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*
Laboratory of Immunology, National Institute for Cancer Research, Genoa, Italy;
Laboratory of Tumor Immunology and
Department of Pathology, Scientific Institute San Raffaele, Milan, Italy;
Laboratory of Clinical Immunology, Division of Infectious Diseases, Centro San Luigi, Milan, Italy; and
¶ Unit of Protein Biology, National Institute for Cancer Research, Genoa, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion. CAMKII is induced via LFA-1: indeed,
oligomerization of LFA-1 leads to CAMKII induction in NK cells.
Moreover, release of lytic enzymes and cytotoxic activity is strongly
reduced by masking LFA-1 or by adding CAMKII inhibitors such as KN62
and KN93, at variance with the inactive compound KN92. NK cell-mediated
lysis of DC and IFN- release by NK cells upon NK/DC contact are
inhibited by exogenous HIV-1 Tat: the protein blocks calcium influx and
impairs CAMKII activation elicited via LFA-1 in NK cells, eventually
inhibiting degranulation. Experiments performed with synthetic,
overlapping Tat-derived peptides showed that the C-terminal domain of
the protein is responsible for inhibition. Finally, both KN62 and Tat
reduced the extension of NK/DC contacts, possibly affecting NK cell
granule polarization toward the target. These data provide evidence
that exogenous Tat inhibits NK cell activation occurring upon contact
with DC: this mechanism might contribute to the impairment of natural
immunity in HIV-1 infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interaction between NK cells and DC is likely to play a complex role in
the regulation of innate immunity. Indeed, contact with DC activates NK
cell cytolytic activity and IFN- secretion, thus amplifying one of
the mechanisms of primary host defense against pathogens
(13). In contrast, it has been recently proposed that DC
can be killed efficiently by autologous NK cells (14, 15, 16);
this process may contribute to the feedback regulation of immune
responses (17, 18). In addition, NK/DC interaction is
supposed to be relevant in the control of antiviral immunity, including
in AIDS, in which DC are a potential virus reservoir (5, 19, 20); finally, production of IFN- by NK cells has been
proposed to participate in the control of viral replication and in the
amplification of the immune response (1, 6, 21).
In this study, we demonstrate that NK cell killing of autologous DC is
mediated by the LFA-1-dependent activation of the calcium-calmodulin
kinase II (CAMKII), resulting in degranulation and extracellular
release of perforin and granzymes. Likewise, IFN- secretion
occurring upon NK/DC contact or LFA-1 engagement is inhibited by
blocking CAMKII.
Interestingly, in the presence of exogenous HIV-1 Tat, several events
that follow NK/DC interaction are affected: the extent of cell to cell
contacts is reduced; calcium entry and degranulation, elicited in NK
cells by binding to DC, are inhibited. Finally, activation of CAMKII
and IFN- secretion are blocked: these suppressive effects eventually
lead to an impairment in the process of DC-mediated activation of NK
cell functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified CD3-CD16+ NK cells were obtained from PBL after immunodepletion of monocytes and T lymphocytes, as described (9), stimulated with 10 µg/ml PHA (Sigma, St. Louis, MO), and cultured in RPMI 1640 medium supplemented with 10% FCS, 1 mM glutamine, 100 U/ml penicillin-streptomycin (all from Biochrom, Berlin, Germany), and 100 U/ml rIL-2 (Cetus, Emeryville, CA). Peripheral blood monocytes were isolated and cultured in the presence of 40 ng/ml GM-CSF plus 100 U/ml IL-4 for 10 days to obtain monocyte-derived DC (12).
