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*
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden;
Division of Clinical Immunology, Department of Laboratory Medicine, and
Division of Experimental Oncology, Karolinska Hospital, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NK cells were initially characterized by their ability to mediate
spontaneous cytotoxicity against tumor cell lines, in particular tumors
of hemopoietic origin (1). Currently, it is understood
that they play a significant role in the host defense against certain
microorganisms, in part through their ability to secrete cytokines such
as IFN-
, TNF-
, and GM-CSF (4, 5). They also take
part in the development and regulation of adaptive immune responses.
The mechanisms underlying the latter processes are less well
understood. They most likely involve cytokines but may also involve
direct interactions with APC such as dendrite cell (DC)3
(6, 7).
DC are specialized APC with a unique ability to prime naive lymphocytes (8, 9). They are distinguishable from other APC due to their typical morphology and constitutive expression of MHC I and II molecules. Immature DC have a high capacity to capture and process Ags at peripheral sites in the body, but a weak capacity to stimulate T cells. After Ag capture, DC migrate from the non-lymphoid tissue to the blood or afferent lymph for transport to regional lymph nodes or other peripheral lymphoid organs. During this time, the DC acquire a "mature" phenotype; they lose their ability to efficiently take up and process Ag and at the same time up-regulate the expression of MHC class I and II, adhesion and costimulatory molecules. These mature DC have a strong capacity to stimulate T cells and initiate immune responses (8, 9).
It was observed recently that murine bone marrow-derived DC could be killed efficiently by syngeneic NK cells in vitro (10). To study whether this effect is specific for the murine system or whether it is observed also with human cells, we have established cultures of human DC from peripheral blood CD14+ monocytes as well as from CD34+ hemopoietic stem cells from cord blood. Using these cells as targets, we demonstrate that freshly isolated human NK cells and human NK cell lines readily kill autologous DC and that this lysis is dependent on granule exocytosis. Susceptibility to lysis by NK cells appears to be a particular property of immature DC. These results are discussed in relation to the ability of DC to interact with NK cells, and the ability of NK cells to regulate development of adaptive immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FITC- and PE-labeled mAbs against CD3 (HIT3a), CD14 (M5E2), CD34 (8G12), CD56 (MY31), CD80 (L307.4), CD86 (2331), HLA-DR (L243), HLA-ABC (G46-2.6), and isotype-matched labeled controls were obtained from Becton Dickinson (Stockholm, Sweden). Anti-CD83 (HB15A) mAb was obtained from Immunotech (Kebolab, Stockholm, Sweden). Anti-CD40 (S2C6) mAb was kindly provided by Dr. S. Pauli (Stockholm University, Stockholm, Sweden). Anti-CD1a mAb (OKT6) was kindly provided by Dr. S. A. Porcelli (Brigham and Womens Hospital, Boston, MA). Abs were used to characterize cell surface phenotypes and purity by flow cytometry. For staining, cells were washed and resuspended in PBS supplemented with 1% heat-inactivated FCS and 0.01% NaN3. Abs were diluted in this buffer and used at a final concentration of between 2–20 µg/ml. Incubations with Abs were conducted for 30 min on ice. Following the final washing step, labeled cells were fixed with 1% formaldehyde solution (Sigma, Stockholm, Sweden), and 10,000 cells were analyzed by flow cytometry on a FACScan flow cytometer using Cellquest software (Becton Dickinson).
Cell lines
K562 (human erythroleukemia), L cells, L cells transfected with CD40L (kindly provided by Dr P. Garrone, Schering Plough, Dardilly, France), RPMI 8866 cells (kindly provided by Dr. G. Trinchieri, Philadelphia, PA), and EBV-transformed B lymphoblastoid cell lines (EBV-BLCL) were cultured in complete medium consisting of RPMI 1640 media (Life Technologies, Täby, Sweden) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). To generate EBV-BLCL, 5 x 106 PBL were added to 1 ml of culture supernatant from the EBV-secreting B95-8 cell line (ECACC, Wiltshire, U.K.) and incubated in a 37°C water bath for 2 h before culturing in complete medium supplemented with 1 µg/ml cycolsporin A (Sandoz, Basel, Switzerland) to prevent outgrowth of EBV-specific T cells. After 2–3 wk of culture, lines were established and used.
