1. No category

OK432-Activated Human Dendritic Cells Kill
Tumor Cells via CD40/CD40 Ligand
Interactions
This information is current as
of June 9, 2014.
Katy S. Hill, Fiona Errington, Lynette P. Steele, Alison
Merrick, Ruth Morgan, Peter J. Selby, Nikolaos T.
Georgopoulos, Dearbhaile M. O'Donnell and Alan A.
Melcher
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
9650 Rockville Pike, Bethesda, MD 20814-3994.
Copyright © 2008 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2008; 181:3108-3115; ;
doi: 10.4049/jimmunol.181.5.3108
http://www.jimmunol.org/content/181/5/3108
The Journal of Immunology
OK432-Activated Human Dendritic Cells Kill Tumor Cells via
CD40/CD40 Ligand Interactions1
Katy S. Hill,2* Fiona Errington,2* Lynette P. Steele,* Alison Merrick,* Ruth Morgan,*
Peter J. Selby,* Nikolaos T. Georgopoulos,3† Dearbhaile M. O’Donnell,3‡
and Alan A. Melcher3,4*
D
endritic cells (DC)5 bridge the innate and adaptive immune systems by sampling the cellular environment,
signaling to immune effector cells via receptor/ligand
interactions or cytokines, and presenting Ag to T cells (1, 2). To
orchestrate an appropriate immune response, DC must sense the
presence of extrinsic threat; they recognize “danger” as pathogenassociated conserved molecules through pattern recognition receptors, including TLR (3). A range of TLR ligands has been shown
to initiate the effective maturation of DC, which is essential for
immune activation. In the absence of full maturation, DC can conversely induce T cell anergy, leading to tolerance rather than innate
and adaptive immune priming (4).
Because DC can now be cultured in large numbers from patients
as well as normal donors, particularly from myeloid precursors,
there is a growing interest in using DC for clinical cellular therapy,
including priming of antitumor immunity (5). In this context, the
choice of reagent for DC maturation in vitro before administration
*Cancer Research U.K., St. James’s University Hospital, Leeds, and †Jack Birch Unit
for Molecular Carcinogenesis, Department of Biology, University of York, York,
U.K.; and ‡Academic Unit of Medical and Clinical Oncology, St. James’s Hospital,
Dublin, Ireland
Received for publication November 19, 2007. Accepted for publication June
29, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Cancer Research U.K.
2
K.S.H. and F.E. contributed equally to the manuscript.
3
N.T.G., A.A.M., and D.M.O. are joint senior authors.
4
Address correspondence and reprint requests to Dr. Alan Melcher, Cancer Research
U.K., St. James’s University Hospital, Beckett Street, Leeds, LS9 7TF, U.K., E-mail
address: alan.melcher@cancer.org.uk
5
Abbreviations used in this paper: DC, dendritic cell; OK-DC, DC matured with
OK432; LPS/IFN-DC, DC matured with lipopolysaccharide and IFN-␥; IDC, immature DC; HFF, human foreskin fibroblast; NHU, normal human urothelial; TRAF,
TNFR-associated factor.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
becomes critical. There is a general consensus that fully activated
DC, expressing appropriate markers of phenotypic activation and
secreting a Th1 profile of inflammatory cytokines, will be optimal
for the priming of specific T cell-mediated antitumor immunity by
Ag-loaded DC (4). Similarly, DC activation can provide the appropriate signals to support initiation of earlier, innate antitumor
immune priming via DC cross-talk with alternative effector cells
including NK, NKT, and ␥␦ T cells (6, 7). When designing protocols for DC culture and activation for patient use, it is clearly
imperative to ensure that DC maturation is optimal and the reagents used are available and suitable for clinical use.
In this regard, OK432 is a clinical-grade, penicillin-inactivated
and lyophilized preparation of Streptococcus pyrogenes, which ligates TLR-2 and/or ⫺4 (8), activates DC, and has potential applications for cancer therapy (9). OK432-matured DC effectively
prime Ag-specific T cell responses in vitro (10), and OK432 has
already been used to activate DC for clinical use (11). Importantly,
OK432 has been used for many years as a direct anticancer agent,
particularly in Japan, and has a well-established clinical safety
profile; hence it represents a promising immunomodulator for incorporation into DC-based trial strategies. However, complete
characterization of DC matured with OK432 (OK-DC), including
assessment of innate immune activation, has not yet been reported.
