The Cooperation between Two CD4 T Cells
Induces Tumor Protective Immunity in
MUC.1 Transgenic Mice
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J Immunol 2005; 175:6551-6559; ;
doi: 10.4049/jimmunol.175.10.6551
http://www.jimmunol.org/content/175/10/6551
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References
Mara Gerloni, Paola Castiglioni and Maurizio Zanetti
The Journal of Immunology
The Cooperation between Two CD4 T Cells Induces Tumor
Protective Immunity in MUC.1 Transgenic Mice1
Mara Gerloni, Paola Castiglioni, and Maurizio Zanetti2
Immunity and tumor protection in mice transgenic for human MUC.1, a glycoprotein expressed in the majority of cancers of
epithelial origin in humans, were induced by vaccination with B lymphocytes genetically programmed to activate MUC.1-specific
CD4 T cells. Their activation required a functional cooperation between two Th cells, one specific for a self (MUC.1) and the other
for a nonself T cell determinant. The immunological switch provided by Th-Th cooperation was sufficient to induce MUC.1-specific
CD4 and CD8 T cell responses in MUC.1-transgenic mice, and protect them permanently from tumor growth. CD4 T cells specific
for MUC.1 lacked cytolytic function, but produced IFN-␥ upon restimulation with Ag. We conclude that immunity against tumor
self-Ags and tumor protection can be regulated exploiting an inherent property of the immune system. The Journal of Immunology, 2005, 175: 6551– 6559.
which, in turn, enables the presentation of and the response to, a
subimmunogenic determinant by a second CD4 T cell (8).
In this report we show that T cell immunity to MUC.1, an Ag
expressed in the majority of tumors of epithelial origin, can be
induced by injection of syngeneic B lymphocytes as APCs genetically programmed (10) to trigger Th-Th cooperation in vivo. Specifically, we demonstrate that mice transgenic (Tg)3 for the human
MUC.1 Ag (11) that are tolerant to MUC.1 and unable to reject
MUC.1-expressing tumors (11–13) respond to the immunization
with Tg B lymphocytes mounting durable tumor-protective T cell
immunity. The key findings of the study can be summarized as
follows: 1) CD4 T cell immunity can be induced by active immunization against a single CD4 T cell determinant of a tumor
self-Ag by gauging the composition of the immunogen to enable
Th-Th cooperation; 2) genetically programmed B lymphocytes are
effective APCs and induce long-lasting tumor immunity; and 3)
antiself CD4 T cells induced by Th-Th cooperation jump-start the
activation of Ag-specific CD8 T cells specific for the same tumor
Ag. These data are relevant to understanding the role of CD4 T
cells in tumor immune surveillance and in the generation of protective tumor immunity.
Materials and Methods
Mice
The MUC.1-Tg mice colony was established at University of CaliforniaSan Diego from founders obtained from Dr. S. Gendler (Mayo Clinic,
Scottsdale, AZ). MUC.1-Tg-positive mice were identified by PCR analysis
as previously described (11). C57BL/6 mice (H-2b) were purchased from
Jackson ImmunoResearch Laboratories. All experimental animals were
housed at the University of California-San Diego animal facility under
standard pathogen-free conditions.
Plasmid DNA, synthetic peptides, and tumor cell lines
Laboratory of Immunology, Department of Medicine and Cancer Center, University
of California-San Diego, La Jolla, CA 92093
Received for publication February 2, 2005. Accepted for publication September
6, 2005.
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 National Institutes of Health Grants RO1CA77427 and
R01CA92119.
2
Address correspondence and reprint requests to Dr. Maurizio Zanetti, University of
California-San Diego, 9500 Gilman Drive, No. 0837, La Jolla, CA 92093-0837. Email address: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
Plasmid ␥1NV2VTSA3, ␥1VTSA3, and ␥1NV2NA3 carry chimeric H chain
genes under the control of a B cell promoter and are formed by the joining
of a human ␥1 constant (C) region gene with a rearranged murine variable
region gene engineered in the CDRs to code for heterologous Th cell determinants as previously described (8). The superscript numbers identify
the CDR in which a given peptide is expressed, e.g., ␥1NV2VTSA3 codes
for the -NVDP- (NV) peptide in CDR2 and the -VTSA- peptide in CDR3.
