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Acquisition of Viral Receptor by NK Cells Through ... Acquisition of Viral Receptor by NK Cells Through ... Document Transcript

  • The Journal of Immunology Acquisition of Viral Receptor by NK Cells Through Immunological Synapse1 Julie Tabiasco,* Alain Vercellone,* Fabienne Meggetto,* Denis Hudrisier,† Pierre Brousset,* and Jean-Jacques Fournie2* ´ Occasional EBV infection of human NK cells may lead to malignant diseases such as naso-pharyngeal NK lymphoma although NK cells do not express CD21, the primary receptor for EBV. Here we show that during early EBV infection in patients, NK cells attacked EBV-infected autologous B cells. In vitro, NK cells activated by conjugation to CD21 B-EBV cell targets transiently acquired a weak CD21 phenotype by synaptic transfer of few receptor molecules onto their own membrane. In the presence of viral particles, these ectopic receptors allowed EBV binding to the novel NK cell host. Hence, trans-synaptic acquisition of viral receptor from target cells might constitute an unsuspected mode of infection for otherwise unreachable lymphoid hosts. The Journal of Immunology, 2003, 170: 5993–5998. E pstein-Barr virus (EBV) is a human herpes virus that in- process by which they bind EBV is unclear (6). It is well estab- fects most of the human population. Infection frequently lished that NK cells are strongly reactive to B-EBV infected cells. occurs during childhood and further remains asymptom- In addition, non-neoplastic EBV-infected NK cells are detected atic, with latent EBV within resting peripheral blood B cells. In very early in IM (7). Hence, early steps of NK cytolytic attack of some individuals, however, EBV may cause infectious mononu- EBV-infected targets could contribute to NK cell infection. Like T cleosis (IM)3 and is present in a variety of lymphocyte and epi- (8) and B lymphocytes (9), NK cells transiently establish a highly thelial neoplasms comprising Burkitt’s lymphoma, Hodgkin’s dis- dynamic immunological synapse with their targets (10). Our re- ease, T or NK cell lymphoma, and NK-cell chronic sults, as well as others, recently showed that through this area, NK lymphoproliferation (1). This shows that EBV may infect B lym- cells acquire membrane fragments from the conjugated cells on phocytes, epithelial cells, and more occasionally T lymphocytes their own cell surface (11–13). All the effector cell types analyzed and NK cells. However, the primary targets are B lymphocytes, in so far mediate a synaptic transfer of patches from the target cell which binding and entry of EBV constitute distinct processes. The membrane before—and independently of—the response (14). viral envelope glycoprotein gp350/220 mediates EBV binding by Here, we demonstrate that NK cell attack of B-EBV targets leads interacting with the EBV receptor CD21 (complement receptor to trans-synaptic acquisition and transient expression of their EBV type-2) (2), while entry results from EBV gp42 binding to the HLA receptor CD21 in a functional state. class II coreceptor (3). A different process is responsible for EBV tropism for epithelial cells, which lack CD21, HLA class II recep- Materials and Methods tor, and coreceptors (4). Although epithelial receptors remain un- Cell cultures identified, in this case EBV adapts the composition of its gp42- Fresh NK cell populations were obtained by PBL depletion of non-NK gH-gL envelope glycoproteins such as to reversibly switch its host cells using NK Cell Isolation Kit (Miltenyi Biotec, Bergisch-Gladbach, cell (5). Germany). Typically, the resulting NK populations comprised 85% of However, the EBV host range also includes NK cells. These CD56 cells and 0.5% CD3 cells. IL-2 activated bulk NK cells were express HLA class II molecules but not CD21, so thus far, the produced as follows. Fresh NK sorted from PBL as above (10,000 cells per well) were cultured in 96-well plates