Cytolytic assay
Cytolytic activity of activated NK cells against autologous DC,
or against the K562 or 721.221 cell lines, was tested in a 4-h
51Cr release assay, as described
(9), at different E:T ratios, from 40:1 to 1:1. In some
experiments, NK cells were pretreated 15 min with 100 or 10 nM Tat
alone or 100 nM Tat plus anti-Tat polyclonal antiserum (1/200
dilution), both from Tecnogen (Piana di Monteverna, Caserta,
Italy), or with normal rabbit Ig (Sigma; 1/200 dilution). In other
samples, the following peptides were used, at 100 to 1 nM
concentration: Tat46–60,
Tat65–80 RGD containing,
Tat20–39, Tat24–52, or
Tat56–70 (Technogen, Piana di Monteverna,
Caserta, Italy) (22, 23). In other experiments, NK
cells were pretreated with the CAMKII inhibitors:
1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine
(KN62) or KN93 or the inactive compound
2-[N-(4-methoxybenzenesulfonyl)]
amino-N-(4-chlorocinnamyl)-N-methylbenzylamine,
phosphate KN92 (Calbiochem-Inalco S.p.A., Milan, Italy; 100 nM), or
with a blocking anti-Fas mAb (ZB4, IgG; MBL, Naka-ku Nagoya,
Japan), or with the anti-perforin mAb 
G9 (PharMingen, San Diego,
CA), both at the concentration of 5 µg/ml. Results are expressed as
percentage of cytotoxicity, calculated as described
(9).
Measurement of perforin and granzyme release
Granzyme release was measured in the supernatants or in cell
lysates of NK cells, according to Shiver et al. (24), as
previously reported (9). Briefly, supernatants or cell
lysates (0.5% Triton X-100) obtained from 2-h cocultures of
106 NK cells plus 105 DC,
with or without 100 nM Tat or 100 nM Tat peptides, were added to 1 mM
dithionitrobenzoic acid (Sigma) and 1 mM
N
-benzyloxycarbonyl-L-lysine
thiobenzylester (Calbiochem), and the reaction run at room temperature
for 30 min. In some experiments, NK cells were treated with the CAMKII
inhibitors KN62 or KN93 (50 nM), or with the inactive analogue KN92 (50
nM). The OD were measured at 414 nm, and results were expressed as
percentage of esterase release calculated as follows:
(OD414 SN/OD414 CL +
OD414 SN) x 100. Perforin release by NK
cells was evaluated by flow cytometry after NK cell/DC interaction as
reduction of perforin-positive NK cells. Cytoplasmic staining was
performed by indirect immunofluorescence, as described
(9). Briefly, aliquots of 105 cells
were fixed with 1% paraformaldehyde and permeabilized with 0.05%
Triton X-100, stained with the anti-perforin mAb G9, followed by
PE-conjugated antiisotype-specific goat anti-mouse serum (GAM;
Southern Biotechnology Associates, Birmingham, AL). NK or DC were
identified using the forward scatter and side scatter
parameters. Samples were analyzed on a flow cytometer (FACSort; Becton
Dickinson, Mountain View, CA) exciting PE at 488 nm, after calibration
with CALIBRITE particles (Becton Dickinson), and gating to exclude
debris. Data were analyzed using the Lysis II computer program
(version 1.1).
Single-cell analysis of calcium fluxes by video microscopy and ratio imaging
Single-cell analysis of calcium fluxes was performed as described (9, 12). Briefly, DC cultured on round coverslips were loaded with 1 µM fura 2-AM (1 h at 37°C), placed in a microincubator (Medical System, Greenvale, NY) on an inverted epi-fluorescence Axiovert 10 microscope (Zeiss, Oberkochen, Germany), and maintained at 37°C by a temperature controller (TC-202; Medical System). NK cells, loaded with 1 µM fura 2-AM, were added to DC at the NK:DC ratio of 4:1 and allowed to bind to DC. DC were stained to allow their identification and follow the interaction with NK cells during the test. The instrument, connected with an intensified charged device-coupled camera (ATTO Instruments, Rockville, MD) and controlled by a Pentium 90 MHz computer, was gated on NK cells, and calcium fluxes were monitored for 30 min. Results were stored as ratio of fura 2 fluorescence at 334 nm divided by the fluorescence at 380 nm excitation. [Ca2+]i was calculated according to Grynkiewicz et al. (25) after calibration of the instrument with fura 2 pentapotassium salt and analysis with AttoView computer program. The [Ca2+]i increases were measured upon NK cell interaction with DC, before or after treatment with 10 µM phenylalkylamine verapamil (VPM; Calbiochem-Inalco), a blocker of L-type calcium channels (26), or 100 nM Tat or Tat peptides, as above.
Measurement of CAMKII activity
CAMKII activity in NK cells was tested, upon NK/DC coculture or
oligomerization of LFA-1 obtained with the specific mAb 70H12 (IgG2a, 5
µg/ml), followed by GAM, as described (9), in the
absence or presence of 100 to 10 nM Tat or 100 to 1 nM Tat peptides.
