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Acquisition of Viral Receptor by NK Cells Through Immunological Synapse¹

Julie Tabiasco^*, Alain Vercellone^*, Fabienne Meggetto^*, Denis Hudrisier, Pierre Brousset^* and Jean-Jacques Fournié^2,*

^* Département Oncogénèse and Signalisation dans les Cellules Hématopoiétiques, Département d’Immunologie, Center de Physiopathologie de Toulouse Purpan, Unité 563 de l’Institut National de la Santé et de la Recherche Médicale, BP3028 Centre Hospitalier Universitaire Purpan, Toulouse, France

	Abstract

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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.

	Introduction

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Epstein-Barr virus (EBV) is a human herpes virus that infects most of the human population. Infection frequently occurs during childhood and further remains asymptomatic, with latent EBV within resting peripheral blood B cells. In some individuals, however, EBV may cause infectious mononucleosis (IM)³ and is present in a variety of lymphocyte and epithelial neoplasms comprising Burkitt’s lymphoma, Hodgkin’s disease, T or NK cell lymphoma, and NK-cell chronic lymphoproliferation (1). This shows that EBV may infect B lymphocytes, epithelial cells, and more occasionally T lymphocytes and NK cells. However, the primary targets are B lymphocytes, in which binding and entry of EBV constitute distinct processes. The viral envelope glycoprotein gp350/220 mediates EBV binding by interacting with the EBV receptor CD21 (complement receptor type-2) (2), while entry results from EBV gp42 binding to the HLA class II coreceptor (3). A different process is responsible for EBV tropism for epithelial cells, which lack CD21, HLA class II receptor, and coreceptors (4). Although epithelial receptors remain unidentified, in this case EBV adapts the composition of its gp42-gH-gL envelope glycoproteins such as to reversibly switch its host cell (5).

However, the EBV host range also includes NK cells. These express HLA class II molecules but not CD21, so thus far, the process by which they bind EBV is unclear (6). It is well established that NK cells are strongly reactive to B-EBV infected cells. In addition, non-neoplastic EBV-infected NK cells are detected very early in IM (7). Hence, early steps of NK cytolytic attack of EBV-infected targets could contribute to NK cell infection. Like T (8) and B lymphocytes (9), NK cells transiently establish a highly dynamic immunological synapse with their targets (10). Our results, as well as others, recently showed that through this area, NK cells acquire membrane fragments from the conjugated cells on their own cell surface (11, 12, 13). All the effector cell types analyzed so far mediate a synaptic transfer of patches from the target cell membrane before—and independently of—the response (14). Here, we demonstrate that NK cell attack of B-EBV targets leads to trans-synaptic acquisition and transient expression of their EBV receptor CD21 in a functional state.

	Materials and Methods

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Cell cultures

Fresh NK cell populations were obtained by PBL depletion of non-NK cells using NK Cell Isolation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). Typically, the resulting NK populations comprised >85% of CD56⁺ cells and <0.5% CD3⁺ cells. IL-2 activated bulk NK cells were 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 µg/ml PHA (Murex Diagnostics, Dartford, U.K.). Three days later, 100 µl of culture medium was replaced in each well by 100 µl of fresh culture medium containing 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 11, these cultures contained >96% CD16⁺CD56⁺ cells and no detectable CD3⁺ cells. The MHC class I^- erythro-myelocytic tumor K562, the Burkitt’s lymphoma Raji, and Daudi cell lines were maintained in culture in RPMI 1640 medium supplemented with 10% FCS (Life Technologies, Gaithersburg, MD) supplemented with 100 U/ml penicillin, streptomycin (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). Unless otherwise stated, all chemicals were from Calbiochem (EMD Biosciences, San Diego, CA).

Analysis of CD21 acquisition

Target cells were distributed in U-bottom 96-well plates (5 x 10⁵ cells/100 µl wells) to which NK cells pulsed for 15 min with 0.5 µM Orange-(5-(and-6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine) (CMTMR; Molecular Probes, Eugene, OR) were added (10⁵ cells/well in additional 100 µl). Culture plates were centrifuged for 1 min at 1200 rpm to promote conjugate formation and were left for 1 h at 37°C in humidified 5% CO₂ atmosphere. For experiments involving Transwell culture plates (Costar, Cambridge, MA) NK cells (4 x 10⁵ cells/400 µl) and Daudi (2 x 10⁶ cells/400 µl wells) cells were left in separated or in similar compartments for 1 h at 37°C in humidified 5% CO₂ atmosphere. Conjugates were then dissociated by washing cells twice in PBS containing 0.5 mM EDTA. Cells were first labeled with mAb DR53 (undiluted culture supernatant) specific for CD21 (provided by G. Delsol, Unité 563 de l’Institut National de la Santé et de la Recherche Medicale, Toulouse, France), washed, and stained with FITC-conjugated goat anti-mouse total Ig (10 µg/ml, Argene Biosoft, Varilhes, France). Samples were run on a FACSCalibur (BD Biosciences, San Jose, CA) and data analyzed with WinMDI software.

