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Department of Dermatology and Allergology, Ludwig-Maximilians-University, 80337 Munich [O. E., K. G., M. R.], and GSF-Institut für Molekulare Immunologie, 81377 Munich [A. D., R. M.], Germany
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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-producing CD4+ T cells [T helper
type 1 (Th1) cells] should be highly efficient for tumor immune
therapy. However, this approach could not be tested because very few
MHC class II-restricted tumor peptides have been defined. Here we show
that stimulation of freshly isolated T helper cells with syngeneic
tumor cells and antigen-presenting cells in the presence of
immunostimulatory CpG DNA allows the generation of large numbers of
strongly polarized, tumor-specific Th1 cells within 3 weeks of culture,
even when T helper cells were derived from tumor-bearing mice. A single
injection of 0.5 x 106 A20-specific Th1
cells even eradicated disseminated A20 lymphomas and provided lifelong
protection without inducing autoimmune disease. The therapy was largely
independent of CD8+ cells but required IFN- and
CD40-CD40L interactions, suggesting that tumor-specific Th1
cells eradicate established tumors by activating proinflammatory
macrophages. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Recent insights into the crucial role of CD4+ T
cells in orchestrating antitumor immunity (4, 5, 6, 7)
and the
power of adoptively transferred IFN--producing Th1 cells in
specifically destroying targeted tissues (8)
suggest that
adoptive transfer of tumor-specific Th1 cell lines could be a safe and
effective approach to tumor therapy. However, only a few MHC class
II-restricted tumor peptide epitopes have been characterized to date
(9)
, a fact that has strongly impaired the exact analysis
of tumor-specific CD4+ T cells, IFN-
-producing Th1 cells, and IL-4-producing Th2 cells in tumor immunity.
Generation of tumor-specific Th1 cell clones in vitro
(7)
or transfection of tumors with antigens recognized by
T-cell receptor-transgenic lymphocytes from healthy mice
(10)
allowed to address some important questions, but
these systems did not analyze the therapeutic potential of naturally
occurring, tumor-reactive Th1 and Th2 cells or the properties of
tumor-specific T cells occurring in tumor-bearing animals.
Because immunostimulatory CpG ODNs promote maturation of APCs and
induce the expression of costimulatory molecules and the production of
IL-12 (11, 12, 13)
, they should be useful as an in
vitro adjuvant for the rapid generation of tumor-specific Th1
cells. Here we report that immunostimulatory CpG ODN 1668
allowed us to generate large amounts of tumor-specific Th1 and Th2
cells within 3 weeks of culture, surprisingly even when
CD4+ T cells were isolated from mice with
advanced tumors. Adoptive transfer of these tumor-specific Th1 cells
efficiently eradicated even established tumors in a CD40L- and
IFN--dependent fashion, whereas in vitro-generated,
IL-4-producing Th2 cells prolonged survival but ultimately failed to
cure animals.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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RII/III. Vaccination with BiV trioma
cells that express the tumor idiotype as an immunoglobulin half
molecule attached to the APC-binding arm confers efficient immunity
against the wild-type tumor (14)
. T-cell lines were
cultured in DMEM supplemented with 10% FCS and 2-mercaptoethanol.
Phosphothioate-modified CpG ODN 1668 (5'-TCCATGACGTTCCTGATGCT-3') was
from MWG (München, Germany). The IL-4 and IFN- content
of culture supernatants was determined using commercially available
ELISA Abs (PharMingen, Hamburg, Germany).
T-cell Lines.