One increasingly recognized property of DC is their ability to
exert direct cytotoxicity against tumor cell targets. This has been
described in rodent as well as human systems, and has been attributed to various mechanisms such as TNF, TRAIL, Fas ligand,
NO, and perforin/granzyme (12–15). Although the physiological
relevance of DC cytotoxicity remains uncertain, there are clear
potential applications for cultured DC with the ability to kill tumor
targets, particularly if this killing is specific to tumor, as opposed
to normal cell, targets.
While analyzing the properties of OK-DC we found that, as
expected, OK432 was an effective DC maturation agent, increasing
the expression of Ag presentation and costimulatory markers and
inducing the production of inflammatory cytokines. Remarkably,
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In vivo, dendritic cells (DC) are programmed to orchestrate innate and adaptive immunity in response to pathogen-derived
“danger” signals. Under particular circumstances, DC can also be directly cytotoxic against tumor cells, potentially allowing them
to release tumor associated Ags from dying cells and then prime antitumor immunity against them. In this study, we describe the
innate characteristics of DC (OK-DC) generated in vitro after exposure of immature human myeloid-derived DC to OK432, a
penicillin-inactivated and lyophilized preparation of Streptococcus pyrogenes. OK-DC produced proinflammatory cytokines, stimulated autologous T cell proliferation and IFN-␥ secretion, expressed CCR7, and migrated in response to MIP-3␤. Moreover,
OK-DC displayed strong, specific cytotoxicity toward tumor cell targets. This cytotoxicity was associated with novel, OK432induced up-regulation of CD40L on the cell surface of OK-DC, and was absolutely dependent on expression of CD40 on the tumor
targets. These data demonstrate that maturation of human DC with OK432, an adjuvant suitable for clinical use, induces direct
tumor cell killing by DC, and describes a novel CD40/CD40L-mediated mechanism for specific DC antitumor cytotoxicity. The
Journal of Immunology, 2008, 181: 3108 –3115.
The Journal of Immunology
OK-DC were significantly more potent at inducing proliferation
and IFN-␥ secretion by autologous T cells than LPS/IFN-␥-matured DC (LPS/IFN-DC). OK-DC/T cell cocultures also displayed
innate killing of a range of tumor targets; however, the majority of
this cytotoxicity resided in neither the CD4 nor CD8 T cell fraction
of these cultures. Instead, OK-DC (but not LPS/IFN-DC) directly
killed tumor, but not normal, cell targets. Target cell death was
cell:cell contact dependent, but was not related to target Fas ligand
or TRAIL sensitivity. Instead, killing was associated with up-regulation of CD40L on OK-DC, and dependent on CD40 expression
by tumor cell targets.
Materials and Methods
Cell culture
Flow cytometry
All Abs were obtained from B.D. Pharmingen except anti-CD40L, which
was from R&D Systems. All analysis was performed using a FACSCalibur
cytometer (BD Biosciences).
DC phagocytosis
IDC, LPS/IFN-DC, or OK-DC were incubated with 1 mg/ml FITC dextran
(Sigma-Aldrich) for 30 min at 37°C and cells were washed three times in
FACS buffer before cell acquisition using a FACSCalibur cytometer (BD
Biosciences). Control IDC (not incubated with FITC dextran) were acquired at the same time to allow background levels of fluorescence to be
determined.
Cytotoxicity assays
Target cells were labeled with 51Cr; cocultured at different E:T ratios with
either T cell/DC cocultures, isolated CD4⫹ or CD8⫹ T cells, or DC for 4 h
(T cell) or 20 h (DC); and a standard 51Cr release assay was performed
(18). For DC, shorter killing assays over 4 h were also performed, which
showed similar results, although overall levels of death were lower (data
not shown). The JAM cytotoxicity test for DNA fragmentation was also
used as previously described (19). To examine the contact dependence of
DC killing, labeled cell targets and DC were separated using a 0.4-␮m
transwell membrane.