EBV binding analysis

Conjugates with 50,000 effector cells and 5,000 target cells were prepared as specified above, dissociated, and NK cells were sorted using CMTMR. These cells were fixed for 10 min at room temperature with 0.1% paraformaldehyde, washed three times with PBS 5% FCS and incubated with EBV viral particles (Advanced Biotechnology, Columbia, MD) in 100 µl during 1 h at 4°C. Samples were washed three times and labeled with 10 µg/ml IgG1 mAb 2L10 specific for EBV gp350/250 (Advanced Biotechnology), or 10 µg/ml IgG1 isotype control (DAKO) washed and stained with FITC conjugated goat anti-mouse total Ig (1:100 dilution, Argene Biosoft). Samples were run on a FACSCalibur (BD Biosciences) and data was analyzed with WinMDI software.

Intracellular Ca²⁺ measurement

NKL cells were loaded with 1 µM 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 emission (_{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 sections 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 endogenous 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-peroxidase three-step reagent (DAKO Strept ABC complex/HRP Duet kit). Endogenous peroxidase was blocked with 1% hydrogen peroxide in methanol for 30 min. Slides were analyzed under mineral oil (Sigma-Aldrich) by microscopy (Nikon TE 200; Nikon, Melville, NY) using a x100 objective (Plan Apo; Nikon).

Confocal microscopy

B-EBV cells were stained with PKH67 as described (13), and bulk NK cells were pulsed for 15 min with 0.5 µM Orange-CMTMR (Molecular Probes). After washing, NK and B-EBV cells were mixed (1:1) in 50 µl RPMI plus 5% human serum in U-bottom 96-well plates and were laid onto polyL-lysine-coated slides with teflon wells (CEL-LINE; Erie Scientific, Portsmouth, NH) for 5 min at 37°C. The cells were fixed for 10 min at room temperature with 3% paraformaldehyde and mounted in 90% glycerol–PBS containing 2.5% 1–4-diazabicyclo (2.2.2) octane (DABCO, Fluka, Buchs, AG). The slides were examined with an LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany) using a x63 Plan-Apochromat objective (1.4 oil, Carl Zeiss). PKH 67 was excited with an argon laser ( 488 nm) and Orange-CMTMR fluorescence was excited with a helium laser ( 543 nm). Images were acquired and treated with the LSM 510 imaging software.

	Results

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NK cells conjugated to B-EBV targets and EBV-infected NK cells in biopsies of lymph nodes from IM patients

Immunostainings were performed on sections of formalin-fixed and paraffin-embedded lymph node samples from patients with primary IM. These were investigated for EBV and NK cell markers. NK (PEN-5⁺) cell membrane and cytoplasm were revealed by 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 cells, frequent uninfected NK cells conjugated to B-EBV targets (Fig. 1a), and also some EBV⁺ NK cells (Fig. 1b, arrow). These images of reactive tissues from early infected donors illustrated the in vivo NK cell-mediated attack of autologous B lymphocytes infected by EBV. Since they were in line with similar observations made from other tonsils biopsies of IM patients (7), this suggested that, despite the lack of known surface receptor for EBV, NK cells are infected early during primary EBV infection.

View larger version (70K): [in this window] [in a new window]   FIGURE 1. Conjugation of NK cells to EBV+ B lymphocytes in vivo and in vitro. a, Microscopy magnification of a lymph node biopsy from a patient with early IM double-stained for EBER probes (blue cell nuclei revealed by alkaline phosphatase–nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate substrate) and anti-PEN5 Ab (stained brown with peroxidase-diaminobenzidine substrate). All fields comprise numerous EBV-infected B cells (EBER+) and frequent NK (PEN-5+) cells bound to autologous B-EBV cells. b, Microscopy magnification of some rare cells from the same biopsies, double stained by the EBER probes (blue) and the PEN5 molecule (brown), revealing their NK-EBV+ phenotype. c, Confocal fluorescence microscopy of in vitro synaptic transfer (arrows) in a 1 h coculture comprising PKH67 (green)-labeled B-EBV cells and CMTMR (red)-stained NK cells.

