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Prepublished online as a Blood First Edition Paper on July 25, 2002; DOI 10.1182/blood-2001-11-0106.
IMMUNOBIOLOGY
From the Department of Cellular Immunology, Flanders
Interuniversity Institute for Biotechnology, Vrije Universiteit
Brussel, St Genesius Rode, Belgium.
Natural killer (NK) cells fulfill essential accessory functions for
the priming of antigen-specific cytotoxic T lymphocytes (CTLs).
On the basis of a NKG2D-ligand-positive tumor model, we obtained
results implicating NK-mediated regulatory as well as NK-mediated
cytolytic activities in the initiation and persistence of CTL activity.
Indeed, CD8+ T-cell-dependent tumor rejection requires NK
cell function in vivo, because tumors will progress both on depletion
of NK cells or in the absence of optimal NK activity. Here we provide
evidence that the absence of NK cells during subcutaneous tumor growth will abrogate generation of antitumor CTL responses and that this process can be linked to the expansion of alternatively activated monocytes. Indeed, our in vitro studies demonstrate that in splenic cultures from NK-deficient tumor-bearing mice, lack of type
1-associated cytokines correlates with the presence of type 2 (alternatively activated) monocytes and the production of type 2 cytokines. Furthermore, these type 2 monocyte-containing splenic
adherent populations potently suppress subsequent memory CTL
restimulation. We evaluated the role of NK lytic effector functions in
the efficient switch of the immune system toward classical (type 1)
activation by including differentially activated monocytic populations
as targets in cytotoxicity assays. The results indicate that the
accessory function of NK cells depends partially on the ability of
activated NK cells to preferentially engage type 2 antigen-presenting
cells. Thus, when the immune system tends to be type 2 oriented, NK
cells can drive an efficient type 2 The crucial role of natural killer (NK) cells in
bridging innate and adaptive immunity through modulation of the
cytokine network is widely acknowledged. Indeed, following stimulation, through NKG2D receptor signaling1 or contact-dependent
interactions with mature dendritic cells,2-5 NK cells are
the major source of the type 1-polarizing cytokine
interferon- Besides an immunoregulatory role of NK cells through cytokine
production, these cells might also perform accessory functions through
cell-cell contact-dependent interaction with antigen-presenting cells
(APCs). Both positive (activatory)15 and negative
(lytic)16-24 regulatory interactions have been
substantiated. In the latter condition, several APC surface molecules
were found to be possibly involved in the lytic interaction, either
activatory such as B7-1/B7-2,16,17,21,22 CD40L,20 CD54,23 HLA,24 or
inhibitory such as CD125 and major histocompatibility
(MHC) class I molecules.26 As such, one
might assume that the functional result of the interaction is
determined by the sum of the opposing signals. Moreover, the outcome of
NK/APC interaction seems to depend on the cytokine milieu.24,26 The implications of these NK/APC interactions for the adaptive immune response however remain speculative.
According to recent studies, different subsets of APCs can develop,
dependent on the resident type 1/type 2 cytokine balance, namely
classically activated APCs (DC1/M1 or type 1-associated) versus
alternatively activated APCs (DC2/M2 or type 2-associated), and
both are antagonistically regulated. DC1/M1 are the final targets and
effectors of proinflammatory processes,27,28 whereas DC2/M2 appear to participate in anti-inflammatory processes, tolerance induction, and wound healing and express a distinct set of molecules and receptors used in innate immunity.29-31 Both subtypes
of APC can, however, exert inducing and/or down-regulatory effects on adaptive immune responses.
