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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 196-206
Interleukin-12-Activated Natural Killer Cells Recognize B7
Costimulatory Molecules on Tumor Cells and Autologous Dendritic Cells
By
Anja B. Geldhof,
Muriel Moser,
Laurence Lespagnard,
Kris Thielemans, and
Patrick De Baetselier
From the Laboratory of Cellular Immunology, Flanders Interuniversity
Institute for Biotechnology, Vrije Universiteit Brussel (VUB); the
Laboratory of Animal Physiology, Université Libre de Bruxelles
(ULB), Sint-Genesius Rode; and the Laboratory of Physiology, Medical
School, VUB, Jette, Belgium.
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ABSTRACT |
Activation of natural killer (NK) cells in the presence of
interleukin-12 (IL-12) augments the capacity of these effector cells to
recognize B7-1- and B7-2-expressing target cells. These effector
cells also efficiently lyse autologous B7-positive progenitor or
organ-derived dendritic cells, suggesting a physiologic regulatory
pathway between IL-12, NK cells, and B7-expressing antigen-presenting
cells. Although IL-12-activated NK cells secreted higher levels of
interferon- , this cytokine did not play a role in
synergistic effects of IL-12 and B7 on NK activation. The
B7-counterreceptor was found to be selectively upregulated on
IL-2/IL-12 as compared with IL-2-activated NK cells. CD28 is
functionally involved in the recognition of B7 on target cells since
IL-2/IL-12-activated NK cells derived from CD28 knockout mice were
strongly reduced in their capacity to lyse syngeneic B7-positive tumor
cells as well as antigen-presenting cells. However, recognition of B7
on allogeneic targets did not require the expression of CD28 on the
IL-2/IL-12-activated NK cells. Hence, IL-12 triggers the expression of
both CD28-dependent and CD28-independent mechanisms that allow NK cells
to eliminate B7-positive target cells including autologous dendritic
cells.
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INTRODUCTION |
NATURAL KILLER (NK) cells constitute
phenotypically and functionally a diverse population of large granular
lymphocytes in peripheral blood and spleen. They are spontaneously
cytotoxic against a variety of target cells, including certain tumor
cell lines and virus-infected cells as well as allogeneic bone marrow
(BM) and lymphoid cells. Although this activity was initially
considered to be nonspecific and non-major histocompatability complex
(MHC) restricted, NK cells are capable to discriminate specifically
between their targets.1 Although NK cell specificity
involves both activating and inhibitory receptors, in general a target
inhibition type of activation mechanism determines this
specificity.2-5 Indeed, we and others have shown that NK
target engagement could be inversely correlated to the expression of
specific MHC class I molecules on the targets.6-12 These
observations have been strengthened by the identification of the MHC
class I-specific Ly49 C-type lectin family of inhibitory receptors on
mouse NK13-16 and KIRs (killer cell inhibitory receptors)
on human NK cells.17-19 Furthermore, recent evidence
suggests that the NK-activating stimuli, delivered by target cells,
could be overruled by class I-mediated inhibition.20
In contrast to the inhibitory pathway of NK regulation, less is
known about the triggering and activation events. In fact,
the target inhibition model could not account for the
observations that some virus-infected, transformed, and even autologous
cells were NK-sensitive notwithstanding a normal expression of class I
molecules.21-23 Hence, appropriate activation signals may
overrule the class I-mediated inhibition and this notion was
strengthened by the observation that expression of B7-1 costimulatory
molecules on NK-resistant tumor cell variants rendered these target
cells NK-sensitive in vivo and in vitro.24-27
NK cell stimulatory factor (NKSF) or interleukin-12 (IL-12) is a
heterodimeric cytokine produced by macrophages, B cells, and other
accessory cell types, which exerts immunomodulatory effects on T and NK
cells.28 In particular, IL-12 has been shown to (1)
synergize with low doses of IL-2 in generation of LAK
activity,29 (2) enhance the cytotoxic activity of NK
cells,30,31 (3) induce the secretion of IFN- by either T
or NK cells,28 (4) mediate direct mitogenic effects on
activated T and NK cells,32 (5) induce the secretion of
tumor necrosis factor (TNF)- and - by purified NK
cells,30 and (6) augment the expression of IL-2R and
TNFR30,31 as well as cell adhesion molecules on human T and
NK cells32 and to negatively regulate the proliferation of
IL-2-stimulated NK cells.31 Interestingly, Kubin et
al33 showed that IL-12 synergized with B7 costimulation to
increase the proliferation of and cytokine release by T cells in vitro.
These observations were confirmed by demonstrating that the antitumor
effect of B7 transfection into a tumor-cell vaccine could be further
amplified by administration of IL-12 at the vaccine site34
or by a combination of B7-1- and IL-12-transfected tumor
cells.35
In these different investigations all the reported effects were
ascribed solely to the interaction between CD28 on T cells and B7-1 on
the target cells.34,35 In view of the involvement of both
B7 and IL-12 in NK cell activation, it was of interest to investigate
whether IL-12 could also play a role in the interaction between B7 (on
target cells) and NK cells. To this end the activity of LAK cells,
generated in the presence of IL-2 with or without IL-12, toward diverse
targets expressing B7-1 and/or B7-2 was compared. Furthermore,
these two different NK effector populations were used to delineate the
NK receptors that may be involved in the target (B7)/NK interaction.
Finally, the differential activated NK cells were tested for their
capacity to recognize B7 on syngeneic normal cells such as dendritic
cells (DCs) aiming at further clarifying the role of NK cells in the
elimination of autologous antigen presenting cells.23,27
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MATERIALS AND METHODS |
Mice.