The conjugation of NK cell to targets cell starts by the establishment 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 discrimination, 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 fluorochrome on red cells. Thus neither CMTMR nor PKH67 fluorochromes diffused out from labeled live lymphocytes in culture, as reported earlier (13, 17, 18). However, after 1 h of coculture confocal 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 infected 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 analyzed for CD21 phenotype by flow cytometry. Dot plots of CMTMR vs CD21 fluorescence allowed clear-cut gating of NK cells (Fig. 2a). The CD21 mean fluorescence intensity (mfi) of the whole NK cell population slightly but reproducibly increased after 1 h of coculture with targets at 37°C (Fig. 2b), but not at 4°C (data not shown). When this experiment was repeated in medium containing latrunculin B, a cytoskeleton inhibitor (15), the NK cell’s 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 activity (13). In contrast, the phorbol ester tetradecanoyl phorbol acetate (TPA) enhances NK cell’s lytic activity and synaptic transfer (13). We thus tested CD21 labeling of NK cells cocultured at 37°C with target cells in medium containing TPA. After 1 h, the increase of the CD21 marker on NK cells was even stronger when TPA was added to the coculture. Adding TPA to the NK cells without target cells, however, did not change their CD21 mfi (Fig. 2b).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. NK cell activation induces the CD21 phenotype upon coculture with CD21+ targets cells. CMTMR+ NK cells cocultured for 1 h at 37°C with unstained Daudi cells, were labeled for CD21. a, Flow cytometry analysis of the coculture discriminates NK (inset) from CD21+ target cells. b, CD21 phenotype of gated NK cells from coculture in the specified conditions (LuB: latrunculin B, PP2: PP2 Src kinase inhibitor), right numbers are the CD21-FITC mfi of at least 5000 gated NK cells.

NK cells acquire CD21 in coculture with CD21⁺ B cell targets

The above experiments could not exclude that CD21 expression was endogenous and up-regulated by NK cells upon exposure to target cells regardless of their CD21 phenotype. To formally rule out the induction of endogenous CD21 expression, NK cells were coincubated with a set of allogeneic cell lines which differed in terms of CD21 expression and susceptibility to lysis. Although the three HLA-I-deficient cell lines Daudi, C1R, and K562 activated the NK cells as demonstrated by their Ca²⁺ fluxes and specific lytic activity, a C1R cell line stably transfected with the protective HLA-B27 allele did not induce Ca²⁺ fluxes in cocultured NK cells (Fig. 3a) and was protected from their lysis (data not shown). In addition, while the B cell lines Daudi, C1R, and C1R-B27 expressed surface CD21, the mono-myelocytic K562 did not (Fig. 3b).

View larger version (33K): [in this window] [in a new window]   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 C1R-B27 B cells but not by the K562 cells (dotted line: isotype control, full line: CD21). c, CD21 acquired by NK cells after coincubation with the above-specified targets (dotted line: isotype control, full line: CD21 on NK cells at t0; red line: CD21 on NK cells after 1 h coculture with target cells).

From ten independent experiments measuring the CD21 acquired by NK cells, the shift of CD21 mfi was always weak but highly reproducible. By (mfi_CD21 of NK cells after transfer) - (mfi_CD21 of NK cells before transfer)/(mfi_CD21 of target cells before transfer), we calculated that within 1 h at 37°C, NK cells acquired on average up to 1% of the CD21 available on activating target cells.

Together, the above observations suggested that CD21 expressed on NK cells was captured from the cell surface of conjugated targets.

The NK cell CD21 phenotype requires contact with CD21⁺ targets and is short-lived

To confirm that CD21 expressed by NK cells originated from the CD21⁺ targets, we measured CD21 acquisition in transwell culture plates. As compared with CD21 mfi acquired by NK cells in 1 h cocultures with CD21⁺ Daudi targets, the NK cell mfi in transwell cocultures remained similar to that of NK cells alone in culture (Fig. 4a). So, the CD21 phenotype by NK cells requires direct contact with CD21⁺ targets.

View larger version (12K): [in this window] [in a new window]   FIGURE 4. CD21 expression by NK cells requires direct contact with CD21+ cell targets and is short-lived. a, CD21 phenotype of NK cells in the specified culture conditions, either alone (top), or mixed with CD21+ Daudi targets in coculture using normal (middle) or Transwell (bottom) 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 value reached in the coculture. (None: no target cell). The disappearance is measured using normalized CD21 mfi of NK cells after increasing times of incubation alone.

So, the NK cells rapidly acquire a small amount of CD21 from their targets and transiently maintain this molecule on their own surface while exposed to novel CD21⁺ targets.