Generation of DC cultures
Monocyte-derived (myeloid) DC were generated as described previously (11). Briefly, human PBMC were isolated from heparinized venous blood or cytopheresis buffy coats from normal volunteer donors by Ficoll-Hypaque density centrifugation (Lymphoprep; Nycomed, Oslo, Norway). Isolated cells were resuspended at a concentration of 3.5 x 106 cells/ml in complete medium, and 10 ml was added to a 25-cm2 flask and incubated for 45 min at 37°C. Nonadherent cells were removed by gentle pipetting, and the plastic adherent cells were rinsed with 20 ml of complete media. The remaining adherent cells were cultured in complete media supplemented with 800 U/ml recombinant human (rh) IL-4 (Genzyme Corporation, Cambridge, MA) and 50 ng/ml rhGM-CSF (Leukomax; Sandoz). For induction of maturation, cultures of monocyte-derived DC were supplemented with 10 µg/ml LPS (Sigma) on day 6 of culture for 48 h before use. Additional cultures were matured using 25% v/v MCM, which was added on day 6 of culture for a 72-h period (12). DC cell cultures were also matured by stimulation through CD40L. Briefly, 1.5 x 105 L cells transfected with CD40L (13) were cultured in 1 ml of complete media in 24-well plates and incubated overnight before irradiation (8000 rad). Day 7 monocyte-derived DC were resuspended at a concentration of 1 x 106 cells/ml, and 1 ml of cells was added to each well onto the monolayers of CD40L-expressing L cells or control L cells. Cells were cocultured for 18 h and were harvested before use by gentle resuspension.
CD34+ stem cell-derived DC cells (Langerhans-like
cells) were prepared from human cord blood mononuclear cells utilizing
the method of Caux et al. (14, 15). Umbilical cord blood
was obtained according to institutional guidelines, and cord blood
mononuclear cells were obtained by density centrifugation. Progenitor
cells bearing the CD34 Ag were isolated from mononuclear fractions by
magnetic cell separation using a CD34 progenitor cell isolation kit
(MiniMacs; Miltenyi Biotech, Malmä, Sweden) according to the
manufacturer’s specification. The purity of the isolated
CD34+ cells was found to be 75–98% as
determined by flow cytometry. The cells were frozen and stored in
-150°C until used. Thawed CD34+ cells were
cultured at a concentration of 50,000 cells/ml in
25-cm2 flasks in RPMI 1640 medium containing 10%
heat-inactivated FCS, 2 mM L-glutamine, 100 IU/ml
penicillin, 100 µg/ml streptomycin, 25 µg/ml gentamicin (all Life
Technologies), and 50 µM 2-ME (Sigma). The media were supplemented
with 1000 U (100 ng)/ml rhGM-CSF (Leukomax), 50 U (2.5 ng)/ml
rhTNF-, and 20 ng/ml rhSCF (Genzyme). The cells were refed on day 3
by adding 50% of the original volume of media to the culture flask.
The media was supplemented with the described cytokines and in addition
with 0.25 U (0.5 ng)/ml rhTGF-ß (Genzyme). The cells were harvested
and used on day 6 or 7,and their phenotype was investigated by flow
cytometry.
Influenza virus infection
Day 7 monocyte-derived DC were collected and washed in serum-free medium. Cells were resuspended in 700 µl serum-free RPMI 1640 media and infected with influenza virus (A/Japan/305/57). One thousand HAU/ml was added to the DC suspension and incubated on ice for 10 min to enable viral binding. Cells were washed in complete media to remove excess virus and incubated in a 37°C water bath for 30 min to allow viral uptake. Infected cells were incubated for an additional 2 h or 24 h at 37°C before use in experiments. To demonstrate viral infection, cells were stained with an anti-hemagglutinin mAb (FC125) (kindly provided by Dr. T. Braciale (University of Virginia, Charlottesville, VA).
