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Distribution in the Target Cell MembraneCytotoxicity Is Controlled by Ligand Cutting Edge: NKG2D-Dependent

Catharina C. GrossEmily Martinez, Joseph A. Brzostowski, Eric O. Long and

http://www.jimmunol.org/content/186/10/5538doi: 10.4049/jimmunol.1002254April 2011;

2011; 186:5538-5542; Prepublished online 4J Immunol 

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4.DC1http://www.jimmunol.org/content/suppl/2011/04/04/jimmunol.100225

Referenceshttp://www.jimmunol.org/content/186/10/5538.full#ref-list-1

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Cutting Edge: NKG2D-Dependent Cytotoxicity IsControlled by Ligand Distribution in the Target CellMembraneEmily Martinez,* Joseph A. Brzostowski,† Eric O. Long,* and Catharina C. Gross*,‡

Although the importance of membrane microdomainsin receptor-mediated activation of lymphocytes hasbeen established, much less is known about the roleof receptor ligand distribution on APC and targetcells. Detergent-resistant membrane domains, into whichGPI-linked proteins partition, are enriched in choles-terol and glycosphingolipids. ULBP1 is a GPI-linkedligand for natural cytotoxicity receptor NKG2D. To in-vestigate how ULBP1 distribution on target cells affectsNKG2D-dependent NK cell activation, we fused the ex-tracellular domain of ULBP1 to the transmembranedomain of CD45. Introduction of this transmembranedomain eliminated the association of ULBP1 with thedetergent-resistant membrane fraction and caused asignificant reduction of cytotoxicity and degranulationby NK cells. Clustering and lateral diffusion of ULBP1was not affected by changes in the membrane anchor.These results show that the partitioning of receptorligands in discrete membrane domains of target cells isan important determinant of NK cell activation. TheJournal of Immunology, 2011, 186: 5538–5542.

Natural killer cells are a subset of cytotoxic lymphocytesthat recognize and kill tumor cells and virus-infectedcells (1). Lysis of target cells is a multistep process

including adhesion of NK cells to target cells, synapse for-mation, polarization of cytolytic granules toward the targetcells, and granule exocytosis (2). Whereas binding of LFA-1on human NK cells to ICAM on target cells induces adhesionand polarization of lytic granules (3), degranulation is trig-gered by low-affinity FcgR CD16 or by synergistic combi-nations of coactivation receptors, such as 2B4 and NKG2D(4, 5).NKG2D is a C-type lectin coactivation receptor expressed

as a disulfide-linked homodimer on NK cells, NKT cells,and some T cells (6). In humans, NKG2D binds to stress-

inducible members of the polymorphic MHC class I-relatedchain A/B (MICA/B) family and the multigene family ofUL16-binding proteins (ULBPs; RAET1A-E). NKG2D ligandsare expressed in multiple types of tumors and play an impor-tant role in immunosurveillance of cancer (7). However, byshedding NKG2D ligands from their cell surface, tumorcells may escape the antitumor response mediated by NKG2D(6).Within the lipid bilayer, proteins and lipids are segregated

laterally, leading to functional subcompartmentalization ofthe plasma membrane (8). Lipid rafts are membrane micro-domains enriched in glycosphingolipids, sphingomyelins,and cholesterol. The role of membrane microdomains in pro-moting receptor-mediated lymphocyte activation has beenwell established (9, 10). Much less is known about how thedistribution of receptor ligands on target cells affects lym-phocyte function, although studies have suggested that it maybe an important parameter for lymphocyte activation. Forexample, the cytoplasmic tail of CD80 (B7-1), a CD28 ligandexpressed in APCs, is required for proper segregation ofCD28 at the immunological synapse and for full T cell ac-tivation (11). Furthermore, although expression of MICA onresistant target cells could overcome MHC class I-dependentinhibitory signaling in NK cells, a truncated form of MICAlacking a potential acylation site could not (12), suggestingthat NKG2D ligand distribution may play a role in over-coming NK cell inhibition.In this study, we tested ULBP1 as a ligand to investigate the