Specific pathogen-free female AKR (Thy 1.1, H-2k), C57BL/6
(Thy 1.2, H-2b), B10.BR (Thy 1.2, H-2k), and
CB-17 SCID/SCID (H-2d) mice were obtained from Harlan CPB
(Zeist, The Netherlands). CD28 /
knockout mice were kindly provided by C. Thompson (Gwen Knapp Center,
University of Chicago, Chicago, IL) to M.M. and approved to be used in
the described experiments.36
Tumor cell lines.
The highly malignant BW-Li (H-2K+, H-2D++) is a
spontaneous metastasizing variant derived from the original,
nonmetastatic BW5147 T-cell lymphoma (AKR origin; Salk Institute, La
Jolla, CA). The BW-LiDhigh(H-2K,
H-2D+++) variant was obtained from BW-Li by successive
fluorescence-activated cell sorter (FACS) sortings for high H-2D and
low H-2K expression. Transfection of BW-Li and
BW-LiDhigh with pcDNAneo1::MoB7-124 or
pcDNA3::MoB7-2 (obtained by cloning mouse B7-2 cDNA in the pcDNA3
expression vector (Invitrogen BV, Leek, The Netherlands) via
EcoRI restriction) gave rise to
BW-Li(B7-1)/BW-LiDhigh(B7-1) and
BW-Li(B7-2)/BW-LiDhigh(B7-2), respectively.6,24
YAC-1 is a Moloney murine leukemia virus-induced line of A/Sn origin.
RMA-S is an antigen-processing defective mutant of RMA (C57BL/6 origin)
and was kindly provided by L. Eisenbach (ILRAD, Israel).
RMA-S(B7-1) was derived from RMA-S after transfection and was kindly
provided by M. Bellone (Instituto Scientifico H. San Raffaele, Milan,
Italy).37 All tumor cell lines were cultured
in RPMI supplemented with 10% fetal calf serum, antibiotics, and
L-glutamine (GIBCO, Grand Island, NY).
Generation of A-LAK cells.
Mouse spleen cells are isolated by flushing mouse spleens with
erythrocyte lysis buffer (8.29 g NH4Cl, 1.0 g
KHCO3, and 0.0372 g EDTA/L, pH 7.4) by means of a needle
and syringe. 2 × 108 spleen cells are loaded on a
0.4-g nylon wool column (Wako Chemicals, Osaka, Japan),
pre-equilibrated with Dulbecco's modified Eagle's medium (DMEM)
containing 5% heat-inactivated fetal calf serum (FCS). After 45
minutes' incubation at 37°C, the nonadherent cell fraction (mainly
T cells) is collected and counted. The cells are brought to a
concentration of 2 × 106/mL in RPMI medium,
supplemented with 0.3 mg/mL L-glutamine, 100 U/mL penicillin, 0.1 mg/mL
streptomycin, 10% FCS, 5 × 105 mol/L
mercaptoethanol (=CM), and 1,000 U/mL IL-2 (Eurocetus, Amsterdam, The
Netherlands), and incubated at 37°C in 5% CO2 and
100% humidity during 5 days. For the generation of IL-2/IL-12 LAK,
recombinant mouse IL-12 (generously provided by Genetics Institute,
Inc, Cambridge, MA) was added to the IL-2 LAK cultures at 100 U/mL, 48
hours after initiation of the cultures for another 72 hours. The total
LAK cell recovery varied from 125% to 150% for IL-2 LAKs and from
60% to 85% for IL-2/IL-12 LAKs as compared with the initial cell
number at the start of the LAK culture in all experiments performed.
The adherent cell fraction, comprising between 20% to 40% of the
total LAK cell population, was harvested with 0.01% EDTA in
phosphate-buffered saline (PBS) and was used as effector cell
population (A-LAK) in [111In]-release cytotoxicity
assays. The viability of the IL-2 A-LAK population was always greater
than 84% for the IL-2 A-LAK and greater than 87% for the IL-2/IL-12
A-LAKs in all experiments performed.
Dendritic cell preparations.
DCs were purified from spleens according to a procedure described by
Crowley et al.38 Briefly, spleens were digested with
collagenase (CLS III; Worthington, Freehold, NJ) and separated into
low- and high-density fractions on bovine serum albumin gradient
(Bovuminar Cohn Fraction V powder; Armour Pharmaceutical Co, Tarrytown,
NJ). Low-density cells were cultured during 2 hours and nonadherent
cells were removed by vigorous pipetting. The same procedure was
repeated with a shorter incubation (1 hour) in serum-free media. After
overnight culture, nonadherent cells were collected and contained at
least 90% of DC as assessed by morphology and specific staining using
N418 monoclonal antibody (MoAb).39 DCs were generated from
BM progenitors according to a procedure modified from a protocol of
Inaba et al.40 Briefly, BM was flushed from tibias and
femurs and depleted of lymphocytes, granulocytes, and Ia+
cells using a cocktail of MoAbs and sheep anti-rat IgG DYNABEADS M-450
(Dynal, Oslo, Norway). The MoAbs were anti-CD8, anti-CD4, GR-1
anti-granulocyte, anti-B220/CD45R, and
anti-I-Ab/I-Eb (Pharmingen, San Diego, CA).
Cells were plated in 24-well culture plates (2.5 ×
105/mL, 1 mL/well) in DMEM supplemented with 10%
heat-inactivated FCS and 200 ng/mL granulocyte-macrophage
colony-stimulating factor (GM-CSF) and cultured for 10 days. The
cultures were usually fed every 2 days by gently swirling of plates,
aspirating 75% of medium, and adding fresh medium with GM-CSF.