For instance, during the process of malignant cancer, tumor immune
escape can be mediated by the activity of tumor suppressor APCs that
belong to either the DC1/M1 or DC2/M2 phenotype. Indeed, inflammatory
macrophages or M1 can exert suppressive activities, for instance, by
the secretion of nitric oxide (NO).32 However, many tumors secrete or induce IL-10 that steers immature dendritic cells toward the DC2 phenotype and are able to induce immune
tolerance.33,34 Furthermore, immunosuppressive macrophages
have been identified in the lymphoid organs of immunosuppressed,
tumor-bearing mice, comprising a population of immature
Gr1+/CD11b+ myeloid
precursors.35-37 Exposure of these
Gr1+/CD11b+ cells to IL-4 (type 2 cytokine)
greatly increased the T-lymphocyte suppressive activity of this
population, whereas type 1 cytokines induced their maturation into
competent mature DC1.38,39 Considering the multiple
possible effects of NK cells on DC/M either via soluble factors or by
direct cellular interactions (see above) might envisage severe
implications of NK-depletion or deficiencies on the cellular composition of the APCs that will concomitantly affect adaptive immune
responses, including CTL activity.
In this study we have analyzed the possible involvement of NK cells
(via either cytokine production and/or cytolytic activity) in
determining the cellular composition of APCs and evaluated the
resulting effects on antitumor CTL responses. Hereby, the BW-Sp3
lymphoma model was adopted because rejection of BW-Sp3 depends on
eliciting strong CTL responses,40,41 and consequently this
model allowed us to examine the involvement of NK cells in early
CD8+-dependent immune reactions. To address the importance
of NK cells for the early antitumor defense, Mice
Tumor cells
CTL induction For the generation of CTLs, splenocytes from 2 mice were restimulated in 75-cm2 flasks in the presence of 107 irradiated (110 Gy) cancer cells (primary CTL cultures). Alternatively, AKR mice were immunized intraperitoneally 3 times with weekly intervals, with 2 × 106 irradiated BW cells and restimulated on top of adherent cells isolated from primary CTL cultures (called secondary cultures). Five days later the cytolytic activity of the viable lymphocytes was tested in a classical 111In-release assay.46In vivo experimental settings For NK depletion, mice were injected intravenously in the tail with 0.02 mL -ASGM-1 antibodies (Wako Chemicals, Osaka,
Japan), 24 hours prior to tumor inoculation, and repeated every
4 to 5 days. In NK depletion experiments, specific depletion was
evidenced by flow-activated cell sorter (FACS) and spontaneous
cytotoxic activity to YAC-1. Mice in groups of 6 were injected
subcutaneously in the right flank with 2 × 106 cancer
cells in 0.2 mL phosphate-buffered saline (PBS). For every treatment,
mortality of the hosts and tumor growth were followed up twice a week.
To test spontaneous NK cell activity of tumor-inoculated hosts after
tumor inoculation (3 or 6 weeks), viable splenocytes were isolated from
mice with regressed versus progressing tumors and immediately, or on
complement-mediated lysis of NK or CTL fractions, included in
111In-release cytotoxicity assays. To deplete NK or
CD8+ cells, 108 splenocytes were incubated with
-DX5 and -ASGM-1 or -CD8 (TIB211, ATCC), respectively,
followed by complement-mediated lysis, using the supplier's protocol
(Harlan Sera-lab, Loughborough, United Kingdom).
Generation of A-LAK cells IL-2-activated LAK cells were prepared as described elsewhere.16 For the generation of IL-2/IL-12 LAK cells, recombinant mouse IL-12 (generously provided by Genetics Institute, Cambridge, MA) was added to the IL-2 LAK cultures at 100 U/mL, 72 hours after initiation of the cultures for another 48 hours. The adherent cell fraction was harvested with 0.01% EDTA (ethylenediaminetetraacetic acid) in PBS and was used as effector cell population in 111In-release cytotoxicity assays.111In-release cytotoxicity assay The target cells were labeled with 111In as described elsewhere.47 After extensive washing, 104 target cells were incubated with 2-fold dilutions of effector cells in a total volume of 200 µL culture medium (CM) in 96-well round-bottom plates. All experiments were performed in triplicates and twice repeated. After 4 hours at 37°C, 100 µL supernatants was collected, and radiation was counted in a gamma-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 targets cells incubated without
effector cells and maximal lysis from target cells in 2% sodium
dodecyl sulfate (SDS).