role of ligand distribution in NKG2D-dependent human NKcell activation. To do so independently of HLA class I ligandsfor inhibitory receptors, we expressed ULBP1 in a mousecell line. A chimera consisting of the extracellular portion ofULBP1 and the transmembrane region of CD45 was gener-ated. Its expression resulted in the localization of the normallyGPI-linkedULBP1 from detergent-resistant membrane (DRM)fractions to detergent-soluble fractions. This redistribution ofULBP1 caused a reduction in cytotoxicity and degranulation

*Molecular and Cellular Immunology Section, Laboratory of Immunogenetics, NationalInstitute of Allergy and Infectious Diseases, National Institutes of Health, Rockville,MD 20852; †Imaging Facility, Laboratory of Immunogenetics, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852;and ‡Department of Neurology—Inflammatory Diseases of the Nervous System andNeurooncology, University Hospital Munster, 48149 Munster, Germany

Received for publication July 7, 2010. Accepted for publication March 11, 2011.

This work was supported by the Intramural Research Program of the National Instituteof Allergy and Infectious Diseases, National Institutes of Health.

Address correspondence and reprint requests to Dr. Eric O. Long or Dr. Catharina C.Gross, Laboratory of Immunogenetics, NIAID-NIH, 12441 Parklawn Drive, Rockville,

MD 20852 (E.O.L.) or Department of Neurology—Inflammatory Diseases of the Ner-vous System and Neurooncology, University Hospital Munster, ICB, Mendelstrasse 7,48149 Munster, Germany (C.C.G.). E-mail addresses: elong@nih.gov (E.O.L.) orcatharina.gross@ukmuenster.de (C.C.G.)

The online version of this article contains supplemental material.

Abbreviations used in this article: DRM, detergent-resistant membrane; MICA, MHCclass I-related chain A; PI-PLC, phosphatidylinositol-specific phospholipase C; TIRF,total internal reflection fluorescence; ULBP, UL16-binding protein.

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by NK cells, implying a role for receptor ligand distribution inthe activation of NK cell responses.

Materials and MethodsCells

Resting human NK cells were isolated from peripheral blood cells by negativeselection using an NK cell isolation kit (Stem Cell Technologies). Freshlyisolated resting NK cells (95–99% CD32CD56+) were resuspended in IMDM(Invitrogen) supplemented with 10% human serum (Valley Biomedical) andused 1–2 d after isolation. Polyclonal IL-2–activated NK cells were cultured asdescribed previously (3). P815 cells were cultured in IMDM supplementedwith 10% heat-inactivated FBS (Thermo Scientific). For phospholipase treat-ment, P815-ULBP1 and P815-ULBP1-CD45TM cells were treated with 2 IU/ml phosphatidylinositol-specific phospholipase C (PI-PLC) (Sigma) for 1 hat 37˚C and 5% CO2, and surface protein levels were measured by flow cyto-metry using an R-PE–conjugated ULBP1 mAb (R&D Systems). The ULBP1mAb was conjugated with a Phycolink R-PE kit (Prozyme).

Transfection of P815 cells

P815 cells were transfected with human ULB1 or ULBP1-CD45TM (Sup-plemental Fig. 1) using the Bio-Rad Gene Pulser (10 mg of each DNA, 260 V,960 mF). Transfected cells were selected in IMDM supplemented with 10%heat-inactivated FBS and 800 mg/ml Geneticin (Invitrogen), and subcloned.Different clones were tested for ULBP1 expression and in functional assays,and representative clones from each cell line were selected for further use.