Cultured cells were readily identified as DCs on the basis of their
distinct morphology and expression of antigens
(anti-I-Ab/I-Eb, N418, anti-B7-1, and
anti-B7-2).24
[111In]-release cytotoxicity assay.
The target cells were labeled with [111In] by incubating
106 cells in 100 µL (RPMI + 5% FCS) with 0.1 mCi
[111In]Cl (Amersham, Gent, Belgium) during 10
minutes.24 After extensive washing, 104 target
cells were incubated with twofold dilutions of effector cells in a
total volume of 200 µL CM in 96-well round-bottom plates. In blocking
experiments, labeled targets were incubated with 0.5 mg/mL anti-B7-1 or
anti-B7-2 F(ab)2 fragments during 30 minutes at room
temperature and immediately included in the
[111In]-release assay. In parallel, F(ab)2
fragments of isotype controls (hamster IgG for anti-B7-1 and rat
IgG2a,  for anti-B7-2) were generated in the lab and
added to the targets as described above. All experiments were performed
in triplicates and at least twice repeated. After 4 hours at 37°C,
the plates are centrifuged 3 minutes at 600 rpm, 100 µL of
supernatants is collected and radiation is counted in a -counter.
The percentage of specific lysis was calculated as ([Experimental
Release  Spontaneous Release]/[Maximal Release
Spontaneous Release]) × 100, where the spontaneous release was
determined from labeled target cells incubated without effector cells
and maximal lysis from target cells incubated 1 hour before harvesting
in 2% sodium dodecyl sulfate (SDS).
FACS staining and analysis of A-LAK cells.
Cell samples comprising 106 adherent LAK cells were
incubated with the appropriate dilutions of antibodies, as suggested by
the distributors, at 4°C for periods of 30 minutes. Mouse
anti-NK1.1-PE and anti-Ly49A-FITC (both with corresponding fluorochrome
labeled mouse IgG2a, [anti-TNP] isotype controls), rat
anti-CD8a-FITC, anti-CD4-PE (RM4-5) and anti-CD8a-PE (with fluorochrome
rat IgG2a, isotype control), rat anti-mouse Pan-NK cell
marker DX-5-FITC (with rat IgM,-FITC isotype control) and hamster
anti-CD28-PE (with syrian hamster [anti-V3 TCR-PE (536)] IgG
isotype control) and anti-CTLA-4-PE (with armenian hamster IgG
(anti-TNP-PE) isotype control) were purchased from Pharmingen. Rat
anti-mouse IL-2R was purchased from the ATCC (cat no. CRL1698). To
prevent FcR-mediated binding of staining antibodies to the LAK cells,
the Fc-receptors were blocked with 2.4G2 derived F(ab)2
antibodies, before the addition of the indicated antibodies. The
isotype controls were included during FACS staining and did not give a
significant aspecific background staining. The proper isotype controls
were used as a reference to delineate the negative populations of the
specifically stained LAKs. The stained cells were subjected to FACS
analysis with the Becton Dickinson (Sunnyvale, CA) FACStar coupled to
an Apple Macintosh FACStation. The results were analyzed with the
CellQuest program.
Cytokine assays.
The production of IL-2 and IFN- was quantified by subjecting culture
supernatants to an IL-2- or IFN--specific sandwich enzyme-linked
immunosorbent assay applying purified rat anti-mouse and biotinylated
rat anti-mouse IL-2 and IFN- (Pharmingen) and comparing the results
with a dilution series of recombinant mouse IL-2 and IFN-.
The production of IFN- in IL-2/IL-12 LAK cultures was blocked by
adding 3 µg/mL of neutralizing anti-mouse IFN- (HB170 [ATCC],
generously provided by A. Darji [GBF, Braunschweig, Germany]), at the
same time that IL-12 was added to the LAK cultures. Up to the time of
harvesting, no IFN- could be detected in the culture supernatant of
the anti-IFN--treated LAK cultures, while the untreated IL-2/IL-12
LAKs produced significant amounts of IFN-, as measured by sandwich
ELISA (see above). In parallel, IL-2/IL-12 LAK cultures were also
treated with isotype control antibodies, purchased from Pharmingen.
Inclusion of the isotype control antibodies did not influence the
cytotoxic activity, nor the production of IFN-, by the LAK cells.
Statistical analysis.
The statistical relevance of differences in specific release was
evaluated with a two-way ANOVA test, applying GraphPad Prism software
(GraphPad Software Inc, San Diego, CA). Differences are considered as
not significant for P  .05, significant for P <
.05, and very significant for P  .01.
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RESULTS |
Presence of IL-12 during LAK cell generation augments B7-1 and B7-2
triggered NK-mediated cytotoxicity toward B7 target cell lines.
To investigate the cytolytic activity of IL-12-activated NK cells
toward BW5147 T-lymphoma variants and thereof derived B7 transfectants,
IL-12-activated LAK cells were generated by culturing nylon wool
purified spleen cells in the presence of 1,000 U/mL IL-2 or IL-12
during 5 days. Because addition of IL-12 at the initiation of the LAK
culture was found to abrogate completely LAK cell generation (data not
shown), conditions were optimized to obtain IL-12-activated LAK cells.
