank undera protocol approved by the Institutional Review Board of the Saint-LouisHospital, Paris, France.

For all purification steps, FcRs on PBMC were blocked using 20 mg/mlhuman IgG (Sigma-Aldrich) for 30 min.

For monocyte isolation, PBMCs were labeled with 20 mg/ml anti-CD14and then positively separated using goat anti-mouse (GAM)-coated mag-netic beads according to the manufacturer’s specifications (Ademtech). Toobtain highly purified CD4+ or CD8+ T cells, PBMCs were labeled withanti-CD4 or anti-CD8 and then positively separated using GAM-coatedmagnetic beads. Positively isolated cells were then incubated overnight at37˚C and repeatedly washed to increase the purity. To obtain negativelypurified CD4+ or CD8+ T cells, PBMCs were labeled with a mixture ofanti-CD14, anti-CD16, anti-CD19, and anti-CD8 or anti-CD4 followed byimmunomagnetic depletion using GAM-coated magnetic beads. ILT2-positive cells were removed using 20 mg/ml purified blocking Ab againstILT2 (GHI/75) and then positively separated using GAM-coated mag-netic beads. ILT2 expression was evaluated by flow cytometry.

Cell lines used were previously described (9).

Cell activation

Purified monocytes were activated with 100 ng/ml LPS (Sigma-Aldrich) for3–5 d. PBMCs or purified T cells were activated for 48 h with 4 mg/mlleucoagglutinin (PHA-L; Sigma-Aldrich) and then cultured in IL-2–sup-plemented medium (100 U/ml; Sigma-Aldrich) for 2–4 more d prior to use.Cells were not used beyond these times.

Abs and flow cytometry experiments

The following Abs were used from Exbio (Prague, Czech Republic): purifiedanti-CD4, -CD8, -CD14, and -CD19, blocking anti–HLA-G 87G Fab, non-blocking anti–HLA-GMem-G/9, and FITC-conjugated anti-CD4, -CD8, and-CD14; from Beckman Coulter: purified anti-CD28, anti-CD16, PC5-con-jugated anti-CD3, and anti-CD25; from BD Biosciences: purified and PE-conjugated anti-ILT2 (clone GHI/75); from Santa Cruz Biotechnology: puri-fied anti-ILT2 (clone VMP55); from Invitrogen: Alexa Fluor 488 GAM IgG1;and from Upstate Biotechnology: anti-phosphotyrosine 4G10. Purified anti-CD3 (OKT3) was provided by Janssen-Cilag.

For flow cytometry experiments, FcRs were blocked in 25% humanserum supplemented with 20 mg/ml human IgG serum prior to staining. AllFcR blocking conditions were maintained during the entire duration of theexperiments. Appropriate isotypic controls were systematically used toevaluate nonspecific binding. PKH67 or PKH26 dyes (Sigma-Aldrich)were used for fluorescent labeling of cell membranes and the sNHS-Biotin kit (Uptima) was used for cell-surface biotinylation according tothe manufacturers’ specifications.

Confocal microscopy

T cells were conjugated with PKH26-labeled monocytes at 37˚C and leftadhered on poly-L-lysine–coated slides for 5 min at 37˚C. The cells werethen fixed for 10 min with 3% paraformaldehyde, stained using an anti-ILT2 (VMP55) followed by GAM Alexa Fluor 488, and analyzed usinga Carl Zeiss LSM 510 confocal microscope (Carl Zeiss).

Trogocytosis assays

Trogocytosis assays were performed as previously described (9). Briefly,highly purified CD4+ and CD8+ T cells were cocultured for 30 min withpurified autologous monocytes at a 1:1 ratio, to a total concentration of 106

to 10 3 106 cells/ml, at 37˚C in a 5% CO2 humidified incubator. Allfurther steps were performed on ice.

For acid wash, cells were washed twice in PBS and resuspended for 4min at 20˚C in citrate buffer (0.133 M citric acid and 0.066 M Na2HPO4

[pH 3.3]) at a density of 5 3 106 cells/ml. The treatment was stopped byaddition of 10% FCS in RPMI 1640 and 10 mM HEPES.

Surface biotinylation experiments

Prior to the coincubationwith autologousTcells, themonocytes’ cell-surfaceproteins were biotinylated using sNHS-Biotin (Uptima). At the end of thecoincubation, T cells were purified, lysed, and the presence of biotinylatedILT2 was investigated by immunoprecipitation of biotinylated proteins withstreptavidin-agarose beads (Sigma-Aldrich) followed by electrophoreticseparation on a 10% SDS-PAGE acrylamide gel, transfer to nitrocellulosemembranes, and immunoblotting with anti-ILT2 (VMP55 clone).