CD4+ T cells were derived from spleen and lymph
node cells from mice immunized twice with 105 BiV
trioma cells (14)
or from mice with a large tumor burden
(>day 30). These CD4+ T cells were enriched by
negative selection over Biotex T-cell columns (TEBU, Frankfurt,
Germany) to 95% purity. No CD8+ T cells were
detectable by fluorescence-activated cell-sorting analysis. The
CD4+ T cells (2 x 105) were stimulated in vitro in
96-well round-bottomed tissue culture plates in the presence of
irradiated (30 Gy) T-cell-depleted syngeneic spleen cells, 3 x 105 irradiated (90 Gy; from a
137Cs source) A20 tumor cells, CpG ODN 1668 (0.2
µM), IL-2 (5 units/ml; a gift from Chiron,
Ratingen, Germany), and anti-IL-4 mAb 11B11 (10 µg/ml) to generate
Th1 lines. To generate Th2 cell lines, IL-4 (1000 units/ml) was added
to the culture instead of anti-IL-4 mAb. Recombinant IL-4 was derived
from X63Ag-653 cells (a gift of F. Melchers; Basel Institute of
Immunology, Basel, Switzerland). On day 10, cells were purified
on a Ficoll gradient and restimulated as described on day 1. The cells
were expanded for another 7 days, and on day 20, these >99% pure
CD4+ T cells were used for adoptive transfer, and
a fraction of these cells were used for functional
characterization.
To generate anti-A20 CD8+ T cell lines, 4 x 106 spleen and lymph node cells from trioma-vaccinated mice were suspended in medium, seeded in 24-well plates, and mixed with 105 irradiated (100 Gy) A20 cells. A restimulation followed at day 6 with an additional 106 naive syngeneic spleen cells/well (irradiated at 30 Gy). On day 8, 30 units/ml IL-2 were added. One day later, T-cell reactivity against irradiated A20 or MPC11 cells was determined by [3H]thymidine incorporation or by measuring granulocyte macrophage colony-stimulating factor secretion in an ELISA. Before in vivo application, the remaining CD4+ T cells were depleted by immunomagnetic separation. This population was >99% CD8+.
Animal Experiments.
Mice were purchased from Bommice (Ry, Denmark). All experiments were
done with groups of five or six female animals. For immunization, mice
were injected i.p. with 105 BiV trioma cells
twice in a 3-week interval (14)
. In the tumor prevention
experiments shown, the animals received a lethal A20 tumor dose
(3 x 105 cells) together with
5 x 105
CD4+ T cells i.p. To evaluate therapy of a
preexisting tumor burden, mice were challenged first with a lethal i.v.
A20 inoculum (3 x 105 cells) and
treated with 5 x 105 Th1 cells
i.v. on day 5 or 7. Mice were euthanized when signs of tumor growth
became visible. CD8+ T cells were depleted
in vivo by five i.p. injections of 0.5 mg of the anti-CD8
mAb RmCD8.2 (14)
every 3–8 days starting 4 days before
Th1 therapy. IFN- was neutralized by injecting 0.5 mg of XMG-1.2 mAb
(15)
i.p. 24 h and again 4 h before Th1 cell
treatment. The anti-CD40L mAb MR-1 (16)
was given at a
dose of 0.25 mg i.p. on day 0, 2, 4, and 8 of Th1 therapy. All animal
experiments were performed at least twice. Statistical survival
analyses were done using the log-rank test.
ELISA.
To evaluate the humoral response against A20 cells after Th1 therapy,
mouse sera were tested for binding to purified A20 immunoglobulin
immobilized on ELISA plates or to A20 cells in a cellular ELISA.
Reactivity was detected by incubating these plates with a
peroxidase-conjugated polyclonal goat antimouse IgM Ab or an anti-IgG
Ab that had been adsorbed against mouse IgG2a. The IL-4 and IFN-
content in the 24 h culture supernatant of stimulated T cells was
determined as described (12)
. All ELISA Abs were from
PharMingen.
Cytotoxicity Assay.