IL-12-activated LAK cells were generated by preactivating nylon wool
purified spleen cells during 2 days with IL-2 and supplementing 100
U/mL of IL-12 for 3 additional days. The cytotoxic activity of the
adherent fraction of IL-2-activated LAK (IL-2 A-LAK) cells as compared
with the adherent fraction of IL-2/IL-12 LAK (IL-2/IL-12 A-LAK) cells
was tested on two different MHC class I-positive BW variants and
thereof derived B7-1 and B7-2 transfectants. In accordance with earlier
observations,24 expression of B7-1 on BW cells strongly
enhanced (P < .0001) the sensitivity of the target
cells to IL-2 A-LAK lysis (Fig 1).
Interestingly, including IL-12 during LAK generation further
potentiated (P < .0001) the cytotoxic activity of LAK
cells toward B7-1-expressing BW cells (Fig 1). Furthermore, even if the
expression of B7-2 on BW-LiDhigh cells was not as potent as
B7-1 to confer sensitivity to IL-2 A-LAK-mediated lysis, activation of
LAK cells with IL-12 increased the potency of these effector cells to
lyse efficiently BW(B7-2) transfectants (P < .0001)
(Fig 1). To show that T cells were not implicated in the B7-induced
cytolytic capacity of IL-2 or IL-2/IL-12 A-LAK cells, LAK populations
were generated from nylon wool purified severe combined
immunodeficiency disease (SCID) spleens. As shown in Fig
2, SCID A-LAKs exerted a similar activity
toward BW(B7) as normal A-LAKs because they also lysed preferentially
B7-1-expressing targets (P < .0001) and this activity could
be further potentiated by incubating the LAK cells with IL-12 during 3
days (P = .0025) (Fig 2). Hence, T cells were not involved in
the lysis of BW(B7) transfectants.
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| Fig 1.
A-LAK cytotoxicity toward BW variants transfected with
B7-1 or B7-2. AKR-derived LAK cells were generated in the presence of
IL-2 alone or in combination with IL-12 and the cytolytic activity of
the respective adherent fractions was tested against BW T-lymphoma
variants and the NK-sensitive tumor YAC-1. The results are represented
as the mean cytolysis of the respective targets as percent specific
release (±SD). (A) Sensitivity of BW-LiDhigh (×),
BW-LiDhigh (B7-1) (+), BW-LiDhigh (B7-2)
( ), and YAC-1 (*) to IL-2 versus IL-2/IL-12 A-LAK effector cells.
(B) Sensitivity of BW-Li ( ), BW-Li(B7-1) ( ), BW-Li(B7-2) ( ),
and YAC-1 (*) to IL-2 versus IL-2/IL-12 A-LAK effector cells.
Spontaneous release in all assays was less than 15%. Both experiments
were performed in triplicate and repeated at least five times, giving
similar results from which one is shown. Standard deviations less than
±3% are not shown for the sake of clarity.
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| Fig 2.
Lytic activity of SCID-spleen-derived A-LAK cells. The
activity of IL-2-activated (filled symbols) versus IL-2/IL-12-activated
(open symbols) A-LAK cells was measured in an in vitro cytotoxicity
assay. The specific lysis of BW-Li (squares) and BW-Li(B7-1) (diamonds)
target cells was compared and here represented as the mean percent
specific [111In]-release of triplicates (±SD).
Spontaneous release was less than 5%. One of two similar experiments
is shown. Standard deviations less than ±3% are not shown for the
sake of clarity.
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Because IL-12 was found to induce the cytolytic activity of NK cells
toward NK-sensitive (P .01 for YAC-1) but not to
NK-resistant targets (P = .08 for BW-LiDhigh) (Fig
1), it was of importance to determine whether the increased activity of
IL-2/IL-12 A-LAKs against BW(B7) targets involved recognition of B7. To
avoid Fc receptor-mediated antibody-dependent cellular cytotoxicity
activities of the A-LAK cells, target cells were treated with the
F(ab)2 fragments of anti-B7-1 or anti-B7-2 antibodies. As
shown in Table 1, blocking B7-1 abrogated
almost completely the B7-1-induced lytic activity of IL-2 A-LAKs at the
different E/T ratios. The increased cytolytic activity of IL-2/IL-12
A-LAKs toward B7-1 and B7-2 transfected, as compared with the
nontransfected BW variants, was reversed with at least 80% at the
highest E/T ratios and strongly reduced at the lowest E/T ratios (Table
1). This specific effect shows that most of the enhanced cytolysis of
B7 transfectants by IL-2/IL-12 A-LAKs is due to the recognition of B7-1
or B7-2 costimulatory molecules by NK cells.
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Table 1.
Effect of Anti-B7-1 or Anti-B7-2 F(ab)2
Treatment on the Lysis of BW Targets by IL-2 A-LAK and IL-2/IL-12 A-LAK
Effector Cells
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IFN- is not required for the increased lytic activity of IL-2/IL-12
A-LAK cells on B7 target cell lines. Comparing IL-2 LAK with IL-2/IL-12
LAK cultures, we observed that addition of IL-12 during LAK generation
induces strongly the production of IFN- (data not shown). Locally
produced IFN- may in turn increase the expression of IL-2R on
IL-2/IL-12 LAK cells and may consequently result in a higher
consumption of IL-2 in these cultures. In fact, IL-2/IL-12 A-LAKs were
found to express higher levels of IL-2R and no IL-2 could be detected
in supernatants of IL-2/IL-12 LAK cell cultures (data not shown).
