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The Journal of Experimental Medicine
JEM © The Rockefeller University Press $8.00
Vol. 202, No. 11, December 5, 2005 1507–1516 www.jem.org/cgi/doi/10.1084/jem.20050956
ARTICLE
1507
Innate NKT lymphocytes confer superior
adaptive immunity via tumor-capturing
dendritic cells
Kang Liu,
1
Juliana Idoyaga,
2
Anna Charalambous,
1
Shin-ichiro Fujii,
1
Anthony Bonito,
1
Jose Mordoh,
2
Rosa Wainstok,
2,3
Xue-Feng Bai,
4
Yang Liu,
4
and Ralph M. Steinman
1
1
Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021
2
Instituto Leloir, Instituto de Investigaciones Bioquimicas and
3
Departamento de Química Biológica, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, 1650 Buenos Aires, Argentina
4
Division of Cancer Immunology, Department of Pathology, Ohio State University Medical Center, Columbus, OH 43210
If irradiated tumor cells could be rendered immunogenic, they would provide a safe, broad,
and patient-specific array of antigens for immunotherapies. Prior approaches have emphasized
genetic transduction of live tumor cells to express cytokines, costimulators, and surrogate
foreign antigens. We asked if immunity could be achieved by delivering irradiated, major
histocompatibility complex–negative plasmacytoma cells to maturing mouse dendritic cells
(DCs) within lymphoid organs. Tumor cells injected intravenously (i.v.) were captured by
splenic DCs, whereas subcutaneous (s.c.) injection led only to weak uptake in lymph node or
spleen. The natural killer T (NKT) cells mobilizing glycolipid
-galactosyl ceramide, used to
mature splenic DCs, served as an effective adjuvant to induce protective immunity. This
adjuvant function was mimicked by a combination of poly IC and agonistic
CD40 antibody.
The adjuvant glycolipid had to be coadministered with tumor cells i.v. rather than s.c.
Specific resistance was generated both to a plasmacytoma and lymphoma. The resistance
afforded by a single vaccination lasted
2 mo and required both CD4
and CD8
T cells.
Mature tumor capturing DCs stimulated the differentiation of P1A tumor antigen-specific,
CD8
T cells and uniquely transferred tumor resistance to naive mice. Therefore, the access
of dying tumor cells to DCs that are maturing to activated NKT cells efficiently induces
long-lived adaptive resistance.
The use of autologous tumor cells as vaccines
dates back to the 1950s when it was found that
chemically induced tumors of inbred mice, if
injected as irradiated cells, could elicit protective
immunity in syngeneic hosts (1). The prospect of
rendering irradiated tumor cells immunogenic is
important because this would deliver a large
spectrum of epitopes to the immune system,
including critical regression antigens that may be
specific to an individual tumor (2). However, it
has been difficult to induce protective immunity
with inactive tumor cells (3–6). The injected cells
can even expand suppressor cells leading to
unresponsiveness (3). It remains a considerable
scientific challenge to learn to improve the im-
munogenicity of safe nonreplicating tumor cell
vaccines.
Tumor cells have been transduced to express
foreign proteins as surrogate antigens (7–10),
but this approach does not address the capacity
of the immune system to respond to a spectrum
of intrinsic tumor antigens. Tumor cells have
also been genetically modified to express individ-
ual costimulatory molecules from the B7 family
(B7.1, B7H, B7-DC) (11–13) or the TNF
superfamily (LIGHT) (14). These modifications
can improve T cell–mediated antitumor re-
sponses, although there is evidence that the
tumor cells must still be presented by host
antigen-presenting cells (15). In addition, tumor
cells have been transduced to secrete cytokines
such as interferons, interleukins, and hemato-
poietins that increase systemic immunity (16–
18). Tumors secreting IL-12 and GM-CSF are
most effective in eliciting T cell–mediated tumor
immunity (19–21). Both IL-12 and GM-CSF
The online version of this article contains supplemental material.
CORRESPONDENCE
Ralph M. Steinman:
steinma@mail.rockefeller.edu
Abbreviations used:
-Gal Cer,
-galactosyl ceramide; CFSE,
carboxyfluorescein diacetate
succinimidyl ester; NKT, natural
killer T.
PROTECTIVE IMMUNITY VIA MATURE, TUMOR-CAPTURING DCS | Liu et al.
1508
can mediate the recruitment of DCs (21–23), which are
specialized antigen-presenting cells for initiating immunity.
However, there are limitations to the clinical usefulness
of genetically modified tumors. These approaches typically
require the administration of live tumor cells, which raises a
safety issue (4, 5). To prioritize responses to patient-specific
antigens, tumor cell lines must be available from the patient
for purposes of genetic transduction, and this may be im-
practical. In addition, genetically modified tumors depend
on a few, often one, immune enhancing product.