DRM preparation

DRM preparation was performed as described previously (13), except thatOptiPrep (Axis-Shield) was used instead of sucrose. Fractions 4–11 wereseparated on 12% SDS NuPAGE gels (Invitrogen) and transferred to poly-vinylidene difluoride membrane (Invitrogen). The membrane was blockedwith Odyssey blocking buffer (LI-COR Biosciences) for 1 h at room tem-perature and incubated with biotinylated goat anti-human ULBP1 Ab (R&DSystem) overnight at room temperature. After washing, the membrane wasstained with IRDye 680-labeled streptavidin (LI-COR Biosciences) for 1 h atroom temperature, and bands were detected using the Odyssey infrared im-aging system (LI-COR Biosciences). In the case of latrunculin A treatment,cells were incubated with either 0.3% DMSO carrier or 3 mM latrunculin A(Calbiochem) for 40 min at 37˚C and 5% CO2.

Total internal reflection fluorescence microscopy

Cell surface ULBP1 or ULBP1-CD45TM on P815 cells was fluorescentlylabeled with PE-conjugated ULBP1 mAb (R&D). For latrunculin A treat-ment, labeled cells were incubated for 40 min at 37˚C and 5% CO2 witheither 0.3% DMSO carrier or 3 mM latrunculin A before analysis by totalinternal reflection fluorescence (TIRF) microscopy. TIRF imaging and anal-ysis was performed as described previously (14).

Cellular assays

Cytotoxicity assays were performed as described previously (15). Degranu-lation assays were performed as described previously (4) with minor changes.In brief, 2 3 105 NK cells were added to 4 3 105 P815 target cells in a totalvolume of 200 ml IMDM medium supplemented with 10% heat-inactivatedFBS, 6 mg/ml monensin (Calbiochem), 20 ml/ml FITC-conjugatedCD107a mAb (Becton Dickinson), and 20 ml/ml PE-conjugated CD56mAb (Becton Dickinson). Cells were mixed and incubated for 1 h at 37˚Cand 5% CO2. Afterward, cells were spun down and expression of CD107a onCD56+ cells was determined by flow cytometry. In case of latrunculin Apretreatment of the target cells, cells were incubated with either 0.3% DMSOcarrier (Sigma) or 3 mM latrunculin A (Calbiochem) for 40 min at 37˚C at5% CO2 and washed extensively before use in the assay.

Results and DiscussionLinking the extracellular portion of ULBP1 to the transmembraneregion of CD45 changes its localization within the membrane

To change the distribution of the NKG2D ligand ULBP1within the plasma membrane, we generated a chimera con-sisting of the extracellular portion of ULBP1 and the trans-membrane region of CD45 (ULBP1-CD45TM) (SupplementalFig. 1). In brief, the extracellular portion of ULBP1 includingthe GPI anchor site (Supplemental Fig. 1B) was fused to the

transmembrane region of CD45 that includes only two ex-tracellular amino acids and four amino acids in the cytosolicportion for anchoring purposes (Supplemental Fig. 1B). TheGPI-linked ULBP1 and the recombinant ULBP1-CD45TMwere transfected into the mouse mastocytoma cell line P815,and clones with similar expression levels were selected (Fig.1A). To verify that ULBP1-CD45TM had lost the GPI an-chor, P815 cells expressing ULBP1 and ULBP1-CD45TMwere treated with PI-PLC, which cleaves GPI anchors.ULBP1 was sensitive to PI-PLC, but ULBP1-CD45TMwas not, indicating a loss of the GPI anchor in the chimera(Fig. 1A). The incomplete cleavage of ULBP1 (Fig. 1A),which could be caused by limited accessibility to phospholi-pase, is consistent with the low amount of ULBP1 sheddingobserved in other target cells (16).To test whether linkage to the transmembrane domain of

CD45 changed the localization of ULBP1, we prepared DRMfractions from P815-ULBP1 and P815-ULBP1-CD45TMcells. Whereas ULBP1 was almost exclusively localized inthe DRM fraction, the ULBP1-CD45TM protein was asso-ciated with the soluble fraction (Fig. 1B). Association ofULBP1 with the DRM fraction was even stronger than that ofthe DRM marker flotillin 1 (Fig. 1B). Therefore, linking theextracellular portion of ULBP1 to the transmembrane regionof CD45 changed its localization from the DRM fraction tothe detergent-soluble membrane fraction.