with irradiated feeder cells (200,000 irradiated peripheral blood lymphoctes per well) in presence of 200 U/ml IL-2 (Proleukin, Chiron, Emeryville, CA), and 10 g/ml PHA (Murex *Departement Oncogenese and Signalisation dans les Cellules Hematopoietiques, ´ ´ ` ´ ´ † Diagnostics, Dartford, U.K.). Three days later, 100 l of culture medium Departement d’Immunologie, Center de Physiopathologie de Toulouse Purpan, ´ Unite 563 de l’Institut National de la Sante et de la Recherche Medicale, BP3028 ´ ´ ´ was replaced in each well by 100 l of fresh culture medium containing Centre Hospitalier Universitaire Purpan, Toulouse, France 200,000 irradiated PBL feeders and 200 U/ml IL-2. NK cell cultures were then split in two every 2 days in IL-2 containing culture medium. By day Received for publication December 30, 2002. Accepted for publication April 11, these cultures contained 96% CD16 CD56 cells and no detectable 14, 2003. CD3 cells. The MHC class I erythro-myelocytic tumor K562, the Bur- The costs of publication of this article were defrayed in part by the payment of page kitt’s lymphoma Raji, and Daudi cell lines were maintained in culture in charges. This article must therefore be hereby marked advertisement in accordance RPMI 1640 medium supplemented with 10% FCS (Life Technologies, with 18 U.S.C. Section 1734 solely to indicate this fact. Gaithersburg, MD) supplemented with 100 U/ml penicillin, streptomycin 1 This work was supported by institutional grants from Institut National de la Sante ´ (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). Un- et de la Recherche Medicale and l’Association pour la Recherche sur le Cancer (Grant ´ less otherwise stated, all chemicals were from Calbiochem (EMD Bio- 5665 to J.-J.F.). sciences, San Diego, CA). 2 Address correspondence and reprint requests to Dr. Jean-Jacques Fournie, Depar- ´ tement d’Oncogènese and Signalisation dans les Cellules Hematopoietiques, Centre ` ´ ´ Analysis of CD21 acquisition de Physiopathologie de Toulouse Purpan, Unité 563, Institut National de la Sante et ´ de la Recherche Medicale, BP3028 Centre Hospitalier Universitaire Purpan, 31024 ´ Target cells were distributed in U-bottom 96-well plates (5 105 cells/100 Toulouse Cedex, France. E-mail address: l wells) to which NK cells pulsed for 15 min with 0.5 M Orange-(5- 3 Abbreviations used in this paper: IM, infectious mononucleosis; EBER, EBV-en- (and-6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine) coded RNA; NK, human natural killer; mfi, mean fluorescence intensity; CMTMR, (CMTMR; Molecular Probes, Eugene, OR) were added (105 cells/well in (5-(and-6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine); TPA, tetrade- additional 100 l). Culture plates were centrifuged for 1 min at 1200 rpm canoyl phorbol acetate. to promote conjugate formation and were left for 1 h at 37°C in humidified Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00
  • 5994 TRANS-SYNAPTIC ACQUISITION OF EBV RECEPTOR BY NK CELLS 5% CO2 atmosphere. For experiments involving Transwell culture plates Results (Costar, Cambridge, MA) NK cells (4 105 cells/400 l) and Daudi (2 NK cells conjugated to B-EBV targets and EBV-infected NK 106 cells/400 l wells) cells were left in separated or in similar compart- ments for 1 h at 37°C in humidified 5% CO2 atmosphere. Conjugates were cells in biopsies of lymph nodes from IM patients then dissociated by washing cells twice in PBS containing 0.5 mM EDTA. Immunostainings were performed on sections of formalin-fixed Cells were first labeled with mAb DR53 (undiluted culture supernatant) specific for CD21 (provided by G. Delsol, Unité 563 de l’Institut National and paraffin-embedded lymph node samples from patients with de la Santé et de la Recherche Medicale, Toulouse, France), washed, and primary IM. These were investigated for EBV and NK cell mark- stained with FITC-conjugated goat anti-mouse total Ig (10 g/ml, Argene ers. NK (PEN-5 ) cell