ILT2 phosphorylation analysis

Cells treated with the phosphatase inhibitor PV (200 mM sodium ortho-vanadate and 200 mM H2O2 at 37˚C for 10 min) were lysed in 1% TritonX-100, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA,0.2 mM sodium orthovanadate, 0.2 mM PMSF, 0.5% Nonidet P-40, pro-tease inhibitors (Sigma-Aldrich), and phosphatase inhibitors cocktails Iand II (Sigma-Aldrich). Lysates were precleared with protein G-Sepharosebeads (Amersham Biosciences) and subjected to immunoprecipitation with2 mg purified anti-ILT2 (clone GHI-75; BD Biosciences) conjugated toprotein G-Sepharose beads. Immunoprecipitates were separated by stan-dard SDS-PAGE and transferred to nitrocellulose membranes (AmershamBiosciences). Immunoblotting was performed with anti-phosphotyrosineAb (4G10; Upstate Biotechnology) or anti-ILT2 followed by GAM-HRP(Sigma-Aldrich) or with streptavidin-HRP (Sigma-Aldrich). The mem-brane was developed using ECL+ Western blotting detection reagents(Amersham Biosciences).

HLA-G–ILT2 blocking procedures

For blocking HLA-G–ILT2 interactions, FcRs were first blocked by in-cubation of the cells 30 min in 25% human serum supplemented with 20mg/ml human IgG. Cells were then incubated with 10 mg/ml blocking Abanti–HLA-G 87G or nonblocking anti–HLA-G MEM-G/9 or blocking Abanti-ILT2 GHI/75, or their isotypic controls, prior to use. All blockingconditions were maintained during the subsequent experiments.

Proliferation assays

Proliferation was measured by tritiated thymidine ([3H]thymidine) in-corporation (1 mCi/well; Amersham Biosciences) on a b-counter (Wallac1450; Amersham Biosciences). All samples were run in triplicate. Ap-propriate proliferation and isotypic controls were included for each ex-periment on each plate.

Measure of the ongoing proliferation of already activated cells. A total of5 3 104 proliferating cells (CD4+ILT2Acq +or CD4+ T cells) was cocul-tured with 2.5 3 104 cells gamma-irradiated HLA-G–expressing cellslymphoblastoid cell line (LCL)–HLA-G1 (75 Gy) and their negative coun-terparts LCL-RSV and plated in a final volume of 200 ml. [3H]thymidinewas added at the time of incubation and proliferation was measured 18 hlater.

Alloproliferative responses of resting cells. A total of 5 3 104 respondernaive PBMCs or highly purified CD4+ T cells from these PBMCs or highlypurified CD4+ T cells incubated with autologous monocytes at a 1:1 ratio(CD4+ T cells plus monocytes) were stimulated with 2.5 3 104 gamma-irradiated LCL-RSV cells or LCL–HLA-G1 cells (75 Gy). Proliferation wasmeasured from day 1–6 every 24 h. Separate plates were set up for each timepoint, and [3H]thymidine was added 18 h before collection.

Cytotoxicity assays

Cytolytic activity of CD8+ T cells was assessed in a standard 4 h [51Cr]release assay against 51Cr-labeled HLA-G–negative melanoma M8 cellstransfected by a control vector (M8-pcDNA) or the same vector containingHLA-G1 cDNA (M8–HLA-G1). (M8 cells and the inhibition of CTLkilling are detailed in Refs. 16, 17). Briefly, effector cells were mixed with5 3 103 51Cr-labeled target cells (Amersham) at different E:T ratios in U-bottom microtiter plates. After 4 h of coincubation at 37˚C in a humidified5% CO2 incubator, supernatant was collected for liquid scintillationcounting (Wallac 1450 Microbeta; Pharmacia). Percentage of specific lysiswas determined as (cpm experimental well 2 cpm spontaneous release)/(cpm maximum release 2 cpm spontaneous release) 3 100. Spontaneousrelease was determined by incubation of labeled target cells with medium.Maximum release was determined by solubilizing target cells in 1% TritonX-100. For each experiment, triplicate samples were used.

Statistical analysis

Data are presented as means 6 SD. Student t test was used, and a p value,0.05 was taken to be significant. For figures showing representativeexperiments, error bars represent SD of triplicates.

ResultsT cells require ILT2-positive cells to upregulate cell-surfaceILT2 upon activation

ILT2 can be expressed at the surface of a minor fraction (0–25%) ofresting CD4+ and CD8+ T cells in healthy individuals (13, 18), andthis proportion is increased in activated and Ag-specific T cells

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(18–20). In the experiment described in Fig. 1, we used freshlyisolated PBMCs for which no ILT2 was detected at the surface ofCD4+ and CD8+ T cells. In agreement with the data describedabove, PHA plus IL-2 activation significantly increased ILT2 ex-pression by CD4+ and CD8+ T cells (Fig. 1A). However, thisupregulation did not occur if ILT2+ cells had been removed fromPBMC prior to PHA plus IL-2 activation (Fig. 1B). In the reverseexperiment, when highly purified (.99%, no monocytes detected)CD4+ or CD8+ T cells were activated by anti-CD3 plus anti-CD28,no ILT2 cell-surface upregulation was observed (Fig. 1C),whereas an additional 30-min coincubation of these purified andactivated T cells with autologous ILT2+ monocytes was sufficientto restore the original activation-dependent ILT2 cell-surface up-regulation. These data show that ILT2 upregulation might not bea feature of T cell activation per se but dependent on the presenceof ILT2+ cells such as autologous monocytes. The absence of ILT2expression by T cells purified prior to activation was confirmed byintracellular flow cytometry (Supplemental Fig. 1A) and confocalmicroscopy using another anti-ILT2 Ab, VMP55 (SupplementalFig. 1B).