Determination of arginase activity Arginase activity was measured in cell lysates as previously described.48 One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol of urea per minute.NO measurement NO was measured as nitrite by using the Griess reagent. Culture supernatant was mixed with 100 µL 1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, and 2.5% H3PO4. Absorbance was measured at 540 nm in a microplate reader.Cytokine assays The production of IL-2, GM-CSF, IL-4, IL-10, monocyte chemoattractant protein 1 (MCP-1), IFN- (BD Biosciences, San Diego, CA), and TNF- (R&D Systems, Minneapolis, MN) was quantified by subjecting culture supernatants to commercially available sandwich enzyme-linked immunosorbent assay (ELISA) tests according to the manufacturers' protocols.
FACS staining and analysis of splenic cells Cell samples comprising 106 cells were incubated with the appropriate dilutions of antibodies, as suggested by the distributors, at 4°C for 30 minutes. Rat anti-CD11b-fluorescein isothiocyanate (FITC), anti-CD4-FITC, anti-CD8-FITC, anti-DX5-FITC, anti-CD80-FITC, anti-CD86-FITC, mouse anti-H-2Dd-FITC, anti-IEd/IAd-FITC, and hamster anti-CD69-FITC and anti-CTLA-4-phycoerythrin (PE) were purchased from BD Biosciences. Anti-NKG2-FITC was kindly provided by K. Van Beneden (UZ-Gent, Belgium). To prevent Fc receptor (FcR)-mediated binding of staining antibodies, cells were blocked with 2.4G2 (ATTC) derived F(ab)2 antibodies prior to the addition of the indicated antibodies. For ASGM-1 staining, the IgG fraction was purified from antiserum (Wako Chemicals) and used in combination with FITC-conjugated mouse/rat/human-preadsorbed goat antirabbit IgG (BD Biosciences). As isotype controls for aspecific binding, control antibodies were purchased from the distributors and included during FACS staining. The stained cells were subjected to FACS analysis with the BDFACS Vantage. The results were analyzed with CellQuest software.Statistical analysis The statistical significance of the differences in subcutaneous tumor growth rate or percentage of specific release was assessed by a 2-sided analysis of variance (ANOVA) test, and significance of differences between experimental groups (presented as bars, mean ± SEM) was interpreted by an unpaired Student t test, applying GraphPad Prism for MAC software (GraphPad Software, San Diego, CA). Differences are considered as not significant for P .05, significant for P < .05, and very
significant for P  .01.
Depletion of NK cells accelerates tumor progression We used as a tumor model the immunogenic mouse T-cell lymphoma (BW-Sp3) that expresses high levels of MHC class I. Following subcutaneous inoculation, BW-Sp3 cells form primary tumors that will either progress or regress (Figure 1).49 We have demonstrated previously that CD8+ T cells are the critical immune effectors involved in the control of BW-Sp3 tumorigenicity.40,41,49 To investigate the importance of NK cells in early local antitumor immune responses, AKR mice were treated with-ASGM-1. In first instance, we verified the NK-specific
effect of this treatment in the spleens of naive mice versus those of
tumor-bearing mice 8 days after tumor injection, of which the
latter are expected to contain activated tumor-specific CTL. Both the
DX5+ and NKG2+ populations, which are almost
exclusively ASGM-1+, are severely reduced by the -ASGM-1
treatment (Table 1 and result not shown).
The -ASGM-1 depletion causes a slight reduction in the number of
ASGM-1+/CD8+ T cells, but not CD4+
T cells, in both the naive and the tumor-bearing condition, indicating that the affected T cells do not represent the activated pool (Table
1). Indeed, IFN--producing CD8+ T cells can only be
detected in the ASGM-1 population, dissociating the
activated and ASGM-1+ phenotype (Figure
2). This finding indicates that
-ASGM-1 depletion primordially affects NK cells but not activated T
cells. In our tumor model this depletion increased tumor growth rate
(P < .0001) and was sufficient to abrogate tumor
regression in all recipients, resulting in early death of 50% of the
hosts after 18 days (Figure 1). Thus, NK cells may be implicated in
local antitumor responses against BW-Sp3.