membrane and cytoplasm were revealed by Biosoft, Varilhes, France). Samples were run on a FACSCalibur (BD Bio- sciences, San Jose, CA) and data analyzed with WinMDI software. peroxidase and diamino-benzidine brown staining, while a blue staining of cell nucleus revealed the EBV-encoded RNA (EBER). All biopsies from reactive lymph nodes showed abundant B-EBV EBV binding analysis cells, frequent uninfected NK cells conjugated to B-EBV targets Conjugates with 50,000 effector cells and 5,000 target cells were prepared (Fig. 1a), and also some EBV NK cells (Fig. 1b, arrow). These as specified above, dissociated, and NK cells were sorted using CMTMR. images of reactive tissues from early infected donors illustrated the These cells were fixed for 10 min at room temperature with 0.1% parafor- in vivo NK cell-mediated attack of autologous B lymphocytes in- maldehyde, washed three times with PBS 5% FCS and incubated with EBV viral particles (Advanced Biotechnology, Columbia, MD) in 100 l during fected by EBV. Since they were in line with similar observations 1 h at 4°C. Samples were washed three times and labeled with 10 g/ml made from other tonsils biopsies of IM patients (7), this suggested IgG1 mAb 2L10 specific for EBV gp350/250 (Advanced Biotechnology), that, despite the lack of known surface receptor for EBV, NK cells or 10 g/ml IgG1 isotype control (DAKO) washed and stained with FITC are infected early during primary EBV infection. conjugated goat anti-mouse total Ig (1:100 dilution, Argene Biosoft). Sam- ples were run on a FACSCalibur (BD Biosciences) and data was analyzed with WinMDI software. Intracellular Ca2 measurement NKL cells were loaded with 1 M Indo-1-acetoxymethyl ester (Indo-1- AM) (Molecular Probes) according to manufacturer’s instructions, mixed at 5:1 E:T ratio with the specified target cells. Cells were centrifuged, incubated 1 min at 37°C, resuspended in medium and analyzed by flow cytometry for intracellular calcium concentration using the ratio of emis- sion ( 405 nm/ 525 nm). For each experiment, 10,000 gated live NK cells were monitored at 37°C in function of time. Immunostaining of biopsies Immunostainings were performed on sections obtained from formalin-fixed and paraffin-embedded lymph node samples from IM patients. Paraffin sec- tions were mounted on glass slides coated with silane (Sigma-Aldrich, St. Louis, MO). Sections were deparaffinized, placed in 10 mmol/L Na-citrate buffer (pH.6), and heated in a microwave oven (Whirlpool model; Philips, Eindhoven, Holland) at 900 watts for cycles of 20 min and 10 min. The slides were then allowed to cool for 30 min at room temperature. Double labeling was conducted with the in situ hybridization technique with FITC- labeled probes (DAKO) as first step. The probes were revealed with an anti-FITC Ab (DAKO) conjugated with alkaline phosphatase while endoge- nous activity was inhibited by Levamisole. Bromo-chloro-indolyl phosphate- nitro blue tetrazolium was used as chromogen. The second step consisted of the immunostaining procedure using the anti-PEN-5 Ab (anti-5H10) (7). Slides were then rinsed in PBS before staining with a streptavidin-biotin-per- oxidase three-step reagent (DAKO Strept ABC complex/HRP Duet kit). En- dogenous peroxidase was blocked with 1% hydrogen peroxide in methanol for 30 min. Slides were analyzed under mineral oil (Sigma-Aldrich) by micros- copy (Nikon TE 200; Nikon, Melville, NY) using a 100 objective (Plan Apo; Nikon). Confocal microscopy B-EBV cells were stained with PKH67 as described (13), and bulk NK FIGURE 1. Conjugation of NK cells to EBV B lymphocytes in vivo cells were pulsed for 15 min with 0.5 M Orange-CMTMR (Molecular and in vitro. a, Microscopy magnification of a lymph node biopsy from a Probes). After washing, NK and B-EBV cells were mixed (1:1) in 50 l patient with early IM double-stained for EBER probes (blue cell nuclei RPMI plus 5% human serum in U-bottom 96-well plates