Each plasmid also carries the enhanced GFP gene inserted at the C terminus of the ␥1 C region. Plasmid pSVneo is the original plasmid forming the
3
Abbreviations used in this paper: Tg, transgenic; DC, dendritic cell.
0022-1767/05/$02.00
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T
umors use various strategies to evade immune surveillance and resist immunotherapy. For tumor Ags that are
true self-Ags, these factors include an intrinsic poor immunogenicity, immune tolerance/anergy, down-regulation of the
MHC molecules, ignorance, secretion of suppressive cytokines,
suppressor-regulatory T cells, and neutralization of immune T cells
by Fas ligand expression. Some of these factors depend on tumorhost interactions, because tumors are composed of neoplastic cells
intimately intermixed with nonneoplastic cells of the host, whereas
others reflect more directly acquired functional defects of the immune system. Activation of T cells requires two signals (Ag and
costimulation) where lack of costimulation prevents activation and
promotes anergy (1). Therefore, in principle, the ability to generate
immunity against tumor Ags rests on a simple immunological principle enabling the host to overcome the poor immunogenicity of
tumor Ags as well as self-tolerance. Promising new approaches
focused on the manipulation of costimulatory molecules have
shown that CTLA-4 blockade or OX40 ligation may enhance
priming of antitumor T cell responses in nontolerant mice (2, 3) or
break peripheral self-tolerance in nontumor model systems (4, 5).
In contrast, with the exception of peptide analogues (6, 7), little has
been proposed with respect to modifications of the Ag that could
strengthen signal 1 and also heighten signal 2.
A response to otherwise subimmunogenic determinants can be
induced by triggering a functional cooperation between two CD4
T help cells, Th-Th cooperation or help for helpers (8). As demonstrated previously, this phenomenon requires associative recognition of Ag (9) and is consistent with a three-cell model in which
the T cell response to a dominant determinant results in the activation of the APC via up-regulation of costimulatory molecules,
6552
backbone of the pNeo ␥1 vector without the human ␥1 C region (14) and
was one of the controls used in the protection experiments shown in Fig.
3. Plasmid DNAs were purified using a Megaprep kit (Qiagen) and were
stored at ⫺20°C until use. Synthetic peptides VTSAPDTRPAP (-VTSA-),
TSAPDTRPA (-VTSA-; 9-mer) NANP NVDP NANP (-NVDP-), and
OVA323–339 (ISQAVHAAHAEINEAGR) were synthesized at the Peptide
Chemistry Facility (California Institute of Technology). B16-MUC.1 and
B16Neo murine (C57BL/6) melanoma tumor lines transfected with a
MUC.1 cDNA or control expression vector (11) were gifts from Dr. S.
Gendler. These cell lines were maintained in DMEM with 10% FBS, penicillin (50 U/ml), and streptomycin (50 ␮g/ml), supplemented with 300
␮g/ml G418. Tumor cells were treated overnight at 37°C with 200 ng/ml
IFN-␥ before use in the CTL assay.
Spontaneous transgenesis of B lymphocytes
In vivo immunization and tumor challenge experiments
MUC.1-Tg mice were immunized by tail vein injection with a suspension
of 5 ⫻ 103 Tg B lymphocytes. In the tumor protection experiments, the
injection of Tg B lymphocytes was repeated on day 21. Four days after the
second immunization, mice were challenged with either 2 ⫻ 104 B16MUC.1 or B16neo tumor cells inoculated s.c. between the scapulae. Mice
were inspected daily, and tumor growth was recorded. Mice were killed
when tumors reached 2 cm in diameter. In the rechallenge experiment,
MUC.1-Tg mice that survived the first challenge were left to rest for 70
days and then rechallenged with either 2 ⫻ 104 B16-MUC.1 or B16neo
tumor cells. The procedures used are in accordance with National Institutes
of Health regulation on laboratory animal welfare according to approved
institutional protocols.