An alternative to genetic transduction would be to
identify mechanisms required to induce immunity to irra-
diated tumor cells. Two principles would seem valuable in
this regard: (a) to learn to deliver dying whole tumor cells
directly to DCs that are abundant in lymphoid organs, and
(b) to render the DCs immunogenic through their differ-
entiation or maturation. A key feature of DCs is the effi-
ciency with which dying cells are processed for presenta-
tion on MHC class I and II products (24, 25). In vivo
dying cells injected intravenously are selectively taken
up by a subset of splenic DCs marked by expression of
CD8
and DEC-205. This leads to the presentation of a
surrogate antigen, ovalbumin, from the dying cells (26, 27).
Such “cross-presentation” of antigens by host cells, rather
than direct presentation by the tumor cells themselves,
primes protective T cells against several experimental tu-
mors (28–30), and this has now been observed with a cel-
lular human tumor vaccine as well (31). However, cross-
presentation of model antigens from dying cells in the
steady state leads to tolerance (26), whereas immunity de-
velops when the DCs simultaneously mature, for example,
in response to innate natural killer T (NKT) lymphocytes
(32, 33). NKT cells not only act as adjuvants by maturing
DCs but they also have additional valuable adjunct func-
tions in tumor resistance. NKT cells can produce IFN-
and lyse tumor targets, whereas the interaction of NKT
cells with DCs leads to the production of IL-12 and the ac-
tivation of other innate NK cells (34–37). Thus, NKT cells
are a potentially attractive means to link innate and adaptive
immunity against tumors.
Prior approaches to inducing immunity with safe irradi-
ated tumor cells have not directly addressed the need for tu-
mor cells to gain access to maturing DCs, to allow cross pre-
sentation and to induce strong T cell–mediated immunity.
Here, we have studied the nonimmunogenic plasmacytoma
cell line, J558, because it expresses a classical tumor antigen
called P1A (38) for which TCR transgenic T cells have been
made (39), and because J558 has an MHC class I–negative
variant that is genetically incapable of direct presentation
(40). We will describe that a single dose of irradiated J558
plasmacytoma cells, as well as A20 lymphoma cells, when
targeted to DCs, leads to long-lasting adaptive immunity as
long as the DCs are matured with innate NKT cells or a
combination of the TLR ligand, poly IC, and agonistic anti-
CD40 antibody.
RESULTS
Dying tumor cells are selectively captured by DCs
when given by the i.v. route
We first verified that DCs in mouse spleen could take up the
MHC class I–negative J558
variant of a mouse plasmacy-
toma. This variant has lost expression of cell surface MHC
class I and multiple antigen presentation genes, including
TAP-1, TAP-2, LMP-2, and LMP-7, due to malfunction of
Figure 1. Intravenous delivery of dying tumor cells to CD8
CD11c DCs in vivo. (A) Kinetics of uptake of 20 106 dying, irradiated,
CFSE-labeled J558 tumor cells injected i.v. or s.c., by CD11c spleen
(SPLN), draining (LN), and distal (dLN) lymph node DCs and analyzed with
flow cytometry as in B. (B) Flow cytometric assays to show selective
uptake of irradiated CFSE-labeled J558 tumor cells by CD8, CD11c
splenic DCs 2 h later (arrows). Comparison of CD11c and CD11c spleen
cells (top), and CD8 CD11c toCD8, CD11c DC subsets (bottom).
(C) Uptake by CD11c DC subsets of graded doses of CFSE-labeled tumor
cells i.v. 5, 20, or 106 irradiated CFSE-labeled J558 tumor cells were
injected i.v. Uptake 2 h after injection is shown in gated CD11c splenic
DCs (arrows).
JEM VOL. 202, December 5, 2005
1509
ARTICLE
the proto-oncogene
pml
(40, 41). Because the tumor cells
lack MHC class I, as we confirmed (Fig. S1 A, available at
http://www.jem.org/cgi/content/full/jem.20050956/DC1),
and also fail to express MHC class II (not depicted), recip-
ient antigen-presenting cells would have to capture and
process J558
cells to elicit CD8
and CD4
T cell re-
sponses. To verify that the J558
cells underwent cell death
after 75 Gy
-irradiation, we placed the cells in culture and
showed that with time, they could be stained with annexin
V and propidium iodide (see Materials and methods and Fig.
S1 B). We followed the uptake of carboxyfluorescein diace-
tate succinimidyl ester (CFSE)–labeled, irradiated, “dying”
J558
cells in vivo by lymph node and splenic DCs after in-
jection by the i.v. and s.c. routes, using flow cytometry. Fig.
1 A shows that the tumor cells, injected immediately after ir-
radiation, were phagocytosed by CD11c
splenic DCs. In
contrast, few CFSE-labeled cells were detected in splenic or
lymph node DCs when the tumor cells were injected by the
s.c. route (Fig. 1 A). CFSE-labeled tumor material was pri-
marily detected in the CD11c
DC-enriched populations
(Fig. 1 B, top) and, as expected from prior work (27), only
the CD8
CD11c
DC subset endocytosed the injected
CFSE-labeled tumor (Fig. 1 B, bottom). In experiments that
are not depicted, we established that few CFSE-labeled cells
were taken up by other CD11c
fractions of spleen, marked
for either CD11b, B220, or CD4. Fig. 1 C shows that the
uptake of J558
cells was limited by the dose of injected tu-
mor. Furthermore, irradiated A20 (MHC I
MHC II
),
J558 (MHC I
MHC II
), and J558
(MHC I
MHC II
)
were phagocytosed by DCs to similar extents (unpublished
data). Therefore, dying tumor cells are selectively captured
by DCs in vivo as long as the i.v. route is used.