ILT2 on CD4+ and CD8+ T cells is acquired from monocytesby membrane transfers (trogocytosis)

Out of all of the possible explanations of the fact that monocytesare required for activation-dependent T cell surface expressionof ILT2, we show in this study that ILT2 is actually not upregu-lated by T cells themselves, but acquired frommonocytes by T cellsthrough the transfer of ILT2-containing membrane patches, a mech-anism known as trogocytosis.

The hallmarks of trogocytosis are kinetics in the order of minutes,transfer of membrane patches and not of individual molecules,cell-cell contact dependence, and a limited lifespan of the acquiredmolecules at the surface of the acquirer cell (1, 2). Fig. 2A shows thatwhen monocytes for which membranes had been labeled with a li-pophilic dye (PKH67) were coincubated with activated autologousCD4+ T cells for 30 min or less, a correlation between T cell posi-tivity for PKH67 and ILT2 was observed. This shows that ILT2-positive T cells had acquired membranes from monocytes. Sup-plemental Fig. 2A shows that ILT2 acquisition was dependent oncell-to-cell contact, and Supplemental Fig. 2B shows the limitedlifespan of acquired ILT2 at the T cell surface, which indicates thatILT2 was not endogenously produced by T cells. Real-time quan-titative PCR experiments also indicated that ILT2-negative acti-vated CD4+ T cells expressed very low amounts of ILT2 mRNAprior to use and that these levels were not significantly increasedafter trogocytosis experiments (data not shown). Moreover, in ac-cordance with previously published data (21), Supplemental Fig. 3shows that CD4+ and CD8+ T cells acquire ∼1% of membranes ofmonocytic origin and between 1 and 0.5% of monocytic ILT2proteins after a 30-min coincubation with autologous monocytes.Fig. 2B illustrates by confocal microscopy that the only ILT2 ex-pressed by CD4+ T cells after coincubation with monocytes colo-calizes with membranes acquired from monocytes.To confirm the exogenous origin of the ILT2 receptors displayed

on T cells, surface-biotinylated monocytes were coincubated withT cells. After coincubation, the presence of biotinylated ILT2was sought using streptavidin-agarose immunoprecipitation andWestern blotting with anti-ILT2. Biotinylated ILT2 receptors wereeasily detected in T cells, demonstrating their monocytic origin(Fig. 2C, lower panel). Immunoprecipitation of ILT2 on controlT cells (Fig. 2C, upper panel) demonstrated that the only ILT2detectable on T cells was transferred ILT2. These data demon-strate that T cells can acquire ILT2 receptors from autologousmonocytes by trogocytosis.

Proper insertion of membrane ILT2

Membranes transferred from a cell to another may not integrate,but may remain affixed onto the acceptor cell (i.e., outside). Obvi-ously such a transfer is unlikely to allow signaling of the ac-quired molecules to the acquirer cell (2, 22). Membranes that areaffixed on acceptor cells can be removed by a mild acid washtreatment (pH 3.3) (23). A pH 3.3 treatment (Fig. 3A) was effi-cient and removed b2-microglobulin, leading to a loss of stainingfor HLA class I Ags using an Ab directed against conformed HLAclass I molecules (W6/32). However, this treatment did not re-move the PKH-labeled membranes of monocytic origin, or theILT2 they contained, from the CD4+ T cell surface. This ruledout the possibility that the acquired membranes were just af-fixed onto the surface of T cells.If properly inserted, transferred membranes and molecules

should spatially behave as endogenously produced ones, and move,or diffuse within the acceptor cell’s plasma membrane. Fig. 3Bshows by confocal microscopy a representative example of ourobservations. Early within the first 30 min of coincubation, mostof the transferred ILT2 at the surface of CD4+ T cells remainedwithin patches of PKH-labeled monocytic membranes. At latertimes, ILT2 receptors were also observed mainly within PKH-labeled monocytic membrane patches. However, an increasingproportion of cells that showed ILT2 receptors outside PKH-labeled membranes of monocytic origin was observed when in-cubation time increased. Representative images are shown in Fig.3B. Three-dimensional images of T cells with PKH-free ILT2 areprovided in Supplemental Video 1.

FIGURE 1. ILT2 expression by T cells requires activation and the presence

of ILT2-expressing APC. Flow cytometry analysis of ILT2 expression by

nonactivated or activated CD4+ and CD8+ T cells. Left panels, CD4+ T cells;

right panels, CD8+ T cells. A, CD4+ T cells and CD8+ T cells within non-

activated or activated PBMC. B, CD4+ T cells and CD8+ T cells within

nonactivated or activated PBMC depleted of ILT2-expressing cells. C, Highly

purified CD4+ and CD8+ T cells from the same PBMCs. D, Highly purified

CD4+ T cells and CD8+ T cells from the same PBMCs after a 30-min coin-

cubation with purified autologous monocytes at a 1:1 ratio. Percentage of

ILT2 expression is indicated. Experiment shown is representative of 15 in-

dependent experiments.