and were laid onto revealed by alkaline phosphatase–nitroblue tetrazolium–5-bromo-4-chloro- polyL-lysine-coated slides with teflon wells (CEL-LINE; Erie Scientific, 3-indolylphosphate substrate) and anti-PEN5 Ab (stained brown with per- Portsmouth, NH) for 5 min at 37°C. The cells were fixed for 10 min at oxidase-diaminobenzidine substrate). All fields comprise numerous EBV- room temperature with 3% paraformaldehyde and mounted in 90% glyc- infected B cells (EBER ) and frequent NK (PEN-5 ) cells bound to erol–PBS containing 2.5% 1– 4-diazabicyclo (2.2.2) octane (DABCO, autologous B-EBV cells. b, Microscopy magnification of some rare cells Fluka, Buchs, AG). The slides were examined with an LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 63 Plan-Apoc- from the same biopsies, double stained by the EBER probes (blue) and the hromat objective (1.4 oil, Carl Zeiss). PKH 67 was excited with an argon PEN5 molecule (brown), revealing their NK-EBV phenotype. c, Confo- laser ( 488 nm) and Orange-CMTMR fluorescence was excited with a cal fluorescence microscopy of in vitro synaptic transfer (arrows) in a 1 h helium laser ( 543 nm). Images were acquired and treated with the LSM coculture comprising PKH67 (green)-labeled B-EBV cells and CMTMR 510 imaging software. (red)-stained NK cells.
  • The Journal of Immunology 5995 NK cells capture membrane patches from the conjugated B-EBV targets in vitro The conjugation of NK cell to targets cell starts by the establish- ment of an immunological synapse and is followed by a trans- synaptic capture of membrane molecules from the target. To test whether EBV-infected B lymphocytes cocultured in vitro with NK cells also enabled such a transfer, we stained a B-EBV cell line with the stable membrane fluorochrome PKH67 and measured its transfer on bulk allogeneic NK cells. For unambiguous discrimi- nation, the cytoplasm of NK cells was stained red with CMTMR before the coculture in complete medium. After 1 h of culture alone or immediately after cell mixing, each cell type harbored its original staining pattern, most notably the absence of green fluo- rochrome on red cells. Thus neither CMTMR nor PKH67 fluoro- chromes diffused out from labeled live lymphocytes in culture, as reported earlier (13, 17, 18). However, after 1 h of coculture con- focal fluorescence microscopy evidenced the acquisition of green patches on the red NK cell surface (Fig. 1c, arrows). Hence, this model confirmed in vitro that NK cells conjugated to B-EBV in- fected targets capture patches from their membranes. CD21 phenotypic switch of NK cells in contact with cocultured B cell targets Since HLA class II CD21 bulk polyclonal NK cells efficiently lyse the HLA class I CD21 B-EBV lymphoma Daudi, we cocultured these cell lines for 1 h to test whether NK cells acquired their target’s CD21. CMTMR-stained NK cells were mixed with Daudi cells (E:T ratio 1:5) for 1 h at 37°C. The culture was ana- lyzed for CD21 phenotype by flow cytometry. Dot plots of CMTMR vs CD21 fluorescence allowed clear-cut gating of NK FIGURE 2. NK cell activation induces the CD21 phenotype upon co- cells (Fig. 2a). The CD21 mean fluorescence intensity (mfi) of the culture with CD21 targets cells. CMTMR NK cells cocultured for 1 h at whole NK cell population slightly but reproducibly increased after 37°C with unstained Daudi cells, were labeled for CD21. a, Flow cytom- 1 h of coculture with targets at 37°C (Fig. 2b), but not at 4°C (data etry analysis of the coculture discriminates NK (inset) from CD21 target cells. b, CD21 phenotype of gated NK cells from coculture in the specified not shown). When this experiment was repeated in medium con- conditions (LuB: latrunculin B, PP2: PP2 Src kinase inhibitor), right num- taining latrunculin B, a cytoskeleton inhibitor (15), the NK cell’s bers are the CD21-FITC mfi of at least 5000 gated NK cells. CD21 mfi did not significantly increase. Adding Rottlerin or PP2 to the cocultures also blocked the CD21 mfi increase on NK cells, in line with the inhibition of NK cell’s synaptic transfer and lytic After 1 h of coincubation at 37°C with these targets, the CD21 activity (13). In contrast, the phorbol ester tetradecanoyl phorbol mfi of gated NK cells only increased after coincubation with the acetate (TPA) enhances NK cell’s lytic activity and synaptic trans- CD21 targets, but not with CD21 target cells or with CD21 fer (13). We thus tested CD21 labeling of NK cells cocultured at cells protected from NK lysis (Fig. 3c). 