weekly with irradiated (100 Gy) bone marrow-derived DC pulsed with the
-VTSA- peptide at a responder-to-stimulator ratio of 10:1. IL-2 (Chiron)
was added to a final concentration of 6 U/ml. After 2 wk, CD4 T cells were
restimulated with -VTSA- peptide (10 ␮g/ml) or control OVA323–339 peptide (1 ␮g/ml) for 6 h at 37°C. Intracellular IFN-␥ detection was performed
using the commercially available Cytofix/Cytoperm kit (BD Pharmingen)
according to the manufacturer’s directions. CD4⫹/IFN-␥-producing T cells
were identified by cell surface staining using the FITC-conjugated Ab antiCD4 (clone L3T4) and PE-conjugated Ab anti-IFN-␥ (clone XMG1.2; BD
Pharmingen).
T cell assays
Proliferation assays were performed as previously described (8). Briefly,
spleen cells were harvested on day 14 and cultured for 3 days at 37°C in
the presence of -VTSA- or -NVDP- peptides (5 ␮g/ml). Tests were run in
triplicate. Results are expressed as cpm or as the stimulation index, calculated as the ratio of (cpm of cells cultured in the presence of synthetic
peptide)/(cpm of cells cultured in the absence of peptide). CD8 CTL were
detected in a conventional 51Cr release assay (15). Briefly, 21 days after
immunization, spleen cells were isolated and restimulated in vitro using the
-VTSA- peptide (5 ␮g/ml) for 6 days. Cultured cells were incubated at the
indicated E:T cell ratio with B16-MUC.1 cells, B16neo cells, EL-4 cells
pulsed with -VTSA- peptide, or EL-4 cells pulsed with the 9-mer -VTSApeptide as target cells. CD4 CTL assay was performed after five rounds of
in vitro restimulation. Cultured cells were incubated at the indicated E:T
cell ratios with B16-MUC.1 cells, B16neo cells, and B6-2 cells pulsed with
either the -VTSA- or the OVA323–339 peptide as targets. Results are expressed as the percentage of specific lysis and were determined as follows:
[(experimental cpm ⫺ spontaneous cpm)/(maximum cpm ⫺ spontaneous
cpm)] ⫻ 100.
Detection of cytokines
IL-2, IL-4, and IFN-␥ were detected in culture supernatants harvested 40 h
after initial seeding using the OptEIA mouse cytokine sets (BD
Pharmingen).
Statistical analysis
Survival curves were constructed using the Kaplan-Meier method. Significance was calculated by Fisher’s exact test, based on binomial distribution, where ␣ is reduced according to the Bonferroni criterion (0.05/number of tests).
Processing of VTSA from full-length MUC.1
To demonstrate processing of the -VTSA- peptide from full-length MUC.1,
the following experimental designed was used.
Preparation of dendritic cells (DC) pulsed with apoptotic B16-MUC.1
cells. Bone marrow-derived DC were cultured with recombinant GM-CSF
and IL-4 (1000 U/ml; BD Pharmingen) for 6 days, followed by overnight
incubation with apoptotic B16-MUC.1 or B16neo tumor cells at a 1:5 (DC
to tumor cell) ratio. Apoptosis was induced by mitomycin-C treatment (100
␮g/ml for 30 min at 37°C), followed by culture at 3–5 ⫻ 105 cells/ml for
48 h in RPMI 1640 containing 0.1% FCS. As a positive control, on day 7
of culture, DC were pulsed with the -VTSA- peptide (13-mer) for 1 h at
3°C. DC were irradiated (3000 rad), washed, and added to the CD4 T cell
culture.
