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Microbiology and Tumor Biology Center, Karolinska Institutet, 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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The so-called costimulatory molecules have been characterized extensively in the interaction between T cells and APC. The interaction between B7 molecules and their ligands CD28 and CTLA-43 is critical in the control of T cell responses (13), and the expression of CD40 on dendritic cells (DC) is important for T cell priming and T cell-mediated effector function (14, 15, 16). The interaction between CD40 expressed on B cells and CD40L expressed on activated T cells is also crucial in enabling B cells to generate germinal centers, produce Abs, and switch Ig isotype (17, 18).
Some previous studies have suggested that costimulatory molecules may interact with NK cells. YT, a human NK-like leukemia cell line that expresses CD28, is able to lyse some human and murine cell lines expressing B7-1 (19). Murine NK cells have been demonstrated to proliferate and produce cytokines in a CD28-dependent manner (20), and the rejection of B7-1-transfected tumors has been suggested to involve NK cells (21). Recent data from our and other laboratories have demonstrated that B7-1 in mice (22, 23) and CD40 in humans (24) can trigger NK cell-mediated cytotoxicity in vitro. Additional data in the murine system have also indicated that B7-2 may trigger NK cell-mediated cytotoxicity in vitro (23).
In the present study, we have addressed the capacity of CD40 to trigger NK cell-mediated cytotoxicity in a mouse model. We have also included B7-2 transfectants in some experiments to extend our results with CD40 transfectants to a different costimulatory molecule within the same experimental system. We show that transfected tumor cell lines expressing CD40 or B7-2 trigger NK cell-mediated cytotoxicity in vitro, and that the triggering effect is observed even in the absence of CD40L and CD28, respectively, expressed on the effector cells. In addition, we show that radiolabeled CD40- or B7-2-transfected cell lines are rapidly eliminated in vivo in an NK cell-dependent manner. The latter response occurs even in CD40L-/- and CD28-/- mice, respectively. Collectively, these data suggest that costimulatory molecules may trigger NK cell-mediated cytotoxicity in vitro and in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) and BALB/c mice (4–8 wk old) were from the Microbiology and Tumor Biology Center, Karolinska Institutet (Stockholm, Sweden). CD40L-/- and CD28-/- mice, generated by homologous gene recombination, have been described previously (18, 25). All mice were maintained at the Microbiology and Tumor Biology Center, Karolinska Institutet. Animal care was in accordance with institutional guidelines.
Cell lines, transfections, and flow cytometry
RMA is a variant of the Rauscher virus-induced T cell lymphoma RBL-5 of B6 origin. RMA-S is an MHC class I-deficient (TAP-2-deficient) variant of RMA. B16.F1 is a subclone of the B16 spontaneous melanoma of B6 background. YAC-1 is a highly NK cell-sensitive, Moloney virus-induced T cell lymphoma of A/Sn background. All cell lines were grown in complete RPMI 1640 medium (5% FCS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 x 10-7 M mercaptoethanol), at 37°C in a humidified 5% CO2 atmosphere in air. Vectors containing mouse CD40 or B7-2 cDNAs were transfected by electroporation, as described (21). Transfected cells were selected in complete medium containing 2 mg/ml of G418 (Life Technologies, Grand Island, NY) for CD40 transfectants, or 20 µg/ml of Mycophenolic acid (Sigma, St. Louis, MO) plus 20 µg/ml of Xantine (Sigma) for B7-2-transfected cell lines. Surviving cells were selected by cell sorting in a FACS Vantage (Becton Dickinson, Mountain View, CA), using rat anti-mouse CD40 mAb FGK-45.5 (kindly provided by Dr. T. Rolink, Basel Institute for Immunology) or the rat anti-mouse B7-2 GL1 mAb (PharMingen, San Diego, CA). The sorted populations were then cloned by limiting dilution. For analysis of MHC class I expression, cells were stained with biotin-labeled anti-H-2Kb or Db mAb (both from PharMingen), followed by FITC-labeled streptavidin (Dakopatts, Glostrup, Denmark). Analysis of the expression of the transfected molecules and MHC class I was performed in a FACScan (Becton Dickinson).
NK cell-mediated cytotoxicity
Two different protocols for activation of NK cells were used.