-Gal Cer injection leads to rapid maturation of phagocytic
DCs in vivo
-Galactosyl ceramide (
-Gal Cer) is a nonmammalian gly-
colipid that is presented by CD1d molecules to an invariant
T cell receptor expressed by innate NKT lymphocytes (42).
A single i.v. dose of
-Gal Cer activates NKT cells to secrete
IL-4 and IFN-
, but this activation also leads to the matura-
tion of DCs in vivo, defined as the ability to initiate com-
bined CD4
and CD8
T cell immunity to coadministered
antigens (32, 33, 43). We established that the coadministra-
tion of
-Gal Cer with both J558 and A20 tumor cells did
not increase the level of tumor cell uptake by DCs; actually,
uptake could be slightly lower, probably because maturing
DCs are known to decrease their level of endocytic activity
(44). Also, in the presence of
-Gal Cer, tumor cell uptake
remained restricted to the CD8
subset of DCs. To evalu-
ate the maturation status of the DCs that had phagocytosed
dying tumor cells, we injected mice with CFSE-labeled, ir-
radiated MHC-negative, J558
cells in the presence or ab-
sence of
-Gal Cer. 5 h later, the DCs were analyzed by
flow cytometry for the expression of a number of cell surface
molecules that change during DC maturation. Injection of
tumor cells alone had little effect on the phenotype of the
total CD11c
splenic population relative to the PBS control
(Fig. 2, compare first and second rows). However, injection
of
-Gal Cer (not depicted) or coinjection of tumor cells
with
-Gal Cer (Fig. 2, third row) resulted in the maturation
of the total CD11c
DC population, as indicated by the up-
regulation of MHC II, CD80, CD86, B7-H1, and B7-DC
5 h later (Fig. 2, third row). When we looked selectively at
those DCs that had captured dying tumor cells (by examin-
ing cells positive for CD11c and CFSE), we found that the
phagocytic cells, which represent
3% of the splenic DCs
(Fig. 1 A), had higher levels of CD1d and other markers (Fig.
2, fourth row). The phagocytic cells strongly up-regulated
the expression of all the measured antigen-presenting and co-
stimulatory molecules in response to
-Gal Cer (Fig. 2, com-
pare fourth and fifth rows). Therefore, the administration of
-Gal Cer allows DCs that capture tumor cells to exhibit
numerous phenotypic changes typical of DC maturation.
Injection of dying tumor cells together with
-Gal Cer
induces tumor immunity
To determine if immunity was induced by delivery of dying
tumor cells to DCs, we used a tumor protection assay. Dif-
ferent groups of naive BALB/c mice were injected with
PBS,
-Gal Cer, 20
10
6
irradiated J558
tumor cells
alone, or J558
together with
-Gal Cer. 7 d after the vacci-
nation, we challenged the mice with 5
10
6
J558 live tu-
Figure 2. -Gal Cer injection rapidly matures CD1d-rich, splenic
DCs capturing dying cells. BALB/c mice were injected with PBS or 20
106 irradiated CFSE-labeled J558 tumor cells i.v. in the presence or
absence of 2 g -Gal Cer. 4.5 h later, bulk spleen cells were prepared
from the mice and stained with CD11c-allophycocyanin and PE-conjugated
mAbs CD40, 80, 86, B7-H1, B7-DC, Ld, I-Ad, and CD1d. The data are shown
for the CD11c total DC population (top three rows) and CD11c CFSE
phagocytic population (bottom two rows).
PROTECTIVE IMMUNITY VIA MATURE, TUMOR-CAPTURING DCS | Liu et al.
1510
mor cells s.c. The tumor grew progressively within 7–10 d
in mice that had received either PBS or
-Gal Cer alone
(Fig. 3 A). Injection with irradiated J558 tumor cells alone
protected only 15% mice from J558 challenge (3 out of 20
mice in four experiments), whereas 88% of the mice (22 out
of 25 in five experiments) were protected and remained tu-
mor-free after vaccination with the combination of irradi-
ated J558
with
-Gal Cer (Fig. 3 A). We were not able to
vaccinate mice with live J558
cells, which grew rapidly and
killed the mice. Vaccination with irradiated J558
and
-Gal
Cer was effective only when tumor cells were injected by
the i.v. and not the s.c. route (Fig. 3 B), as predicted by the
data in Fig. 1 A in which tumor cells had to be injected i.v.