Preparation of immune CD4 T cells. Mice immunized by injection of
5000 B lymphocytes Tg for ␥1NV2VTSA3 twice on days 0 and 21 were
killed 48 h after the booster injection. Spleens were pooled, and CD4 T
cells were negatively selected using the StemSep kit (StemCell Technologies) comprising a mixture of biotinylated Abs to the following surface
Ags: CD11b, CD45R, CD8, TER119, and Ly-6G. The recovered CD4 T
cells were then cultured in a 96-well plate (2 ⫻ 105 cell/well) with bone
marrow-derived DC at a ratio of 40:1.
Determining Ag presentation. The ability of immune CD4 T cells to be
restimulated by MUC.1-pulsed DC was assessed by [3H]thymidine incorporation after 72 h of culture. Culture supernatants were harvested 40 h
after initial seeding, and IFN-␥ in the culture supernatants was detected by
ELISA.
Generation and analysis of a CD4 T cell line
A CD4 T cell line specific for -VTSA- was generated in MUC.1-Tg mice
immunized twice with ␥1NV2VTSA3-Tg lymphocytes (5 ⫻ 103). Four
days after the second immunization, mice were killed, and spleens were
harvested. CD4 T cells were negatively enriched from pooled spleen cells
using anti-CD8 and anti-CD19 magnetic beads following the manufacturer’s instructions (Miltenyi Biotec). Enriched CD4 T cells were restimulated
Results
Tg B lymphocytes trigger Th-Th cooperation in vivo
First we established that Ag-presenting B lymphocytes Tg for plasmid ␥1NV2VTSA3 could generate an anti-MUC.1 CD4 T cell response attributable to Th-Th cooperation in vivo. To this end,
C57BL/6 mice were immunized by i.v. injection of primary syngeneic B lymphocytes rendered Tg for ␥1NV2VTSA3. This codes
for the subimmunogenic MUC.1 determinant VTSAPDTRPAP
(-VTSA-) and a dominant Th cell determinant NANPNVDPNANP
(-NVDP-) from a malaria parasite Ag (8). In this approach to immunization, B lymphocytes serve as APCs for both Ag synthesis
and presentation (16) and can be considered the sole APC because
contaminant DC represent ⬍0.2%. The participation of host APCs
in CD4 T cell priming is ruled out by experiments showing that Tg
B lymphocytes from MHC class II knockout mice fail to immunize
(10). Previously, we also showed that after spontaneous transgenesis ex vivo, naive B lymphocytes up-regulate costimulatory molecules and become de facto functional APCs (16).
C57BL/6 mice immunized with B lymphocytes Tg for
␥1NV2VTSA3 generated a vigorous (ⱖ30 stimulation index) response against both the -NVDP- and -VTSA- determinants (Fig.
1A). Immunization with lymphocytes Tg for -VTSA- only did not
generate a detectable response, proving that, as demonstrated previously using direct DNA immunization (8) and -VTSA- peptide
in IFA (our unpublished observations), -VTSA- per se in unable to
induce a CD4 T cell response. Because immunity was generated
using B lymphocytes Tg for the dual-epitope transgene
␥1NV2VTSA3, the effects observed in vivo are consistent with
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This was performed according to the procedure described previously (10).
Briefly, spleen cells (4 ⫻ 106) in 200 ␮l of PBS without Ca2⫹ and Mg2⫹
(CellGro) were incubated with 25 ␮g of plasmid DNA for 1 h at 37°C
together with 5 ␮g of PMACS Kk plasmid (Miltenyi Biotec), which codes
for a truncated mouse H-2 Kk molecule as a selectable cell surface marker.
The cells were then washed and incubated in RPMI 1640 medium (Irvine
Scientific) supplemented with HEPES buffer, glutamine, and 10% FCS at
37°C in 5% CO2 atmosphere overnight. The next day, the cells were harvested, and Tg cells were enriched by positive selection on a column MS⫹/
RS⫹ mounted on the magnetic field of a MACS separator (Miltenyi Biotec). Tg cells were enumerated by flow cytometry on a FACSCalibur (BD
Biosciences).