For activation with IL-2, 25 x 106
erythrocyte-depleted splenocytes were resuspended in 10 ml of 
MEM
medium containing 10 mM HEPES, 2 x 10-7 M
mercaptoethanol, 10% FCS (all reagents from Life Technologies), and
1000 U/ml human rIL-2 (Pepro Tech, Rocky Hill, NJ). Cells were cultured
in 25-cm2 flasks in 10% CO2, at 37°C. After
4 days, nonadherent and adherent cells were collected, washed once,
resuspended in complete medium without IL-2, and then used as
effectors. These effector cells are referred to as IL-2-activated NK
cells. For activation of NK cells with the IFN-inducer Tilorone
(Sigma), 0.2 ml of a 10 mg/ml solution of Tilorone was given i.p. per
mouse 24 h before sacrifice and removal of spleen. Single cell
suspension of erythrocyte-depleted splenocytes was used as effector
cells. These effectors are referred to as IFN-induced NK cells. The
cytotoxic assays were performed in standard 4-h 51Cr
release assay.
In blocking experiments, target cells were preincubated with 1 µg of rat F(ab')2 anti-mouse CD40 per 106 cells or with saturating amounts of the CTLA-4-Ig fusion protein. The incubation was performed on ice for 20 min, after which excess of Ab or soluble molecule was washed away with PBS.
Generation of Con A-activated T cell blasts
For the generation of Con A-activated T cell blasts, 107 erythrocyte-depleted splenocytes were cultured in complete RPMI medium supplemented with 3 µg/ml of Con A (Sigma) for 48 h, and then used as targets in a standard 4-h 51Cr release assay.
In vivo studies
Cell lines transfected with CD40 or B7-2 were labeled with 51Cr for 1 h at 37°C, washed three times in PBS, and resuspended in PBS. A total of 106 cells per mouse was injected i.v. Twenty-four hours after inoculation, mice were sacrificed, and the remaining radioactivity in the lungs was measured in a gamma counter. The results are expressed as percentage of the total activity inoculated. To deplete NK cells in vivo, 200 µg of the anti-NK1.1 PK136 mAb (American Type Culture Collection (ATCC), Manassas, VA) was injected i.p. 48 h before inoculation of the transfectants (26). Control mice received 200 µg of nonimmune mouse IgG (Sigma). CD28-/- mice were pretreated with the hamster anti-mouse CTLA-4 UC10-4F10-11 mAb (PharMingen), 200 µg/mouse, 48 and 24 h before inoculation of the radiolabeled cells.
Purification of splenic DC
Splenic DC were enriched by plastic adherence, as described (27). Briefly, spleens from B6 mice were cut into small fragments and digested with collagenase type IV (Sigma) at 37°C for 60 min to release DC. Low density cells were selected by centrifugation on a 35% BSA gradient (Sigma), cultured in plastic dishes for 90 min, after which nonadherent cells were washed away. The adherent cells were cultured in complete RPMI medium. During this time, the DC detached from the plates. After overnight culture, the cells were stained with FITC-labeled anti-B220 mAb RA3-6B2 (PharMingen), or with rat anti-MHC class II mAb M5/114 (ATCC) plus hamster anti-CD11c mAb N418 (ATCC), followed by biotin-labeled goat anti-rat (Caltag, South San Francisco, CA) plus FITC-labeled goat anti-hamster (PharMingen), and PE-labeled streptavidin. DC were identified as B220-, CD11c+/MHC class IIhigh, and at least 80% of the cells were DC by these criteria.
Statistical analysis
Statistical analysis from the in vivo experiments was performed using a paired Student’s t test. A p value of <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genes encoding for CD40 and B7-2 were transfected into the
CD40-, B7- RMA, and B16.F1 cell lines, and
clones expressing the transfected gene products were selected. Care was
taken to select clones with unaltered expression of MHC class I. The
expression of H-2 Kb, Db, CD40, and B7-2 on the
transfected cell lines generated and used in the present study is shown
in Table I
. For comparison, MHC class I
expression on freshly isolated splenic DC is shown. MHC class I
expression on mock-transfected cell lines (RMA-neo, RMA-gpt, and
B16.F1-neo) was similar to that of wild-type cells (data not shown).
The expression levels of CD40 and B7-2 were monitored every second to
third week, and no alteration was observed over time.