According to different reports, the effect of IL-12 on NK activation
has been ascribed to IFN-.41,42 Therefore, the possible
contribution of IFN- on the increased cytotoxic activity of
IL-2/IL-12 A-LAKs toward BW(B7-1) and BW(B7-2) was investigated. To
this end, neutralizing amounts of anti-IFN- antibodies were added
to the LAK cultures simultaneous with IL-12. Blocking IFN- in the
LAK cultures, as verified in enzyme-linked immunosorbent assay (ELISA)
(data not shown), did not significantly influence BW(B7-1) (P =
.5) or BW(B7-2) (P = .05) directed lysis (Fig
3). These results refute a direct
involvement of IFN- on the induced cytotoxic activity of IL-2/IL-12
A-LAKs.
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| Fig 3.
Blocking of IFN- during generation of IL-2/IL-12 LAK
cells does not reverse the IL-12-promoting effect on LAK cell lysis of
B7-1- or B7-2-expressing tumor cells. The cytolytic activity of
AKR-derived IL-2 A-LAKs is compared with the cytolytic activity of
untreated or anti-IFN- MoAb-treated IL-2/IL-12 A-LAKs on BW-Li,
BW-Li(B7-1), and BW-Li(B7-2) targets. The results are represented as
the percentage specific [111In]-release (±SE) at
different E/T ratios. Standard deviations less than ±3% are not
shown for the sake of clarity. Spontaneous release was less than 4%.
The experiment was repeated twice. ( ), IL-2 A-LAK; (), IL-2 +
IL-12 A-LAK; ( ), IL-2 + IL-12 A-LAK + -IFN-.
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Presence of IL-12 during LAK cell generation alters the morphotype
and the membrane phenotype of A-LAK cells.
The morphotype of IL-2/IL-12 A-LAK cells was strikingly different from
IL-2 A-LAK cells because these populations contained a larger
proportion of bigger/granulous cells (Fig
4A and B), which showed an upregulation of
the side scatter signal as evaluated by FACS scatter signal analysis
(Fig 4C through E). These cells have the tendency to stick stronger to
plastic than IL-2 A-LAK because longer incubation times and higher EDTA
concentrations were required to detach efficiently the adherent
IL-2/IL-12 LAK cell fractions from the culture plates.
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| Fig 4.
Presence of IL-12 during LAK cell production generates an
adherent subpopulation with a specific morphotype. In contrast to IL-2
LAKs (A), IL-12 induces the formation of granulous, large cells (B).
Adherent AKR-derived LAK cells were stained with Crystal violet and
photographed (original magnification × 100) in culture plate. This
morphological shift can also be visualized by FACS scatter analysis of
AKR-derived IL-2 A-LAK cells (C) as compared with IL-2/IL-12 A-LAK
cells (E). The population with highest cellular density is situated in
region 1 (R1) for IL-2 A-LAK and in region 2 (R2) for IL-2/IL-12 A-LAK.
The overlay histogram (D) compares the side scatter signals of both LAK
populations.
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Because BW(B7) transfectants were lysed more efficiently by IL-2 and
IL-2/IL-12 A-LAK cells, the expression of B7 counterreceptors on the
effector cell populations was analyzed. In accordance with Nandi et
al,43 A-LAK cells express higher levels of CD28. Before the
addition of IL-2 to the nylon wool-purified spleen cells, CD28 was
expressed solely on a CD4/CD8-positive population (Fig
5A). Yet after 5 days' culture in the
presence of IL-2, a CD4/CD8
population positive for CD28 expression appeared (Fig 5B). Adding IL-12
to the LAK cultures on day 3 induced drastically the expression of CD28
on the CD4/CD8 population (Fig
5C). This population of
CD4/CD8/CD28+ cells
corresponded to the subpopulation of IL-2/IL-12 A-LAK cells with high
granulosity. This result was confirmed by analyzing the expression of
CD28 on A-LAK cells derived from SCID mice and as shown in Fig 5D
through F, the profile of CD28 expression was comparable with that of
the CD4/CD8 subpopulation of
A-LAK cultures generated from immunocompetent spleen cells. The effect
of IL-12 on the expression of CD28 and other T and NK cell antigens on
IL-2 A-LAK cells are compiled in Fig 6A
through C. Although the expression of CD28 was strongly induced by
addition of IL-12, the expression of CTLA-4, the second counterreceptor
for B7-1 and B7-2, was not found to be elevated during LAK cell
generation either with IL-2 or with IL-2 and IL-12 (data not shown). To
evaluate the expression of the commonly known NK cell markers such as
Ly49 and NK1.1 (not expressed in the H-2k haplotype,
syngeneic with BW tumor cells), IL-2 and IL-2/IL-12 LAK were generated
from spleens of allogeneic C57BL/6 (H-2b haplotype) and
congenic B10.BR (H-2k haplotype) mice strains. The
expression of Ly49A and NK1.1 was strongly upregulated during IL-2 LAK
culture (result not shown), yet adding IL-12 during LAK cell generation
did not further induce the expression of these NK cell-specific
membrane antigens (Fig 6B and C). The expression of the NK cell marker
DX5 also refutes any positive role of IL-12 in the enrichment of NK
cells in the LAK preparations (Fig 6A and C).
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| Fig 5.
IL-12 induces the expression of CD28 on a subpopulation
of adherent LAK cells. The expression of CD28 is represented in
function of CD4/CD8 expression for AKR-derived nylon wool purified
spleen (A), IL-2 A-LAK (B), and IL-2/IL-12 A-LAK (C) cells as compared
with SCID-derived nylon wool purified spleen (D), IL-2 A-LAK (E), and
IL-2/IL-12 A-LAK (F) cells. The A-LAK cells were pretreated with 2.4G2
antibodies to block Fc receptors and subsequently incubated with
phycoerythrin (PE)-labeled CD28 and fluorescein isothiocyanate
(FITC)-labeled CD8 antibodies. The dot plots indicate the percentage
of positive cells calculated via quadrant statistics using the suitable
isotype controls.