Targeting of ULBP1 to the detergent-soluble membrane fractionreduces the sensitivity of target cells to lysis by NK cells

It has been shown that redistribution of ICAM-2 on tumorcells via ezrin renders these cells more sensitive to lysis by NKcells (17). To test whether distribution of ULBP1 in either the

FIGURE 1. Fusion of the ULBP1 extracellular domain with the trans-

membrane domain of CD45 alters its distribution in P815 cells. A, P815 cellstransfected with GPI-linked ULBP1 (left panel) or ULBP1-CD45TM (rightpanel) were either untreated (solid line) or PI-PLC–treated (dashed line), and

analyzed with a PE-conjugated ULBP1 Ab or an IgG2A isotype control

(shaded). Data are representative of nine individual experiments. B, Fractions4–11 of DRM preparations from P815 cells expressing either GPI-linked

ULBP1 (left panel) or ULBP1-CD45TM (right panel) were analyzed by im-

munoblotting with Abs to ULBP1 (upper lane), raft-associated Flotillin-1

(Flot-1, middle lane), and detergent-soluble membrane transferring receptor

(TfR, bottom lane). Data are representative of two individual experiments.

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DRM fraction or the detergent-soluble membrane fraction hadany functional consequence for sensitivity to NKG2D-depen-dent cytotoxicity, P815-ULBP1 and P815-ULBP1-CD45TMcells were used as targets in a 2-h lysis assay (Fig. 2). Expres-sion of ULBP1 on P815 cells rendered them more sensitive tolysis by primary, resting NK cells (Fig. 2A), and IL-2 acti-vated NK cells (Fig. 2B). Expression of ULBP1-CD45TM onP815 cells resulted in a lower sensitivity to lysis by NK cells,as compared with ULBP1 on P815 cells (Fig. 2). These re-sults indicate that distribution of ULBP1 within the mem-brane may be important for proper NK cell function.We next tested which step in NK cell cytotoxicity was

sensitive to changes in the distribution of ULBP1. Expressionof ULBP1 in the detergent-soluble membrane fraction of P815cells resulted in reduced degranulation of resting NK cells (Fig.2C) and of IL-2–activated NK cells (Fig. 2D). Expression ofULBP1 and ULBP1-CD45TM on P815 cells had only aminor enhancing effect on the polarization of lytic granulestoward the NK–P815 cell contact (not shown). We concludethat a change of the localization of ULBP1 from the DRMdomains to the detergent-soluble membrane fraction reducescytotoxicity of NK cells at the level of degranulation.

Clustering and lateral diffusion of ULBP1 and ULBP1-CD45TM inthe plasma membrane are similar

We next tested how the segregation of ULBP1 into DRM anddetergent-soluble domains affected the extent of ULBP1 clus-tering and lateral mobility within the plasma membrane. Toinvestigate these parameters, we labeled ULBP1 and ULBP1-CD45TM with a PE-conjugated Ab to ULBP1, and theirdistribution and mobility were visualized by TIRF micro-

scopy. TIRF microscopy is a spatially limited, high-contrasttechnique that eliminates interference from bulk fluores-cence, which may be present within cells, and selectively detectsfluorophores proximal to and within the plasma membraneof cells on glass coverslips (18). Both ULBP1 and ULBP1-CD45TM were distributed into small clusters at the surfaceof P815 cells (Fig. 3A). Although individual ULBP1 andULBP1-CD45TM proteins were labeled with a single PE-fluorophore, photobleaching characteristics (the presence ofmultistep bleaching events over long track length, data notshown) of fluorescent PE-labeled particles suggested thatULBP1 and ULBP1-CD45TM were observed primarily asclusters and not single molecules. Cluster analysis by fluo-