Anti-CD40L blocking Ab experiments
51
Cr cytotoxicity assays of OK-DC against CD40-expressing RT112 were
performed as above at an E:T ratio of 10:1, with addition of an anti-CD40L
Ab (1 ␮g/ml or 5 ␮g/ml), or 1 ␮g/ml isotype control (R&D Systems). DC
were preincubated with Ab for 30 min before addition of tumor cells to the
killing assay.
Results
OK432 induces maturation of human myeloid DC
Human myeloid IDC were treated with OK432 for 48 h to generate
OK-DC, or LPS/IFN-␥ to generate LPS/IFN-DC, and the surface
expression of MHC class I and class II, CD1a, CD80, CD86,
CD83, and CD54 were examined by flow cytometry. Fig. 1A demonstrates that, in general, OK432 stimulated equivalent or greater
phenotypic DC maturation than LPS/IFN-␥. No changes in CD56
and CD11c expression were identified (data not shown). To confirm that OK432 induced DC maturation, phagocytosis of FITC
dextran by IDC, LPS/IFN-DC, and OK-DC was determined, as
mature DC are less effective at phagocytosis than IDC. Consistent
with phenotypic maturation, we found that IDC were more efficient at phagocytosis than either LPS/IFN-DC or OK-DC
(Fig. 1B).
To further determine whether OK-DC were functionally active,
we next examined the ability of OK432 to induce the secretion by
DC of a range of proinflammatory cytokines. OK432, like LPS/
IFN-␥, induced production of TNF-␣, IL-6, and IL-12, which were
not secreted by IDC (Fig. 1C). In contrast, IL-10 (an immunosuppressive cytokine that can inhibit the generation of successful antitumor immunity) was not induced by OK432, although it was by
LPS/IFN-␥ (Fig. 1C).
OK432 enhances DC migration
In brief, 2 ⫻ 105 DC were seeded into transwells (Nunc, Fisher Scientific)
in triplicate in a 24-well plate (Corning Lifesciences, Schiphol-Rijk, Netherlands), above wells containing X-Vivo medium/1% human AB serum
with or without 0.5 ␮g/ml MIP-3␤ (R&D Systems). Plates were incubated
for 3 h. Transwells were then carefully removed and discarded, and all well
contents harvested. Cells were stained for CD11c and analyzed using a
FACSCalibur cytometer as above; data was acquired from each tube for
exactly 1 min. The number of CD11c⫹ events over this time for each
replicate and condition was calculated.
For DC-based immunotherapy to be effective, it is important for
DC to traffic to appropriate sites for interaction with immune effector cells. In particular, DC migration to lymph nodes, where
they can prime innate and adaptive immune responses, from sites
of Ag acquisition such as the tumor itself, will be essential for
successful immunotherapy. One cell surface marker of DC migratory capacity is CCR7, which can promote DC migration from
peripheral tissues to lymph nodes (20). Fig. 2, A and B shows that,
although both LPS/IFN-␥ and OK-432 up-regulated CCR7 on DC,
only OK-DC efficiently migrated toward a gradient of MIP-3␤/
CCL-19, a potent chemoattractant for DC (21). The poor correlation between CCR7 expression on LPS/IFN-DC and their migratory capacity in this assay may be due to the complexity of the
functional interactions between chemokines and their multiple potential receptors. Nevertheless, these data show that OK-DC are
likely to migrate effectively to lymph nodes in vivo.
T cell proliferation assay
OK-DC potently stimulate autologous T cells
IDC, LPS/IFN-DC, and OK-DC were cocultured at a 1 DC: 10 T cell ratio
for 5 days. Cells were pulsed with [3H]thymidine (0.5␮Ci/per well) for
18 h and harvested onto filter mats using a TOMTEC harvester 96
MachIIIM. [3H]Thymidine incorporation was determined using a Wallac
Jet 1459 microbeta scintillation counter and microbeta Windows software
(Wallac).
Previous studies have demonstrated that peptide-pulsed OK432treated DC can prime Ag-specific T cells (10), a result we have
also been able to replicate in our laboratory (data not shown).