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| Fig 6.
NK and T-cell markers on H-2k- and
H-2b-derived IL-2 and IL-2/IL-12 A-LAK populations. Flow
cytometric analysis of (A) AKR (H-2k), (B) C57BL/6
(H-2b), and (C) B10.BR (H-2k)-derived IL-2
versus IL-2/IL-12 A-LAK preparations, stained with FITC (Y axis) and PE
(X axis)-labeled MoAbs as described in Materials and Methods. The
cells were preincubated with 2.4G2 for 30 minutes before staining. The
results are represented in dot plots indicating the percentage of
positive cells. Positive populations were calculated via quadrant
statistics applying the proper isotype controls to outline the negative
populations (top panels represent FITC-labeled rat IgM and PE-labeled
syrian hamster Ig, other isotype controls are not shown).
|
|
CD28-dependent and -independent recognition of B7 target cell lines
by A-LAK cells.
Because IL-2/IL-12 A-LAKs lysed more efficiently B7 target cells and
expressed more strongly CD28, it was of interest to test whether
CD28-B7 interactions are functionally involved in the increased
cytolysis. To this end IL-2 and IL-2/IL-12 LAK cultures were generated
from spleens of CD28/ knockout mice
(H-2b haplotype). FACS analysis confirmed that CD28 was not
expressed on spleen cells derived from these mice and that CD28 was not
induced during culture of nylon wool purified fractions in the presence
of IL-2 or IL-2 and IL-12 (data not shown). Testing the
CD28/ A-LAK activity toward NK-sensitive
YAC-1 targets, a reduced cytolytic activity that reached between 75%
and 50% of normal A-LAK activity, was systematically observed
(P < .0001) (Fig 7). Comparing
the sensitivity of BW(B7-1) target cells to either syngeneic (AKR,
H-2k), congenic (B10.BR, H-2k), or allogeneic
(C57BL/6, H-2b) effectors, with
CD28/ (H-2b) IL-2/IL-12 A-LAK
cell effectors, absence of CD28 did not influence strongly the
B7-1-mediated effect (P < .0001 for all effectors). Indeed,
taking into account that CD28/ IL-2/IL-12
A-LAK cells showed an overall reduced cytolytic activity (as tested on
YAC-1 cells), these effector cells still recognized more efficiently
B7-1 target cells (P < .0001). These results indicate that
CD28 is not solely responsible for the B7-1-mediated recognition and
lysis of B7-1 target cells by IL-2/IL-12 A-LAK cells. However, it
should be emphasized that the target cells and effector cells were
allogeneic in this experimental set-up. Therefore, B7-1 positive,
syngeneic tumor cells (RMA-S) were also tested and as shown in Fig 7,
the lysis of RMA-S(B7-1) cells was completely reduced to the background
lysis (RMA-S target cells) when CD28/
IL-2/IL-12 A-LAK cells were used (P > .1). Hence, depending
on the target cell population, B7 recognition by IL-2/IL-12 A-LAK cells
appears to be CD28-dependent or -independent.
View larger version (23K):
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[in a new window]
| Fig 7.
Involvement of CD28 expression in the A-LAK-mediated
lysis of B7-1 expressing tumor cells. (A) The lytic activity of AKR
(H-2k haplotype), (B) B10.BR (H-2k haplotype),
(C, E) C57BL/6 (H-2b haplotype), and (D, F)
CD28/ knockout mice (H-2b
haplotype)-derived IL-2/IL-12 A-LAK cells was tested against (A
through D) parental and B7-1 transfected BW-Li (H-2k
haplotype) and (E through F) RMA-S (H-2b haplotype) tumor
cell variants in an [111In]-release assay. The specific
lysis of YAC-1 was used as a reference for the lytic capability of the
IL-2/IL-12 A-LAK. Spontaneous release was 10% for all cell lines.
One representative of three experiments is shown, indicating the mean
percentage specific release of targets in triplicate (±SD). Standard
deviations less than ±3% are not shown for the sake of clarity.
(), BW-Li; (), BW-Li(B7-1); (*), YAC-1; (), RMA-S; (×),
RMA-S(B7-1).
|
|
Activation with IL-12 endows LAK cells with the capacity to lyse
syngeneic DCs via a CD28-dependent recognition.
So far the B7 target cell lines used in this study were transformed
cells. To test whether high expression of B7-molecules could trigger
the lytic machinery of NK cells toward lysis of nontransformed
syngeneic B7+ cells, mature DCs were isolated in vitro and
confronted with IL-2 and IL-2/IL-12 A-LAKs. Mature DCs, generated from
BM (progenitor DCs) or spleen (spleen DCs), were purified and analyzed
for expression of B7-1 and B7-2. Both dendritic cell preparations
expressed high levels of B7-1 and B7-2 (Fig
8) and their susceptibility to A-LAK lysis
was tested. Although a high expression of B7-1 and B7-2 was recorded on
the DCs, IL-2-activated A-LAK cells did not significantly lyse
autologous DCs at similar E/T ratios used for the lysis of
B7-expressing tumor cells (Table 2). Yet
when the LAKs were activated in the presence of IL-12, spleen-derived
(P < .0001) as well as progenitor (P < .0001)
autologous DCs became susceptible to NK-mediated destruction (Table 2).
Furthermore, the lysis of DCs by IL-2/IL-12 A-LAKs was completely
abrogated when the LAKs were generated from
CD28/ mice (P > .5 for spleen DCs,
P > .05 for progenitor DCs). Hence, here also recognition and
lysis of B7-positive targets by IL-2/IL-12 A-LAKs is CD28-dependent.