FIGURE 2. Exclusion of ULBP1 from the DRM fraction of target cells

reduces the cytotoxicity and degranulation of NK cells. Susceptibility of P815

(open circles), P815-ULBP1 (black circles), and P815-ULBP1-CD45TM

(gray circles) cells by freshly isolated resting (A) or IL-2–activated NK cells (B)was measured in a 2-h assay. Bars indicate SD of independent experiments

with three NK cell donors. Degranulation of freshly isolated resting NK cells

(C) or IL-2–activated NK cells (D) in response to P815, P815-ULBP1, and

P815-ULBP1-CD45TM cells was measured in a 1-h degranulation assay with

CD107a mAb. Each symbol represents an individual donor among three (C)or five (D), which were tested in independent experiments. Paired t test wasperformed. *p # 0.01.

FIGURE 3. Lateral diffusion of ULBP1 and ULBP1-CD45TM is similar.

A, Pictures of P815-ULBP1 (left panel) and P815-ULBP1-CD45TM (rightpanel) cells labeled with PE-conjugated Ab to ULBP1. Scale bars, 5 mm. B,The intensity of ULBP1 (blue) and ULBP1-CD45 (green) particles was

measured in a 5 3 5-pixel grid centered over the peak of the Gaussian dis-

tribution calculated for the particle in the first frame in which it appeared in

the tracking algorithm. Average particle intensities are shown in cumulative

probability plots. Median intensities are indicated in parentheses. C, Themovement of ULBP1 and ULBP1-CD45TM particles labeled with PE-

conjugated Ab to ULBP1 was tracked by capturing TIRF images at 35

frames/s for 100 frames. Diffusion coefficients of ULBP1 (blue, n = 3532)

and ULBP1-CD45TM (green, n = 2295) particles are shown in cumulative

probability plots. Data are from one of seven representative experiments. D,Movement of DMSO-treated P815-ULBP1 (blue, n = 2826) and P815-

ULBP1-CD45TM (green, n = 2921), and latrunculin A–treated P815-

ULBP1 (red, n = 4114) and P815-ULBP1-CD45TM (aqua, n = 3240) cells

was tracked by TIRF as described in B. Data are from one of eight in-

dependent experiments. E, Degranulation of IL-2–activated NK cells stimu-

lated with P815-ULBP1 and P815-ULBP1-CD45TM cells pretreated with

DMSO carrier or 3 mM latrunculin A, as described in Fig. 2B. Each symbol

represents an individual donor among six, which were tested in independent

experiments. Paired t test was performed. **p # 0.0025, ***p # 0.0004.

5540 CUTTING EDGE: CYTOTOXICITY CONTROLLED BY LIGAND DISTRIBUTION

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rescence intensity measurements revealed no significant dif-ference in ULBP1 and ULBP1-CD45TM clusters, with amedian intensity of 1283 and 1204, respectively (Fig. 3B).Therefore, the distribution of ULBP1 in different membranedomains did not have a detectable impact on the number ofULBP1 molecules per cluster. Furthermore, the cluster in-tensity for both molecules was homogenous (Fig. 3B). Weconclude that the difference in sensitivity to NKG2D-depen-dent cytotoxicity is not due to a change in the number ofULBP1 molecules per cluster.The lateral movement of labeled ULBP1 and ULBP1-