NK lytic activity is suboptimal in recipients with progressing tumors To further substantiate the possibility that NK cells are involved in the immune response to BW-Sp3, we monitored the general activation status of peripheral NK cells during subcutaneous tumor growth by incubating splenocytes, freshly isolated from tumor-challenged mice, with the NK-sensitive target YAC-1 and by evaluating lytic activity. We observed that in hosts in which the local tumor was regressing (Figure 3Ai,ii) or had regressed (Figure 3B), NK activity was higher than in naive mice. Furthermore, in spleens of mice with progressing tumors, 3 (Figure 3Aiii) or 6 weeks (Figure 3B) after tumor inoculation, NK activity was substantially depressed as compared with those of naive mice and regressors (Figure 3). The effect, observed with the total splenocyte fraction, was substantially reduced after removal of DX5+/ASGM-1+ cells from the effector population (Figure 3A). On the contrary, in vitro depletion of CD8+ splenocytes enriched for YAC-1-reactive effectors, corroborating that the lytic cells reside within the NK population. These results suggest that ex vivo NK activity correlates with BW-Sp3 tumor outcome.
NK depletion results in impaired CTL activity Because BW-Sp3 tumor rejection relies on both CD8+ cytolytic T cells40,41,49 and NK cells (see earlier section), the role of NK cells in early antigen-specific CTL activity in tumor-bearing mice was investigated. Splenocytes of tumor-inoculated immunocompetent and NK-depleted mice were restimulated in vitro and subsequently tested for their CTL activity. Within 5 to 8 days after tumor inoculation, immunocompetent mice generated substantial levels of specific effector CTLs that progressively reached a maximal activity around day 12 (Figure 4A). This time point coincides with the time when tumor regression is initiated (Figure 1). On the contrary, NK cell depletion completely prevents the generation of CTL effector function. Indeed, as shown in Figure 4A, the residual CTL activity of NK-depleted splenocytes does not exceed the CTL activity of restimulated naive spleens up to 12 days following tumor injection (P .5). The inferior CTL activity of in
vitro-restimulated splenic cultures, derived from -ASGM-1-treated
mice, could be correlated with a lower percentage
(P .01 for all time points except for day 5 (P = .06) (Figure 4B) and a lower level of activation of
CD8+ T cells (Figure 4B). The latter was evidenced by a
smaller fraction of CD8+CTLA4+ and
CD8+CD69+ T lymphocytes in the
-ASGM-1-depleted conditions as compared with the immunocompetent
conditions (21% versus 35% [day 8] and 18% versus 28% [day 12]
of the CD8+ T cells are double-positive for CTLA4, whereas
12% versus 18% [day 8] and 17% versus 27% [day 12] are
double-positive for CD69; Figure 4B). The absence of NK cells does not
significantly influence the number of CD4+ splenocytes
(P .06 for all time points) or their activation status
(Figure 4C). Again, the relative contribution of ASGM-1+
cells to the CD8+ T-cell pool is unchanged, regardless of
the presence and expansion of antitumor CTLs (Table
2), corroborating that BW-Sp3-activated CTLs are not typically characterized by ASGM-1 expression.
NK cell depletion promotes type 2 cytokine responses Because NK cells can be active producers of cytokines, it was appropriate to monitor the presence of type 1/type 2 cytokines in the different CTL cultures. In addition, because the chemokine MCP-1 was reported to fuel the development of type 2 immune responses, its production was also evaluated. As shown in Figure 5A, on subcutaneous inoculation of BW-Sp3 cells, type 1 cytokines (ie, IFN-, TNF-, IL-2, and GM-CSF) are
progressively induced (.001 P < .03 in all cases)
and reach a maximum within 8 days. In contrast in -ASGM-1-treated tumor-bearing animals, the production of type 1 cytokines barely rises
above background levels and is always significantly lower (.001 P .04) than in the corresponding immunocompetent
conditions (Figure 5A). However, the production of the type 2 cytokine
IL-4 was significantly higher in cultures derived from NK-depleted mice, at least at 8 days (P = .03) to 12 days
(P = .027) after tumor inoculation (Figure 5B). This
finding was, however, not the case for IL-10, when we did not measure
substantial differences in any situation (results not shown). Finally,
as shown in Figure 5B, the constitutive production of the type
2-associated chemokine MCP-1 became progressively reduced in
tumor-conditioned immune splenocytes from day 5 (P = .03)
and day 8 (P = .005) through day 12 after receiving the
tumor load (P < .001), and its expression remained high
through day 12 in the absence of NK cells (P = .4) as
compared with the naive control.
