of September 26, 2014.This information is current as

Ig-like Receptor in Human NK CellsEndocytosis of an Inhibitory Killer Cell The AP-2 Clathrin Adaptor Mediates

CampbellOshinsky, Ashley M. James, Ilya Serebriiskii and Kerry S. Amanda K. Purdy, Diana A. Alvarez Arias, Jennifer

ol.1303406http://www.jimmunol.org/content/early/2014/09/19/jimmun

published online 19 September 2014J Immunol 

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DCSupplemental.htmlhttp://www.jimmunol.org/content/suppl/2014/09/19/content.1303406.

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

The AP-2 Clathrin Adaptor Mediates Endocytosis of anInhibitory Killer Cell Ig-like Receptor in Human NK Cells

Amanda K. Purdy,* Diana A. Alvarez Arias,*,1 Jennifer Oshinsky,* Ashley M. James,*

Ilya Serebriiskii,†,‡ and Kerry S. Campbell*

Stable surface expression of human inhibitory killer cell Ig-like receptors (KIRs) is critical for controlling NK cell function and

maintaining NK cell tolerance toward normal MHC class I+ cells. Our recent experiments, however, have found that Ab-bound

KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late

endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the m2 subunit of the AP-2

clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/m2 interaction,

either by mutation of the ITIM tyrosines in 3DL1 or mutation of m2, significantly diminished endocytosis and increased surface

expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is

diminished upon Ab engagement with the receptor, as compared with untreated cells. Thus, we have identified AP-2–mediated

endocytosis as a mechanism regulating the surface levels of inhibitory KIRs through their ITIM domains. Based on our results, we

propose a model in which nonengaged KIRs are internalized by this mechanism, whereas engagement with MHC class I ligand

would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Fur-

thermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors.

The Journal of Immunology, 2014, 193: 000–000.

Natural killer cells selectively recognize and kill virus-infected and transformed cells while remaining toler-ant of normal cells (1, 2). Their activation is controlled

by a balance of signals from activating (aNKR), adhesion, andinhibitory (iNKR) surface receptors (3). Activation is dominantlysuppressed upon engagement of iNKRs (especially the humankiller cell Ig-like receptors [KIRs]) with MHC class I (MHC-I)expressed on normal cells. With few exceptions, normal cellselicit NK cell tolerance through their high expression of MHC-Iand low expression of ligands for aNKRs (4). However, followinggenotoxic stress (5) or virus infection (6), aNKR ligands can beupregulated and/or MHC-I downregulated on target cells to tip thebalance toward NK cell activation and targeted cytotoxicity.KIR inhibitory function centers around their cytoplasmic ITIMs

[(I/V)xYxx(L/V)] (3). KIR engagement with MHC-I ligands

results in 1) phosphorylation of ITIM tyrosine residues withsubsequent recruitment of SHP-1 and SHP-2 protein tyrosine

phosphatases that dominantly suppress aNKR signaling pathways,

and 2) induced tyrosine phosphorylation of the adaptor Crk, which

relocalizes from activating to inhibitory complexes (7–9). These

events terminate early NK cell activation signaling and establish

tolerance toward normal MHC-I–expressing cells.The surface levels of KIRs or their cognate ligands can directly

impact the activation thresholds of NK cells (10, 11), but little is

known regarding the mechanisms regulating the surface expres-

sion of KIRs. Generally, receptor surface expression can be con-

trolled by de novo protein synthesis, endocytosis, recycling back

to the cell surface, and protein degradation. With respect to KIRs,

both KIR3DL2 and KIR2DL4 can relocalize from the cell surface

to endosomes to mediate intracellular functions (12, 13). Fur-

thermore, polymorphic sequence variants of KIRs can exhibit

wide disparities in surface expression (14, 15). Protein kinase C–

dependent phosphorylation of Ser394 also appears to stabilize the

surface expression of KIR3DL1 (3DL1), and other sequence

motifs, including the first ITIM tyrosine have been implicated in

regulating surface expression (16, 17). These reports demonstrate

a need for better mechanistic understanding of KIR endocytosis

and intracellular trafficking.Mammalian cells can internalize receptors constitutively or

in response to specific stimuli via either clathrin-dependent or

-independent endocytosis (18–20). Clathrin forms a triskelion

structure that drives endocytic vesicle formation but requires

adaptors to bind surface receptors. The AP-2 clathrin adaptor is

directly implicated in the internalization of many receptors, in-

cluding transferrin receptor (TfR), low-density lipoprotein recep-

tor, and epidermal growth factor receptor (21–23). AP-2 is a

heterotetrameric complex composed of a- and b-adaptin that in-

teract with clathrin and the plasma membrane, m2, which asso-

ciates with cargo containing tyrosine-based motifs, and s2, which

is involved in binding cargo-containing dileucine-based motifs

*Immune Cell Development and Host Defense Program, Institute for Cancer Re-search, Fox Chase Cancer Center, Philadelphia, PA 19111; †Developmental Thera-peutics Program, Institute for Cancer Research, Fox Chase Cancer Center,Philadelphia, PA 19111; and ‡Kazan Federal University, Kazan 420008, Russia

1Current address: Janssen Pharmaceutical, Spring House, PA.

Received for publication December 20, 2013. Accepted for publication August 21,2014.

This work was supported by National Institutes of Health Grants CA083859 (to K.S.C.),CA009035 (to A.K.P. and D.A.A.A.), and CA06927 (to the Fox Chase Cancer Center),a Health Research Formula Fund (CURE) grant from the Pennsylvania Department ofHealth (to K.S.C.), which specifically disclaims responsibility for analyses, interpreta-tions, or conclusions, and by a subsidy of the Russian government to support theProgram of Competitive Growth of Kazan Federal University (to I.S.).