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The independent movements of dyed lipids and ILT2 show that

at least some ILT2+ monocytic membranes integrated properly,

even though we cannot know whether ILT2 molecules exited

the original membrane patch, or whether labeled lipids diffused

out of the area beneath ILT2.

Transferred ILT2 can access the intracellular machinery oftheir new cell host

It was shown that ILT2 phosphorylation is induced in T cells bythe protein tyrosine phosphatase inhibitor PV (14). Thus, we coin-cubated activated CD4+ T cells and biotinylated monocytes, CD4+

FIGURE 2. ILT2 display by activated T lymphocytes is due to trogocytosis. A, Activated CD4+ T cells that display ILT2 also display membranes acquired

from autologous monocytes. Flow cytometry was performed on purified activated CD4+ T cells prior to or after a 30-min coincubation with autologous

monocytes, the membranes of which had been labeled with the lipophilic dye PKH67. Data show the presence of membranes of monocytic origin versus ILT2

on CD4+ T cells. Percentage of ILT2+PKH67+ double-positive CD4+ T cells is indicated. Experiment shown is representative of three. B, ILT2 andmembranes

acquired from monocytes are colocalized on activated CD4+ T cells. Confocal microscopy was performed on purified activated CD4+ T cells after a 30-min

coincubation with autologous monocytes, the membranes of which had been labeled with the lipophilic dye PKH26. Blue, nuclear staining (DAPI); green,

ILT2; red, monocyte membranes. Scale bars, 5mm. Data show the colocalization of monocytic membranes and ILT2, indicative of a monocytic origin of ILT2.

Left panel, Monocyte–lymphocyte conjugate. Right panel, CD4+ T lymphocyte after coincubation. Arrows point to areas of interest. Experiment shown is

representative of three. C, Biotinylated ILT2 is acquired by autologous activated CD4+ T cells. Upper panel, Total ILT2 content in lysates of purified CD4+

T cells and autologous monocytes at the time of coincubation. Immunoprecipitation with anti-ILT2 (clone GHI/75), blotting with anti-ILT2 (VMP55 clone).

Lower panel, Detection ofmonocytic ILT2 in T cell lysates after coincubationwith biotinylatedmonocytes. Immunoprecipitationwith streptavidin beads, blot-

ting with anti-ILT2 (VMP55 clone). Biotinylated Monocytes, control biotinylated monocytes prior to coincubation with CD4+ T cells; CD4+-ILT2BiotinAcq+,

CD4+ T cells after a 30-min coincubation with biotinylated monocytes; CD4+ T cells, CD4+ T cells prior to coincubation with biotinylated monocytes.

FIGURE 3. ILT2-containing membranes of monocytic origin integrate into CD4+ T cell plasma membrane. A, ILT2-containing membranes of monocytic

origin on activated CD4+ T cells are not removed by acid wash. Flow cytometry analysis for ILT2 and PKH67 was performed on CD4+ T cells that had

acquired ILT2-containing PKH67-labeled membranes from autologous monocytes. ILT2 cell-surface expression and PKH67 positivity was evaluated prior

to and after acid wash. Percentage of ILT2+PKH67+ double-positive CD4+ T cells is indicated. The experiment shown is representative of three. B, ILT2 of

monocytic origin can rapidly be observed as PKH-free clusters at the surface of CD4+ T cells. Confocal microscopy for ILT2 and PKH26 was performed on

purified activated CD4+ T cells that had acquired ILT2-containing PKH26-labeled membranes from autologous monocytes. Scale bars, 5 mm. Blue, nuclear

staining (DAPI); red, membranes of monocytic origin (PKH26); green, ILT2 (of monocytic origin). Left panel, CD4+ T lymphocyte prior to trogocytosis.

Middle panel, CD4+ T lymphocyte during a coincubation with autologous monocytes. Right panel, Examples of PKH26 versus ILT2 localization on CD4+

T cells. The arrows point to areas of interest.

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T cells that had acquired ILT2 receptors (CD4+ILT2BiotinAcq+)were then sorted, treated with PV, lysed, and the phosphorylationof acquired ILT2 receptors was determined by Western blottingwith streptavidin-HRP or antiphosphotyrosine. Fig. 4 shows onceagain that CD4+ T cells did not express endogenous ILT2 priorto coincubation with monocytes and acquired biotinylated ILT2during the coincubation with biotinylated autologous monocytes.Fig. 4 also shows that PV treatment had no effect on the level ofacquired ILT2 in T cells, but was able to induce ILT2 phosphor-ylation in CD4+ILT2BiotinAcq+ T cells. This demonstrates thatacquired ILT2 receptors were accessible to the biochemical ma-chinery of the acceptor T cells. Purity of the T cell population andcontamination controls ensured that the observed ILT2 phos-phorylation was not related to the presence of contaminating mo-nocytes.Thus, these data demonstrate that acquired ILT2 receptors are

properly inserted within the plasma membrane of T cells, candiffuse within the plasma membrane, and that transferred ILT2 wasaccessible to components of the T cell intracellular machinery.