NK cell depletion promotes the development of alternatively activated monocytes (M2) Our earlier studies have indicated a physiologic interaction between NK cells and APCs.16 In the present study we also observed that the number of splenic adherent cells expressing CD11b was substantially higher in CTL cultures derived from NK-depleted animals (Figure 6A). Microscopic examination of the above mentioned CTL cultures, revealed a striking difference in the morphologic appearance of the adherent fractions from the CTL cultures derived from NK-depleted tumor-bearing mice as compared with those derived from immunocompetent tumor bearers (Figure 6B), suggesting a role of NK cells in the regulation of the phenotype and/or activation status of adherent splenic cells. This difference is also expressed by the fact that in our culture conditions, M2 have a substantial induction in CD11b expression level (Figure 6B). Moreover, ASGM-1 is uniformly expressed on both M1 and M2, excluding a preferential engagement of one population by-ASGM-1 (Table 2).
Hence, taking these observations into account together with our
aforementioned results demonstrating a switch from a type 1- to a type
2-dominating cytokine response in the absence of NK cells, it was
appropriate to test whether in the present experimental conditions
different types (M1/M2) of macrophages are elicited. Thus, NO was
measured in the supernatant from CTL cultures, and the adherent
fractions were tested for arginase activity. The results compiled in
Figure 7A show that the adherent
fractions of CTL cultures, derived from BW-Sp3 tumor-bearing AKR mice,
produce important quantities of NO, whereas, on in vivo depletion of NK
cells, the NO production in the CTL cultures drops to background
levels. Furthermore, in contrast to the up-regulation of NO production
under immunocompetent conditions, the adherent fractions derived from
NK-deficient cultures preferentially express the arginase pathway
(Figure 7B). Indeed, the arginase activity is significantly higher in
these cell populations as compared with populations derived from naive
(P .02) or immune tumor-bearing (P .01)
AKR. Hence, NK depletion of tumor-bearing mice favors the expansion of
splenic M2 cells.
Adherent splenic cells conditioned by NK depletion can suppress memory CTL activity To determine whether the adherent cells, including M2 cells, identified in the NK-depleted cultures, can directly influence CTL activity, memory T cells from BW-Sp3-immunized mice were restimulated in the presence of the aforementioned adherent populations. Respecting physiologic T/monocyte proportions as they are present during primary CTL restimulation, the results depicted in Figure 8A show that the adherent fraction of splenocytes from day 5 and day 8 tumor-bearing NK-deficient mice can completely block memory CTL restimulation (P < .0001). In contrast, although cultured adherent splenocytes from mice, injected with BW-Sp3 on day5, suppress CTL activity with 71% ± 13%
(P = .0007), this suppressive activity disappears in
adherent splenocytes from mice injected with BW-Sp3 on day 8.
Notably, cultured adherent cells from naive spleens can also block
efficiently CTL effector function (P < .0001) (Figure 8A). Furthermore, there was a perfect correlation between suppression of CTL activity and arginase activity in the adherent fractions of the
secondary cultures (Figure 8B). To exclude that the suppressive activity solely depends on quantitative effects (the number of adherent
cells is substantially higher in NK-depleted conditions), we repeated
these experiments by restimulating BW-Sp3-specific memory T cells in
the presence of equal numbers of adherent splenocytes derived from
primary cultures. Again, the suppressive activity remains predominantly
associated with the condition of NK depletion (Figure 8C) and
correlated to arginase activity (Figure 8D). These results suggest that
adherent splenic populations from both naive mice and NK-depleted
tumor-bearing mice are activated during the secondary CTL cultures
toward M2 cells that are highly suppressive on CTL activity. In
contrast adherent splenic populations from tumor-bearing mice are less
prone to develop into suppressive M2 cells.