Address correspondence and reprint requests to Dr. Kerry S. Campbell, Institute forCancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA19111. E-mail address: kerry.campbell@fccc.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: AA, Y377/407A; aNKR, activating NKR; 3DL1,killer cell Ig-like receptor 3DL1 (or KIR3DL1); DN, dominant negative; iNKR,inhibitory NKR; KIR, killer cell Ig-like receptor; MFI, mean fluorescence intensity;MHC-I, MHC class I; PV, pervanadate; TfR, transferrin receptor; WT, wild-type.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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(19, 21). Although the mechanism of KIR endocytosis is un-known, the CD94/NKG2A iNKR is reportedly internalized bya macropinocytosis-like pathway, although the sequence elementsinvolved remain undefined (24).In this study, we demonstrate that the ITIM sequences of iKIRs,

in addition to their role in negative signaling, also provide a handlefor 3DL1 internalization. This internalization occurs through in-teraction with m2 of the AP-2 clathrin adaptor complex. Our dataalso suggest that AP-2 association may occur more readily whenKIRs are not engaged with MHC-I ligand, whereas interactionwith MHC-I ligand may reduce AP-2 association, which wouldpromote stable KIR surface expression to prolong inhibitoryfunction.

Materials and MethodsCells and culture

KHYG-1, NKL, Jurkat, HEK293T and LentiX 293T cells (Clontech,Mountain View, CA) were cultured as described (8, 25, 26). Healthyvolunteer blood donors were recruited by informed consent as approved bythe Fox Chase Cancer Center Institutional Review Board. Primary CD56+

CD32KIR3DL1+/2 NK cells were sorted by FACS and cultured in RPMI1640 medium plus 5% human serum, 10% FBS, and 500–1000 U/mlrecombinant human IL-2 (Roche, provided by the National Cancer Insti-tute Biologic Resources Branch, Frederick, MD). Some primary NK cellswere restimulated with either irradiated RPMI 8866 cells or irradiatedallogeneic PBMCs as described (27).

Microscopy

Following attachment to prewarmed poly-L-lysine slides (BD Pharmingen,San Jose, CA), NKL cells expressing 3DL1-cherry and EYFP-Rab4 orEGFP-Rab7 (1 d after passage and stimulation with IL-2) were cooled onice for 15 min and then labeled with DX9–brilliant blue 421 (BioLegend,San Diego, CA) for 30 min. Slides were subsequently washed three timesin room temperature PBS to remove excess mAb, warmed to 37˚C for 0–30min, and washed three times in PBS at room temperature again. Cells werefixed in prewarmed PBS containing 3% paraformaldehyde for 15 min atroom temperature. Slides were mounted with 0.16 mm coverslips (ThermoFisher, Pittsburgh, PA) in ProLong Gold antifade reagent (Life Technol-ogies, Eugene, OR). Images were acquired using a 603 oil objective withEZ-C1 3.80 software on an inverted Nikon TE2000 with a C1 confocalscanhead (Nikon, Melville, NY). Z-stacks (0.3-mm step size) were col-lected from at least 25 cells expressing high levels of Rab and 3DL1 foreach time point. 3DL1 internalization was analyzed by quantifying thecolocalization of DX9 mAb and Rab4 or Rab7. Colocalization wasquantified using a rigorous method developed by Manders et al. (28, 29),which measures the degree of overlap of pixels in two separate fluorescentchannels independent of their intensity and relative to the total intensitywithin each channel. Manders coefficients were quantified for each0.3-mm slice of each z-stack with the Just Another Colocalization Plugin inImageJ after thresholding (http://imagej.nih.gov/ij/, http://rsbweb.nih.gov/ij/plugins/track/jacop.html). The resulting coefficients were compared forsignificance with the Wilcoxon rank sum test using R software (R Foun-dation; http://www.r-project.org). A Manders coefficient of 0 correspondsto nonoverlapping distribution, whereas a value of 1 signifies 100%colocalization. To better visualize the colocalization in Fig. 1D, six of the0.3-mm slices from the center of representative cells were merged asa maximum projection to generate the images shown.

Cytotoxicity assay

The CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison,WI)was performed according to the manufacturer’s instructions. Briefly,721.221 cells (1 3 105; lacking or expressing the 3DL1 ligand [HLA-B*51]) were mixed with NKL cells to achieve NK cell/target cell ratios of1.25:1 to 10:1. Cells were incubated together in a U-bottom plate at 37˚Cin a humidified atmosphere of 7% CO2. After 4 h, supernatants wereharvested, exposed to substrate, and absorbance (490 nm) was measuredon a BioTek EL808 microplate reader (BioTek, Winooski, VT). Back-ground absorbance from a blank media control was subtracted from allvalues. Target cell maximum was determined after target cell incubationwith lysis buffer, whereas spontaneous values were computed from NK ortarget cells alone. Specific lysis was calculated as: 100 3 [(experimentalabsorbance 2 NK cell spontaneous 2 target cell spontaneous)/(target cellmaximum 2 target cell spontaneous)].

Yeast two-hybrid screen

A LexA-based yeast two-hybrid screen was performed as described (25).Bait for the screen was the human 3DL1*0010101 cytoplasmic domain (aa340–423, http://www.ebi.ac.uk/ipd/kir) fused with the LexA-DNA bindingdomain, which was expressed from the pEG202 plasmid. Control baitconstructs encoded the cytoplasmic domains of CD5 (aa 402–495) ormurine CD4 (aa 418–457) (30, 31). The 3DL1 ITIM tyrosines were mu-tated to alanines (Y377A, Y407A, and Y377/407A) using QuikChange II(Stratagene, Santa Clara, CA).

GST pull-down assay

GST-3DL1 fusion proteins (wild-type [WT] and Y to A mutant 3DL1 cy-toplasmic domains; aa 340–423) were generated from pGEX-4T1 plasmid(GE Healthcare, Piscataway, NJ) in BL21 bacteria, purified on glutathione-agarose (Thermo Scientific, Rockford, IL), incubated with HEK293T celllysates, and washed as previously described (16, 32). Adsorbed proteinswere separated by SDS-PAGE, transferred to polyvinylidene difluoride, andimmunoblotted with anti-m2 Ab (Sigma-Aldrich, St. Louis, MO) or anti–a-adaptin mAb (BD Biosciences, Mountain View, CA).