View larger version (30K):
[in this window]
[in a new window]
| Fig 8.
Mature splenic (top) and progenitor (bottom) DCs express
high levels of B7-1 and B7-2 on their surface. In vitro-activated DCs
were, upon harvesting, treated with 2.4G2 antibodies to block Fc
receptors and subsequently incubated with FITC-labeled B7-1 or B7-2
antibodies. The cells were analyzed by FACS and their fluorescence
signals are represented in overlay histograms.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
IL-12 Induces the Capability of A-LAK Cells to Kill
Autologous DCs When CD28 Is Expressed on the Effector Cell
Population
|
|
| |
DISCUSSION |
We herein confirm that NK cells, though being subjected to MHC class
I-mediated inhibitory signals, can still be effectively activated to
destroy tumor targets when appropriate activation signals are provided.
Indeed, we and others have recently reported that expression of B7-1 on
tumor cells can activate NK cell-mediated lysis. This was shown in the
mouse: (1) in vitro via cytotoxicity assays with IL-2-activated LAK
cell fractions and polyI:polyC-activated NK cells,24,25,27
and (2) in vivo where immune effectors rejecting B7-1 expressing tumors
were shown to reside primarily in the nonadaptive NK arm of immune
response.24-26 Similar observations were made for a human
NK leukemia cell line, YT2C2, because these cells could lyse
B7-1-expressing targets in vitro.44 In the present study we
have extended these observations by showing that activation of NK cells
with IL-12 synergizes with the B7-induced effect. Furthermore, evidence
is provided that IL-12 induces, besides the B7-1-, also the
B7-2-mediated NK responses. Consequently, NK cells can recognize the
B7-2 antigen for engagement and subsequent cytolysis of diverse
targets, provided appropriate activation signal(s) (ie, IL-12) are
supplemented. Collectively, these observations stress the importance of
specific activation signals in NK cell function and indicate that NK
cells can cope with inhibitory and activating stimuli in such a way
that strong activating signals can overrule the MHC class I inhibition.
Although the effect of IL-12 on NK cell activity has been extensively
investigated, several questions concerning the mechanism of enhanced
cytolysis remain unsolved. In accordance with other
studies,45 IL-12 was found to enhance the lytic activity of
LAK cells toward NK-sensitive BW variants, but not toward NK-resistant
variants (data not shown). IL-12 was proposed to augment the NK
cell-mediated cytotoxicity at a postbinding level of the lytic
interaction by increasing the maximal level of granule
exocytosis.46 We observed that the granulosity of
IL-2/IL-12 A-LAK cells is increased as compared with IL-2 A-LAKs.
IL-12, in the presence of IL-2, can also upregulate NK cell activation
markers and surface adhesion molecules, including CD69, CD71, CD56,
LFA-1, ICAM-1 and CD2 and it has been speculated that the subsequent
enhanced conjugate formation is sufficient to trigger a cytolytic
response.32,46 Yet other investigators failed to confirm
this significant upregulation in adhesion molecules or related
enhancement in E/T conjugate formation.47 We found that the
expression of certain surface molecules, among which CD28 and the
IL-2R, was induced on IL-2/IL-12 A-LAK cells, yet other membrane
markers such as CD4, CD8, or NK1.1 were not modulated. Furthermore,
blocking of B7-2 or B7-1 was sufficient to abrogate the B7-specific
induced lytic activity of IL-2 and IL-2/IL-12 A-LAK cells, indicating
that the enhanced lysis of B7-1/B7-2 expressing targets is not simply
due to an overall stimulating effect of IL-12 on the cytolytic
machinery of NK cells, but rather that engagement of B7 appears to be
the limiting step.
The present report is not the first one to show that IL-12 can
synergize with B7-1-mediated effects. In vitro it has been reported
that IL-12 synergizes with B7/CD28 interactions by inducing (1)
efficient proliferation and cytokine production (mostly IFN-, but
also TNF- and GM-CSF) by human T cells48 and (2)
IL-2R expression by mouse Th1 clones.49 In vivo, B7-1
and IL-12 were reported to cooperate in the induction of effective
antitumor immunity and therapy. This cooperative effect was ascribed to
the presence of CD4+ and CD8+ T cells
and/or involved IFN-.34,35,50 Coughlin et
al34 observed an effect of anti-asialoGM1 antibody
treatment on the IL-12/B7 cooperative antitumor activity. These
experiments enforce the possible relevance of our observations in
immune responses and cancer therapy.
In general, these different studies proposed that the IL-12-induced
lysis of B7-expressing targets involved both T cells and IFN-
production. As far as IFN- is concerned, antibody neutralization
experiments have shown that IFN- was essential for the in vivo
antitumor efficacy of IL-12 alone.41,42 Also, the IFN
regulatory factor-1 (IRF-1) seemed to play a crucial role in the
induction of NK cell-mediated cytotoxic and effector functions in
vivo.51 In fact, IFN- was shown to be implicated in
inducing NK proliferation, converting a noncytolytic NK precursor cell
into a functional NK cell, increasing the recycling of already existing
NK cells, enhancing target-binding capacity of NKs, and increasing the
lytic efficiency of conjugated effector cells.52 Although
we observed a strong elevation of IFN- production during IL-2/IL-12
LAK cell generation, neutralization of this cytokine during LAK cell
generation did not significantly reduce the cytolysis of B7-positive
targets, indicating that IFN- was not required for the B7-mediated
induction of LAK lysis. Similarly, IFN- was not found to be involved
in the cytolytic activity of short time cultures of NK +
IL-12.53 Finally, because IL-2/IL-12 A-LAK cells generated
from SCID spleens or normal mice manifested similar features, T cells
were clearly not required for the particular activities of these
effector cells.