Preliminary kinetics experiments showed that the optimal time for
CAMKII activation was 12 min in NK/DC coculture and 4 min in LFA-1
oligomerization (Table I
). CAMKII was
measured with the CAMKII assay kit, using the specific substate and
[-32P]ATP, after immunoprecipitation
with the specific anti-CAMKII Ab (Upstate Biotechnology, Lake
Placid, NY) and chromatography. Results are expressed as cpm x
10-3 and are the mean ± SD from triplicate
samples.
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Effector/Target cell interaction was evaluated by transmission electron microscopy as well; to this purpose, cells were fixed in 2% glutaraldehyde for 20 min at 4°C and washed three times in phosphate buffer. Postfixation was performed in 1% osmium tetroxide. Samples were then dehydrated in ethyl alcool and propylene oxide and embedded in epon-araldite resin. Thin sections (80 nm) were then obtained, stained with uranyl acetate and lead citrate, and analyzed under a Zeiss CEM 902 electron microscope.
IFN- production
Cell-free supernatants were collected 24 h after NK/DC
contact (4:1 ratio) or oligomerization of LFA-1 obtained with the
specific mAb, or with GAM alone, as described above. In some
experiments, NK cells were pretreated with the
F(ab')2 of the 70H12 mAb (9) or of
the isotype-matched TA181H12 mAb (12), both at 5 µg/ml,
CAMKII inhibitor KN62 (50 nM), or with HIV-1 Tat (50 nM). IFN-
concentrations in culture supernatants were measured following the
manufacturer’s recommended procedure using the human IFN- ELISA kit
(R&D Systems, Minneapolis, MN), and results were expressed as
nanograms/106 NK cells/milliliter. The limit of
detection was 1 ng/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now accepted that DC activate NK cells, which, in turn, can
lyse autologous DC (13, 14, 15, 16). However, the signal
transduction pathway(s) underlying NK cell activation in this system is
still poorly defined. To investigate this point, we first studied the
mechanism of lethal hit delivery by NK cells. Fig. 1A shows that an
anti-perforin mAb blocked the cytotoxicity of DC by autologous NK
cells, at variance with an anti-Fas mAb; this indicates that, in
this system, lytic enzymes released upon calcium-dependent exocytosis
of NK cell granules are the prevalent effectors of target cell damage,
in agreement with previous observations that DC lysis by NK cells is
inhibited by calcium chelators (14, 16). Granule
exocytosis in epithelial cells is regulated by calcium-dependent
activation of CAMKII (27, 28); thus, we asked whether this
kinase was also involved in regulating degranulation in NK cells, As
shown in Fig. 1B, NK cell treatment with the CAMKII
inhibitors KN62 or KN93, but not with the inactive KN92, leads to the
inhibition of perforin depletion, which occurs upon binding to DC. In
keeping with this, the CAMKII inhibitors KN62 or KN93 impaired DC
killing (Fig. 1C); this inhibition was dose dependent
(D), and the specificity of CAMKII involvement was confirmed
by the finding that the inactive compound KN92 had no effect on NK cell
cytolytic activity (Fig. 1, C and D). However,
IL-2-cultured NK cells efficiently lysed HLA-I-negative target cells,
represented by the lymphoblastoid 721.221 and the erythroleukemia K562
cell lines (Fig. 1, E and F), and this lysis was
not affected by CAMKII inhibitors. Interestingly, the anti-LFA-1
mAb also was ineffective in blocking the killing of K562 (Fig. 1F) or 721.221 cells (Fig. 1E).
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CAMKII is activated by extracellular calcium entry that induces
calmodulin conformational changes, allowing its interaction with the
kinase (28). Because calcium fluxes can be elicited via
LFA-1, the 
2 integrin that mainly mediates NK
cell binding to target cells (2, 7, 9), we investigated
the role of LFA-1 triggering in CAMKII induction. Fig. 2 shows that interaction with DC elicits
in NK cells calcium fluxes (Fig. 2A) and CAMKII activation,
which is blocked by KN62 and by masking LFA-1, with the
F(ab')2 of the specific mAb, thus inhibiting cell
to cell contact (Fig. 2B). This suggests that CAMKII is
induced through the engagement of LFA-1 integrin in NK cells during
interaction with the target: indeed, CAMKII activation was also
achieved by oligomerization of LFA-1 obtained with the specific mAb
70H12 (Fig. 2, C and D, hatched columns).