Immunoprecipitation and immunoblotting

For experiments in Fig. 2, 3DL1+ KHYG-1 cells were lysed in mRIPAbuffer (1% IGEPAL CA-630, 0.5% sodium deoxycholic acid, 150 mMNaCl, 10 mM Tris [pH 7.5], 0.1% SDS, 2 mM sodium orthovanadate, and1 mg/ml each of aprotinin, leupeptin, and soybean trypsin inhibitor[Sigma-Aldrich]. 3DL1 was immunoprecipitated with protein G–agarose(Millipore, Billerica, MA) precoupled with DX9 mAb. Proteins wereseparated as above, immunoblotted with anti–a-adaptin mAb (BD Trans-duction Laboratories) and rabbit anti-KIR Ab (16), followed by HRP-conjugated secondary Ab. The resulting blots were visualized with ECL(Millipore) and exposure to autoradiography film (Denville Scientific,Metuchen, NJ). The intensities of m2, a-adaptin, and KIR bands werequantified using ImageJ software. The ratio of m2 or a-adaptin/KIR proteinlevels for WT was arbitrarily set to 100%. In experiments for Fig. 5,KHYG-1 cells (4 d after passage and IL-2 stimulation) were stimulatedwith 1 mM pervanadate alone, 7.5 ug DX9 alone (a saturating concen-tration), or both in combination for 10 min on ice prior to lysis, as de-scribed (33). These cells were lysed in IP buffer (50 mM Tris-HCl [pH7.5], 150 mM NaCl, 1 mg/ml protease inhibitors as above, 2 mM sodiumorthovanadate, 1 mM NaF, 2 mM EGTA, and 0.5% Triton X-100). 3DL1was immunoprecipitated from these lysates with 5.133 mAb coupled tocyanogen bromide–activated Sepharose beads (GE Healthcare). Proteinswere separated as above and immunoblotted with anti–a-adaptin poly-clonal Ab (Proteintech, Chicago, IL), 4G10 mAb (Millipore), anti–SHP-1polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX), and anti-KIRpolyclonal Ab (no. ARP53462; Aviva, San Diego, CA) followed by anti-mouse IR680 and anti-rabbit IR800 (LI-COR Biosciences, Lincoln, NE).Proteins were visualized as described (26). Band intensity was quantifiedwith ImageJ.

Retroviral and lentiviral constructs and transduction

To disrupt human m2 (NM_001025205; American Type Culture Collec-tion, Manassas, VA) binding to tyrosine-based motifs, residues D176 andW420 were mutated to alanine (m2-dominant negative [DN]). To visualizeKIR localization within the endosomal compartment, 3DL1*001 fused in-frame to mCherry (Genewiz, South Plainfield, NJ) and EYFP-Rab4 orEGFP-Rab7 (gift from Dr. Mario Zerial, Max Planck Institute, Dresden,Germany) were subcloned into pBMN-NoGFP to generate retrovirusas described (26, 34). For primary NK cell expression, constructs weresubcloned into pCDH-EF1-MCS-T2A-copGFP (System Biosciences,Mountain View, CA) to generate lentivirus by transfecting LentiX293T cells with pCDH, pMD2.G (VSV-G), and psPAX2 (gag/pol) plas-mids (from Dr. Sam Kung, University of Manitoba, Winnipeg, MB,Canada). Lentiviral supernatants were harvested 48–72 h later, filtered, andconcentrated by ultracentrifugation or polyethylene glycol precipitation.Viral titers were determined in LentiX 293T cells (35). Primary NK cellswere infected on 2 consecutive days with lentivirus (multiplicity of in-fection of 20–40 in 8 mg/ml Polybrene). To alleviate compensatorymechanisms resulting from long-term expression of exogenous proteins,cells were assayed 48–72 h after the second infection.

FACS-based KIR internalization assay

NK cell lines and primary NK cells were always assayed 3–4 d after passageinto fresh IL-2–containing medium to improve consistency of the results.NK cells were stained with anti-3DL1 (PE-conjugated DX9) or anti-CD71(TfR; BioLegend) mAbs at 4˚C. A sample was left on ice (0 min) and

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remaining cells were incubated at 37˚C for 5–180 min. Cells were movedto ice-cold HBSS plus 1% FBS plus 0.1% NaN3, stained with Alexa Fluor647–conjugated anti-mouse IgG (Invitrogen, Grand Island, NY) at 4˚C,and analyzed by flow cytometry on a BD LSR II (BD Biosciences). Cellaggregates were gated out by forward scatter height versus forward scatterarea analysis, and viable NK cells were gated by forward scatter heightversus side light scatter and lack of propidium iodide (Invitrogen) stain-ing. Lentiviral-transduced cells were subgated into GFP2 (uninfected) andGFP+ (infected) populations. For each sample, the percentage internali-zation = 100 – [(2˚ Ab mean fluorescence intensity (MFI) at t min/2˚ AbMFI at 0 min) 3 100]. Jurkat T cells (see Fig. 3C) were subjected to anacid wash stripping assay as described (16). Briefly, following internali-zation, cells were treated with 200 ml HBSS containing 100 mM glycineand 100 mM NaCl (pH 2.5) at 4˚C to remove surface-bound Ab. Cells werewashed twice with HBSS plus 1% FBS, and 3DL1 expression was ana-lyzed by FACS.

ResultsAfter Ab engagement, 3DL1 moves to early/recycling and lateendosomes

Previously, we provided evidence that 3DL1 internalizes andrecycles back to the cell surface in NK cells (16). In that work, weshowed that turnover of surface 3DL1 on transduced NK-92 cellswas not changed during a 4-h assay when 50 mg/ml cycloheximidewas added to the cells, indicating that 1) surface expression isquite stable, 2) recycling is occurring, and 3) trafficking to deg-radation pathways is minimal in the absence of de novo proteinsynthesis. To better define the subcellular distribution of 3DL1 anddetermine whether 3DL1 relocalizes to endosomal compartmentsfollowing internalization from the cell surface, we expresseda fluorescently tagged version of 3DL1 (3DL1-Cherry) in NKLcells. First, we confirmed that the C-terminal tag did not disruptinhibitory function (Fig. 1A), which is consistent with a previousreport using 3DL1-EGFP (36). Next, we quantified the amount of3DL1 expressed on both the cell surface and within the endosomalcompartment in fixed NKL cells coexpressing 3DL1-Cherry andeither EYFP-Rab4, which marks early/recycling endosomes, orEGFP-Rab7, a late endosomal marker. As expected, a signifi-cant fraction of 3DL1-Cherry localized to the plasma membrane(median Manders coefficient of 0.3165 and 0.466 in EYFP-Rab4– and EGFP-Rab7–expressing cells, respectively), consistentwith its established role in inhibitory signaling (Fig. 1B) (3). Wealso found a significant amount localized to punctuate internalstructures, with a small pool coinciding with Rab4+ endosomes(median Manders coefficient of 0.0615, Fig. 1C, left) and a moresizeable fraction colocalizing with Rab7+ endosomes (medianManders coefficient of 0.222, Fig. 1C, right). Taken together,these data indicate that 3DL1 traffics from the cell surface throughthe endosomal compartments.To visualize internalization specifically, we labeled 3DL1