Generation of large granular lymphocytes (LGL), polyclonal NK cell populations, and NK cell lines
For preparation of LGL, mononuclear cells were isolated by
Ficoll-Hypaque density centrifugation (see above). PBMC were depleted
of monocytes by plastic adherence for 1 h at 37°C; then the
flask was turned over and incubated for one further hour. The
nonadherent cells were collected and resuspended in 2 ml of warm
(37°C) complete media, and the cells were loaded onto a nylon wool
column (Polysciences, Warrington, PA). Columns were incubated for
1 h at 37°C before 50 ml of warm media was run through the
column. Collected cells were pelleted by centrifugation and then
resuspended in 4 ml of a 30% Percoll solution (Sigma) in PBS with 10%
FCS. This was overlaid onto a 40% Percoll solution and centrifuged for
30 min at 2000 rpm. Cells at the LGL interface were harvested and
washed three times with PBS before being resuspended in complete media
and used immediately as effector cells. For preparation of lymphokine
activated killer (LAK) cells, mononuclear cells were isolated as above,
and PBMC were depleted of monocytes by plastic adherence for 45 min at
37°C. The recovered cells were resuspended in complete medium
supplemented either with 1000 U/ml rhIL-2 or 2000 U/ml rhIFN-
(Peprotech, London, U.K.) and cultured at 37°C for 48 h. NK cell
lines were generated by resuspending nonadherent cells in IMDM medium
(Life Technologies) supplemented with 5% heat-inactivated FCS, 5%
heat-inactivated AB+ human serum, and 10%
leukocyte-conditioned medium (16). Cells (2.5 x
105 cells/well) were cultured in 24-well plates
at 37°C, in the presence of 0.5 x 105
irradiated (5000 rad) RPMI 8866 cells. Following 7 days of culture,
cells were collected and enriched for NK cells by incubating for 45 min
at 4°C with saturating amounts of anti-CD3 (OKT3), anti-CD4
(OKT4), and anti-CD8 (OKT8) hybridoma supernatants. Cells were
washed twice with PBS before resuspending in rabbit complement
(Pel-Freez, Brown Deer, WI) and incubated at 37°C for 1 h. After
washing, 1 x 105 recovered cells were
further expanded in 24-well plates in complete medium supplemented with
200 U/ml rIL-2, in the presence of 1 x 105
RPMI 8866 cells to generate NK cell lines. Typical NK cell lines were
>99% positive for the human NK cell marker CD56 and negative for CD3
and other T cell markers.
NK cell-mediated cytotoxicity
Cytotoxicity of cells was measured in a standard 4-h 51Cr-release assay using Na251CrO4-labeled cells in triplicate at various E:T ratios. To assess extracellular Ca2+ requirements, cytotoxicity assays were performed in media supplemented with 8 mM EGTA and 6 mM Mg2+ (EGTA/Mg2+). The percentage specific 51Cr release was calculated according to the formula: % release = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. The spontaneous release was usually less than 20% of the maximum release.
Statistical analysis
Statistical analysis was performed using the Student two sample t test, where p < 0.05 is significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a typical experiment, after 7 days of culture with GM-CSF and
IL-4, peripheral blood-derived leukocytes appeared as loosely adherent
clumps with typical dendritic morphology with 
80–95% purity as
assessed by flow cytometry (data not shown). The cell surface phenotype
of such cells exhibited a classic "immature" DC phenotype (Table I
). The cells expressed MHC class I and
class II as well as CD40, but low levels of CD80, CD86, CD1a, and CD83.
Expression of CD14 was negligible. Following maturation by culturing
the cells with MCM, LPS, or culture on CD40L-expressing L cells, cell
surface Ag expression was modulated. Expression of MHC class I and
class II molecules, CD1a, and the costimulatory molecules CD40, CD80,
and CD86 was increased. Expression of the known DC maturation marker
CD83 was also up-regulated (Table I).
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Peripheral blood-derived LAK cells, representing short-term
activated polyclonal NK cells, do not efficiently kill EBV-BLCL. In
contrast, day 7 monocyte-derived DC were readily killed by allogeneic
rhIL-2 or rhIFN--activated LAK cells (Fig. 1). It was important to verify that this
lysis was not simply a result of NK cell activation due to allogeneic
stimulation; thus, LAK cell cultures and DC were generated from the
same donors. It was found that autologous DC were highly susceptible
even to autologous LAK cells (Fig. 2). In
this context, it is important to note that activation of the NK cells
was necessary to obtain efficient lysis of DC. Using LGL as effector
cells, in the absence of stimulation, only low levels of lysis of
autologous DC was observed (Fig. 3). Cell
to cell contact appeared to be required for NK cell-mediated lysis of
DC since supernatant from the NK cell cultures had no cytolytic
activity (data not shown).
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LAK cell cultures could be a potential source of T cells activated
by IL-2. Thus, it was of importance to determine whether the population
of cells responsible for DC lysis resided within the NK cell subset.
For this purpose, five independent
CD3-/CD56+ NK cell lines
and corresponding autologous monocyte-derived DC were generated.
Although the CD3-/CD56+ NK
cell lines spared the autologous EBV-BLCL, they specifically lysed the
DC targets and the NK-sensitive K562 cell line (Fig. 4).