TUMOR PROTECTION BY Th-Th COOPERATION
The Journal of Immunology
6553
FIGURE 1. Activation of Th-Th cooperation by immunization with Tg B lymphocytes. A, CD4 T cell response induced via Th-Th cooperation in
C57BL/6 mice. Mice were immunized by single injection of 5 ⫻ 103 spleen B lymphocytes Tg for ␥1NV2VTSA3. B lymphocytes Tg for plasmid ␥1VTSA3
served as the control. Values represent the mean stimulation index of four mice per group ⫾ SD. B, Titration of the two CD4 T cell responses. C57BL/6
mice were immunized by a single injection of B lymphocytes Tg for ␥1NV2VTSA3. Two mice per group received an injection of decreasing amounts
(10,000 to 1) of Tg B lymphocytes. Mice were killed on day 14, and spleen CD4 T cells were restimulated in vitro with the -VTSA- (Œ) or the -NVDP(䡺) peptide. Tests were run in triplicate, and proliferative responses were expressed as cpm. The data shown are from a representative experiment of two
independent experiments performed, yielding a similar dose-response profile.
B lymphocytes genetically programmed to trigger Th-Th
cooperation immunize MUC.1-Tg mice
Next, we determined whether MUC.1-specific CD4 T cell immunity could be induced in mice Tg for the human MUC.1 Ag (11).
MUC.1-Tg mice are reportedly tolerant to MUC.1 and unable to
reject MUC.1-expressing tumors (11–13). In these mice, Abs
against MUC.1 do not provide immunity to tumor (12). MUC.1-Tg
mice given a single injection of ␥1NV2VTSA3-Tg lymphocytes
developed a CD4 T cell proliferative response against -VTSA(Fig. 2). No response was generated by injecting lymphocytes Tg
for plasmid control ␥1VTSA3, suggesting that the CD4 T cell response against -VTSA- after immunization with lymphocytes Tg
for ␥1NV2VTSA3 is due to the “help for helpers” effect of Th-Th
cooperation. Additional experiments were performed to ensure that
MUC.1-Tg mice are unable to respond against -VTSA- or MUC.1
using a variety of immunization conditions. As shown in Table I,
the synthetic peptide -VTSA- in immunological adjuvant with or
FIGURE 2. Th-Th cooperation breaks CD4 T cell tolerance to the
MUC.1 Ag. Groups of four MUC.1-Tg or C57BL/6 mice were immunized
by single injection of 5 ⫻ 103 Tg B lymphocytes as indicated. B lymphocytes Tg for plasmid ␥1VTSA3 served as the control. The data shown are
from a representative experiment of two independent experiments performed. Results are expressed as stimulation indexes.
without helper peptide -NVDP-, MUC.1⫹ B16 tumor cells, or B
lymphocytes Tg for ␥1VTSA3, all failed to induce a detectable
CD4 or CD8 T cell response to the -VTSA- peptide. Thus, the CD4
T cell response against -VTSA- in MUC.1-Tg mice requires Th-Th
cooperation, which is based on linked recognition of endogenously
processed Ag. A helper effect by epitopes of the backbone H chain
transgene was similarly ruled out. Consistent with previously published data on the type of cytokines produced after immunization
with Tg B lymphocytes (10), we found that after restimulation in
vitro with the -VTSA- peptide, only cultures from mice immunized
with the dual-determinant transgene produced IL-2, IFN-␥, and
IL-4 (Table II), making this a Th0 response.
Despite the fact that CD8 T cell tolerance to MUC.1 has been
broken in monkeys after DNA vaccination (17), and in MUC.1-Tg
mice immunized with armed DC (18 –20), the induction of CD4 T
cells specific for a MUC.1 peptide in MUC.1-Tg mice has not been
shown previously.
Active CD4 T cell immunity to MUC.1 protects against tumor
The possibility of generating specific anti-MUC.1 immunity in
MUC.1-Tg mice prompted us to investigate whether this was sufficient to protect from MUC.1-expressing tumors in vivo. We used
transplantable murine B16 (H-2b) melanoma cells transfected with
the human MUC.1 cDNA (12). MUC.1-Tg mice injected with 2 ⫻
104 B16-MUC.1 tumor cells s.c. develop tumors in 100% of mice
in ⬃20 days. In a pilot experiment we found that only two of eight
MUC.1-Tg mice were tumor protected if immunized by a single
injection of Tg lymphocytes, suggesting that priming per se is
insufficient to establish protective immunity (data not shown).