Alternative activation renders macrophages susceptible to NK-mediated lysis As mentioned above, depletion of NK cells, besides favoring the development of M2 cells, leads to an increase in the number of adherent cells in the splenic cultures (Figure 6B). In earlier studies, we and others have demonstrated that autologous antigen-presenting cells can become targets for LAK-mediated cytolysis, provided the NK cells are properly activated.16,17 Thus, it was of interest to test whether M1 versus M2 cells differ in their susceptibility toward activated NK cells (Figure 9A). In the first instance, mouse macrophagelike RAW264.7 cells were steered toward a M1 (cultured in the presence of IFN-) or M2 (cultured in the
presence of IL-4 and IL-10) phenotype, using the NO/arginase balance as
a read-out system to verify the M1 versus M2 phenotype of the
cytokine-treated RAW264.7 cells (Figure 9B). Subsequently, both
RAW264.7 populations were tested as targets in LAK cell-mediated
cytotoxicity experiments. The results depicted in Figure 9A indicate
that steering of RAW264.7 to M1 strongly reduces the susceptibility of
the targets to LAK lysis (23% specific lysis at an effector-target
ratio of 40:1) as compared with Il-4/IL-10-treated targets (54%
specific lysis at an effector-target ratio of 40:1;
P < .0001). Because we described several antigens
contributing to NK recognition in earlier studies,16 we
evaluated the surface expression of MHC and B7 antigens on the
differentially activated RAW264.7 cells by FACS. The expression of MHC
class I and II is considerably higher on RAW264.7 (IFN-) as compared
with RAW264.7 (IL-4/IL-10) (Table 3),
supporting a role for MHC class I in conferring resistance to NK lysis.
Moreover, the expression of NK (co)stimulatory B7-1 and B7-2 is twice
as high on RAW264.7 (IL-4/IL-10) versus RAW 264.7 (IFN-) cells. Secondly, the adherent fractions of the different primary CTL cultures
(see above) were also tested for their susceptibility to
IL-12-activated LAK cells. As shown in Figure 9C, consistent results
could be obtained: M2-like cells from NK-deficient conditions were
significantly better lysed (P .0001) than M1-like cells from immunocompetent cultures. Hence, NK cells can be involved in
preferential physical elimination of M2 in vivo.
The contribution of NK cells in the regulation of CTL activity has
been documented in a number of experimental models, including xenospecific CTL generation,8,50 induction of influenza
virus-specific CTLs,51 and priming of tumor-specific
CTLs.52,53 Overall, in these studies the mechanisms
underlying NK-mediated regulation of CTL activity were not exactly
defined and remain controversial. Indeed, Smyth and Kelly8
and Smyth et al50 demonstrated that, although
CD4+ T cells are critical for a mouse antihuman
xenospecific CD8+ CTL response, NK1.1+ cells
play an IFN- Difficulties in analyzing the NK dependence of CTL activation, relies
partially on the NK specificity of the antibodies that are widely
applied to deplete NK cells in vivo. Indeed, NK/T cells express T-cell
markers as well as NK cell markers, but several studies have
in addition demonstrated that antigen-specific T cells can express any
of the specific NK cell markers (DX5, NK1.1, ASGM-1, Ly49) at selective
steps during CTL differentiation or activation.44,54-59
Also ASGM-1, used in our study to specifically target NK cells for in
vivo depletion, has had its controversies. Early studies demonstrated
that cytotoxic treatment of splenocytes with anti-ASGM-1 specifically
removed NK cells and was ineffective in removing alloreactive killer
and helper T cells.43,58,60,61 Other studies on
virus-specific CTLs have suggested an influence of The results presented herein unequivocally link NK activity and CTL-mediated antitumor responses and furthermore provide evidence for novel regulatory mechanisms possibly implicated in the NK/CTL interaction. Indeed our analysis of the involvement of NK cells in the BW-Sp3 tumor model revealed the following main findings: (1) Progression of BW-Sp3 tumors leads to both impaired anticancer cell CTL and NK cell activities, and depletion of NK cells during the early phase of BW-Sp3 tumor development impairs the development of CTL responses and increases tumor progression; (2) NK cell depletion during early BW-Sp3 tumor formation alters the tumor-elicited splenic cytokine production in favor of a type 2 cytokine microenvironment and promotes the development of alternatively activated splenic macrophages (M2 cells); (3) M2-enriched splenic adherent cells suppress potently memory CTL activity; (4) M2 cells are more susceptible to NK-mediated lysis as compared with classically activated macrophages (M1 cells). The induction of type 1 cytokines by BW-Sp3 cancer cells, as documented
in this and previous reports,41 may reflect the inherent
capacity of these malignant cells to activate the innate immune system.