Fresh spleen cells (2 x 106) were
stimulated with ConA (2 µg/ml) for 36 h to obtain ConA
blasts. The ConA blasts, A20 lymphoma cells, and MPC11 plasmacytoma
cells were labeled with [3H]thymidine (5
µCi/ml) for 8 h at 37°C. These target cells were washed twice
with medium and then mixed (5 x 104 cells/well) with the indicated numbers of
effector cells in 96-well round-bottomed plates. After incubation for
2 h at 37°C, cells were harvested on glass fiber filters, and
[3H]thymidine uptake was measured using an
automatic filter counting system (TRACE96; Inotech, Dottiuon,
Switzerland). The percentage of specific lysis was calculated as
described previously (17)
.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. These CD4+ T cells produced about
50 times more IFN- than IL-4 when exposed to A20 cells. No specific
cytokine production was observed when the cells were exposed to
syngeneic APCs or to an unrelated lymphoma, MPC11 (Fig. 1B)
.
Because these CD4+ T cells specifically killed
A20 lymphomas in vitro in a
Ca2+-independent fashion (Fig. 1D)
,
these cells were A20-specific Th1 cells.
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. Adoptive transfer of Th1 cells was more efficient than
adoptive transfer of A20-specific CD8+ cytotoxic
T-cell lines (Fig. 2B)
. Moreover, Th1 cells protected even
CD8-depleted mice from the A20 lymphoma (Fig. 2A)
,
suggesting that CD8-independent mechanisms play an important role in
Th1 cell-mediated tumor protection. The treatment provided lifelong
protection (up to 2 years), and no tumor cells were detected in spleen
or bone marrow by fluorescence-activated cell-sorting analyses. More
importantly, no dormant tumor cells were detectable in long-term
cultures from spleen or bone marrow cells of Th1 cell-treated mice
(data not shown).
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suggested that
A20-reactive Th1 cells predominantly recognized tumor-associated
antigens. Because adoptive transfer of autoreactive T cells can induce
severe autoimmune disease (8
, 13)
, and because the
tumor-reactive Th1 lines generated in vitro may recognize
surface proteins shared between normal tissue and A20 cells, we
performed extensive pathological examination of the Th1 cell-treated
animals. Peripheral blood cell counts and histology of spleen, lymph
nodes, bone marrow, liver, kidney, heart, lung, small bowel, and brain
were all normal and showed no signs of autoimmune disease.
Adoptive Transfer of A20-specific Th1 Cells Eradicates Disseminated
Lymphomas by a CD40L- and IFN--dependent Mechanism.
Adoptive transfer of A20-reactive Th1 cells was safe and effective in
preventing tumors. Therefore, we analyzed the cells’ capacity in the
therapy of established tumors. Within 5 days, injection of
0.3 x 106 A20 lymphoma cells
establishes a tumor burden that responds only to the most efficient
immunization protocols (14
, 18)
, and in the following 3
days, the splenic tumor burden increased again at least 100-fold
(analyzed in four mice). When given 5 days or even 7 days after
establishing the lymphoma, adoptive transfer of 0.5 x 106 A20-reactive Th1 cells still cured up to 70%
of the animals (Fig. 3A).
The therapy provided long-lasting immunity because 10 of
10 mice rejected a lethal A20 rechallenge on day 100 or 125. Effective
therapy was dependent on Th1 cell recognition of tumor-associated
peptides because identically generated, ovalbumine-specific Th1
cells provided no protection (Fig. 3A)
. Importantly, the
therapy required a high IFN-:IL-4 ratio. Th0 cells producing both
IL-4 and IFN- as well as IL-4-producing Th2 cells, which can induce
strong immunoglobulin production and attract eosinophils (6
, 7)
, significantly prolonged survival. However, all
IL-4-producing Th0 and Th2 cell lines tested were unable to cure mice
of the lymphoma (shown for Th2 cells in Fig. 1C
and
Fig. 3A
). The high therapeutic efficacy of A20-reactive Th1 cells
was surprising because it was shown that naive tumor-specific
CD4+ T cells from transgenic animals are
immediately silenced and provide no protection when transferred into
tumor-bearing mice (10)
. In contrast, transfer of
tumor-specific Th1 cell lines established persistent, A20-specific Th1
responses in vivo because spleen cells from Th1-treated mice
produced large amounts of IFN- when stimulated directly ex
vivo with A20 cells (Fig. 3B)
. It is not known whether
this IFN- was produced by CD4+ or
CD8+ T cells and whether the responding Th1 cells
are derived from the donor or the recipient. However, a series of
unrelated experiments suggests that most of the IFN- is derived from
the transferred Th1
cells.5
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-dependent monocyte/macrophage-mediated cytotoxicity (4
, 7)
, or induce production of antitumor immunoglobulin by B cells
(5)
. All three mechanisms are dependent on CD40-CD40L
interactions (16
, 20)
. To determine the contribution of
CD40-CD40L interactions to Th1 cell-mediated therapy, we blocked this
interaction in Th1 cell-treated animals with the anti-CD40L mAb MR-1.