to be picked up well by DCs. Corresponding to the uptake
data in Fig. 1 C, the level of protection varied with the dose
of injected tumor cells with 10
6
cells being ineffective (Fig. 3
C). To evaluate the specificity of the antitumor immune re-
sponse, we challenged the mice with either live Meth A sar-
coma cells s.c. or live A20 lymphoma cells after vaccination
with irradiated J558 with
-Gal Cer. Vaccination with dying
J558 L
d
and
-Gal Cer protected mice against a challenge
of J558, but not Meth A or A20 (Fig. 3 D). Likewise, immu-
nization with dying A20 with -Gal Cer led to protection
against only A20, but not J558 (Fig. 3 E). To compare -Gal
Cer with other DC maturation stimuli, we studied agonistic
anti-CD40 monoclonal antibody as well as the TLR3 ligand,
poly IC. The glycolipid was the only adjuvant that could in-
dependently induce protective immunity to irradiated tumor
injected i.v., but the combination of anti-CD40 and poly IC
was also active (Fig. 3 F). To assess memory, we verified that
Figure 3. Acquired resistance to J558 after vaccination with MHC I
tumor and -Gal Cer. (A) Mice were vaccinated with PBS, 2 g -Gal Cer,
20 106 irradiated MHC I–negative J558 cells with or without 2 g -Gal
Cer i.v. 1 wk later, mice were challenged with a lethal tumorogenic dose of
5 106 live MHC I J558 tumor cells s.c. (B) Mice were vaccinated with PBS,
20 106 irradiated J558 cells alone, or 20 106 irradiated J558 cells with
2 g Gal Cer i.v. or s.c. and, 1 wk later, challenged with 5 106 live MHC
class I J558 cells s.c. (C) Mice were vaccinated with 1, 5, or 20 106
irradiated J558 cells plus 2 g -Gal Cer i.v. and, 1 wk later, challenged with
5 106 J558 s.c. (D) Mice were vaccinated with 20 106 irradiated J558
cells plus 2 g -Gal Cer i.v. and, 1 wk later, challenged with 5 106 J558 or
to establish tumor specificity, with 5 106 Meth A sarcoma or 5 106 A20
lymphoma cells s.c. (E) Mice were vaccinated with 5 106 irradiated A20
cells plus 2 g -Gal Cer i.v. and 1 wk later, challenged with 5 106 J558 or
5 106 A20 cells s.c. (F) Mice were vaccinated with 5 106 irradiated J558
cells and either 2 g -Gal Cer i.v., 50 g CD40 i.p, 50 g polyI:C i.p., or
both CD40 and poly IC and, 3 wk later, each group was challenged with
5 106 J558 s.c. (G) 8 wk after vaccination, mice were challenged with a
lethal dose of 5 106 live J558 tumor cells. (H) To detect a therapeutic effect,
mice were injected with 1 or 5 106 live J558 tumor cells s.c., and 3 d later
treated with PBS, or 20 106 irradiated J558 cells with 2 g -Gal Cer i.v.
In all experiments, mice were monitored every other day for tumor growth
and scored positive when the tumors were palpable. Each group included at
least five mice, and one representative experiment of three is shown.
Figure 4. CD4 or CD8 T cell depletion abrogates tumor immunity
at the time of challenge. (A) Mice were vaccinated with 20 106 irradiated
J558 tumor cells plus -Gal Cer. 7 d after immunization, mice were
challenged with 5 106 MHC class I positive or negative J558 cells s.c.
(B) Wild-type BALB/c mice or J18/ mice were vaccinated with 20 106
irradiated J558 tumor cells plus -Gal Cer and, 3 wk after vaccination,
the mice were challenged with 5 106 J558 cells s.c. (C) 8 wk after
vaccination with 20 106 irradiated J558 tumor cells and -Gal Cer, mice
received 1 mg anti-CD4, anti-CD8, or control rat IgG. 1 d later, all mice
were challenged with 5 106 J558 tumor cells and monitored every other
day for tumor growth. Mice were scored positive when the tumors were
palpable. Each group included five mice; one representative experiment
of two is shown.
JEM VOL. 202, December 5, 2005 1511
ARTICLE
vaccinated mice were protected against J558 challenge 2 mo
(Fig. 3 G) and 4 mo (not depicted) after vaccination. We also
induced tumor resistance if we performed an immunother-
apy-type experiment and vaccinated 3 d after the injection of
the tumor cells (Fig. 3 H). However, therapeutic immunity
was manifest only when the tumor dose was 106 and not 5
106 cells, whereas protective immunity was evident in mice
challenged with both doses. Thus, long-lived tumor immu-
nity can be elicited by a single i.v. vaccination with irradiated
J558 and -Gal Cer, which also has a therapeutic effect.