37°C with target cells in medium containing TPA. After 1 h, the From ten independent experiments measuring the CD21 ac- increase of the CD21 marker on NK cells was even stronger when quired by NK cells, the shift of CD21 mfi was always weak but TPA was added to the coculture. Adding TPA to the NK cells without highly reproducible. By (mfiCD21 of NK cells after transfer) target cells, however, did not change their CD21 mfi (Fig. 2b). (mfiCD21 of NK cells before transfer)/(mfiCD21 of target cells be- Collectively, these results demonstrated that the CD21 pheno- fore transfer), we calculated that within 1 h at 37°C, NK cells type acquired by NK cells is correlated to their activation by acquired on average up to 1% of the CD21 available on activating CD21 targets. target cells. Together, the above observations suggested that CD21 ex- NK cells acquire CD21 in coculture with CD21 B cell targets pressed on NK cells was captured from the cell surface of conju- The above experiments could not exclude that CD21 expression gated targets. was endogenous and up-regulated by NK cells upon exposure to target cells regardless of their CD21 phenotype. To formally rule The NK cell CD21 phenotype requires contact with CD21 out the induction of endogenous CD21 expression, NK cells were targets and is short-lived coincubated with a set of allogeneic cell lines which differed in To confirm that CD21 expressed by NK cells originated from the terms of CD21 expression and susceptibility to lysis. Although the CD21 targets, we measured CD21 acquisition in transwell cul- three HLA-I-deficient cell lines Daudi, C1R, and K562 activated ture plates. As compared with CD21 mfi acquired by NK cells in the NK cells as demonstrated by their Ca2 fluxes and specific 1 h cocultures with CD21 Daudi targets, the NK cell mfi in trans- lytic activity, a C1R cell line stably transfected with the protective well cocultures remained similar to that of NK cells alone in cul- HLA-B27 allele did not induce Ca2 fluxes in cocultured NK cells ture (Fig. 4a). So, the CD21 phenotype by NK cells requires direct (Fig. 3a) and was protected from their lysis (data not shown). In contact with CD21 targets. addition, while the B cell lines Daudi, C1R, and C1R-B27 expressed Because conjugation to a cell target is a transient event, we then surface CD21, the mono-myelocytic K562 did not (Fig. 3b). analyzed the kinetics of CD21 appearance and of CD21 clearance
  • 5996 TRANS-SYNAPTIC ACQUISITION OF EBV RECEPTOR BY NK CELLS FIGURE 3. NK acquire CD21 from conjugated targets. a, The MHC class I Daudi, C1R, and K562 targets but not the C1R-B27 cells activate Ca2 flux in NK cells. Without targets, the Ca2 flux in NK cells alone was below the baseline (arrow). b, CD21 is expressed by Daudi, C1R, and FIGURE 4. CD21 expression by NK cells requires direct contact with C1R-B27 B cells but not by the K562 cells (dotted line: isotype control, full CD21 cell targets and is short-lived. a, CD21 phenotype of NK cells in line: CD21). c, CD21 acquired by NK cells after coincubation with the the specified culture conditions, either alone (top), or mixed with CD21 above-specified targets (dotted line: isotype control, full line: CD21 on NK Daudi targets in coculture using normal (middle) or Transwell (bottom) cells at t0; red line: CD21 on NK cells after 1 h coculture with target cells). culture plates. b, CD21 