Interestingly, more recent studies have shown that a distinct, nonAg-specific innate killing by T cells can alternatively be stimulated
ELISA
DC were seeded at 200,000 cells/ml, and supernatant collected after 48 h.
IL-12p40, TNF-␣, IFN-␥, IL-10, and IL-6 were detected using matched
paired Abs (BD Pharmingen) following standard protocols.
DC migration
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CD14⫹ cells, isolated from human PBMC by MACS selection (Miltenyi
Biotec), were cultured with 800 U/ml GM-CSF (Schering-Plough) and
0.05 mg/ml IL-4 (R&D Systems) in X-Vivo medium (Cambrex) with 1%
human AB serum (Sigma-Aldrich) for 5 days to generate immature DC
(IDC), and matured for a further 2 days in 10 ␮g/ml OK432 (Chugai
Pharmaceuticals) to generate OK-DC. Reference maturation stimuli tested
comprised 250 ng/ml LPS (Sigma-Aldrich) and 1000 U/ml IFN-␥ (SigmaAldrich). Autologous T cells were isolated from the CD14 negative PBMC
fraction using The Pan T Cell Isolation Kit II (Miltenyi Biotec); CD8 and
CD4 T cells were positively selected using CD4/CD8 microbeads (Miltenyi Biotec). T2 (T cell) and K562 (CML) cell lines were grown in RPMI
1640 (Life Technologies), 10% FCS (Harlan Sera-Labs), and 1% glutamine (Life Technologies). Bladder tumor cell lines EJ, RT112, and 253J
cells were grown in a 50:50 ratio of RPMI 1640:DMEM (Life Technologies), 5% FCS and 1% glutamine; SW480 cells (colorectal tumor cells)
were grown in DMEM, 10% FCS, and 1% glutamine; parental RT112 and
SW480 cell lines were transduced to express CD40 using a retroviral vector (16, 17). Human foreskin fibroblast (HFF) and normal human urothelial
(NHU) cells were grown as previously described (16).
3109
3110
DC CYTOTOXICITY VIA CD40-CD40L INTERACTIONS
upon short term culture of T cells with cytokines (22, 23). We
therefore set out to address whether our cytokine-secreting OK-DC
could similarly stimulate innate T cell functions, including cytotoxicity. First, T cell proliferation stimulated by autologous IDC,
FIGURE 2. OK432 enhances DC migration. A, Surface expression of CCR7 on IDC,
LPS/IFN-DC, and OK-DC was analyzed by
FACS (representative of n ⫽ 3). B, Two ⫻
105 DC were placed in the upper chamber of
an 8-␮m transwell and migration toward 0.5
␮g/ml MIP-3␤ determined (representative of
n ⫽ 3; error bars ⫽ SD).
OK-DC, or LPS/IFN-DC was investigated using [3H]thymidine
incorporation. LPS/IFN-␥ was taken forward as the maturation
comparator in these coculture experiments as we had established
this as our previous optimal combination for DC phenotype
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FIGURE 1. OK432 activates human monocyte derived DC. Human monocyte derived DC were either left untreated (IDC), treated with 250 ng/ml
LPS/1000U/ml IFN-␥ (LPS/IFN-DC), or treated with 10 ␮g/ml OK432 for (OK-DC) for 48 h. A, DC were harvested and surface expression of CD1a, CD80,
CD86, CD83, CD54, MHC class I, and MHC class II analyzed by FACS (data representative of four independent donors is shown). B, Control IDC (no
FITC dextran), IDC, LPS/IFN-DC, and OK-DC (all with FITC dextran) were incubated for 30 min, washed, and uptake of FITC dextran was determined
by flow cytometry (representative of n ⫽ 3). C, DC supernatants were harvested and the production of TNF-␣, IL-6, IL-10, and IL-12 was examined by
ELISA (representative of n ⫽ 3; error bars ⫽ SEM).
The Journal of Immunology
3111
activation/cytokine induction during head-to-head comparisons of
a number of reported DC maturation agents (CD40L, LPS alone,
CpG, poly I:C, TNF-␣, and a cytokine mixture comprising
TNF-␣, IL-1␤, IL-6, and PGE2 (Ref. 24 and data not shown).