Innate and adaptive T cells are required to generate
resistance after vaccination with dying tumor and -Gal Cer
To identify resistance mechanisms for the observed protec-
tive tumor immunity, we vaccinated mice with dying MHC
class I J558 plus -Gal Cer and challenged them with live
MHC class I–negative or positive J558 tumor cells. The vac-
cinated mice were protected against the MHC class I–posi-
tive J558 cells, but not the MHC class I negative J558 tu-
mor cells (Fig. 4 A), suggesting that CD8 T cells were
required for resistance. As expected, NKT cells were also re-
quired for effective vaccination because J281/ mice (also
termed J18/), which cannot respond to -Gal Cer be-
cause they lack NKT cells (45), failed to develop immunity
to dying cells plus glycolipid (Fig. 4 B). To further evaluate
the type of T cells required for protective immunity 8 wk af-
ter a single vaccination, the immune mice were injected with
depleting antibodies specific for CD4, CD8, or control IgG
and challenged with J558 cells 1 d later. We verified by
FACS that anti-CD4 and anti-CD8 antibodies depleted the
respective cell populations within 2 d, and that the depletion
lasted 2 wk, when they began to repopulate slowly as re-
ported (46). As shown in Fig. 4 C, mice injected with con-
trol IgG remained resistant to J558 challenge. However, de-
Figure 5. Proliferation and differentiation of tumor antigen-specific
T cells in response to dying tumor cell delivery in vivo. (A) 3 106
CFSE-labeled, naive CD8 P1CTL T cells were injected i.v. into BALB/c
mice. 1 d later, 20 106 J558 cells were injected i.v. with or without
2 g -Gal Cer coinjection. 3 d later, mice were killed and spleen cells
were stained with CD8-allophycocyanin and CD25 or CD62L-PE. (B) Spleen
cells obtained as in A were cultured at 107/ml with 1 g/ml P1A 35–43
peptide (LPYLGWLVF) for 4 h at 37C in the presence of 5 g/ml BFA. The
depicted cells were from gated CD8CFSE P1A transgenic T cells, and
intracellular IFN- and IL-2 were identified with PE-conjugated mAb
after fixation and permeabilization. Black lines are staining with isotype
control antibody; gray lines are staining with anti–IFN- or IL-2. Percentages
are the fraction of transgenic T cells expressing intracellular cytokine
above background.
PROTECTIVE IMMUNITY VIA MATURE, TUMOR-CAPTURING DCS | Liu et al.1512
pletion of either CD4 or CD8 T cells from vaccinated
mice significantly abrogated tumor immunity elicited by J558
with -Gal Cer (Fig. 4 C). Therefore, both innate NKT cells
and adaptive CD4 and CD8 T cells contribute to the tumor
resistance induced by DCs capturing dying cells in vivo.
-Gal Cer coinjection with dying cells better activates
antigen-specific CD8 T cells
To document the consequences of -Gal Cer for the quality
of an antigen-specific T cell response to irradiated tumor, we
took advantage of the P1CTL mouse, a CD8 TCR trans-
genic line specific for P1A tumor antigen presented on Ld
MHC class I molecules (39). 20 106 irradiated MHC class
I–negative, J558 cells with or without -Gal Cer were in-
jected into the mice that had received CFSE-labeled P1CTL
cells 1 d earlier. T cell proliferation and phenotype were an-
alyzed 3 d later with flow cytometry. In the absence of
-Gal Cer, DCs could cross-present P1A from dying tumor
cells to CD8 P1CTL T cells, driving the T cells into multi-
ple cycles of proliferation (Fig. 5, top row). This is consistent
with the capacity of CD8 DCs to present antigens on both
MHC class I and II products from dying cells in the steady
state (26, 27). However, the proliferating P1CTL T cells re-
tained markers typical of naive cells, i.e., low CD25 and
high CD62L (Fig. 5 A, white arrows). In contrast, in the
mice that had received J558 plus -Gal Cer, the T cells
proliferated more extensively and many began to show an
activation phenotype within 3 d, as indicated by the up-reg-
ulation of CD25 and down-regulation of CD62L (Fig. 5 A,
compare black and white arrows). Furthermore, T cells that
had been stimulated in the presence of -Gal Cer adjuvant
in vivo were able to produce significantly more IFN- and
IL-2 upon brief restimulation with P1A peptide in vitro
(Fig. 5 B, compare right with middle and left panels). There-
fore, DCs process antigens from tumor cells and induce the
proliferation of antigen-specific T cells in vivo, but a matu-
ration stimulus is required for the differentiation of effector
T cells (Fig. 5) as well as protective immunity (Figs. 3 and 4).
Evidence that mature DCs present tumor antigen
and transfer tumor immunity
To verify that mature DCs were responsible for the presenta-
tion of antigens from the captured dying tumor cells and also
elicited tumor immunity, we first isolated DCs from mice in-
jected with dying J558 tumor cells without or with -Gal
Cer. The CD11c DC-enriched and CD11c DC-depleted
Figure 6. Maturing DCs mediate P1A antigen presentation and elicit
tumor immunity in vivo. Mice were given -Gal Cer or PBS together with
irradiated J558- cells. 4 h later, CD11c and CD11c cells were isolated and
used to stimulate CD8 TCR transgenic T cells from P1CTL mice in vitro (A)
or in vivo (B). In A, in vitro T cell proliferation was measured by a [3H]thymidine
pulse at 40–50 h. In B, in vivo proliferation of CFSE-labeled P1CTL T cells
was measured 3 d later. (C) Splenic CD11c and CD11c cells were isolated
from mice injected with irradiated J558 cells and -Gal Cer 4 h earlier.
106 CD11c or 5 106 CD11c cells were then transferred i.v. into naive
BALB/c mice. 1 wk later, the mice were challenged with live 5 106 J558
cells. Mice were monitored every 3 d for tumor growth. Each group includes
12–17 mice pooled from three experiments.