phenotype of NK cells after various durations of coculture with CD21 Daudi targets (full dots) or CD21 K562 targets (empty dots). For clarity, the CD21 mfi were normalized to the highest from the surface of NK cells transiently cocultured at 37°C with value reached in the coculture. (None: no target cell). The disappearance is the activating Daudi target. For clarity, we normalized the results measured using normalized CD21 mfi of NK cells after increasing times of to the maximal CD21 mfi reached by NK cells during the whole incubation alone. experiment (i.e., 1.3% of the CD21 mfi from target cells). Al- though no shift in CD21 mfi was observed in presence of the CD21 cell targets K562, CD21 progressively increased on the surface of NK cells coincubated from 15 min till 2 h to the CD21 and B cells, we nevertheless tested the functionality of ectopic Daudi cell targets, and its intensity peaked by 1 h. Further, CD21 CD21 by measuring the EBV binding to NK cells. NK cells co- clearance from NK cells previously coincubated 1 h with Daudi, incubated for one hour with Daudi cells as above, dissociated and dissociated, sorted, and incubated alone for various times before sorted to 99% purity, were then exposed to EBV particles (mar- CD21 analysis permitted to measure CD21 disappearance. In these moset-derived B95-8 strain) at various ratio, before extensive wash conditions, the CD21 phenotype of NK cells progressively de- and stain for bound virus. EBV binding was measured by flow clined, was reduced by half after 90 min, and totally disappeared cytometry of the NK cells stained with anti-viral gp350 mAb and after 3 h (Fig. 4b). with isotype-matched control. In the same experiments, we also So, the NK cells rapidly acquire a small amount of CD21 from controlled the CD21 acquisition by NK cells. The comparison of their targets and transiently maintain this molecule on their own gp350 mAb mfi vs control isotype mfi demonstrated that before surface while exposed to novel CD21 targets. EBV addition, the NK cell surface did not contain any virus. Ac- cordingly, the EBV did not bind to NK cells without preliminary Ectopic CD21 enables EBV binding to the NK cell surface exposure to targets. After NK cells exposure to CD21 targets, CD21 is a transmembrane Ig-like monomer with EBV-binding do- however, the results differed. Little -if any- EBV binding was re- main located on its extracellular amino-terminal repeat domain peatedly observed on NK cells exposed to low EBV doses. In (16). Recognition by mAb of CD21 molecules expressed by targets presence of higher viral concentrations, however, EBV did bind to and acquired by NK cells indicated that the CD21 orientation has NK cells previously exposed to CD21 targets (Fig. 5a). The low not changed during transfer. We therefore hypothesized that the EBV binding to NK cells only resulted from the low numbers of newly acquired receptor was functional on NK cells. Although the CD21 receptors on the NK cell surface, because the same EBV/ molecular steps responsible for EBV entry in NK cells are un- CD21 ratio (mfi gp350/mfi CD21 0.7 for all doses tested) was known and may differ from those required for entry into epithelial found for Daudi and NK cells. In contrast, when such experiments
  • The Journal of Immunology 5997 Although the physiological function of synaptic transfer in im- munity could promote either affinity maturation (20, 21) or extinc- tion of response (for review see Ref. 14), we suggest here it could also be subverted by pathogens. Under defined circumstances, syn- aptic transfer enables a previously unattended attack of receptor- negative lymphoid cells. Here we find that upon conjugation to their targets, NK cells actively transfer on their own membrane a small amount of the viral receptor CD21 in functional orientation. Synaptic transfer on NK cells is a physiological event, strictly controlled by their activation state, and