Fig. 3Ai shows that OK-DC were significantly better at stimulating autologous T cell proliferation than IDC or LPS/IFN-DC.
Illustrative photographic images of IDC and OK-DC T cell cocultures are shown in Fig. 3Aii; greater expansion of the lymphocyte population upon coculture with OK-DC is clearly visible. Similarly, OK-DC were more effective at stimulating the
production of IFN-␥ in these cocultures than both IDC and LPS/
IFN-DC (Fig. 3B).
Next, cytotoxicity after T cell coculture with IDC or OK-DC, in
the absence of Ag, was examined by 51Cr release assay. Killing of
T2 and K562 tumor cell targets after coculture of T cells with
IDC or OK-DC for 5 days suggested that OK-DC stimulated
greater innate T cell cytotoxicity against these tumor cell targets
(Fig. 3C). OK-DC/T cell cocultures also killed a range of other
tumor cell lines, while poorly proliferative LPS/IFN-DC/T cells
were as ineffective as IDC/T cells (data not shown). Because
generation of innate T cell cytotoxicity upon coculture with DC
has not previously been reported, we were next interested in
determining which T cell population was responsible for tumor
cell killing. CD4 and CD8 T cells were isolated after coculture
with OK-DC and their cytotoxicity against T2 or K562 tumor
cell targets determined (Fig. 3D). Surprisingly, it appeared that
neither the CD4 nor CD8 T cells were responsible for the majority of the innate killing observed, as a significant cytotoxic
component was always found in those cells remaining after
CD4 or CD8 isolation. Because the OK-DC themselves always
remained in the cytotoxic fraction, and direct killing by DC has
been previously reported (12–15), this led us to question
whether the OK-DC were directly responsible for tumor cell
target death. In initial experiments, we positively selected
CD86-expressing cells from the T/OK-DC cocultures (as a
marker expressed by DC but not T cells), and found that indeed
significant cytotoxicity resided in this CD86⫹ fraction (data not
shown). We therefore went on to investigate further the cytotoxic potential of OK-DC alone, without any prior culture with
T cells.
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FIGURE 3. OK-DC potently stimulates autologous T cells. DC were cocultured with autologous T cells for 5 days at a 1:10 DC:T cell ratio. Ai,
Overnight proliferation was measured by addition of tritiated thymidine (representative of n ⬎ 4). ii, Images of IDC/T cells and OK-DC/T cells after
coculture for 6 days. B, IFN-␥ secretion in DC:T cell cocultures was measured by ELISA (representative of n ⬎ 4). C, Killing of T2 and K562 tumor cell
targets by IDC/T cell (diamonds) and OK-DC/T cells (squares) cocultures was determined by 51Cr release assay (representative of n ⬎ 4). D, Either CD4
or CD8 T cells were positively selected from IDC/T cell and OK-DC/T cell cocultures and killing of T2 and K562 tumor cell targets measured using 51Cr
release assay (at a 100:1 E:T ratio). Killing of positively selected CD4⫹ and CD8⫹ isolated cells and remaining nonselected cells was determined
(representative of n ⫽ 2; all error bars ⫽ SD).
3112
DC CYTOTOXICITY VIA CD40-CD40L INTERACTIONS
range of tumor cell targets (T2, K562, EJ, and 253J cells) and
normal cells (HFF and NHU) was examined using a 51Cr release
assays (Fig. 4, A and B, respectively). OK-DC effectively killed all
four tumor cell targets investigated while the cytotoxicity of IDC
was negligible (Fig. 4A). Some killing by LPS/IFN-DC was observed, but only at low levels. In contrast to killing of tumor targets, no DC killed normal NHU or HFF cell targets (Fig. 4B).
These studies demonstrate, for the first time, that OK432 stimulated human myeloid DC to specifically kill tumor, but not normal
cell, targets.