expressed on the cell surface with the DX9 mAb at 4˚C andquantified the colocalization of the anti-3DL1 mAb with EYFP-Rab4 (Fig. 1D) and EGFP-Rab7 (Fig. 1E) at 0–30 min of inter-nalization at 37˚C (37). We found a significant increase ofanti-3DL1 mAbs colocalizing in Rab4+ endosomes at 15 min, whichstabilized to a similar degree at 30 min (Fig. 1F, top). In contrast,significant colocalization of anti-3DL1 mAbs in Rab7+ lateendosomes did not occur until the 30 min time point (Fig. 1F,bottom). Similarly, we also observed a significant increase in3DL1-Cherry colocalized with Rab4+ endosomes at 15 min andwith Rab7+ endosomes at 30 min (Fig. 1G, top and bottom, re-spectively). We also observed a significant increase in anti-KIRmAbs colocalizing with 3DL1-Cherry at 15 and 30 min (Fig. 1H),presumably due to an accumulation of the Ab-bound receptor withdenser pools in endosomal compartments after internalization.Taken together, these data are consistent with a slow rate of in-

ternalization of Ab-labeled KIR3DL1 moving from the cell sur-face at time 0 min to merge with intracellular compartments thatinclude Rab4+ endosomes by 15 min and Rab7+ late endosomesby 30 min.

The m2 subunit of AP-2 interacts with the cytoplasmic domainof 3DL1

As a means to identify proteins responsible for 3DL1 internali-zation, we performed a yeast two-hybrid screen using the cyto-plasmic domain of 3DL1 as bait. In this screen, we identified fiveclones encoding the m2 component of the AP-2 clathrin adaptorcomplex (residues 146–435; data not shown). The 3DL1/m2 in-teraction was subsequently confirmed in yeast, along with theinteraction of m2 with the cytoplasmic domain of CD5 (whichdirectly interacts with m2) (38) but not CD4 (which can only in-teract indirectly with m2 through the HIV protein Nef) (39)(Fig. 2A). The m2 protein interacts with cargo containing tyrosine-based motifs (Y-X-X-f; X is any amino acid and f is a hydro-phobic residue) (19). 3DL1 has two potential m2 binding siteslocated within the N- and C-terminal ITIMs, that is, VTY377AQLand ILY407TEL, respectively. We mutated each tyrosine to alanineand assayed their interactions with m2 in a yeast two-hybrid re-porter assay. Individual Y377A and Y407A mutants exhibitedsignificantly decreased interaction with m2, with the Y377A mu-tant being most affected (Fig. 2B). Disruption of both tyrosinescompletely abrogated the m2/3DL1 interaction.We next engineered WTand mutant 3DL1 cytoplasmic domains

as recombinant GST fusion proteins and probed 293T (Fig. 2C) orKHYG-1 cell lysates (data not shown) for interaction with the AP-2 complex. Consistent with the yeast two-hybrid results, 3DL1-WT interacted with both m2 and a-adaptin of AP-2. In this assay,consistent with the yeast reporter assay, mutation of either Y377alone or both tyrosines to alanine eliminated interaction with m2or a-adaptin, whereas the Y407A mutation only partially dis-rupted binding (Fig. 2C). Taken together, these in vitro data andthe in vivo findings in yeast indicate that the m2 subunit of AP-2interacts with the cytoplasmic ITIM tyrosines of 3DL1. WhereasY377 is crucial for interaction with m2, Y407 contributes but isless imperative to binding.We also tested whether AP-2 could be coimmunoprecipitated

with full-length 3DL1 from NK cells. 3DL1 was isolated froma sorted subset of either 3DL12 or 3DL1+ KHYG-1 cells andprobed for AP-2 by immunoblot. Consistent with the GST pull-down data, a-adaptin coimmunoprecipitated with 3DL1 (Fig. 2D).

AP-2 promotes 3DL1 internalization through interaction withITIM tyrosines

In view of our observations by confocal microscopy that DX9 mAbcauses endocytosis of 3DL1, we quantified endocytosis by firstlabeling cell surface 3DL1 with PE-conjugated DX9 at 4˚C, in-cubating the cells for various times at 37˚C, and then staining witha fluorophore-tagged secondary Ab to determine the amount ofDX9 retained on the cell surface (see Materials and Methods). Todetermine whether disruption of the 3DL1/m2 interaction affectsinternalization of 3DL1, we compared the endocytic rate of WTand Y377/407A (AA) receptor upon Ab binding in NKL cells. Therate of internalization of 3DL1-WT was slow, with ,25% endo-cytosed by 30 min (Fig. 3A), consistent with our microscopystudies (Fig. 1). In contrast, internalization of the 3DL1-AA mu-tant was significantly delayed in NKL cells as compared with3DL1-WT (Fig. 3A). Also, surface expression of 3DL1-AA wasconsistently higher than 3DL1-WT (Fig. 3B), indicating thatdisruption of association with the AP-2 clathrin adaptor results inaccumulation of 3DL1 on the NK cell surface. In contrast, the rate