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C),
and this effect was neutralized by an anti-Tat antiserum (not
shown). To determine which domain of HIV-1 Tat protein was involved in
this inhibition, different Tat-derived synthetic peptides (22, 23) were used in LFA-1-dependent CAMKII activation assay. The
C-terminal Tat peptide, spanning the 65–80 aa, exerted an inhibitory
activity comparable with that of the whole Tat protein (Fig. 2C), which was still evident at 10 nM concentration (Fig. 2D). Conversely, the Tat46–60
peptide, containing the basic domain of the protein, or the
Tat20–39 or Tat24–51
peptides, containing the cysteine-rich and the core domain of Tat
(22), did not exert any effect (Fig. 2C).
Finally, a slight inhibition was observed using the
Tat56–70 peptide, partially overlapped to
Tat65–80 (Fig. 2, C and
D).
Contact with DC elicits CAMKII-dependent IFN- secretion by NK
cells, which is inhibited by Tat
Another important antiviral tool is represented by IFN-, which
is believed to be a mediator of NK cell cytotoxicity (21).
IFN- can be produced and released by NK cells upon interaction with
DC (13); therefore, we analyzed the effect of CAMKII
inhibitors and of HIV-1 Tat on IFN- secretion upon NK/DC contact. As
shown in Table II, coculture of NK cells
and DC, as well as oligomerization of LFA-1, induced the secretion of
IFN-, which was detectable in the culture supernatant by ELISA;
interestingly, pretreatment of NK cells with KN62 or KN93, but not with
KN92, inhibited the release of IFN-, raising the possibility that
the calcium-dependent activation of CAMKII controls more than one
effector mechanism in NK cells. Interestingly, a comparable inhibition
was observed when NK cells were exposed to synthetic Tat (Table II).
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Fig. 3 shows that NK/DC interaction
elicited an increase in the
[Ca2+]i of NK cells
starting from 5 min after their binding to DC; several waves of calcium
fluxes were observed during the following 20–30 min, similarly to what
was reported in NK/tumor cell interaction (29). This
intracellular calcium rise was mainly due to extracellular calcium
influx, as it was strongly reduced by the addition of the L-type
calcium channel blocker VPM (Fig. 3A). Interestingly, a
similar inhibitory effect was observed using synthetic Tat (Fig. 3B) and its C-terminal peptide
Tat65–80 (Fig. 3C), at variance with
the basic Tat46–60 peptide (Fig. 3D)
or the other Tat-derived peptides (not shown).
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A)
was maintained in the presence of Tat (Fig. 4C). However,
the extent and complexity of cell to cell contacts were strongly
reduced upon exposure of NK cells to Tat (Fig. 4, C vs
A). More importantly, in untreated NK cells, most granules
were detectable along the membrane portion in contact with DC (Fig. 4, A and B), while in Tat-treated NK cells, granule
were distributed throughout the whole cytoplasm (Fig. 4C).
Interestingly, a similar effect was observed using the CAMKII inhibitor
KN62 (Fig. 4D). The analysis of 100 cell to cell contacts
for each sample showed that >80% of untreated NK cells had filopodia
or irregular cytoplasmic processes that formed many interdigitations
with the cytoplasmic protrusions of DC; moreover, electron-dense
granules localized in the cytoplasm toward DC in >70% of NK cells. In
Tat-treated NK cells, the number of such interdigitations and the
length of filopodia were reduced by 50%, and granules were always
spread in the cytoplasm. Altogether, these data suggest that calcium
waves occurring upon NK/DC binding are needed for CAMKII activation,
which, in turn, contributes to the stabilization of cell to cell
contacts, granule polarization, and the delivery of the lethal hit:
this mechanism is impaired by exogenous Tat.