CD45TM particles recorded by TIRF microscopy was trackedautomatically using an algorithm developed for MatLabsoftware (19), which was further modified to refine particlepositioning with a two-dimensional Gaussian fit (20). Short-range mean square displacements were determined from po-sitional coordinates of particles tracked for five frames (over160 ms) (20) and were linearly dependent on time underall conditions measured, consistent with a simple diffusionmodel for this range of movement. Short-range diffusioncoefficients were then determined for thousands of particlesin multiple cells and graphed either in cumulative probabi-lity plots (also known as cumulative distribution function) torepresent the frequency of diffusion coefficients for the entirepopulation of tracked particles (Fig. 3C), or median scatteredplots (Supplemental Fig. 2A). Each one of several thousandparticles is represented as a separate point in the cumulativeprobability plot. This type of graph can visually resolve smalldifferences between samples even when extensive overlapoccurs. ULBP1 clusters displayed a high lateral mobility at thesurface of P815 cells, with a median diffusion coefficient of0.122 mm2/s. Lateral mobility of ULBP1-CD45TM, witha median diffusion coefficient of 0.075 mm2/s, was reducedcompared with ULBP1. Single-particle tracking experimentshave shown that diffusion rate at the plasma membrane isreduced when proteins associate with lipid rafts (21) orwith protein complexes (22). The distribution of ULBP1-CD45TM in the detergent-soluble membrane fraction mayhave resulted in intermolecular interactions that reducedULBP1 mobility even further than the association of ULBP1with DRM domains.To test whether the lateral mobility of ULBP1 and ULBP1-

CD45TMwas controlled by the actin cytoskeleton, we trackedmobility on P815 cells treated with either DMSO carrier aloneor 3 mM latrunculin A (Fig. 3D, Supplemental Fig. 2B).Whereas the mobility of ULBP1 did not change after treat-ment with latrunculin A, the mobility of ULBP1-CD45TMincreased from a median diffusion coefficient of 0.065 mm2/sto a diffusion coefficient of 0.093 mm2/s, which was close tothe mobility of ULBP1 (0.118 mm2/s) after treatment (Fig.3D). A higher dose of latrunculin A (10 mM) did not increasethe mobility of ULBP1-CD45TM any further and had noeffect on the mobility of ULBP1 (data not shown).Previous work from our group has shown that immobili-

zation of ICAM on target cells, rather than its clustering,promotes proper LFA-1–dependent conjugate formationand granule polarization in primary NK cells (14). To testwhether changes in lateral diffusion of ULBP1 were respon-sible for the difference in sensitivity to lysis by NK cells (Fig.2), we took advantage of the similar lateral diffusion ofULBP1 and ULBP1-CD45TM after treatment with latrun-

culin A. If lateral diffusion of ULBP1 was the main deter-minant of the functional difference (i.e., greater ULBP1mobility leading to increased NKG2D-dependent degranu-lation), treatment of the target cells with latrunculin A shouldequalize the response to P815-ULBP1 and P815-ULBP1-CD45TM cells. As seen earlier in the absence of DMSO (Fig.2), in the presence of DMSO, ULBP1 induced significantlygreater degranulation than ULBP1-CD45TM (Fig. 3E).Treatment of P815 cells with latrunculin A did not equalizethe response induced by ULBP1 and ULBP1-CD45TM:degranulation induced by ULBP1 did not change, but that in-duced by ULBP1-CD45TM was reduced even further (Fig.3E). Therefore, NKG2D-dependent cytotoxicity is controlledby the distribution of NKG2D ligands into separate mem-brane domains, independently of the number of ligand mol-ecules per cluster, and of the lateral mobility of clusters in theplasma membrane.The molecular basis for the change in NKG2D-dependent