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Several studies have shown that DC can be further induced to
mature by treatment with different stimuli, such as MCM
(12) and LPS (17), or through CD40L
stimulation (8). To address the effect of DC maturation
with respect to their susceptibility to lysis by autologous NK cell
lines, monocyte-derived DC cultures were induced to mature before being
used as targets. DC treatment with LPS or MCM or stimulation through
CD40L resulted in mature populations of cells (Table I
) that were less
susceptible to NK lysis than corresponding untreated day 8 DC (Fig. 5).
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It has recently been demonstrated that DC infected with influenza
virus are potent APC for the initiation of specific antiviral CTL
responses (18, 19). This function suggests that infected
DC may display a mature Ag presenting rather than an Ag uptake
phenotype. To study how influenza virus infection affects
susceptibility to NK cells, immature day 7 monocyte-derived DC were
infected with the A/Japan/305/57 influenza virus strain. Productive
virus infection was verified by expression of hemagglutinin on the cell
surface (data not shown). Cell viability was not affected by influenza
virus infection as determined by trypan blue exclusion. In line with
observations above on the reduced NK cell susceptibility of mature DC,
these virally infected cells were rendered less sensitive to autologous
NK cells than similar uninfected DC. A reduction in killing was
observed after infection and culture for only 2 h (Fig. 6A). Twenty-four hours after
infection, the DC were almost completely resistant to autologous NK
cell-mediated lysis (Fig. 6B).
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Calcium ions are necessary for degranulation and perforin
polymerization (20). To investigate the calcium dependence
of NK cell-mediated killing of DC, EGTA/Mg2+, a
calcium chelator, was used. LAK or NK cell line-mediated lysis of
autologous DC was almost entirely dependent on the presence of calcium
ions, since all 51Cr release was abrogated in the
presence of EGTA/Mg2+ (Fig. 7). This dependence on extracellular
Ca2+ suggests that LAK cells and NK cell lines
lyse autologous DC using perforin and granzyme action. Although our
data indicate that perforin-mediated killing is the predominant
cytolytic mechanism of DC killing, we cannot exclude an additional role
for the Fas-mediated cytolytic pathway. Using flow cytometry, we
detected the expression of CD95 (Fas) on the DC cell surface. This was
not altered by DC maturation, and Fas-Ligand expression was detected on
NK cell lines (data not shown).
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To investigate whether NK cell susceptibility was restricted to
peripheral blood-derived DC, an alternative source of DC was utilized,
namely CD34+ umbilical cord blood-derived
Langerhans-like cells. The DC cultured from CD34+
stem cells from umbilical cord blood were found to have a phenotype
similar to that described previously (14, 21, 22). These
DC were for susceptibility to lysis using allogeneic LAK cells or
allogeneic NK cell lines as effectors. Like the monocyte-derived DC,
these Langerhans-like cells were demonstrated to be highly susceptible
to lysis (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytotoxic T lymphocytes and NK cells kill targets by two mechanisms, namely the perforin and Fas-Fas ligand (FasL) pathways. The relative contribution of granule exocytosis was studied using the calcium chelator EGTA. Degranulation and perforin polymerization are entirely dependent on the presence of extracellular calcium (20, 23). EGTA totally abrogated LAK and NK cell line-mediated lysis of dendritic cell targets, confirming that, in this case, cytotoxicity is almost entirely granule dependent. This is in line with previous experiments where it was found that perforin-/- mice could not kill autologous DC (10). This differs slightly from studies using the YT leukemic cell line and its derivatives, since it has been suggested that YT cells may use both perforin- and Fas-mediated pathways (23, 24). We do not formally exclude a Fas-mediated component of DC killing by NK cells since it is well documented that NK cells express Fas Ligand (25) and DC express CD95. Furthermore, it has been suggested that there may be preferential activation of either the Fas or perforin mechanisms, depending on the type of target cell (20).