Mechanism of OK-DC tumor specific killing
Tumor specific killing by OK-DC
DC were again either left untreated (IDC), treated with LPS-IFN-␥
(LPS/IFN-DC), or OK432 (OK-DC), and cytotoxicity toward a
FIGURE 5. OK-DC express CD40L and kill via a contact dependent mechanism. A, IDC and OK-DC were cocultured with 51Cr labeled T2 cell targets
(10:1 E:T ratio) for 20 h either in the presence or absence of a 0.4 ␮m transwell membrane. % killing of T2 was determined using a 20 h 51Cr release assay
(representative of n ⫽ 2; error bars ⫽ SD). B, Surface expression of CD40 on T2, K562, EJ, and 253J cells was analyzed by FACS. C, Surface expression
of CD40L on IDC, LPS/IFN-DC, and OK-DC was analyzed by FACS (representative of n ⫽ 4).
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FIGURE 4. Tumor specific killing by OK-DC. IDC, LPS/IFN-DC and
OK-DC were cocultured at different ratios with 51Cr labeled T2, EJ, K562,
and 253J targets (A), or labeled normal HFF and NHU cell targets (B) for
20 h. DC cytotoxicity was determined using a standard 51Cr release assay
(representative of n ⫽ 4; all error bars ⫽ SD).
Various cellular mechanisms have been implicated in DC cytotoxicity, including via TRAIL and Fas, both of which are members of
the TNF superfamily (12–14). In the current studies, however, levels of OK-DC killing of TRAIL-sensitive (EJ) vs TRAIL-insensitive (253J) (16), or Fas-sensitive (T2) (25) vs Fas-insensitive
(K562) (26) cell lines (Fig. 4A) were similar, suggesting that a
different mechanism was responsible for OK-DC cytotoxicity. Secreted factors, such as TNF-␣, have also been implicated in DC
killing (12). Therefore, to determine whether OK-DC killing was
contact dependent or mediated by the release of a soluble factor(s),
OK-DC and tumor targets were cultured in the presence or absence
of a transwell membrane. OK-DC killing of T2 tumor cell targets
is shown in Fig. 5A, which demonstrates that cytotoxicity is clearly
dependent on cell:cell contact.
When considering alternative surface molecules that might account for OK-DC contact-dependent, tumor-specific killing, we
noted that CD40, like Fas and TRAIL, a member of the TNF
receptor superfamily, has also been implicated in cell death pathways, in addition to its central role in immune activation. The
functional outcome of CD40/CD40L interactions is complex and
often dependent on cell lineage and differentiation (27). For example, CD40-expressing epithelial cells can secrete proinflammatory cytokines and proliferate in response to CD40 ligation (28,
29). In contrast, in carcinoma cells, membrane-presented, surface
CD40L induces tumor cell-specific apoptosis (17, 30) via a signaling mechanism involving TNFR-associated factor (TRAF)3
The Journal of Immunology
3113
and JNK/AP-1 activation (19). Significantly, CD40 ligation on the
normal NHU and HFF cell targets resistant to OK-DC killing in
Fig. 4B is known not to be associated with cell death (19, 30). The
tumor-specific nature of epithelial CD40/CD40L-mediated killing
led us to question whether this could be the mechanism responsible
for OK-DC cytotoxicity. To address this we first determined
whether each of the tumor cell targets from Fig. 4A expressed
CD40, and would therefore potentially be responsive to CD40Linduced killing. CD40 expression on K562, T2, 253J, and EJ tumor cell targets is shown in Fig. 5B, which confirms that each cell
line expresses CD40 on their surface. For OK-DC cytotoxicity to
occur via CD40/CD40L interactions up-regulation of surface
CD40L on DC in response to OK432 is also required; Fig. 5C
demonstrates that this does indeed occur. Potentially consistent
with their poor killing by this mechanism (Fig. 4A), LPS/IFN-DC,
in contrast to OK-DC, expressed only very low levels of CD40L.
To confirm that CD40 expression was absolutely required for
OK-DC killing, we tested two CD40-negative tumor cell lines,
SW480 and RT112, and their equivalent stable CD40-expressing
transductants (Fig. 6A) as targets in OK-DC cytotoxicity assays.