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of endocytosis and surface level of TfRs were consistent in thesesame cells expressing 3DL1-WT or 3DL1-AA (Fig. 3A, 3B).These data demonstrate that the methodology used to generateKIR-expressing NKL cells (e.g., retroviral transduction) did notglobally affect receptor endocytosis and that the 3DL1-AA mu-tation specifically impacted 3DL1. To confirm that the resultsrepresented receptor internalization, rather than dissociation of

primary DX9 or TfR Ab, we compared the changes in MFIs ofdifferentially fluorophore-conjugated primary and secondary Absthroughout the time course of the assay. Consistent with inter-nalization, we observed a significant decrease in secondary Absurface staining fluorescence over time, which did not track witha similar decrease in primary Ab fluorescence during the sametime course (Supplemental Fig. 1). We also compared the inter-

FIGURE 1. 3DL1 localizes to early and late endosomes after Ab engagement. (A) Nonradioactive colorimetric assay comparing the cytotoxicity of either

KIR2 (squares; NKL) or 3DL1-Cherry+ NKL cells (circles; KIR+ NKL) against 721.221 target cells lacking (No KIR ligand) or expressing the 3DL1 ligand

HLA-B*051 (KIR ligand) at various NK cell/target cell (NK:T) ratios. The means 6 SD of data from five independent experiments are shown. For each

experiment, the data were first divided by the “NKL, No KIR ligand” sample (arbitrarily set to 100%) to generate the percentage of total cytotoxicity.

Statistical analysis comparing matched samples for each NK cell line was calculated using the Student t test. *p # 0.05, **p # 0.01. (B) 3DL1 and Rab4/

Rab7 localization in representative NKL cells 1 d after IL-2 stimulation. Following fixation, cells expressing 3DL1-Cherry and either EYFP-Rab4 or EGFP-

Rab7 were labeled with brilliant blue 421–conjugated DX9 (BV421-DX9). Shown are maximum projected images of six 0.3-mm slices through the center

of representative individual cells. Cherry, 3DL1-Cherry (red); aKIR, brilliant blue 421–conjugated DX9 mAb bound to cell surface 3DL1 (blue); Rab4,

EYFP-Rab4 (green); Rab7, EGFP-Rab7 (green). (C) Compilation of data as in (B). Thresholded Manders coefficients of 3DL1-Cherry colocalized with

DX9 mAb (cell surface), Rab4 (Rab4+ endosome), and Rab7 (Rab7+ endosome). The median of each data set is indicated by a horizontal bar. Data are

pooled from two independent experiments where each icon represents a separate cell. Black-filled icons designate values derived from the representative

NK cells shown in panel (B). (D and E) Intracellular localization of 3DL1 with Rab4 (D) or Rab7 (E) is shown in representative NKL cells after 0–30 min

exposure to DX9 mAb. 3DL1-Cherry+ NKL cells were stained with brilliant blue 421–conjugated DX9 mAb on ice to label the receptor at the cell surface

and kept on ice (time 0) or incubated at 37˚C for 15 or 30 min. Shown are maximum-projected images of six 0.3-mm slices through the center of rep-

resentative individual cells. (F–H) Compilation of data as in panel (C) Thresholded Manders coefficients of DX9 mAb colocalized with Rab4 or Rab7 (F, top

and bottom panels, respectively), 3DL1-Cherry colocalized with Rab4 and Rab7 (G, top and bottom panels, respectively), and DX9 mAb colocalized with

3DL1-Cherry in the same cell populations (H). Black-filled icons designate values derived from the representative NK cells shown in panels (D) or (E).

Statistical analysis used the Wilcoxon rank sum test. *p# 0.05, **p# 0.01. Data are pooled from two independent experiments where each icon represents

a separate cell. All images were taken using a 603 oil objective.

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nalization of both 3DL1-WT and 3DL1-AA in Jurkat T cells usingan acid-stripping protocol. Again, 3DL1-AA was internalized ata slower rate and expressed at higher surface levels, as comparedwith 3DL1-WT (Fig. 3C). Unfortunately, primary NK cells andNK cell lines were found to be extremely sensitive to acid wash,which restricted the use of this assay to Jurkat cells.Next, we compared surface expression and internalization rates

in human primary NK cells. We used lentiviral transduction andsorting to express 3DL1-WT or 3DL1-AA in CD32CD56+3DL12

human primary NK cells. 3DL1-AA internalization was alsosignificantly delayed in primary NK cells, and surface expressionwas significantly elevated compared with 3DL1-WT (Fig. 3D,3E). Collectively, we conclude that the endocytosis of 3DL1depends, at least partially, on the cytoplasmic ITIMs, becausetyrosine mutation significantly slowed internalization and in-creased surface expression in cell lines and primary NK cells.

Expression of DN AP-2 reduces 3DL1 internalization

We next tested the impact of expressing a DN form of the m2subunit of AP-2 on 3DL1 surface expression and internalization inprimary NK cells. A D176A/W421 mutant of m2 (designated m2-DN) disrupts the interaction of m2 to Y-X-X-f–bearing cargo(similar to TfRs and KIRs) without affecting either the formation

FIGURE 2. The m2 subunit of AP-2 interacts with the ITIM tyrosine

residues of 3DL1. (A) EGY50 yeast cells were cotransformed with prey

plasmid encoding m2 (aa 146–435) and bait plasmids encoding 3DL1,

CD5, or CD4 cytoplasmic domains or empty vector (vector). Yeasts were

plated on leucine-deficient medium (+ 2% glucose [Glu, left] or 2% ga-

lactose + 0.2% raffinose [Gal, right]) to induce protein expression. Results

are representative of two independent transformations. (B) Quantitative

b-galactosidase assay of EGY50 cells cotransformed with prey plasmid

containing m2 and bait plasmid with cytoplasmic domains of WT or ala-

nine mutant 3DL1 (YA, AY, or AA). Data are means 6 SD of b-galac-

tosidase production from three experiments. (C) GST fusion proteins

exposed to 293T cell extracts were immunoblotted with anti-m2 or anti–a-

adaptin Ab. The degree of m2 and a-adaptin pulldown was determined by

quantifying the ratio of m2 or a-adaptin/KIR protein levels for Y to A

mutants relative to WT (set to 100%). Data are representative of three

independent experiments. (D) 3DL12 (lane 1 only) and 3DL1+ KHYG-1

cells were lysed, immunoprecipitated with anti-3DL1 mAb, and immu-

noblotted for a-adaptin, SHP-1, and KIR. E, empty lane; L, ladder; WCL,

whole-cell lysate. Approximate protein molecular mass is indicated on the

left-hand side of each immunoblot. Data are representative of two inde-

pendent experiments.