Acquired ILT2 inhibits the function of acceptor T cells afterligand engagement

It was shown that engagement of T cell ILT2 with HLA-G inducesinhibition of CD4+ T cell proliferation (9, 24) and CD8+ T cellcytolytic function (25). We investigated whether engagement ofacquired ILT2 with HLA-G was able to cause T cell functionalinhibition.We first showed that transferred ILT2 receptors were able to

mediate the inhibition of CD4+ T cell ongoing proliferation upon

engagement with HLA-G. For this, we generated purified, poly-clonally activated ILT2-negative CD4+ T cells and had them ac-quire or not ILT2 from autologous monocytes (CD4+, CD4+

ILT2Acq+ T cells, respectively). These T cells were already pro-liferating at the start of the experiment, and their original lack ofILT2 was assessed by flow cytometry. HLA-G–expressing cellswere then added and T cell proliferation measured. Fig. 5A showsthat the proliferation of ILT2-negative CD4+ T cells was notinhibited by HLA-G, but that of CD4+ILT2Acq+ T cells wascompletely stopped in the presence of HLA-G. The inhibition ofCD4+ILT2Acq+ T cells was directly due to the interaction betweenacquired ILT2 receptors and HLA-G. Indeed, anti–HLA-G (87G)and anti-ILT2 (GHI/75) blocking Abs, but not nonblocking anti–HLA-G Ab (Mem-G/9), restored the ongoing proliferation ofCD4+ILT2Acq+ T cells. Furthermore, proliferation of CD4+

ILT2Acq+ T cells was unaffected when HLA-G-negative controlcells were used in the coincubation.We next showed that acquired ILT2 receptors could inhibit

CD8+ T cell cytotoxic function. For this, we set up cytotoxicityassays between purified activated CD8+ T cells that originallydid not express ILT2 and had acquired ILT2 or not from auto-logous monocytes (CD8+ and CD8+ILT2Acq+ T cells, respectively)and HLA-G–positive targets. Fig. 5B shows that ILT2-negativeCD8+ T cells lysed their targets regardless of HLA-G. CD8+

ILT2Acq+ T cells lysed HLA-G–negative targets but their cytotoxicfunctions were blocked by HLA-G–expressing target cells. Thisinhibition was directly due to HLA-G–ILT2 interaction, as shownby the use of blocking Abs against HLA-G (87G) or ILT2. Iso-typic controls or the nonblocking anti–HLA-G Mem-G/09 did notrestore the cytotoxic activity of CD8+ T cells against HLA-G–positive target cells.Thus, these data show that activated ILT2-negative CD4+ and

CD8+ T cells are intrinsically insensitive to ILT2 ligands (e.g.,HLA-G) until they can borrow ILT2 from ILT2-expressing cellssuch as monocytes by trogocytosis of ILT2-containing mem-branes. Given that resting T cells have little or no trogocytic ca-pability (9), these data also indicate that T cell inhibition throughILT2 engagement should concern only fully activated T cells.

Inhibition of CD4+ T cell alloreactivity through ILT2 isdirected by ILT2 trogocytosis

We next demonstrated that trogocytosis-dependent ILT2-medi-ated inhibition held true in situations in which resting T cellswere involved. For this demonstration, we studied ILT2-medi-ated inhibition of resting T cell responsiveness to allogeneicstimulation using HLA-G as ILT2 ligand, because it is knownthat HLA-G inhibits the alloproliferative response of PBMC,as measured by thymidine incorporation at day 6 or 7 afterallostimulation (9, 26, 27).Thus, we set up alloproliferative experiments between irradiated

HLA-G–positive or HLA-G–negative LCL as stimulator cells andfreshly isolated PBMC or highly purified CD4+ T cells from thesePBMC (CD4+ T cells), or highly purified CD4+ T cells from thesePBMC mixed with autologous monocytes (CD4+ T cells plusmonocytes), as responder cells. PBMC used in these experimentswere chosen for their lack of ILT2 expression on T cells by flowcytometry performed at the time of isolation. In this system,allostimulation was provided by control or HLA-G–transfectedLCL cells, and ILT2 was provided by autologous monocytes. Tocorrelate ILT2 acquisition with alloproliferation inhibition, theILT2 expression and the proliferation of the responder cells weremeasured every 24 h for 6 d.For the first 3 d of the experiment, no difference was observed in

the phenotype and proliferation of CD4+ T cells. Thus, in Fig. 6,

FIGURE 4. Acquired ILT2 can access the intracellular machinery of

acceptor T cells. Purified CD4+ and Purified Monocytes, purified CD4+

T cells and biotinylated autologous monocytes prior to coincubation; Pu-

rified CD4+ILT2BiotinAcq+, purified CD4+ T cells after incubation with bio-

tinylated autologous monocytes; Contamination control, purified CD4+

T cells to which biotinylated autologous monocytes were deliberately added

at a proportion superior to that measured for purified CD4+ILT2BiotinAcq+ to

model the contribution of contaminant monocytes to the results. Top panel,

Endogenously produced ILT2 is detected in monocytes but not in autolo-

gous CD4+ T cells prior to coincubation. Middle panel, No effect of PV

treatment on the amounts of biotinylated ILT2 in CD4+ILT2BiotinAcq+ T cells

and controls. Bottom panel, Phosphorylation of ILT2 in CD4+ILT2BiotinAcq+

and controls after PV treatment.