Indeed BW-Sp3 cells express a ligand (ie, Rae-1; J.A.V.G., unpublished
observation, 2001) for the recently identified stimulatory
lectinlike NKG2D receptor that is expressed by NK cells and activated
macrophages in mice.1,63,64 Interaction between NKG2D and
ligands (Rae-1, H60) expressing tumor cells triggers NK cytotoxicity
and IFN- The differential generation of M1 versus M2 cells depends on the
resident type 1/type 2 cytokine balance that, in turn, can be
determined by the genetic background of the host. Indeed in mice
certain strains are more prone to develop type 1 or type 2 cytokine
responses and, respectively, M1 or M2 cells.66 In this
context it is appropriate to mention that the mouse AKR strain used in
this study is strongly type 2 oriented (J.A.V.G., unpublished results,
2001) and, hence, biased toward the development of M2 cells.
According to our results, provision of strong type 1 cytokine triggering signals, such as NKG2D ligand-expressing BW-Sp3 cells, may
overcome a constitutive type 2-oriented immune system, resulting in
the local production of type 1 cytokines (Figure 5) and hereby associated NO-producing M1 cells (Figure 7). In the absence of NK cells
this increased production of type 1 cytokines drops to background
levels, and concomitantly arginase-producing M2 cells dominate over
NO-producing M1 cells. This shift in cell phenotype in the absence of
NK cells is further supported by a striking difference in adherent cell
morphotype and by a marked increase in expression level and number of
CD11b+ cells (Figure 6). Hence, in our experimental tumor
model NK cells play a crucial role in determining whether an ongoing
antitumor response will support alternative or classical activation of
macrophages. In agreement with other reports, the occurrence of M2
cells correlated with a limited production of type 1 cytokines and a
sustained (IL-10) or increased (IL-4) production of type 2 cytokines.27,66-68 Our cytokine pattern analysis further
revealed an intriguing role for MCP-1, a C-C( Besides playing an indirect role in directing type 1-oriented responses and consequently altering the macrophage activation status toward the M1 phenotype, NK cells may also directly control the M1/M2 balance through their selective cytolytic activity. Indeed, our results indicate that M2 cells are better targets for activated NK (LAK) cells than M1 cells. In earlier studies, we and others have shown that NK cells can successfully engage autologous antigen-presenting cells, provided that the appropriate activation signals are present.16-18,71 This process can involve both soluble factors such as IL-1216 and/or optimal ratios of NK-associated activatory and inhibitory signals.16,17,20-26,71,72 Accordingly, we observed in the present study that as compared with M1, M2 cells express lower levels of inhibitory MHC class I, likely involved in conferring resistance to NK lysis26,73; higher expression patterns of B7-costimulatory molecules, implicated in reverting MHC class I-mediated inhibition of NK lytic activity16,17; and higher levels of activating CD11b (CD18) adhesion molecules, formerly described to confer NK sensitivity.71 Thus, M2 cells express a pattern of surface molecules allowing successful engagement by NK cells. As such, our results confer a positive effect of NK-mediated elimination of M2 cells to CTL generation. This could be expected in view of the capacity of M2 cells to counteract proinflammatory signaling, which is crucial during the early phase of CTL responses. The M2-enriched splenic adherent populations that develop in our
experimental conditions suppressed potently the in vitro generation of
tumor-specific CTLs. Although M1 cells were amply documented to
suppress antitumor responses,74-76 more recent
investigations reported on an inhibitory population of adherent
CD11b/Gr-1 double-positive cells that cause reversible impairment of
tumor-specific CD8+ T-cell responses on direct cell-to-cell