FIGURE 3. Mutation of the ITIM tyrosines slows 3DL1 internalization

in NK cells. (A) Internalization assay and (B) surface expression of 3DL1

(left panels) and TfR (CD71, right panels) in NKL cells expressing WT (n)

or AA mutant (N) 3DL1. Cells were labeled with PE-conjugated anti-3DL1

or anti-CD71 mAbs at 4˚C and incubated for 0–30 min at 37˚C. The

remaining surface-bound mAb was labeled with Alexa Fluor 647–conju-

gated anti-mouse IgG at 4˚C. The percentage internalized was quantified

by FACS. Surface expression (DX9 mAb MFI) was measured at time

0 min. Results in (A) and (B) are from the same four experiments, and

paired values derived from individual experiments are connected by lines.

(C) KIR internalization was assessed using an acid-stripping assay in

Jurkat T cells (see Materials and Methods). The fraction of 3DL1 inter-

nalized was quantified during a 2-h time course (left panel), and surface

expression was assessed at time 0 min (right panel) in Jurkat T cells that

were retrovirally transduced to express 3DL1-WT or 3DL1-AA. Results

are individual determinations from four independent experiments, and

paired values derived from individual experiments are connected by lines.

(D) Internalization assay [performed as in (A), left panel] and surface

expression at time 0 min (right panel) of 3DL1-WT or 3DL1-AA

expressed in primary NK cells by lentiviral transduction. 3DL12 NK cells

sorted from three healthy donors were transduced to express 3DL1-WT or

3DL1-AA. Presented data are from seven experiments using cells derived

from six independent transductions. Paired values derived from individual

experiments are connected by lines, and values from individual donors are

represented by distinct icons. Statistical analysis used the Student t test.

*p # 0.05, **p # 0.01. (E) Representative histogram of 3DL1-WT and

3DL1-AA surface expression from an experiment shown in (D).

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of the AP-2 complex or the internalization of dileucine motif–based cargo (21, 40, 41). Because primary NK cells express verylow levels of TfRs (our unpublished observations), we first showedthat m2-DN expression effectively delayed internalization andincreased surface expression of TfRs in KHYG-1 cells (Fig. 4A).We also measured surface levels of 3DL1 in NKL cells followingexpression of m2-DN. Importantly, m2-DN expression causeda significantly greater increase in the surface levels of TfRs than inKIR surface levels on NKL cells. Furthermore, the impact wastransient, as the elevation of cell surface expression for bothreceptors was lost after 1 wk of culture (Supplemental Fig. 2).From these results, we conclude that KIR surface expression levelsare more tightly regulated than TfRs, and compensatory mechanismsrapidly diminish the efficacy of m2-DN in NK cell lines. To avoidthese compensatory mechanisms, we next analyzed the impact ofshort-term m2-DN expression on 3DL1 internalization in primaryNK cells. To this end, m2-WT or m2-DN were next expressed bylentiviral transduction in 3DL1+ human primary NK cells, and thetransduced populations were identified by coordinate GFP expres-sion (Fig. 4B). Expression of m2-DN significantly delayed endocy-tosis (Figs. 4C, 4D) and increased surface expression of 3DL1 (Fig.4D) as compared with expression of m2-WT (Fig. 4C) or controltransduction with empty vector (Fig. 4D). In contrast, lentivirus in-fection and resulting GFP expression alone did not impact 3DL1internalization or surface expression (Supplemental Fig. 3). Thesedata confirm that the AP-2 clathrin adaptor can significantly con-tribute to the endocytosis of 3DL1 and thereby influence the levels ofreceptor surface expression on NK cells.

The KIR/AP-2 interaction is regulated by Ab binding to 3DL1

The ability of KIRs to inhibit NK cell cytotoxicity is dependent ontyrosine phosphorylation of the ITIM tyrosines. Because we havefound that AP-2 associates with KIRs through these same tyrosines,we next tested whether the KIR/AP-2 interaction is regulated by thephosphorylation state of these tyrosines. We hypothesized thatbecause m2 binds unphosphorylated tyrosines (19), the KIR/AP-2interaction would be enhanced when the KIR ITIMs are notphosphorylated, but decreased when the ITIM tyrosines are phos-phorylated. To test this hypothesis, we immunoprecipitated 3DL1

from unstimulated cells or cells stimulated with 1) pervanadate(PV) alone to induce robust and stable tyrosine phosphoryation, 2)DX9 mAb alone, which mimics MHC-I engagement and shouldtransiently increase ITIM phosphorylation (36, 42), or 3) bothtogether. In accordance with previous publications (8, 43), PV-treated cells exhibited a high degree of KIR tyrosine phosphory-lation and SHP-1 association. Furthermore, we found that theassociation of the a-adaptin subunit of AP-2 was significantlydiminished following receptor engagement with DX9 in thepresence or absence of PV, whereas PV alone reduced a-adaptinassociation only modestly, which did not reach statistical signifi-cance (Fig. 5). Collectively, these data show that the KIR/AP-2association is most pronounced in unmanipulated cells, and al-though tyrosine phosphorylation can reduce the association, Abengagement seems to further promote AP-2 displacement. In fact,DX9 mAb engagement alone significantly displaced a-adaptinbinding but did not induce tyrosine phosphorylation above base-line in this assay. We were unable to reproducibly observe dif-ferences in 3DL1 expression levels on NK cells that had beenconjugated with target cells bearing or lacking HLA-B*51 ligand(data not shown). This could be due to inefficiency of ligandengagement under these conditions, however, resulting in onlya minor fraction of the total surface 3DL1 being affected, therebylimiting detection of changes in surface levels on a per cell basis.This is consistent with the work of Treanor et al. (44) that showedthat only a small fraction of KIRs in an immune synapse arephosphorylated in microclusters. In contrast, DX9 Ab has thepotential to bind all of the 3DL1 on the cell surface, and if DX9binding is consistent with ligand engagement, our data suggestthat KIRs may be more susceptible to AP-2–dependent internal-ization when not engaged with ligand, whereas engagement withligand would displace AP-2 to stabilize the receptor on the sur-face, where it can mediate prolonged inhibitory signaling tomaintain NK cell tolerance.