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time points until day 3 are shown as a unique 0→3 d time point.For illustration, day 3 data were arbitrarily chosen but do not differfrom day 0, showing in particular no ILT2 expression on CD4+

T cells.As shown by the first two plots in Fig. 6A, CD4+ T cells within

a responder population of total PBMC remained negative for cell-surface ILT2 for 4 d. Consistently, as shown by the first two bargraphs, these T cells remained insensitive to inhibition by HLA-Gduring the first 4 d of allostimulation. As a consequence, the dailylevels of proliferation of these PBMCs were identical regardless ofwhether HLA-G was present in their environment or not. Thesedata on the first days of PBMC allostimulation clearly show thatILT2 does not inhibit the Ag selection and the initial activationsteps of resting T cells or even possibly their first proliferationcycles. After day 4, a proportion of CD4+ T cells displayed cell-surface ILT2, and this proportion increased through day 6(rightmost two plots of Fig. 6A). This means that a proportion ofCD4+ T cells became trogocytic as of day 4 and that this pro-portion increased through day 6. The proportion of ILT2-positiveCD4+ T cells was nevertheless much lower than in Figs. 1–5, butthis can be explained by the fact that allogeneic activation suchas in Fig. 6 concerns only a fraction of resting T cells, whereaspolyclonal activation as in Figs. 1–5 concerns all. The ILT2-positive T cells in Fig. 6 would then correspond to effectorsgenerated through Ag-specific activation. This also means that ittook 4 d from a resting T cell to become trogocytic upon allos-timulation, which matches the kinetics of trogocytic capabilityinduction through allostimulation and polyclonal activation de-scribed in this study and elsewhere (9). As can be seen in thecorresponding last two bar charts of Fig. 6A, it is from the pointwhen T cells started acquiring ILT2 that they reacted to thepresence of HLA-G and that their proliferation decreased com-pared with controls. At day 6, the HLA-G–mediated alloprolif-eration inhibition that could be observed was typical of what hasbeen reported elsewhere (26–28). The observed alloproliferationinhibition was directly due to HLA-G–ILT2 interaction and there-fore to ILT2 transfer from monocytes to activated T cells, becauseit could be prevented by blocking Abs directed against HLA-G(87G) or ILT2, respectively, whereas a nonblocking anti–HLA-GAb (MEM-G/9) had no effect.By contrast with the results obtained when total PBMCs were

allostimulated, highly purified CD4+ responder T cells, which hadno access to ILT2 exogenously produced by monocytes, remainedcell-surface ILT2 negative throughout the 6 d of the experiment(Fig. 6B, plots) and also remained mostly insensitive to HLA-G–mediated proliferation inhibition (Fig. 6B, bar graphs). However,sensitivity to HLA-G could be restored if the very same highlypurified ILT2-negative CD4+ T cells were coincubated with au-tologous ILT2+ monocytes at a 1:1 ratio (Fig. 6C). In this case, the

FIGURE 5. Acquisition of ILT2 is responsible for proliferation and

cytolytic inhibitions of activated T lymphocytes. A, Acquisition of ILT2 by

activated CD4+ T cells inhibits their proliferation in presence of HLA-G

molecules. As shown on flow cytometry plots, proliferation assays were

performed between activated ILT2-negative CD4+ T cells, which did not

acquire ILT2 (top left panel), or activated ILT2-negative CD4+ T cells,

which had acquired ILT2 from monocytes (top right panel), with HLA-

G1–positive or –negative LCL cells. Proliferation of activated CD4+

T cells was quantified by thymidine incorporation during 18 h of coculture.

Bottom left panel, Proliferation of activated CD4+ T cells is not inhibited in

presence of HLA-G1 molecules. Bottom right panel, ILT2-positive acti-

vated CD4+ T cells stop to proliferate only in presence of LCL–HLA-G1

cells. Proliferation of ILT2-positive CD4+ T cells can be restored using

blocking Ab (87G Fab and anti-ILT2) for the HLA-G1–ILT2 interaction

compared with nonblocking Ab (MEM-G/9), which cannot restore their

proliferation. B, Acquisition of ILT2 by activated CD8+ T cells inhibits

their cytolytic activity in presence of HLA-G molecules. As shown on flow

cytometry plots, cytotoxic assays were performed between activated ILT2

negative CD8+ T cells, which did not acquire ILT2 (top left panel), or

activated ILT2 negative CD8+ T cells, which had acquired ILT2 from

monocytes (top right panel), with HLA-G1–positive or negative M8 cells.

Cytotoxic function of activated CD8+ T cells was quantified at a 50:1 E:T

ratio. Bottom left panel, Cytotoxic function of activated CD8+ T cells is not

inhibited in presence of HLA-G1 molecules. Bottom right panel, ILT2-

positive activated CD8+ T cells are no longer cytotoxic in presence of

LCL–HLA-G1 cells. Cytotoxicity of ILT2-positive CD8+ T cells can be

restored using blocking Ab (87G Fab and anti-ILT2) for the HLA-G1–

ILT2 interaction compared with nonblocking Ab (MEM-G/9), which

cannot restore their cytotoxic function. This figure consists of three in-

dependent experiments. *Significant statistical analysis.