JEM VOL. 202, December 5, 2005 1513
ARTICLE
cells from spleen were added as stimulators in culture of na-
ive CD8 P1CTL TCR transgenic T cells without further
antigen. When -Gal Cer had been coadministered, the iso-
lated DCs were much more effective at stimulating prolifera-
tion of naive CD8 T cells in culture, whereas CD11c cells
were inactive (Fig. 6 A, right, closed squares). Next, we iso-
lated CD11c DCs from spleens 4 h after immunization and
transferred the DCs to naive animals to test their capacity to
stimulate proliferation of CFSE-labeled CD8 P1CTL T
cells in vivo (Fig. 6 B). 3 d later, we detected P1CTL prolif-
eration only in response to DCs from mice given irradiated
tumor cells with -Gal Cer (Fig. 6 B, black arrow). CD11c
non-DCs from the same mice were not able to stimulate
P1CTL T cells, and DCs from mice that had received J558
without -Gal Cer failed to stimulate P1CTL above the
background (Fig. 6 B). Finally, to test if the antigen-present-
ing mature DCs were critical for inducing protective tumor
immunity, we transferred DCs or non-DCs from the vacci-
nated mice into naive mice and then challenged them with
live J558 tumor cells. As shown in Fig. 6 C, when naive
mice had been given 1.5 106 CD11c DCs from donor
mice injected with dying J558 cells together with -Gal
Cer, 58% of mice (10 out of 17 mice tested; P 0.01) were
fully protected. CD11c non-DCs and CD11c DCs from
mice injected with PBS or dying J558 cells alone, failed to
transfer protection to naive mice (0/12, Fig. 6 C). These data
provide direct evidence that mature antigen-capturing DCs
are responsible for presentation of tumor antigen and the
adjuvant action of -Gal Cer in vivo.
DISCUSSION
Genetic transduction of tumor cells is being used to increase
the adaptive immune response to tumor antigens, including
strategies in which cytokines and chemokines are introduced
to recruit DCs to the tumor. An alternative would be to
learn to deliver dying whole tumor cells to maturing DCs
within lymphoid tissues because these cells have dozens of
specializations pertinent to the initiation of immunity, in-
cluding ready access to the recirculating pool of T cells.
Here, we provide some mechanisms to address this goal.
The earliest attempts to increase the immunogenicity of
whole tumor cells and stimulate T cell–mediated tumor immu-
nity used bacterial components such as BCG or C. parvum as
adjuvants (47–49). Although these approaches have achieved
only limited success (50), they imply a need for coordination
between innate immunity and adaptive immunity against tu-
mors. Interestingly, innate NKT cells are required for the anti-
tumor effects of both IL-12 and GM-CSF (45, 51), the two cy-
tokines that are most often successful in improving immunity to
genetically modified tumor cells. Our findings indicate that
NKT cells can serve as superior adjuvants for protective antitu-
mor immunity. In prior studies, we studied the capacity of
NKT cells to mature DCs that were presenting the foreign anti-
gen, ovalbumin (32, 43), but in the current paper, we have ex-
amined the generation of long-lived T cell memory and
protection to a poorly immunogenic syngeneic tumor.
We find that a single dose of irradiated nonmodified tu-
mor cells, when directed to maturing DCs in vivo, here by
the i.v. route, leads to long-lived protective and combined
CD4 and CD8 T cell immunity. We compared the effi-
cacy of targeted i.v. delivery of J558 tumor to maturing DCs
with the use of DCs that were derived from bone mar-
row precursors and loaded with J558 tumor ex vivo as de-
scribed previously (6). However, two doses of 200,000 tu-
mor loaded DCs given 1 wk apart by the s.c. route, failed to
protect the mice to challenge with J558 s.c. The -Gal Cer
maturation stimulus we used to mature splenic DCs in vivo
induced superior protective immunity relative to other DC
stimuli, such as ligation of CD40, TLR4, and TLR3. This
glycolipid has been manufactured in a nontoxic form for hu-
man use (52). It is presented on CD1d molecules to activate
NKT lymphocytes (42, 45), which then mature the DCs as
mentioned (32, 33). When NKT cells are activated with a
single dose of -Gal Cer, there is a production of immune
enhancing cytokines, specifically TNF and IL-12 by the
DCs, and IFN- by NKT and NK cells, and there is an up-
regulation of CD40L on the NKT cells. To elicit protective
immunity to a syngeneic tumor, we find that the combina-
tion of a proinflammatory TLR ligand, poly IC, and agonis-
tic anti-CD40 antibody is able to mimic the effects of -Gal
Cer. Interestingly, each stimulus (poly IC, anti-CD40,
-Gal Cer) induces similar phenotypic changes of matura-
tion; i.e., increased expression of CD40, CD86, MHC class
II, B7-H1, B7-H2, and decreased interferon- receptor or
CD119, but for protective immunity, either -Gal Cer or
the combination of poly IC and CD40 is required.