responses when ULBP1 is moved to a different membraneenvironment is still unknown. This change could be relevant,as the related NKG2D ligand ULBP2 is expressed as botha GPI-linked form and a transmembrane form (23). However,expression of the transmembrane form of ULBP2 on the NK-sensitive CHO cell line had a similar small enhancing effecton sensitivity to lysis by NK cells, as expression of both forms(23). Potential cis interactions of ULBP1 with cell surfaceproteins are different in the DRM domains than in the restof the plasma membrane. The slower mobility of ULBP1-CD45TM, as compared with ULBP1, and the recovery toa similar lateral mobility after inhibition of F-actin suggestthat ULBP1-CD45TM interacts with molecules tethered tothe cytoskeleton. Receptor ligands on target cells may oftenexist within the context of larger protein complexes, and therole of these complexes in ligand recognition should be givengreater consideration. The overall extent of basal ULBP1clustering, before contact of target cells with NK cells, wasvirtually identical for ULBP1 and ULBP1-CD45TM. Nev-ertheless, despite a similar number of molecules per cluster,the molecular and biophysical properties of ULBP1 andULBP1-CD45TM clusters may be different because of theunique properties of membrane subdomains. Such differencesmay change the interaction of receptor NKG2D with its li-gands at immunological synapses, the organization of whichplays an important role in lymphocyte responses (24). Dis-tribution of ULBP1 either within or outside of DRM do-mains may also affect trogocytosis, a process by whichcell surface proteins are transferred between target cells andlymphocytes (25–27). Whether intercellular transfer of a li-gand for an activation receptor from a target cell to an ef-fector cell leads to amplification of the response or todesensitization of the receptor is unclear. It would be in-teresting to investigate whether the distribution of ULBP1 indifferent membrane domains has an impact on its transfer toNK cells.Our data suggest that ligand distribution into distinct mem-

brane domains in general may play an underappreciated rolein the activation of NK cells. Given the potential of tumorcells or virus-infected cells to alter ligand distribution at theplasma membrane and to escape immune responses, it will beimportant to investigate how the distribution of other ligandsimpacts the activation of lymphocytes.

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AcknowledgmentsWe thank M. Vales-Gomez and H. Reyburn for advice and comments and

M. Peterson and M. March for discussion.

DisclosuresThe authors have no financial conflicts of interest.

References1. Lanier, L. L. 2008. Up on the tightrope: natural killer cell activation and inhibition.

Nat. Immunol. 9: 495–502.2. Orange, J. S. 2008. Formation and function of the lytic NK-cell immunological

synapse. Nat. Rev. Immunol. 8: 713–725.3. Barber, D. F., M. Faure, and E. O. Long. 2004. LFA-1 contributes an early signal

for NK cell cytotoxicity. J. Immunol. 173: 3653–3659.4. Bryceson, Y. T., M. E. March, D. F. Barber, H. G. Ljunggren, and E. O. Long.

2005. Cytolytic granule polarization and degranulation controlled by differentreceptors in resting NK cells. J. Exp. Med. 202: 1001–1012.

5. Bryceson, Y. T., H. G. Ljunggren, and E. O. Long. 2009. Minimal requirement forinduction of natural cytotoxicity and intersection of activation signals by inhibitoryreceptors. Blood 114: 2657–2666.

6. Champsaur, M., and L. L. Lanier. 2010. Effect of NKG2D ligand expression onhost immune responses. Immunol. Rev. 235: 267–285.

7. Guerra, N., Y. X. Tan, N. T. Joncker, A. Choy, F. Gallardo, N. Xiong,S. Knoblaugh, D. Cado, N.M. Greenberg, and D. H. Raulet. 2008. NKG2D-deficientmice are defective in tumor surveillance in models of spontaneous malignancy.[Published erratum appears in 2008 Immunity 28: 723.] Immunity 28: 571–580.

8. Lingwood, D., and K. Simons. 2010. Lipid rafts as a membrane-organizing prin-ciple. Science 327: 46–50.

9. Dykstra, M., A. Cherukuri, H.W. Sohn, S. J. Tzeng, and S. K. Pierce. 2003. Locationis everything: lipid rafts and immune cell signaling. Annu. Rev. Immunol. 21: 457–481.

10. Harder, T., C. Rentero, T. Zech, and K. Gaus. 2007. Plasma membrane segregationduringTcell activation: probing theorder of domains.Curr.Opin. Immunol.19: 470–475.

11. Tseng, S. Y., M. L. Liu, and M. L. Dustin. 2005. CD80 cytoplasmic domaincontrols localization of CD28, CTLA-4, and protein kinase Ctheta in the immu-nological synapse. J. Immunol. 175: 7829–7836.