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Synthetic Tat or its C-terminal peptide
Tat65–80, at variance with the Tat-basic
(Tat46–60) peptide, inhibited the release of
granzymes from NK cells (Fig. 5A), as well as the
intracellular perforin depletion (Fig. 5B), elicited by
binding to DC, and they were effective up to 10 nM concentration (Fig. 5, A and B). These findings indicate that Tat
inhibits the degranulation and enzyme release occurring upon NK cell/DC
interaction and needed for NK cytolytic activity. Indeed, synthetic Tat
significantly reduced the lysis of DC by NK cells, and this inhibition
could be prevented by a neutralizing anti-Tat antiserum, at
variance with normal rabbit Ig (Fig. 6A). Again, the C-terminal
Tat65–80 peptide exerted an inhibitory activity
on the lysis of autologous DC by NK cells (Fig. 6B)
superimposable to that of the whole Tat protein (Fig. 6A),
and this inhibition was detectable from 100 to 10 nM (Fig. 6C). Conversely, little or no inhibition of cytotoxicity was
observed when the Tat46–60,
Tat20–39, or Tat24–51
peptides were used (Fig. 6B). Finally,
Tat56–70 peptide, partially overlapped to
Tat65–80, slightly reduced (by 10%) NK-mediated
DC killing at the highest concentration (100 nM; Fig. 6, B
and C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by NK cells, following DC contact, also is CAMKII
dependent. All these events are inhibited by exogenous HIV-1 Tat by
blocking calcium entry and CAMKII activation.
It is well known that variations in
[Ca2+]i regulate many
secretory events, such as neurotransmitter release and exocytosis of
secretory granules (30); furthermore, increasing evidence
indicates that Ca2+-regulated exocytosis is not
restricted to specialized secretory cells, but is present in many cell
types, including hemopoietic cells (31). In immune cells,
this regulated secretion provides a tight control over the delivery of
highly bioactive effector proteins, thus representing a major mechanism
of control of immune responses (32). However, the
biochemical events that follow receptor signaling leading to exocytosis
are still unclear. In epithelial tissues, granule secretion is induced
by the activation of CAMKII (27, 28); in this study, we
show that also in NK cells, degranulation needs the induction of this
kinase, which is triggered by calcium mobilization via LFA-1 upon
effector/target cell adhesion. Along this line, we found that release
of IFN- by DC-activated NK cells is inhibited by blockers of CAMKII.
Thus, the activation of CAMKII occurs in different cell types, possibly
representing a common step in the control of exocytosis. However, the
lysis of the lymphoblastoid 721.221 and the erythroleukemia K562 cell
lines by IL-2-cultured NK cells was apparently independent of CAMKII
activation as well as of LFA-1 engagement. Because CAMKII induction can
be achieved in NK cells via LFA-1, it is possible that this enzyme is
not triggered when LFA-1 is not the main surface structure involved in
NK cell activation.
Our data show that the lysis of DC by NK cells is due to perforin and granzymes release: this mechanism of killing has been reported as an essential step in the control of primary virus infections, including HIV-1, by CTLs (33, 34, 35). In keeping with these reports, Fas/Fas ligand interaction is not involved in the NK-mediated lysis of DC. Of note, we found that Tat acts on natural killing by inhibiting the release of both perforin and granzymes, which occurs upon NK/DC binding. It has been proposed that the lysis of DC by autologous NK cells is involved in the regulation of immune responses (14, 15, 16). This mechanism could play a role also in viral diseases, in which the killing of autologous DC can help the clearance of infected cells (5, 6); conversely, a decreased NK cell function would allow the survival of infected DC. Support to this hypothesis comes from the finding that NK cells can be isolated from influenza virus-infected lungs before the detection of virus-specific T lymphocytes in mice (36). Recently, hemagglutinins of influenza or parainfluenza virus have been identified as ligands for the recently described natural cytotoxicity receptor (NCR) NKp46 and reported to activate NK cells (37), thus confirming the antiviral potential of this lymphocyte population. This may be of relevance also in the case of HIV-1 infection, in which follicular DC represent a reservoir of the virus (19, 20).
We have reported that during interaction between NK cells and DC, several activating and inhibiting molecules, expressed on either cell types, can be engaged and determine the final functional result. In particular, ligation of NKp30 and NKp46 on those NK cells that express these receptors leads to autologous APC lysis through the activation of the phosphatidylinositol 3 kinase (38). Interestingly, the lysis of HLA-I-negative targets seems to depend on the engagement of NCR: indeed, activated NK cells brightly expressing NKp30 and NKp46 can lyse the 721.221 cell line, at variance with NK cells with a dull expression of NCR (39). In contrast, we found that this lysis is not dependent on CAMKII and on LFA-1 (different from NK cell-mediated lysis of DC). Thus, two enzymatic pathways can be responsible for the release of perforin and granzymes, possibly due to the preferential engagement of NCR or LFA-1 or other adhesion receptors.