Because tumor cells do normally not express B7-molecules on their
surface, the physiologic relevance of our observations was explored by
testing the cytotoxic activity of IL-2 versus IL-12/IL-2 A-LAK cells
against normal cells that express abundantly B7-1 and B7-2. DCs,
following purification and maturation in vitro, were found to be
relative resistant to IL-2 A-LAK cells, while significant lysis was
observed with IL-2/IL-12 A-LAK cells. These results differ to some
extent from a recent report showing that BM-derived
cells, in contrast to resting peritoneal DCs and
macrophages, were efficiently lysed by autologous IL-2 LAK
cells.27 Different reasons may account for this opposed
result, such as (1) differences in the preparation of LAK cells, (2)
the effector/target (E/T) ratio used that was rather low in our study
yet more physiologically relevant, and (3) the activation status of the
target cells used. The last aspect may be important since during
activation/maturation of DCs, an evolution in surface marker expression
occurs, in particular the expression of B7-1 and B7-2 on immature DCs
is extremely low compared with fully activated DCs.38-40
Collectively, both studies provide sufficient evidence to postulate a
role for NK cells in controlling immune responses via interacting with
antigen-presenting cells. In fact, a negative regulatory effect of NKs
on antigen-presenting cells (APC) was previously suggested in studies,
showing that NK cells can lyse activated monocytes54 and
inhibit the generation of CD8+ cytotoxic T lymphocytes
(CTLs) by suppressing or eliminating DCs.55 Besides
negative regulation of NK cells on APC, a positive regulation may exist
as well. Indeed, it was recently reported that IFN-/ can induce
the trafficking of NK cells from the BM to the marginal zone of the
spleen where the macrophages, DCs, and monocytes reside. Subsequently,
the NK cells would promote the maturation of these cells via cytokine
production, when the NK cells get properly activated. Also, it was
shown here that IL-12 was implicated in the activation but that full
activation may require both exposure to soluble IL-12 and delivery of a
costimulatory signal through NK-APC cell contact.56 Our
results suggest that these events will be deleterious for APC that are
already fully matured or hyperactivated, closing the regulatory loop
between APC and NK interactions. Similar regulatory interactions have
recently also been established between NK cells and CTLs.57
The role of CD28 expression on NK cells in the lysis of B7-positive
target cells is rather controversial. Because B7 expressing targets
were more efficiently lysed by IL-2/IL-12 A-LAK cells, which express
higher levels of CD28, the importance of CD28 in this phenomenon was
addressed using effector cell populations from
CD28/ knockout mice. We could conclude that
CD28 plays an important role in the NK-mediated lysis of B7-expressing
targets, since CD28/ IL-2/IL-12 A-LAKs were
no longer able to lyse B7-1/B7-2-positive DCs as well as
B7-1-expressing syngeneic tumor cells. However, the B7/CD28
interaction seemed not to be primordial in the lysis of allogeneic
B7-1/B7-2-expressing tumor cells. These observations may indicate that
CD28 is not always involved in the recognition of B7 targets, implying
that NK cells may express a third B7-counterreceptor, capable to
transduce costimulatory signals and whose expression is controlled by
appropriate activation/differentiation signals (such as IL-12). In
fact, using other NK cell populations, Chambers et al27
failed to show any effect of CD28 in the lysibility of B7-expressing
targets. In contrast, in the human system it was demonstrated that CD28
was functionally responsible for the destruction of B7-expressing
targets because anti-CD28 treatment of the FcR-negative YT2C2 NK line
blocked the B7 mediated lysis of B7 expressing targets.44
Alternative costimulatory pathways and new receptors were also
substantiated in other systems. Indeed, it was reported that
CD28-deficient mice exhibit normal cytotoxic T-lymphocyte
activity36 and, furthermore, that CTLA-4 does not fully
substitute for all functions of CD28.58 Furthermore,
blocking of B7-1 and B7-2 does not interfere with the activation of
CD8+ cells by allogeneic MHC class I molecules expressed on
Epstein-Barr virus (EBV)-transformed B cells.59 Finally, it
was reported that anti-B7 or anti-CD28 antibodies do not always
completely block the lytic activity of the human NK-like line YT on
B7-positive EBV-transformed cells.60
Collectively our experimental evidence suggests that IL-12 activation
of NK cells increases the capacity of these effector cells to lyse
B7-expressing target cells via CD28-dependent and CD28-independent
mechanisms.
| |
FOOTNOTES |
Submitted April 10, 1997;
accepted August 29, 1997.
Supported by grants for the Sportvereniging tegen de Kanker (A.B.G.),
the GOA (P.D.B., K.T.), the Governmental Vlaams Actieprogramma
Biotechnologie (P.D.B.), and the Vereniging voor Kankerbestrijding
(P.D.B.).
Address reprint requests to Anja B. Geldhof, PhD,
Laboratory of Cellular Immunology, VIB-VUB/IMOL II, Paardenstraat 65,
B-1640 Sint-Genesius Rode, Belgium.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank L. Brijs, M. Gobert, E. Vercauteren, E. Omasta, and F.
Thielemans for an excellent technical assistance and both
Hoffman-LaRoche, Inc and Genetics Institute, Inc
(Cambridge, MA) for supplying the recombinant IL-12 used for this work.
| |
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Abstract
Introduction
Abstract