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;
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.
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