It is probable that the DC cell surface expression of several different molecules contributes to the NK cell-sensitive phenotype of these cells. Alterations in the expression of some these molecules during DC maturation may contribute to the more NK cell-resistant phenotype observed. Expression of HLA-A, -B, and -C are increased upon monocyte-derived DC maturation and also when these cells are infected with influenza virus. Since NK cells express inhibitory receptors with specificity for MHC class I, it is not unlikely that the increased levels of MHC class I expressed by mature DC may in part account for the reduction of NK cell susceptibility (3, 26). The role of costimulatory molecules in NK cell killing has recently been highlighted in a number of publications (10, 27, 28, 29, 30, 31). Briefly, in murine tumor models it has been demonstrated that the introduction of CD80 (B7-1), CD86 (B7-2), and CD40 is associated with enhanced susceptibility to NK cell lysis (10, 29, 30, 31). In the human system, similar results have been observed in different models involving CD80- or CD40-expressing cells (32, 33). Given these results, the present observations were somewhat surprising. The expression of the costimulatory molecules CD80, CD86, and CD40 and other adhesion molecules increase upon DC maturation; in fact, one of the hallmarks of mature DC is the high expression of costimulatory molecules (8). If these are the only molecules that control NK cell susceptibility, one might expect that mature DC would be even more NK cell sensitive than immature DC, although the opposite pattern was observed. In relation to these results, it is of interest to note that EBV-BLCL have extremely high expression of all costimulatory molecules yet are among the most resistant NK cell target documented. Although the results generated with tumor cell targets transfected with costimulatory molecules suggest that these molecules can trigger NK cells, they do not necessarily imply that these molecules alone are responsible for the NK cell-sensitive phenotype of DC. Most likely, modulation of other molecules may counteract the (possible) triggering effect. In line with a possible triggering role for costimulatory molecules on DC, Carbone and colleagues observed recently that NK cell recognition of DC was partially blocked by incubating targets with mAbs against CD40, suggesting that this molecule may be one of several ligands involved in NK cell lysis of DC (E. Carbone, manuscript in preparation). On the other hand, recent studies by Chambers have demonstrated that murine DC from CD80, CD86, or CD80/CD86 and CD40 deficient mice are still susceptible to lysis by autologous murine NK cells at levels similar to wild type DC (B. J. Chambers, personal communication). Taken together, several different cell surface molecules, costimulatory and MHC class I molecules most likely contribute to render DC, and in particular immature DC, highly susceptible to lysis to autologous NK cells. Further studies should focus on identifying key molecules in these interactions.
One implication of the present observations is that there is a molecular match between receptors and ligands on DC and NK cells. This suggests that, under certain conditions, DC and NK cells may be able to communicate with each other, either to stimulate or prevent immune responses. Indeed, indirect evidence for a functional role of DC-NK cell interactions can be found in the literature. In short, NK cells have in different models been demonstrated to affect the outcome of adaptive immune responses (6, 7, 34), and, in certain in vitro experimental models, a direct role for DC-NK cell interactions has been suggested (35, 36, 37, 38). Hypothetically, NK cells could, under certain circumstances, have a role in activating DC cells. Such a role has recently been described for CD4+ T cells in their interactions with DC (39, 40, 41). Likewise, DC may, under certain circumstances, play a role in the activation of NK cells. Such dialogue may occur either directly via cell surface receptor-ligand interactions or via cytokines.
In conclusion, the present studies demonstrate that human DC can be
targeted by NK cells. The present results should stimulate studies
aimed at identifying molecules involved in the interactions between DC
and NK cells and the physiological relevance of such interactions
between DC and NK cells in vivo. A particular subject of interest is
the NK cell-mediated control of development of adaptive immune
responses and the role of DC in this event. In this respect, it is of
interest to note that a recent study in a murine model suggested that
cell-to-cell contact between DC and resting NK cells resulted in a
substantial increase in both NK cell cytolytic activity and IFN-
production (42).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hans-Gustaf Ljunggren, Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm, Sweden. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; MCM, monocyte-conditioned media; EBV-BLCL, EBV-transformed B lymphoblastoid cell line; LAK, lymphokine-activated killer cell; LGL, large granular lymphocyte; CD40L, CD40 ligand; rh, recombinant human.
Received for publication May 5, 1999. Accepted for publication September 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Adv. Exp. Med. Biol. 417:21.[Medline]
. II. Functional analysis. Blood 90:1458.. J. Exp. Med. 184:695.This article has been cited by other articles:
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) and day 7 monocyte-derived DC (•). Data represent
one of five independent experiments.
). Data represent one of five
independent experiments.
) or LPS (
), or through CD40L
stimulation (
). Data represent one of four independent experiments.
Immature DC vs MCM matured DC, p < 0.02; immature
DC vs LPS matured DC, p < 0.005; immature DC vs
CD40L matured DC, p < 0.005. Statistical analysis
made from data at the 50:1 E:T ratio.
) or DC
infected with influenza virus (
). DC were infected for 2 h
(A) or for 24 h (B). Data represent
one of four independent experiments.