When DCs were isolated from mice injected with irradi-
ated tumor cells, we could establish that the DCs from gly-
colipid-treated mice were better stimulators of CD8 T cells
specific for the P1A tumor antigen in vitro. Importantly,
transfer of these maturing DCs to naive mice protected
60% of them, whereas direct vaccination of dying tumor
cells with -Gal Cer protected 80–100% of the mice. The
DC transfer experiments might not have been optimal be-
cause they entailed the transfer of only 10-30,000 DCs
bearing tumor cells (only a small percent of the CD8 DCs
take up tumor cells in Fig. 1), which also might not lodge in
the recipient lymphoid tissues with high efficiency.
The harnessing of DCs in this paper offers some advan-
tages over genetic modification of tumor cells to improve
immunogenicity. First, maturing DCs express a plethora of
cytokines, chemokines, and accessory molecules (32, 53, 54)
as illustrated in Fig. 2. Second, because of the capacity of
DCs to cross-present cell-associated antigens in vivo (26,
55), the tumor cells become an effective source of antigen,
potentially a spectrum of antigens for a broad immune at-
tack. Third, by delivering tumor cells to the DCs, one en-
hances antigen presentation beyond the presenting capacities
of the tumor cells themselves, because DCs express such effi-
cient processing pathways for MHC class I (26), class II (55),
and CD1 (56), even if the tumor cells have dampened their
own antigen-presenting activities, as is often the case (57).
PROTECTIVE IMMUNITY VIA MATURE, TUMOR-CAPTURING DCS | Liu et al.1514
This is illustrated by our findings with the presentation of
P1A. This is a classical tumor antigen, one of the first specific
antigens to be described by Van den Eynde et al. (38). The
experiments in Fig. 5 reveal the processing and presentation
of the nonmutated P1A antigen from the tumor cells.
Nonetheless there are limitations to our experiments.
We would like to mention three. First, the delivery of tumor
cells to DCs, while enhanced by the use of the i.v. route of
injection, requires a relatively large dose of irradiated tumor
in mice, at least 5 106 cells. Efficacy might be further in-
creased, for example, by opsonizing the tumor or otherwise
increasing the frequency of antigen-capturing DCs. Like-
wise, human research will be needed to determine the effec-
tive dose that will result in immunization of patients (e.g.,
with hematologic malignancies like myeloma). Second, we
have tested only one CD1d-binding glycolipid, -Gal Cer,
and there are newer synthetic and natural glycolipids that
may prove to be more effective (58, 59). As mentioned,
-Gal Cer has already been found to be nontoxic in humans,
even at relatively high doses. Third, our studies do not ad-
dress the potentially important regulation imparted by sup-
pressor T cells which, beginning with the work of R. North
and colleagues, has been shown to be able to regulate the
function of protective or effector T lymphocytes (60, 61).
The approach in this paper takes full advantage of the pos-
itive feedback between NKT cells and DCs that occurs when
a single dose of -Gal Cer is given together with irradiated tu-
mor cells. We have observed specific immunity to two differ-
ent hematopoietic tumors, the J558 plasmacytoma and the
A20 lymphoma. We would like to suggest that vaccination
with dying tumor cells, under conditions where the tumor
cells are captured by DCs maturing in response to innate
NKT cells, be evaluated in humans, and that this be initiated
with hematologic malignancies such as the tumors tested here.
MATERIALS AND METHODS
Mice. 6-8-wk-old BALB/c females were obtained from Taconic. BALB/c
transgenic mice expressing a TCR specific for the tumor antigen P1A35–
43:Ld complex have been described previously (39). J18/ mice on
BALB/c background were gifts from M. Tsuji (New York University
School of Medicine, New York, NY). Mice were maintained under spe-
cific pathogen-free conditions. All experiments were conducted according
to institutional guidelines.
Cell lines. The plasmacytoma J558 and the MHC class I mutant cell line
J558Ld have been described previously (40). The Meth A fibrosarcoma
was provided by Z. Li (University of Connecticut Health Center, Farming-
ton, CT). The A20 lymphoma was provided by H.I. Levitsky (Sidney Kim-
mel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD).
The cell lines were cultured in RPMI 1640 medium supplemented with
10% FCS, 100 g/ml penicillin/streptomycin, and 2 mM glutamine. All
lines tested negative for Mycoplasma by Hoechst staining and PCR reaction
(American Type Culture Collection).
Reagents. Rat mAbs for MHC class II (TIB120, M5/114.15.2), granulo-
cytes (RB6-8C5, Gr-1), B220 (TIB146, RA3-3A1), F4/80 (HB198), CD4
(GK 1.5), and CD8 (TIB211, 3.155) were obtained from the American
Type Culture Collection. Anti-CD16/32, PE-conjugated anti-CD8,
CD11b, B220, CD4, V8.3, IFN-, IL-2, H-2Ld/H-2Db, CD62L, CD69,
B7-H1, B7-DC, and allophycocyanin-CD11c were obtained from BD Bio-
sciences or eBioscience. Sheep anti–rat IgG conjugated to magnetic beads
were obtained from Dynal. Anti-CD11c and CD8 microbeads were ob-
tained from Miltenyi Biotec. The other reagents were RPMI 1640
(GIBCO BRL), FCS (GIBCO BRL), CFSE (GE Healthcare), ACK buffer
(BioSource30% BSA solution (Sigma-Aldrich). -Gal Cer (2S, 3S, 4R-
1-O(-galactopyranosyl)-2(N-hexacosanoylamino)-1,3,4-octadecanetriol) was
provided by Kirin Brewery and diluted in PBS.