Ectopic CD21 enables EBV binding to the NK cell surface

CD21 is a transmembrane Ig-like monomer with EBV-binding domain located on its extracellular amino-terminal repeat domain (16). Recognition by mAb of CD21 molecules expressed by targets and acquired by NK cells indicated that the CD21 orientation has not changed during transfer. We therefore hypothesized that the newly acquired receptor was functional on NK cells. Although the molecular steps responsible for EBV entry in NK cells are unknown and may differ from those required for entry into epithelial and B cells, we nevertheless tested the functionality of ectopic CD21 by measuring the EBV binding to NK cells. NK cells coincubated for one hour with Daudi cells as above, dissociated and sorted to 99% purity, were then exposed to EBV particles (marmoset-derived B95-8 strain) at various ratio, before extensive wash and stain for bound virus. EBV binding was measured by flow cytometry of the NK cells stained with anti-viral gp350 mAb and with isotype-matched control. In the same experiments, we also controlled the CD21 acquisition by NK cells. The comparison of gp350 mAb mfi vs control isotype mfi demonstrated that before EBV addition, the NK cell surface did not contain any virus. Accordingly, the EBV did not bind to NK cells without preliminary exposure to targets. After NK cells exposure to CD21⁺ targets, however, the results differed. Little -if any- EBV binding was repeatedly observed on NK cells exposed to low EBV doses. In presence of higher viral concentrations, however, EBV did bind to NK cells previously exposed to CD21⁺ targets (Fig. 5a). The low EBV binding to NK cells only resulted from the low numbers of CD21 receptors on the NK cell surface, because the same EBV/CD21 ratio (mfi _gp350/mfi _CD21 = 0.7 for all doses tested) was found for Daudi and NK cells. In contrast, when such experiments involved CD21^- targets instead of Daudi, EBV did not bind to NK cells (Fig. 5b).

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Coculture with CD21+ target cells enables EBV binding to NK cells. a, fluorescence histograms and mfi for EBV gp350 (right) vs isotype control (left) of NK cells treated as specified (numbers above: mfi from 5000 sorted NK cells). b, Result from a similar experiment but with prior NK cell coculture with CD21- or CD21+ target cells.

	Discussion

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Several recent studies have demonstrated that B cells (9), CD8 T cells (17), T cells (18), and NK cells (11, 12, 13) capture and internalize from their target cells a small amount of surface molecules across the immunological synapse. Recent electron microscopy of CD8 T cells conjugated to target cells evidenced membrane fusions at the synapse which physically bridge both cell types (19). Thus, lateral diffusion along the fused membrane could provide some clues to explain the synaptic transfer of surface markers molecules without changing their orientation. Several issues remain enigmatic. How membrane fusion occurs, how it is disrupted during conjugate dissociation, and why this transfer is polarized from target to effector cell and is restricted to membrane molecules remain unclear.

Although the physiological function of synaptic transfer in immunity could promote either affinity maturation (20, 21) or extinction of response (for review see Ref.14), we suggest here it could also be subverted by pathogens. Under defined circumstances, synaptic 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 immunological synapse. Confocal pictures of large patches from target 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 effectors comprise particulate Ags (20), MHC-peptide complexes (22), mIgM (18), MHC class I (11), and CD4 (our unpublished observations). 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 interrupted synapses, it might last longer in vivo, where NK cells serially 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 several 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 functional 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 apply in other viral diseases. Among these, our hypothesis could prove useful to account for the recently discovered NK cell reservoir for HIV in AIDS patients (24, 25). We believe that validation of this novel model deserves future investigation.

Note.

While submitting this manuscript, the trans-synaptic host cell-to-effector T lymphocyte transmission of HTLV-I was reported (26).

	Acknowledgments

 
We thank the expert technical assistance of Fatima L’Faqihi, useful comments from E. Espinosa, and sustained encouragements from E. Vivier and G. Delsol.

	Footnotes

 
¹ This work was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale and l’Association pour la Recherche sur le Cancer (Grant 5665 to J.-J.F.).

² Address correspondence and reprint requests to Dr. Jean-Jacques Fournie, Département d’Oncogènèse and Signalisation dans les Cellules Hématopoiétiques, Centre de Physiopathologie de Toulouse Purpan, Unité 563, Institut National de la Santé et de la Recherche Médicale, BP3028 Centre Hospitalier Universitaire Purpan, 31024 Toulouse Cedex, France. E-mail address: fournie{at}toulouse.inserm.fr

³ Abbreviations used in this paper: IM, infectious mononucleosis; EBER, EBV-encoded RNA; NK, human natural killer; mfi, mean fluorescence intensity; CMTMR, (5-(and-6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine); TPA, tetradecanoyl phorbol acetate.

Received for publication December 30, 2002. Accepted for publication April 14, 2003.

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