Fig. 6B shows that CD40⫺ SW480 and RT112 cells were not sensitive to OK-DC killing, while SW480 and RT112 CD40⫹ transfectants were. We confirmed this result, using the JAM test as an
alternative measure of cytotoxicity (19) (Fig. 6C). Using this assay
killing, as assessed by DNA fragmentation, was again greater
against the CD40 expressing tumor targets than the parental CD40
negative cells. Finally, we were able to show that OK-DC cytotoxicity against CD40-expressing RT112 targets was significantly
reduced in the presence of an anti-CD40L blocking Ab, compared
with an isotype control Ab (Fig. 6D). These studies demonstrate
that OK432 stimulates DC to become tumor-specific killers via
ligation between CD40L on the DC and CD40 on the tumor cell,
representing a novel mechanism of DC-mediated cytotoxicity.
Discussion
Depending on their microenvironment DC may dampen, modulate,
or activate various immune responses. Among the many maturation protocols used to activate DC in vitro, OK432 has shown
recent promise as a clinically viable TLR agonist (9). As expected
for a DC maturation agent, in this study OK432 up-regulated expression of the DC activation markers MHC I, MHC II, CD80,
CD86, CD54, and CD83 (Fig. 1A), reduced DC phagocytosis (Fig.
1B), and induced secretion of the inflammatory cytokines IL-12,
TNF-␣, and IL-6 (Fig. 1C). OK-DC also displayed increased surface expression of CCR7, migrated toward MIP-3␤ in vitro (Fig.
2), and stimulated proliferation and IFN-␥ production upon coculture with autologous T cells in the absence of Ag (Fig. 3, A and B).
This OK-DC-induced T cell activation was significantly greater
than that induced by DC matured with LPS/IFN-␥.
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FIGURE 6. Killing by OK-DC is CD40-CD40L mediated. A, Expression of CD40 on RT112 and SW480 cells and corresponding CD40 transductants
was determined by FACS. B, OK-DC were cocultured at different ratios with 51Cr labeled RT112 (CD40⫹/⫺) and SW480 (CD40⫹/⫺) cell targets for 20 h.
Cytotoxicity of OK-DC was determined using a standard 51Cr release assay (representative of n ⫽ 4; error bars ⫽ SD). C, Percentage of DNA fragmentation
of RT112 (CD40⫹/⫺) and SW480 (CD40⫹/⫺) after coculture with OK-DC for 20 h was determined examined using the JAM cytotoxicity assay (representative of n ⫽ 3; error bars ⫽ SEM). D, Blockade of RT112⫺CD40⫹ cell killing by OK-DC in the presence of anti-CD40L, but not isotype control, Ab
(representative of n ⫽ 2; error bars ⫽ SEM).
3114
There are clear potential applications for cytotoxic OK-DC. DC
can acquire tumor-associated Ags from within tumors and prime
therapeutic antitumor immunity, particularly when used in combination with an additional modality, such as chemotherapy or radiotherapy, to trigger additional cell death (33, 34). If OK-DC can
be delivered intratumorally or locoregionally into involved sites,
their direct and specific killing of tumor cells may release tumorassociated Ags for T cell priming; further work is required to address these questions in animal models. Critically, however, the
clinical practicality of OK432 should allow early translation to
testing in patients. In this regard, we have recently completed a
pilot clinical study, injecting OK-DC intralymphatically into the
feet of patients with advanced cancer (but not with involved lymph
nodes), in which we showed efficient tracking of injected cells to
draining pelvic nodes (E. West, R. Morgan, K. Scott, A. Merrick,
A. Lubenko, D. Pawson, P. Selby, P. Hatfield, R. Prestwich, S.
Fraser, et al., submitted for publication). We are now planning to
develop this strategy to target tumor-affected nodes, where OK-DC
may be able to kill and engulf malignant cells to generate systemic
antitumor immunity.
In summary, this study describes a preparation of mature, myeloid-derived human DC (OK-DC) which exert specific cytotoxicity against tumor cell targets via a novel CD40/CD40L-mediated
mechanism. Tumor cell killing by OK-DC may be particularly
suitable for clinical application and development, as OK432 has
been safely administered to cancer patients for many years
(35, 36).
Acknowledgments
We thank Chugai Pharmaceuticals, Japan for providing OK432.