Induction of cell death. Tumor cells were harvested, washed twice with
RPMI 1640, resuspended to 10 106/ml in RPMI 1640, and irradiated
with 75 Gy. Detection of apoptotic tumor cells used the annexin V-FITC
Apoptosis Detection Kit (BD Biosciences), after which flow cytometry
(FACS Vantage SE, Becton Dickinson) was performed. Within 24 h, 24%
of the tumor cells were apoptotic, i.e., annexin V positive but PI negative
(Fig. S1 B). By 48 h, 57% of the irradiated cells underwent secondary ne-
crosis; and 72 h later, 84% of them were necrotic (PI). Therefore, we refer
to the irradiated cells that we injected as “dying cells.”
Tumor-specific TCR transgenic T cells. CD8 P1A-specific, T cells
were prepared from meshed cell suspensions of TCR transgenic lymph
nodes and spleen by depleting B220, CD4, F4/80, and MHC class II–
expressing cells using sheep anti–rat IgG Dynabeads. For CFSE labeling (GE
Healthcare), the cells at 107/ml in PBS were incubated with 5 M CFSE for
10 min at 37C. The reaction was stopped by washing three times with PBS.
In vivo delivery of dying tumor cells, DC maturation stimuli, and
tumor protection assay. 2 107 irradiated J558-Ld cells were injected
i.v. or s.c. into BALB/c mice with or without -Gal Cer as a DC matura-
tion stimulus. In some experiments, we compared -Gal Cer to agonisitic
anti-CD40 mAb (1C10, 25 g i.p.) or the Toll-like receptor ligands, poly
IC (50 g i.p., Invivogen) or lipopolysaccharide (20 g i.p., Sigma-
Aldrich). The mice had been given CFSE-labeled P1CTL CD8 T cells i.v.
1 d earlier, or were naive animals. In some experiments, mice were killed 3 d
later, and T cell division and activation in spleen were analyzed by flow cy-
tometry. Additionally, 7 d or 2 mo later, 5 106 live J558 tumor cells were
inoculated subcutaneously. 5 106 Meth A fibrosarcoma was used as a
control tumor for challenge. Tumor cell growth was measured with calipers
every other day. Mice were scored positive for tumor as soon as tumors be-
came palpable and grew progressively. Mice were killed when tumor size
exceeded 400 mm2.
Flow cytometry. T cell division, phagocytosis of CFSE-labeled tumor
cells, and acquisition of cell surface–activation markers were determined by
flow cytometry. In brief, spleens were harvested and low density spleno-
cytes were stained with CD11c-allophycocyanin and CD8-PE, CD11b-
PE, B220-PE, or CD4-PE for the up-take examination. For in vivo prolif-
eration of P1CTL T cells, spleens were harvested and the splenocyte
suspension was staining with CD8-Cy, V8.3-PE, CD25-PE, and CD62L-
PE. For intracellular cytokine staining, splenocytes were stimulated in vitro
with 1 g/ml P1A peptide in 5 g/ml brefeldin A (Sigma-Aldrich) at 37C
for 4 h. The cells were first stained with CD8-Cy-Chrome, fixed, perme-
abilized with cytofix-cytoperm buffer (BD Biosciences), and stained with
PE-conjugated mAbs to IL-2 or IFN-.
In vivo depletion of CD4 and CD8 T lymphocytes. Mice vacci-
nated with J558 and -Gal Cer were injected with ascites containing 1 mg of
rat monoclonal anti-CD4 (clone GK 1.5) or anti-CD8 (clone 53–6.72). The
mice received three daily injections, the first one i.v. 1 d before the challenge,
and two i.p. injections on the day of the challenge and 1 d after the challenge.
Control mice received 1 mg of rat IgG (Jackson ImmunoResearch Laborato-
ries). The depletion was monitored by staining with anti-CD4 and anti-CD8
antibodies followed by flow cytometry (BD Biosciences).
Online supplemental material. Fig. S1 A shows lack of MHC class I
expression on J558 cells relative to parental J558 plasmacytoma. Fig. S1 B
JEM VOL. 202, December 5, 2005 1515
ARTICLE
depicts death of J558 cells, untreated or 24/48/72 h after -irradiation, as
assessed by staining with annexin V and propidium iodide. Online sup-
plemental material is available at http://www.jem.org/cgi/content/full/
jem.20050956/DC1.
K. Liu was supported by a predoctoral fellowship from the Cancer Research Institute
and R.M. Steinman was supported by National Institutes of Health grant nos. AI
13013 and AI 51573.
The authors have no conflicting financial interests.
Submitted: 11 May 2005
Accepted: 26 October 2005
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