proceeds via the NK im- munological synapse. Confocal pictures of large patches from tar- get membrane smearing to the NK surface suggested that this transfer is qualitatively non-selective (13). So far, the different surface molecules found synaptically captured by lymphoid effec- tors comprise particulate Ags (20), MHC-peptide complexes (22), mIgM (18), MHC class I (11), and CD4 (our unpublished obser- vations). As recently found for NK and T cells (13, 18), the extent of trans-synaptic CD21 acquisition merely reflects the level of NK cell activation by targets. Although in vitro, the ectopic expression on effector cells is relatively short-lived (1–3 h) after in- terrupted synapses, it might last longer in vivo, where NK cells seri- ally engage several targets and express their markers for hours (11). NK cells are physiologically reactive to B-EBV targets, so they are highly prone to acquire their surface receptors. The in vivo relevance of these findings could deal with infectiology, since sev- eral clinical reports pinpoint the unexplained EBV (23) or HIV (24) infection of NK cells. We postulate a trans-synaptic infection model of receptor-negative lymphoid cells which involves four steps: 1) activation of (receptor-negative) lymphoid effectors by targets expressing the viral-receptor, 2) establishment of a func- tional immunological synapse between these cells, 3) synaptic transfer of the receptor on effectors, 4) viral binding to the ectopic receptors of the lymphoid effector. This model implies a transient effector cell susceptibility to infection, primarily defined by the ectopic receptor persistence on its cell surface. Because various lymphoid cell subsets make immunological synapses with recognized cell targets, trans-synaptic acquisition of viral receptors by other lymphoid effectors might conceivably ap- FIGURE 5. Coculture with CD21 target cells enables EBV binding to ply in other viral diseases. Among these, our hypothesis could NK cells. a, fluorescence histograms and mfi for EBV gp350 (right) vs prove useful to account for the recently discovered NK cell reser- isotype control (left) of NK cells treated as specified (numbers above: mfi voir for HIV in AIDS patients (24, 25). We believe that validation from 5000 sorted NK cells). b, Result from a similar experiment but with of this novel model deserves future investigation. prior NK cell coculture with CD21 or CD21 target cells. Note. While submitting this manuscript, the trans-synaptic host cell-to-effector T lymphocyte transmission of HTLV-I was re- involved CD21 targets instead of Daudi, EBV did not bind to NK ported (26). cells (Fig. 5b). So, the CD21 acquired by NK cells upon exposure to CD21 Acknowledgments targets is still functional on the NK cell surface and enables bind- We thank the expert technical assistance of Fatima L’Faqihi, useful com- ing of EBV. ments from E. Espinosa, and sustained encouragements from E. Vivier and G. Delsol. Discussion Several recent studies have demonstrated that B cells (9), CD8 References T cells (17), T cells (18), and NK cells (11–13) capture and 1. Rickinson, A. B., and E. Kieff. 2001. Epstein-Barr virus. In Fields Virology, Vol. internalize from their target cells a small amount of surface mol- 2. D. M. Knipe, and P. M. Howley, eds. Raven, Philadelphia, p. 2575. 2. Tanner, J., J. Weis, D. Fearon, Y. Whang, and E. Kieff. 1987. Epstein-Barr virus ecules across the immunological synapse. Recent electron micros- gp350/220 binding to the B lymphocyte C3d receptor mediates adsorption, cap- copy of CD8 T cells conjugated to target cells evidenced mem- ping, and endocytosis. Cell 50:203. brane fusions at the synapse which physically bridge both cell 3. Li, Q., M. K. Spriggs, S. Kovats, S. M. Turk, M. R. Comeau, B. Nepom, and L. M. Hutt-Fletcher. 1997. Epstein-Barr virus uses HLA class II as a cofactor for types (19). 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