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Two different types of activated NK cells were used to address the
question as to whether NK cells could be triggered by the expression of
the costimulatory molecules on target cells. In a first set of
experiments, splenocytes were activated in vitro with IL-2. In the
second set of experiments, splenocytes from mice that had been injected
previously with the IFN-inducer Tilorone were used. B6-derived IL-2- or
IFN-activated NK cells did not kill RMA, RMA-neo, or RMA-gpt cell lines
efficiently (Fig. 1, A and
B). In contrast, similar effector cells readily killed RMA
cells transfected with CD40 (RMA-CD40 clone.16) and B7-2 (RMA-B7-2
clone.39) (Fig. 1, A and B). Different clones of
cells transfected with CD40, expressing similar levels of CD40 and MHC
class I as the RMA-CD40 clone.16 (data not shown), were killed equally
well by IL-2-activated NK cells (Fig. 1C). RMA cells
transfected with B7-2, expressing similar levels of B7-2 as the
RMA-B7-2 clone.39, were also killed equally well. In contrast, the
RMA-B7-2 clone.35, expressing low levels of B7-2, was not killed
efficiently (Fig. 1D). RMA-CD40 clone.16, RMA-B7-2 clone.39,
and RMA-B7-2 clone.35 were selected for the rest of the experiments
shown in this study.
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E, IL-2-activated NK cells killed B16.F1-CD40 clone.7 at
higher levels than the parental and the B16.F1-neo cell lines. This
result confirms that the effect of CD40 is not restricted to a single
tumor cell line.
Triggering of NK cells was not restricted to syngeneic B6 effectors.
BALB/c-derived NK cells killed RMA-CD40 clone.16 more efficiently than
RMA-neo (Fig. 2B), and with
these effectors, the RMA-CD40 clone.16 was as susceptible as YAC-1
cells. Notably, the difference between RMA-neo and RMA-CD40 clone.16
was, if anything, larger when BALB/c-derived NK cells were used.
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The IL-2-activated spleen cell culture contains a mixture of
different cell subpopulations, and it cannot formally be excluded that
the observed effects were mediated by effector cells other than NK
cells. To confirm that the killing of the transfectants was mediated by
NK cells, NK1.1+/TCR- cells were sorted from
IL-2-activated B6 splenocytes, and assayed against RMA-CD40 clone.16
and RMA-B7-2 clone.39. As shown in Fig. 3, only NK cells
(NK1.1+/TCR-), but not T cells
(TCR+/NK1.1-), were able to kill the RMA-CD40
clone.16 and the RMA-B7-2 clone.39. The results were not restricted to
specific subpopulations of NK1.1+ cells according to their
expression of specific Ly49 receptors (data not shown). Yet, it is
still an open question as to whether the current triggering effect
observed is an effect attributed to a particular subpopulation of NK
cells, or the whole population per se.
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To test critically whether the observed effects were due
specifically to the expression of the transfected molecules on the cell
surface, blocking experiments were performed with rat anti-mouse
CD40 F(ab')2 fragments or with CTLA-4-Ig fusion protein
before the cytotoxic assay. As shown in Fig. 4A, the killing of the
RMA-CD40 clone.16 by IL-2-activated NK cells was reduced to wild-type
levels when the target cells had been incubated with rat anti-mouse
CD40 F(ab')2 fragments, while addition of CTLA-4-Ig fusion
protein had no effect. Conversely, the NK-mediated killing of RMA-B7-2
clone.39 was reduced to wild-type levels when target cells were
preincubated with CTLA-4-Ig, but not when incubated with
F(ab')2 anti-CD40 (Fig. 4B). These data
demonstrate that the increased sensitivity of the transfected cell
lines to lysis by NK cell effectors is dependent on the presence of
CD40 and B7-2 at the cell surface.
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Is triggering of cytotoxicity by CD40 and B7-2 dependent on CD40L
and CD28 expressed by the NK effectors? To address this question, we
performed experiments in which IL-2-activated NK cells were prepared
from CD40L-/- and CD28-/- mice. As shown in
Fig. 5, A and B,
IL-2-activated NK cells from both B6 and CD40L-/- mice
killed efficiently RMA-CD40 clone.16. In a similar manner,
IL-2-activated NK cells from both B6 and CD28-/- mice
killed RMA-B7-2 clone.39 (Fig. 5, C and D).
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RMA-CD40 clone.16 and RMA-B7-2 clone.39 are rapidly eliminated in vivo in an NK cell-dependent manner
Previous studies have shown that MHC class I-deficient RMA
(H-2b) cell lines are efficiently eliminated in vivo in an
NK cell-dependent manner when injected into B6 mice, whereas MHC class
I-positive RMA cells are not (29; this study). As shown in Fig. 6, B6 mice efficiently eliminated RMA
cells transfected with CD40 and B7-2, whereas control cells were
spared. NK cell depletion resulted in an increase in the remaining
radioactivity in the lungs, indicating that the elimination of the
transfected cell lines is diminished in the absence of NK cells (Fig.
6).