This mAb completely abolished the capacity of anti-A20 Th1 cells to
cure established lymphomas (Fig. 3C)
. Although other models
of CD40-mediated tumor immunity suggest a crucial role for
tumor-specific CD8+ T cells (18)
and
for Abs directed against tumor epitopes (5)
, these two
modes of antitumor immunity seem to be of minor importance in our
model. Transfer of A20-reactive Th1 cells controlled the tumor in about
3 days, a time too short to provide help for immunoglobulin production
or cytotoxic T-cell responses. Consequently, injection of anti-CD8 mAb
barely influenced the efficacy of Th1 cell-mediated tumor therapy (data
not shown), and significant Ab titers against A20 cells could not be
detected (data not shown). Thus, Th1 cells may kill A20 cells through
direct CD40-CD40L interactions or indirect, IFN--dependent
mechanisms (4
, 7)
. To address the role of Th1 cell-derived
IFN- in adoptive tumor therapy, we inhibited IFN- with the
XMG-1.2 mAb in vivo according to a protocol that inhibits
Th1 cell-mediated protection in murine leishmaniasis (21)
.
The therapeutic efficacy of A20-reactive Th1 cells was completely
abrogated (Fig. 3D)
, strongly suggesting that adoptive
therapy with Th1 cells was dependent on both CD40-CD40L interactions
and IFN-.
Th cells express CD40L (14
, 20)
, and A20-reactive Th1
cells kill A20 lymphomas in vitro in a
Ca2+-independent fashion (Fig. 1D)
.
Because binding of CD40 may lead to direct tumor elimination
(18)
, our findings can be interpreted as direct killing of
the A20 lymphoma by CD40-binding Th1 cells. However, the data showing
that A20-specific Th0 cells producing both IL-4 and IFN- were
ineffective in curing mice from established lymphomas and that
anti-IFN- mAb abolished the therapeutic effect strongly suggest that
other mechanisms were critical for tumor elimination by Th1 cells.
Signaling through CD40 and IFN- activates APCs such as macrophages
and primes these cells for initiating inflammatory immune responses
(4
, 7 , 17
, 21, 22, 23)
. Because this priming of APCs is
inhibited by IL-4 or anti-IFN-, the IL-4-producing Th2 or Th0 cells
may have inhibited tumor elimination by suppressing the development of
proinflammatory macrophages, a mechanism originally described for
inflammatory autoimmune and parasitic diseases (8
, 21)
.
CpG ODNs Allow the Rapid Generation of Tumor-specific and
Protective Th1 Cells from Mice with Large Tumor Burden.