Moreover, the interaction of NK cells with DC contributes to the
amplification of innate immunity, before the onset of acquired immune
responses: in particular, upon contact with DC, NK cells become
cytotoxic, secrete lytic enzymes, and produce IFN-, one of the
effectors of NK cell function (1, 2, 13, 21).
Interestingly, we found that all these events are activated by CAMKII,
triggered via LFA-1 upon NK/DC contact, and HIV-1 Tat can impair this
key biochemical mechanism by interfering with calcium entry. Indeed,
the effect of Tat on NK cell-mediated killing of DC is not due to the
block of effector/target cell binding, as the number of NK cells
adhering to DC is not decreased upon exposure to Tat. However, the
extent of membrane engaged in cell to cell contacts is deeply reduced,
and granules are spread in the cytoplasm, instead of being localized at
the cell to cell contact, upon treatment of NK cells with Tat. As NK/DC
binding induces a Ca2+ influx that is blocked by
Tat or VPM, it is conceivable that calcium entry is needed to
strengthen the interactions between NK cells and DC, and drive CAMKII
activation. In the whole, our observations indicate that Tat interferes
with the earliest signaling events that follow NK/DC by blocking one
important biochemical mechanism that leads to NK cell activation. Thus,
early release of Tat by HIV-1-infected neighboring cells might hamper
the triggering of the effectors of innate immunity and delay the
amplification of an efficient antiviral response.
In conclusion, we have described one of the biochemical mechanisms underlying the activation of NK cells; as DC are the professional APCs, this mechanism might also contribute to bridge innate with acquired immunity. Finally, our present data add new evidence for a role of extracellular Tat as a viral tool to escape a first-line immune response.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Maria Raffaella Zocchi, Laboratory of Tumor Immunology, Scientific Institute San Raffaele Via Olgettina 60, I-20132 Milan, Italy. E-mail address: zocchi.maria{at}hsr.it
3 Abbreviations used in this paper: [Ca2+]i, intracellular free calcium concentration; CAMKII, calcium-calmodulin kinase II; DC, dendritic cell; GAM, goat anti-mouse; NCR, natural cytotoxicity receptor; VPM, verapamil.
Received for publication July 30, 2001. Accepted for publication October 22, 2001.
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References |
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M. Jinushi, T. Takehara, T. Kanto, T. Tatsumi, V. Groh, T. Spies, T. Miyagi, T. Suzuki, Y. Sasaki, and N. Hayashi Critical Role of MHC Class I-Related Chain A and B Expression on IFN-{alpha}-Stimulated Dendritic Cells in NK Cell Activation: Impairment in Chronic Hepatitis C Virus Infection J. Immunol., February 1, 2003; 170(3): 1249 - 1256. [Abstract] [Full Text] [PDF]
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) or the anti-perforin
mAb (
), at the concentration of 5 µg/ml, were added at the
onset of the cytotoxic assay. C, Lysis of DC was tested
in the absence or presence of 100 nM KN62, KN92, or KN93, or in the
presence of saturating amounts of the anti-LFA-1 mAb 70H12 (5
µg/ml). D, NK cell-mediated lysis of autologous DC at
20:1 in the presence of 100, 10, 1 nM KN62 or KN93. Nil: lysis in the
absence of drugs. E and F, Cytolytic
activity against 721.221 (E) or K562 (F)
target cells was assayed without (nil) or with 100 nM KN62 or KN92 or
KN93 or in the presence of saturating amounts of the anti-LFA-1
mAb. B, NK cells were coincubated 2 h with DC at
the E:T ratio of 10:1, in the absence or presence of 50 nM KN62, KN93,
or KN92, as indicated. Cells were then permeabilized and stained with
the anti-perforin mAb (
) or pretreated 15 min with 100 nM Tat (
) is also shown.
B and C, Effect of the different Tat
peptides (at 100 nM concentration in B, from 100 to 1 nM
in D) on NK cell-mediated DC lysis at the E:T ratio of
20:1. Results are expressed as percentage of 51Cr-specific
release and are the mean ± SD from four independent experiments
for each panel.