12. Eleme, K., S. B. Taner, B. Onfelt, L. M. Collinson, F. E. McCann, N. J. Chalupny,D. Cosman, C. Hopkins, A. I. Magee, and D. M. Davis. 2004. Cell surface or-ganization of stress-inducible proteins ULBP and MICA that stimulate human NKcells and T cells via NKG2D. J. Exp. Med. 199: 1005–1010.

13. Cherukuri, A., S. J. Tzeng, A. Gidwani, H. W. Sohn, P. Tolar, M. D. Snyder, andS. K. Pierce. 2004. Isolation of lipid rafts from B lymphocytes. Methods Mol. Biol.271: 213–224.

14. Gross, C. C., J. A. Brzostowski, D. Liu, and E. O. Long. 2010. Tethering of in-tercellular adhesion molecule on target cells is required for LFA-1-dependent NKcell adhesion and granule polarization. J. Immunol. 185: 2918–2926.

15. Peterson, M. E., and E. O. Long. 2008. Inhibitory receptor signaling via tyrosinephosphorylation of the adaptor Crk. Immunity 29: 578–588.

16. Fernandez-Messina, L., O. Ashiru, P. Boutet, S. Aguera-Gonzalez, J. N. Skepper,H. T. Reyburn, and M. Vales-Gomez. 2010. Differential mechanisms of sheddingof the glycosylphosphatidylinositol (GPI)-anchored NKG2D ligands. J. Biol. Chem.285: 8543–8551.

17. Helander, T. S., O. Carpen, O. Turunen, P. E. Kovanen, A. Vaheri, and T. Timonen.1996. ICAM-2 redistributed by ezrin as a target for killer cells. Nature 382: 265–268.

18. Axelrod, D. 2001. Total internal reflection fluorescence microscopy in cell biology.Traffic 2: 764–774.

19. Douglass, A. D., and R. D. Vale. 2005. Single-molecule microscopy reveals plasmamembrane microdomains created by protein-protein networks that exclude or trapsignaling molecules in T cells. Cell 121: 937–950.

20. Tolar, P., J. Hanna, P. D. Krueger, and S. K. Pierce. 2009. The constant region ofthe membrane immunoglobulin mediates B cell-receptor clustering and signaling inresponse to membrane antigens. Immunity 30: 44–55.

21. Kusumi, A., C. Nakada, K. Ritchie, K. Murase, K. Suzuki, H. Murakoshi, R. S.Kasai, J. Kondo, and T. Fujiwara. 2005. Paradigm shift of the plasma membraneconcept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. Annu. Rev. Biophys. Biomol.Struct. 34: 351–378.

22. Vale, R. D. 2008. Microscopes for fluorimeters: the era of single molecule mea-surements. Cell 135: 779–785.

23. Fernandez-Messina, L., O. Ashiru, S. Aguera-Gonzalez, H. T. Reyburn,and M. Vales-Gomez. 2011. The human NKG2D ligand ULBP2 can be expressedat the cell surface with or without a GPI anchor and both forms can activate NKcells. J. Cell Sci. 124: 321–327.

24. Taner, S. B., B. Onfelt, N. J. Pirinen, F. E. McCann, A. I. Magee, and D. M. Davis.2004. Control of immune responses by trafficking cell surface proteins, vesicles andlipid rafts to and from the immunological synapse. Traffic 5: 651–661.

25. Sprent, J. 2005. Swapping molecules during cell-cell interactions. Sci. STKE 2005:pe8.

26. Davis, D. M. 2007. Intercellular transfer of cell-surface proteins is common and canaffect many stages of an immune response. Nat. Rev. Immunol. 7: 238–243.

27. Daubeuf, S., A. Aucher, C. Bordier, A. Salles, L. Serre, G. Gaibelet, J. C. Faye,G. Favre, E. Joly, and D. Hudrisier. 2010. Preferential transfer of certain plasmamembrane proteins onto T and B cells by trogocytosis. PLoS ONE 5: e8716.

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