In the experiments described above, A20-reactive Th1 cells were derived
from preimmunized animals. To mimic a situation more close to the
clinic, we investigated whether anti-A20-reactive Th1 cells could also
be generated from mice that had to be euthanized because of tumor
wasting (>day 30). In some models, tumors eliminate or silence
(10)
tumor-reactive T cells, whereas in other transgenic
models, tumor-reactive T cells could be readily derived from
tumor-bearing animals (24)
. We first analyzed whether
A20-reactive CD4+ T cells could be detected in
lymphoma-bearing mice. Surprisingly, using CpG ODNs, we could readily
generate A20-specific, cytotoxic T cells, even from animals with a
large tumor burden (Fig. 4A)
. Whereas few or no tumor-specific Th1 cells were obtained
from cultures initiated in the absence of CpG ODNs (Fig. 4B)
, the addition of CpG ODN 1668 as an in vitro
adjuvant allowed us to generate large numbers of A20-reactive Th1 cells
even from animals with large tumors. These Th1 cells specifically
recognized A20 lymphomas and produced the same cytokine pattern as Th1
cells derived from immunized animals (Fig. 4C)
. Importantly,
these Th1 cells also rejected A20 lymphomas (Fig. 4D)
,
although preliminary results suggest that Th1 cell lines from
tumor-bearing animals may be less effective than Th1 cells derived from
BiV trioma-immunized animals (compare Fig. 2A
and
Fig. 4D
).
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:IL-4 ratio,
CD40-CD40L interactions, and IFN-. These data suggest that Th1 cells
eradicated the established tumors predominantly through an indirect
mechanism. Because Th1 cells control intracellular pathogens through
the activation of macrophages (21)
, and because activation
of macrophages by IFN- was recently shown to be important in tumor
prevention (7)
, it is likely that Th1 cells use this
CD40L- and IFN--dependent mode of cell-mediated immunity for
the elimination of established tumors. Surprisingly, these mechanisms of tumor elimination differ substantially from those recently reported by Nishimura et al. (25) , who found that both Th1 and Th2 cells are equally effective in tumor therapy. Moreover, in the system reported by Nishimura et al., Th1 cells strictly required tumor-specific CD8+ T cells to eradicate the tumor and killed lymphoma cells exclusively in a Ca2+-dependent fashion. Importantly, these two systems differ profoundly in their approach. Nishimura et al. used T-cell receptor-transgenic Th cells directed against OVA-transfected tumor cells. In contrast, we used no artificial rejection antigen and derived tumor-specific Th1 cells from nontransgenic, tumor-bearing animals by stimulating CD4+ T cells in the presence of CpG ODNs. Establishing whether these fundamentally different modes of tumor immunity are determined by the source of the Th, by the immunostimulatory ODNs, or by the routes of tumor application (s.c. versus i.p. or i.v.) may provide important data for the development of tumor therapies.
Importantly, tumor elimination was never associated with autoaggressive disease, showing that tolerance to self-proteins, whose expression pattern is shared between A20 cells and normal tissue, was not broken, even when A20-specific Th1 cells were derived from trioma-immunized animals. Because tumor-reactive Th1 cells can be generated not only from immunized animals but also from tumor-bearing animals, adoptive transfer of tumor-specific Th1 cells may provide a novel and safe therapeutic approach for hematopoietic tumors and perhaps even solid tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Sander Foundation (97.041.1),
SFB 217 and 456. 
2 O. E. and R. M. contributed equally to this work.
3 To whom requests for reprints should be
addressed, at: (a) GSF-Institut für
Molekulare Immunologie, Marchioninistrasse 25, 81377 Munich, Germany.
Phone: 49-89/7099-313; Fax: 49-89/7099-300; E-mail: mocikat{at}gsf.de
4 The abbreviations used are: APC,
antigen-presenting cells; ODN, oligodeoxynucleotide; Th, T helper; IL,
interleukin; mAb, monoclonal antibody; Ab, antibody; CD4OL, CD40
ligand; GnA, concanavalin A; OVA, ovalbumine.
5 O. Egeter and M. Röcken, unpublished
data.
Received 11/15/99. Accepted 2/ 3/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. Proc. Natl. Acad. Sci. USA, 96: 8633-8638, 1999.This article has been cited by other articles:
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) or in
combination with 0.5 x 106 Th1 cells (
).
On day 6 and day 13, spleen cells were stimulated in
vitro in the presence or absence of irradiated A20 cells. The
IFN-