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). These data show that
the in vivo elimination of the transfectants can occur in the absence
of CD40L (for RMA-CD40 clone.16) or CD28 (for RMA-B7-2 clone.39) on the
host effector cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In recent years, it has been understood that many NK cell-mediated effector functions are controlled by triggering as well as inhibitory signals (7, 8, 9, 10). The inhibitory receptors and signals are fairly well characterized, and there is now a more intensive interest in the characterization of molecules involved in the triggering of NK cells. It has been demonstrated, both in the murine and in the human system, that the expression of costimulatory molecules by different tumor cell lines can result in an increased susceptibility to lysis by NK cells. The transfection of murine B7-1 in different tumor cell lines triggers NK cell-mediated cytotoxicity in vitro (22, 23). Additional data have indicated that the same effect occurs with the murine B7-2 molecule (23). Moreover, human NK cell lines are triggered to kill in vitro the mastocytoma cell line P815 transfected with human CD40 (24).
The present study shows that the transfection of murine CD40 and B7-2 into the MHC class I+ thymoma cell line results in an increased sensitivity to NK cells. That the transfected cell lines are killed but the mock transfectants are spared indicates that the inhibitory signal delivered through MHC class I is overcome by the positive signal delivered by CD40 or B7-2.
In humans, a relatively high proportion of melanomas has been shown to express CD40 (30). The killing of the murine melanoma cell line B16.F1 transfected with CD40 extends the finding to a second tumor cell line, other than RMA, and opens the possibility that this cell type can be targeted by NK cells in vivo when expression of costimulatory molecules occurs.
B7 molecules expressed on APC interact with CD28 and CTLA-4 expressed on T cells (13). In a similar manner, CD40 expressed by B cells and DC interacts with CD40L expressed on activated T cells (14, 15, 16, 17, 18). Expression of CD28/CTLA-4 or CD40L on NK cells has been shown to be dependent on the maturation state, the method of activation, and the origin of the NK cells. Although CD28 is expressed on human fetal NK cells (31), it has been claimed to be lost after maturation and is absent on peripheral blood NK cells in adults. In mice, data regarding the expression of CD28 are controversial. CD28 expression has been reported on bone marrow-derived NK cells upon activation with IL-2 (32), but has not been detected on 4-day IL-2-activated splenic NK cells (22). In humans, CD40L has been reported to be expressed upon short-term culture of NK cells (24). In our system, CD40L expression was not detected on resting or on in vitro IL-2-activated NK cells, and no evidence for expression of CD40L on murine NK cells is available to our knowledge. In this context, the in vitro killing of RMA-CD40 and RMA-B7-2 cells by CD40L-/-- and CD28-/--derived NK cells, as well as the in vivo elimination of RMA-CD40 clone.16 and RMA-B7-2 clone.39 by CD40L-/- and CD28-/- mice, are not entirely surprising. The NK cell-mediated killing of tumors expressing costimulatory molecules in the absence of the "expected" ligand on effector cells has already been described. Chambers and coworkers (22) found that IL-2-activated NK cells derived from CD28-/- mice were able to kill tumor cells expressing B7-1 molecule. Moreover, NK cells derived from patients with a mutation on CD40L are able to kill in vitro the mastocytoma cell line P815 transfected with the human CD40 molecule (E. Carbone, unpublished data). Taken together, these and previous results do not exclude a role for CD28 and CD40L on NK cells under conditions in which these molecules are expressed, but they also indicate that receptors other than CD28 and CD40L, with a B7-1/B7-2- and CD40-binding capacity, respectively, may be expressed on NK cells.
Our results show that the triggering of cytotoxicity in vitro is a direct consequence of the presence of CD40 and B7-2 on the cell surface of the tumor cells, since blocking of these molecules results in reduced sensitivity to NK cell-mediated lysis. This and other observations has led to the suggestion of the existence of alternative CD40- and B7-binding molecules expressed on NK cells. In this scenario, the triggering would imply a direct contact between CD40 and B7-2, and these alternative receptors. Such CD40 and B7-2 receptors would then be expressed on NK cells after IL-2 or IFN activation, as effectors obtained in both ways mediate cytotoxicity against CD40 or B7-2 transfectants. An alternative hypothesis is that the observed effects are mediated by unrelated molecules. In this situation, CD40 and B7-2 would interact with molecules on the surface of the target cell, and these molecules would in turn trigger the NK cells. The contact between such molecules and CD40 or B7-2 would be disrupted when incubating the transfectants in vitro with anti-CD40 mAb or with CTLA-4, respectively.