DiscussionOur results show that 3DL1 can be slowly internalized, first toearly/recycling endosomes and subsequently to late endosomes(Fig. 1). Moreover, at least part of this endocytic process is me-

FIGURE 4. Expression of DN m2 (m2-DN) delays internalization and increases surface expression of 3DL1 in primary NK cells. (A) TfR internalization

(top panel) and surface expression (bottom panel) were determined as in Fig. 3 in control (2), m2-WT–expressing, or m2-DN–expressing KHYG-1 cells.

Results are from individual determinations at 10 min (top panel) or 0 min of internalization (bottom panel) from four independent experiments, with values

derived from individual experiments connected by lines. (B) Infected primary NK cells are marked by GFP expression following lentiviral transduction, and

m2-DN–expressing cells exhibit reduced surface expression of 3DL1. 3DL1+ primary human NK cells were infected with lentivirus containing m2-WT or

m2-DN. The percentage of GFP2 and GFP+ cells for each condition is indicated. Bottom panels: 3DL1 surface expression in the GFP+ populations at time

0 and following 150 min at 37˚C with MFI of 3DL1 is shown. (C) Data from three experiments performed as in (B) comparing 3DL1+ primary cells infected

with lentivirus containing m2-WTor m2-DN. Paired values derived from individual experiments are connected by lines, and different donors are represented

as distinct icons. (D) 3DL1 internalization (left panel) and surface expression (right panel) are shown in primary NK cells infected with lentivirus (Lenti)

generated with empty vector (2) or m2-DN (DN) lentivirus. Shown are 12 experiments with NK cells from five healthy donors (separate icon/donor), and

paired values derived from individual experiments are connected by lines. Statistical analysis used the Student t test. *p # 0.05, **p # 0.01.

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diated by interaction between 1) the m2 subunit of the AP-2 cla-thrin adaptor complex and 2) the ITIM motifs in the KIR cyto-plasmic domain (Figs. 2–4). Our findings are consistent witha previous report by Chwae et al. (45) that found AP-2 interactionwith a chimeric 3DL1 receptor construct in Jurkat T cells; how-ever, an ITIM-mediated basis for the interaction was not defined.The same group also provided evidence that the N-terminal ITIMand several additional sequence elements in the 3DL1 cytoplasmicdomain are involved in endocytosis of this chimeric receptor inresponse to treating the Jurkat cells with protein kinase C agonists(17). We cannot rule out alternative mechanisms that can alsomediate KIR endocytosis, but our experiments studied the full-length receptor to characterize the role of AP-2 binding to ITIMtyrosines in primary NK cells. Furthermore, it is important toemphasize that endocytosis of TfRs, which is considered to pri-marily involve AP-2/clathrin (21), was diminished to a similardegree as 3DL1 by expression of m2-DN in our experiments(Fig. 4). Alternatively, m2-DN expression resulted in significantlygreater elevation of TfR surface expression as compared with3DL1 (Fig. 4A, Supplemental Fig. 2). Based on these observa-tions, although AP-2/clathrin can mediate similar degrees ofendocytosis of both receptors, we conclude that 3DL1 surface ex-pression is more tightly controlled than TfRs, presumably throughmore efficient recycling of KIRs back to the cell surface. The moreefficient retention of iKIR expression on the cell surface is in ac-cordance with the critical role that these receptors play in tolerizingNK cells from attacking normal MHC-I+ cells in the body, whereasthe primary function of TfRs is to internalize iron.

Following engagement with MHC-I at the immune synapse,KIRs are phosphorylated on ITIM tyrosines in aggregated micro-clusters (44), leading to the recruitment of SHP-1/SHP-2 andinhibitory signaling (3, 7, 8). Because m2 associates withunphosphorylated tyrosine-based motifs, we expected m2 to in-teract with 3DL1 and induce endocytosis only when not engagedwith ligand. A similar mechanism has been described for CTLA-4,on which phosphorylation disrupts recruitment of m2 to a cyto-plasmic tyrosine to regulate endocytosis (46, 47). Although wewere surprised that PV-induced tyrosine phosphorylation of 3DL1did not significantly displace a-adaptin binding, it is possible thatthe pool of 3DL1 associated with AP-2 was not efficiently phos-phorylated under these conditions. Instead, we found that the KIR/AP-2 interaction is most profoundly diminished following en-gagement with DX9 Ab in the presence or absence of PV (Fig. 5).The lack of significant detectable tyrosine phosphorylation of3DL1 by treatment with DX9 alone suggests that Ab-mediateddisplacement of AP-2 may result through a mechanism indepen-dent of ITIM tyrosine phosphorylation. It is possible that Abbinding induces additional changes in the receptor cytoplasmicdomain (in addition to just tyrosine phosphorylation) to moreeffectively dissociate the clathrin adaptor. Although this mecha-nism has not been defined, if Ab binding is characteristic of ligandengagement, our results suggest that engaged KIRs are maintainedat the target cell interface to mediate prolonged inhibitorysignaling and sustained self-tolerance toward normal MHC-I–bearing cells. Furthermore, although DX9 engagement for 10 mindecreased the interaction of KIRs with AP-2 (Fig. 5), the Ab-engagedreceptor was ultimately slowly internalized by an ITIM/AP-2–dependent process during a longer time course, as shown in ourinternalization assays (Figs. 3, 4) and microscopy studies (Fig. 1).In contrast, our data further imply that AP-2–mediated endocy-tosis of 3DL1 would presumably occur more readily when NKcells are engaged with MHC-I–-deficient cells, thereby more ef-ficiently removing the iKIRs from the immune synapse to allowmore efficient cytotoxicity. aKIRs (KIR2DS, KIR3DS) lack fullITIMs and would therefore not be able to directly recruit AP-2. Itis possible that the aKIRs can be endocytosed through anothermechanism, however, including potential m2 binding to the ITAMtyrosines of the associated DAP12 adaptor, similar to a recentreport of AP-2–mediated BCR internalization through interactionwith an ITAM on CD79b (48).Sequence motifs in KIRs that exist outside of the cytoplasmic