contact. Immune suppression depended entirely on chronic GM-CSF
production that recruited these immature myeloid progenitors to the
spleen. Exposure to IL-4 alone increased inhibitory activity, whereas
Th1 cytokines sustained classic activation to APC.37-39,77
Besides those functional similarities, the myeloid suppressor cells
show some important differences from M2 described by others and
us.39 The researchers acknowledge the dissimilarities
between their Gr-1+/CD11b+-adherent population
and M2, stressing the immature phenotype of their suppressor myeloid
cells. Moreover, we can add some additional deviating features: First,
our activatory, but not our suppressor, macrophages support the
production of GM-CSF in the CTL cultures. This finding coincides with a
study that demonstrates that the presence of GM-CSF before vaccination
can induce a Th2 response, whereas administration of GM-CSF after
vaccination will predominantly enhance Th1 immunity.78
Secondly, none of the studies report the production of arginase in
their suppressor cells. Finally, the M2 cells in our model do not
express F4/80 (A.B.G., unpublished result, 2001) but do
express Fas-L, FcR Collectively, our results corroborate the crucial role of NK cells in the development and maintenance of antitumor CTLs. Our study further points to a new regulatory mechanism possibly implicated in the NK/CTL interaction, namely NK-mediated control of suppressive M2-like populations. This control function may rely on both NK-mediated regulatory (ie, initiation of type 1-oriented signaling) as well as NK-mediated cytolytic (ie, elimination of M2 cells) activities. Hereby, it should be emphasized again that our used strain is strongly type 2 oriented and as such more suitable to uncover NK cell/M2/CTL interactions. Indeed, other mouse strains such as C57bl/6 tend to support type 1 cytokine responses that do not favor M2 cell development.66 Incidentally, numerous studies on NK cell-mediated regulation were performed in the C57bl/6 (B6) background in which anti-NK1.1 antibodies are applicable for NK cell depletion. However, the human immune system tends to be type 2 oriented,79-83 and, furthermore, poor NK cell function is frequently observed in patients with advanced tumors.84,85 Hence, a regulatory pathway linking NK cells, M2 cells, and CTLs may be as well operative in human cancer.
We thank L. Brijs, M. Gobert, E. Vercauteren, and E. Omasta for excellent technical assistance; K. Van Beneden for providing anti-NKG2 antibodies; and both Hoffman-LaRoche, and Genetics Institute, for supplying the recombinant IL-12 used for this work.
Submitted November 30, 2001; accepted July 12, 2002.
Prepublished online as Blood First Edition Paper, July 25, 2002; DOI 10.1182/blood-2001-11-0106.
Supported by grants from the Foundation of Scientific Research-Flanders (FWO-no. 1.5.213.00). A.B.G. is a Postdoctoral Fellow of the Foundation of Scientific Research-Flanders (FWO).
A.B.G. and J.A.V.G. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Anja B. Geldhof, Department of Cellular Immunology, VIB-VUB/IMOL II, Paardenstraat 65, B-1640 St-Genesius Rode, Belgium; e-mail: abgeldho{at}vub.ac.be.
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Top
Abstract
Introduction
type 1 switch in the population
of antigen-presenting cells to provide signaling for the generation of CTLs.
(Blood. 2002;100:4049-4058)
], granulocyte-macrophage
colony-stimulating factor [GM-CSF], macrophage inflammatory
protein-
, 2 of
6 mice), immunocompetent mice with progressing BW-Sp3 tumors (
, 4 of
6 mice), or NK-depleted mice with progressing tumors (
, 6 of 6 mice). *Indicates the time point at which 50% of the
) in in vitro cytotoxicity experiments. (B) Splenic
effectors from naive mice (+), mice with regressed tumors (
) or BW-Sp3(B7-1) (
) target cells.
Effector-to-target ratio for the presented results is 100:1. (B)
Percentage of CD8+ T cells in restimulated CTL cultures.
indicates CD4+CD69