Disclosures
The authors have no financial conflict of interest.
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On finding that T cells cocultured with OK-DC were cytotoxic
against tumor cells (Fig. 3C), we initially suspected that this represented innate T cell killing as previously reported on T cell stimulation with cytokines such as IFN-␥, IL-2, and IL-15 (22, 23).
However, as the main cytotoxic fraction of the cocultures was in
neither the CD4 nor CD8 fraction (Fig. 3D), we considered instead
whether the OK-DC were themselves directly cytotoxic.
Recently, several reports have shown that, under special circumstances or during an intermediate phase, DC can, like NK cells, be
directly cytotoxic (14, 15, 31, 32). When OK-DC were cocultured
with a range of tumor cell targets (T2, K562, EJ, and 253J) they
too were cytotoxic (Fig. 4A); neither IDC nor LPS/IFN-DC
showed significant killing ability. Significantly, OK-DC did not
kill normal NHU or HFF (Fig. 4B). These cytotoxic OK-DC are
distinct from recently described IFN-␥-producing killer DC (6), as
they do not directly secrete IFN-␥ themselves, and do not express
any surface NK markers (data not shown). The tumor specificity of
OK-DC killing (Fig. 4), its contact dependence (Fig. 5A), and its
lack of discrimination between Fas and TRAIL insensitive and
sensitive targets (Fig. 4A), led us to seek an alternative mechanism
for OK-DC cytotoxicity, including other potential members of the
cytotoxic TNF ligand family.
CD40/CD40L engagement has previously been shown to specifically kill tumor cells, although not in the context of DC effectors. Membrane-presented, but not soluble, CD40 agonists can induce apoptosis in carcinoma, but not normal, CD40-expressing
homologous epithelial cells (19). Membrane-presented CD40L
triggers cell death in malignant human urothelial cells via a direct
mechanism involving rapid up-regulation of TRAF3 protein, without concomitant up-regulation of TRAF3 mRNA, followed by activation of the JNK/AP-1 pathway and induction of the caspase9/caspase-3-associated intrinsic apoptotic machinery. The current
study clearly shows that OK-DC killing is restricted to CD40expressing (K562, T2, 253J, EJ; Fig. 5B), as opposed to CD40negative (RT112, SW480; Fig. 6A), tumor targets. Critically, and
consistent with OK-DC expression of CD40L (Fig. 5C), transfected RT112 and SW480 expressing CD40 acquire sensitivity to
OK-DC (Fig. 6, B and C), and OK-DC-mediated killing of CD40expressing RT112 cells is blocked by an anti-CD40L Ab (Fig. 6D).
We therefore conclude that CD40/CD40L interactions represent a
novel pathway by which DC activated in this way can specifically
kill tumor cells. CD40/CD40L is not likely to be the only/exclusive
mechanism for OK-DC killing (note low-level target cell death
with LPS/IFN-DC in Fig. 4A, and some killing by OK-DC of
CD40⫺ RT112 targets in the JAM assay in Fig. 6C). Nevertheless,
the clear differential between killing of CD40 positive and negative
targets in Fig. 6 validates CD40 activation by membrane CD40L as
the major and novel killing mechanism for these cytotoxic DC. It
should also be noted that, although OK-DC express both CD40 and
CD40L, they do not kill each other (i.e., there is no evidence of cell
fratricide); this is demonstrated by low-level uptake of tritiated
thymidine by OK-DC when cultured alone (data not shown), suggestive of limited proliferation rather than cell death.
To date, we have been unable to find another DC maturation
protocol which up-regulates CD40L and confers target CD40-dependent tumor cytotoxicity. Our main maturation comparator so
far has been LPS/IFN-␥, as we have previously established this, in
our hands, as optimal for DC phenotypic activation and cytokine
production; however neither LPS/IFN-DC nor DC matured with a
cytokine mixture comprising TNF-␣, IL-1␤, IL-6, and PGE2 (24)
were cytotoxic (data not shown). We are currently testing DC matured with a wider variety of TLR ligands, cytokines, and CD40L,
as well as further subtypes of both mouse and human DC, such as
plasmacytoid DC.
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