domain have also been implicated in contributing to endocytosis.Upon binding to CpG oligodeoxynucleotides, KIR3DL2 report-edly relocalizes from the cell surface to early endosomes, therebytransporting the CpG to interact with TLR9 at that location (12).Surprisingly, relocalization in that context was reportedly inde-pendent of the cytoplasmic domain, because truncation distal tothe transmembrane domain had no impact upon KIR3DL2 inter-nalization. In that report, 3DL1 internalization was also observedupon binding with CpG DNA, although to a lesser extent thanKIR3DL2. Furthermore, KIR2DL4, a unique activating receptorthat contains a single ITIM (49), can also internalize to earlyendosomes, where it can mediate intracellular signaling or bedegraded following ubiquitylation (13, 25). Published data, how-ever, suggest that internalization of KIR2DL4 is independent ofthe transmembrane and cytoplasmic domains, because a chimericreceptor consisting of the extracellular domain of KIR2DL4 andtransmembrane/cytoplasmic domains of the plasma membrane-localized gp49B receptor was also targeted to endosomes (50).These studies further reinforce the functional relevance of KIRendocytosis and the roles of sequence elements outside of thecytoplasmic domain in mediating internalization. Our work has

FIGURE 5. The KIR/AP-2 interaction is regulated by KIR engagement

and ITIM phosphorylation. (A) 3DL1 was immunoprecipitated (IP) from

unstimulated (Unstim) 3DL1+ KHYG-1 cells or the same cells after

treatment for 10 min on ice with PV, DX9 mAb (DX9), or PV and DX9

mAb (PV+DX9). IPs were immunoblotted for phosphotyrosine (pY),

a-adaptin, SHP-1, and KIR. (B) Compilation of data from five experiments

performed as in (A). Band intensities were quantified by ImageJ, and the

relative band intensity was calculated as a ratio to the intensity of the KIR

band in each lane. Each icon represents an independent experiment with

the mean value shown as a horizontal bar. The immunoblot shown in (A)

was used to generate the band intensity data designated by the gray-filled

square icons in (B). Statistical analysis used the Student t test. *p # 0.05,

**p # 0.01.

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identified the interaction of the AP-2/clathrin complex with thecytoplasmic ITIMs of 3DL1 as one mechanism shuttling iKIRsfrom the cell surface. Although this mechanism is expected to alsobe operational for other iKIRs, further analysis is warranted tospecifically examine the contributions of ITIM/AP-2 interactionson the endocytosis of KIR3DL2 and KIR2DL4.Given that KIR-dependent inhibition of NK cell activation is

rapid (tyrosine phosphorylation and SHP-1/SHP-2 recruitmentoccur within minutes of ligand engagement) (51), whereas the rateof 3DL1 internalization in human primary NK cells is slow (only19.7 6 7.25% internalized by 30 min; Figs. 3D, 4C, 4D), it isunlikely that AP-2–mediated endocytosis of 3DL1 contributesdirectly to inhibitory function. It is possible, however, that thisAP-2–mediated mechanism may also be involved in the KIR-dependent physical transfer of HLA molecules from target cellsinto NK cells that has previously been reported (52). Alternatively,our data and the consistent expression levels of 3DL1 on thesurface of NK cells suggest that KIRs normally undergo consti-tutive internalization and recycling to maintain inhibitory capacityand tolerance. Although we cannot rule out involvement of otherendocytic mechanisms regulating KIR surface expression, ourfindings define a new functional role for the ITIMs on 3DL1.CTLA4 has been shown to recruit AP-2 through a non-ITIM ty-rosine (46, 47, 53), but to our knowledge, our data are the first todemonstrate that AP-2 can internalize an inhibitory receptorthrough an ITIM binding site. This mechanism may more gener-ally target additional ITIM-bearing receptors for endocytosis.

AcknowledgmentsWe thank Drs. David Wiest and Alana O’Reilly for constructive critique of

the manuscript, Drs. Sam Kung and Erica Golemis for reagents and advice,

Drs. Alexander MacFarlane IVand Sam Litwin for help with the Wilcoxon

rank sum test, and the DNA Sequencing, Flow Cytometry, Bioinformatics

and Biostatistics, and Cell Culture Facilities at the Fox Chase Cancer

Center for materials and technical support.

DisclosuresThe authors have no financial conflicts of interest.

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Supplemental Figures – Purdy et al.

Figure S1. Decrease in secondary antibody fluorescence is not due to a loss of primary antibody staining during internalization assay. Compilation of data from 3DL1-WT experiments in Fig. 3A comparing the MFI of PE-conjugated DX9 or TfR (primary Ab; Bottom) and AlexaFluor 647-conjugated anti-mouse IgG (secondary Ab; Top) in NKL cells at 0-30 min of internalization. The mean of 4 independent experiments is represented by a black line with filled in icons. p values were generated from the paired Students t-test, n.s. = not significant, * designates ≤ 0.05 and ** denotes ≤ 0.01.

Figure S2. µ2-DN expression results in a transient elevation in surface levels of TfR and KIR on NKL cells. Mean fluorescence intensity (MFI) measurements of TfR and 3DL1 surface levels on NKL cells were determined by FACS on the indicated days after infection with retrovirus to express µ2-DN (open bars) or in control (uninfected; filled bars). The mean ±S.D. of triplicate samples are shown for each time point, with corresponding p values generated from the paired Students t-test where * designates ≤ 0.05, ** denotes ≤ 0.01, and n.s. = not significant.

Figure S3. Lentivirus infection alone does not significantly affect the rate internalization or surface expression of 3DL1. Compilation of data from experiments in Fig. 4 comparing the percent internalization (Left panel) or MFI of surface expression (Right panel) of 3DL1 in primary NK cells infected with lentivirus generated with empty pCDH vector (GFP+ = infected, GFP- = not infected). Differences between the groups were not significant (n.s.) using the Student’s t test.