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FIGURE 6. Acquisition of ILT2 inhibits alloproliferation of CD4 T cells. Mixed lymphocytes reactions were performed between T lymphocytes and

stimulatory cells during 6 d. Proliferation was measured at the indicated time points, and ILT2 expression on CD4+ T cells was concomitantly analyzed by

flow cytometry. Mixed lymphocyte reactions were set up with no stimulatory cells (none), irradiated LCL stimulator cells (LCL), or irradiated LCL-HLA-

G1 stimulator cells in medium alone (LCL–HLA-G1) or supplemented with an isotype-matched irrelevant Ab (LCL–HLA-G1 + Isotypic Control),

a nonblocking anti–HLA-G1 Ab [LCL–HLA-G1 + Anti–HLA-G1 (MEM-G/9)], a blocking anti–HLA-G1 Ab as F(ab9)2 [LCL-HLA-G1 + Anti–HLA-G1

(87G Fab)], or a blocking anti-ILT2 Ab (LCL-HLA-G1 + Anti-ILT2). The responder cell populations used were total resting PBMCs (A), purified CD4+

resting T cells (B), purified CD4+ resting T cells plus autologous purified monocytes (C) at a 1:1 ratio. Data presented are from one out of three independent

experiments performed. *Significant statistical analysis.

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kinetics of ILT2 display by CD4+ T cells and those of allopro-liferation inhibition matched those of control PBMC.Thus, these data show that membrane transfers are required for

the in vitro ILT2-mediated inhibition of T cell alloproliferativeresponses and that the parameters of trogocytosis dictate which cellis concerned as well as the kinetics of ILT2-mediated inhibition.

DiscussionA minor proportion of T cells have been shown to be able to ex-press ILT2 at the resting stage, a proportion that was shown toincrease with activation and even more so in cloned T cells (29).Because endogenous expression of ILT2 by T cells would pre-clude any investigation on transferred ILT2, we used ILT2-negativeresting CD4+ and CD8+ T cells and autologous monocytes puri-fied from resting PBMCs of healthy donors as the only ILT2-expressing cells. The absence of ILT2 cell-surface and intra-cellular expression was systematically verified for the T cellswe used, prior to each experiment, before activation and againafter activation, right before use (Fig. 1A, Supplemental Fig. 1).Trogocytosis differs from other mechanisms of intercellular

Ag transfers, because it concerns entire portions of a donor cell’splasma membrane and not individual molecules. Thus, trogocy-tosis has the potential of being a transfer of functional units fromone cell to another. Thus far, we and others have demonstrated thatacquired molecules can carry on their outward-oriented function(i.e., can act as a ligand and signal from the acquirer cell to an-other cell), but the possibility of a transferred receptor signalinginward has not been investigated in humans. In theory, this hasalways been a possibility on the condition that the originallyfunctional membrane patch integrates properly within the plasmamembrane of the acceptor cell and finds within its new cell abiochemical environment that it can reach and that is compatiblewith its functional requirements (e.g., signaling pathways). Thedata we presented in this study demonstrate that this is not onlya possibility, but also a requirement for the well-described in vitrofunction of the inhibitory receptor ILT2.The key findings of this article, as well as the trogocytosis-based

mode of action of ILT2, are summarized in Fig. 7, which is onlya schematic representation based on the data presented in Fig. 6.As depicted, during the first phase of the immune response, restingT cells do not express ILT2 and cannot acquire it from surroundingautologous monocytes for lack of Ag-independent trogocytic ca-pability. It is possible that trogocytosis occurs during this phase, asreported in other systems. However, trogocytosis by resting cellsis dependent on Ag recognition (5) and thus concerns moleculesexpressed by the presenting APC, not molecules expressed bybystander environmental cells, which do not present the selectingAg. Immune regulation through molecules transferred from thestimulating APC may also happen at this time point but cannot bedetected in our experimental system. At one point (day 4 in ourcase of allostimulation), activated Ag-specific T cells becomecapable of performing Ag nonspecific trogocytosis and acquiremembranes from surrounding cells, including ILT2-containingmembranes from autologous monocytes. The set of moleculesinvolved in this type of acquisition is still unknown but clearlydifferent from those used in Ag-specific trogocytosis by restingcells (9). Thus, through trogocytosis, activated T cells acquiremultiple sensitivities from their microenvironment, which includesensitivity to inhibition by ILT2 ligands, as shown by our data. Inthe second phase of the response, T cells have become sensitive toa new array of immunomodulatory molecules. If these immuno-modulatory molecules are present within the local environment,they may now signal to their transferred receptors and alter T cell