Clones expressing high levels of B7-2 are killed, while the one expressing low levels is not, indicating that the costimulatory molecule must be present at a certain level on the cell surface to trigger NK cell-mediated cytotoxicity in vitro. We did not obtain transfectants expressing different levels of CD40, and therefore we cannot conclude that the observed effect is a general feature of the costimulatory molecules in its interaction with NK cells. One possibility to explain the different susceptibility of B7-2 clones is that the response is governed by a balance between positive and negative signals. With low levels of expression, the positive signal delivered through the costimulatory molecule is not sufficient to overcome the inhibitory one delivered through MHC class I. Alternatively, the outcome of the response is the result of a threshold effect: in this scenario, a certain level of costimulatory molecule would be required to trigger killing, irrespective of MHC class I expression.
The question of whether NK cells can interact directly with costimulatory molecules has been addressed mainly in in vitro experimental systems. These have demonstrated that different tumor cell lines transfected with such molecules trigger NK cell-mediated cytotoxicity. The role of NK cells in in vivo immune responses has to date been studied by depletion of this lymphocyte population with mAbs. These data support the notion that, indeed, NK cells are involved in the in vivo elimination of tumors lacking MHC class I expression (29), and in clearance of viruses and parasites (3, 4). Moreover, recent data have demonstrated their involvement in the course of autoimmune and inflammatory diseases (33, 34). A novel finding in our study is that the expression of the costimulatory molecules on tumor cells also results in their rapid elimination after i.v. inoculation into mice, and that mice depleted of NK cells before inoculation have an impaired capacity to clear the transfected cells. The effect is therefore dependent, at least in part, on NK cells, although we cannot exclude the possibility that other cells of the innate immune system could also be involved. In this sense, granulocytes have shown to be important in the antitumor immune response induced by B7-transfected tumors (21, 35). Furthermore, the fact that RAG-/- mice eliminate B7-1 transfectants as efficiently as normal B6 mice do (B. J. Chambers, unpublished data) indicates that the rapid elimination is independent of the presence of NK/T or T cells.
Another in vivo evidence on the role of costimulatory molecules in triggering NK cells comes from in vivo experiments with tumor cells transfected with B7 molecules. The transfection of B7 molecules into tumors has indeed become a useful tool to induce strong antitumor cytotoxic T cells (36), and in some particular experimental models, the involvement of NK cells in the antitumor response has also been implicated (21, 35).
To which extent the interaction between costimulatory molecules and NK cells is a relevant event in the course of the immune response is presently unknown. It is important to keep in mind that costimulatory molecules are expressed almost exclusively by APC, which have a key role in initiating, developing, and terminating immune responses. Therefore, it is tempting to speculate that the interaction between NK cells and APC could be a mechanism to control the generation of adoptive immune responses. In line with this idea, previous reports have suggested that NK cells might interfere with DC function (37, 38). In addition, NK cells mediate the killing of DC in a partially CD40-dependent manner.4 However, results proving a role for NK cell-mediated killing of APC in vivo are lacking. Finally, we should also consider the possibility that interaction of NK cells with APC through costimulatory molecules can lead to production of cytokines (19) or augmentation of NK cell proliferation (20).
Collectively, our report extends previous data on the capacity of costimulatory molecules to trigger NK cell-mediated cytotoxicity, and shows for the first time that murine CD40 is able to trigger murine NK cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alfonso Martín-Fontecha, Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, Box 280, S-171 77 Stockholm, Sweden. E-mail address:
3 Abbreviations used in this paper: CTLA-4, cytolytic T lymphocyte-associated antigen-4; CD40L, CD40 ligand; DC, dendritic cells.
4 E. Carbone et al. Recognition of autologons dendritic cells by human NK cells. Submitted for publication.
Received for publication December 28, 1998. Accepted for publication March 3, 1999.
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G. Sconocchia, J. A. Titus, A. Mazzoni, A. Visintin, F. Pericle, S. W. Hicks, F. Malavasi, and D. M. Segal CD38 Triggers Cytotoxic Responses in Activated Human Natural Killer Cells Blood, December 1, 1999; 94(11): 3864 - 3871. [Abstract] [Full Text] [PDF]
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J. L. Wilson, J. Charo, A. Martin-Fontecha, P. Dellabona, G. Casorati, B. J. Chambers, R. Kiessling, M.-T. Bejarano, and H.-G. Ljunggren NK Cell Triggering by the Human Costimulatory Molecules CD80 and CD86 J. Immunol., October 15, 1999; 163(8): 4207 - 4212. [Abstract] [Full Text] [PDF]
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), RMA-neo (•), RMA-gpt (
), RMA-CD40 clone.16 (
), RMA-B7-2
clone.39 (
); C, RMA-neo (•), RMA-CD40 clone.16
(