behavior. In the case presented in this study, T cells became sen-sitive to ILT2 ligands, and because HLA-G was present within thelocal environment, these T cells stopped acting as effector cells.Thus, according to this model, the requirements for inhibition ofT cells through ILT2 are: 1) Ag-selection followed by activation;2) presence of ILT2-expressing monocytes; and 3) presence of anILT2 ligand, HLA-G. To the best of our knowledge, it is the firsttime that transfers of sensitivity to immune modulatory moleculesare shown and that this mechanism is proven crucial to an alreadyacknowledged immune-regulation mechanism.On a purely mechanistic standpoint, the data presented in this

study show that membrane patches and molecules acquired bytrogocytosis do not remain foreign patches, but integrate and, ifpossible, behave as would endogenously produced molecules. Inour case, exogenously produced ILT2 was able to send an inhibitorysignal strong enough to block the functions of the acceptor T cells,as would have endogenously produced ILT2. In our experimentalconfiguration, donor and acceptor cells are both known to be able tosignal through ILT2. It is therefore reasonable to postulate that theacquired patch integrated properly within the T cell plasma mem-brane and could use the compatible biochemical machinery of theacceptor cell to function. However, for other configurations inwhich donor and acquirer cells are biochemically very different,some signaling pathways might not be enabled, possibly leading toa partial or a lack of inward-oriented function of the acquired patch,even after integration of the transferred membrane. This limitationmight be considered as a regulation mechanism for sensitivitytransfers, allowing some but not all acquirer cells to acquire somebut maybe not all sensitivities of the donor cell. Transfers ofsensitivities would then be regulated at the cell-type level (tro-gocytic cell versus non trogocytic cell), molecular level (transferredversus not transferred molecules, integrated versus nonintegratedmembrane patch), microenvironmental level (presence versusabsence of a triggering ligand), and biochemical level (enabledversus disabled function of the transferred molecules). The pos-sibility of inward signaling by acquired molecules through com-patible biochemical pathways also raises two important but yetunresolved points: 1) if a signal is sent through a given membrane

FIGURE 7. ILT2 acquisition confers sensitivity to immune suppression

by HLA-G. Proliferative response and ILT2 acquisition by CD4 T cells was

analyzed during 6 d of allostimulation by coincubating PBMCs with either

HLA-G–negative cells or –positive cells. Initially, CD4+T cells did not express

ILT2 receptors, shown on flow cytometry analysis and confocal micro-

scopy, and proliferative response of CD4 T cells to HLA-G–positive or –nega-

tive cells is similar until 4 d of allostimulation. Then, activated CD4+ T cells

began to acquire exogenous ILT2 receptors, as shown by flow cytome-

try and confocal microscopy, and became susceptible to HLA-G expression.

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patch, will the consequences of this signal be different for the donorand the acceptor cells?; and 2) when considering only one signalingpathway and its consequences for a given cell, is it possible tobroaden the range of triggering events for this signaling pathwaythrough acquisition of new sensibilities?From an immunological standpoint, our data show that trogo-

cytosis is involved in a mechanism of in vitro immune regulationthat is well known and may be important should it be confirmedin vivo. This is a key point, because if an immune regulation mech-anism has been studied, agreed upon, and validated, so that it isregarded as a reliable fact, and if it is now proven that trogocytos-is is required for this immune regulation mechanism, it means thattrogocytosis might very well be involved in other immune mech-anisms and thus may constitute a normal step and/or a generalmechanism of immunity. This is first a problem: indeed, 1) if tro-gocytosis is a general mechanism of immune responses, the directlink among gene expression, phenotype, and function might be toomuch of a shortcut, and origin and traceability of molecules mightbecome parameters (difficult ones) to seriously consider. Further-more, 2) if trogocytosis is involved in or responsible for immuno-logical phenomena currently studied or relied upon (such as ILT2-mediated inhibition of T cells), it means once more that the sim-plification of experimental systems by removal of bystander cells toobtain clean models might sometimes be just the wrong thing to do.Yet, if intercellular transfers of cell-surface proteins, sensitiv-

ities, and functions constitute a generic mechanism in immunology,it means that there exists a level of powerful immune regulation thatwe hope is now clearly evidenced that is still vastly ignored andcompletely unexploited. This level of immune regulation wouldconstitute one of the last ones in a sequence of many. Its purposecould be the adaptation of one given immune response to variouslocal contexts. This would be a very flexible and cheap way for theimmune system tocheck thefitnessof a generic (centrally generated)response against particular (local) environmental constrains. Be-cause trogocytosis-mediated regulation might dramatically alterthe outcome of immune responses (9, 28, and current study) itshould be exploitable for the benefit of the patients. Acting on tro-gocytosis might include enabling it, stopping it, or using it byacting on microenvironmental cellular parameters to endow im-mune cells with new desirable sensitivities and functions that wouldbe location and time restricted.

AcknowledgmentsWe thank Niclas Setterbald and the Confocal Imagery Department (Institut

de Formation et de Recherche 105) of the Institut Universitaire d’Hemato-

logie, Hopital Saint-Louis, Paris, France, for technical help and Agnes

Lemas, Maria Loustau, and Emilie Lesport from Commissariat a l’Energie

Atomique-Service de Recherches en Hemato-Immunologie for technical

contributions.

DisclosuresThe authors have no financial conflicts of interest.

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