Therefore, protection experiments were conducted in mice immunized twice at a 3-wk interval. Four days after the second injection,
mice were challenged with B16-MUC.1 tumor cells or B16neo
cells as a control. As expected, none of the 12 mice immunized
with B lymphocytes Tg for -VTSA- only and challenged with B16MUC.1 tumor cells were protected. In contrast, all 23 mice vaccinated with B lymphocytes Tg for ␥1NV2VTSA3 and challenged
with B16-MUC.1 cells survived tumor free (Fig. 3, top panel).
Mice challenged with B16neo cells were not protected, suggesting
that protection is Ag specific. Protection was also not seen in mice
immunized with B lymphocytes Tg for control plasmids and subsequently challenged with B16-MUC.1 tumor cells.
The longevity of protection was assessed in protected mice allowed to rest for 70 days after the initial tumor challenge and then
subsequently rechallenged with either 2 ⫻ 104 B16-MUC.1 or
B16neo cells. We reasoned that if protection was Ag specific, only
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those described for Th-Th cooperation (8). Furthermore, the experiment demonstrates that a key requirement in Th-Th cooperation is that the two Th determinants (e.g., -NVDP- and -VTSA-)
need to be presented by the same APC (linked recognition of Ag)
to enable the antiself immunity.
The responses against the two Th cell determinants followed a
similar dose-response curve (Fig. 1B), suggesting that the thresholds of activation for the two responses are similar and that T cell
activation by the nonself-determinant dictates the threshold of T
cell activation against the subimmunogenic MUC.1 determinant.
6554
TUMOR PROTECTION BY Th-Th COOPERATION
Table I. MUC.1 Tg mice are tolerant to MUC.1
CD 4 T Cell Responseb
Vehicle
No.
Mice
Medium
(cpm)
VTSAc
(cpm)
␥1VTSA3 transgenic product
Tg B cells
3
-VTSA- 11 mer peptide
CFA
4
-VTSA- 11 mer ⫹ -NVDP- peptides
CFA
3
B16-MUC.1 tumor cells
Saline
3
115
601
75
115
241
493
297
994
529
971
547
1885
4460
23
40
48
50
78
49
45
143
156
92
710
590
2771
Immunogen
a
CTL Responseb
SI
Total
responders
EL-4
(% lysis)
EL-4 ⫹
VTSAd
(% lysis)
Total
responders
⬍1
0/3
0
0.2
0/3
⬍1
0/4
0
0.8
0/4
⬍1
0/3
0
0
0/3
⬍1
0/3
0
0
0/3
mice rechallenged with B16-MUC.1 cells would be protected. In
line with this prediction, all mice rechallenged with B16-MUC.1
cells survived tumor free for the duration of the observation period
(160 days), whereas mice rechallenged with B16neo cells died, as
did naive mice challenged with the B16-MUC.1 cells (Fig. 3, bottom panel). Taken together, these results demonstrate unambiguously that protective immunity against a tumor self-Ag enabled by
Th-Th cooperation is long lasting. It is noteworthy that the absence
of protection against the B16neo challenge implies that no epitope
spreading had occurred as a result of the first tumor injection.
cessing of the 11-mer -VTSA- peptide, which generated a 9-mer
peptide recognized by CD8 T cells. Examples of both CD4 and
CD8 epitopes within the same peptide structure have been reported
for self (26, 27) and nonself (28) Ags. Finally, while the characteristics and specificity of killing rule out lysis by NK cells, a role
for cross-priming by DCs is highly unlikely, because Tg B lymphocytes induce both CD4 and CD8 T cell responses in mice lacking functional DCs (10).
CD4 T lymphocytes against MUC.1 are noncytolytic, but
produce IFN-␥
Generation of CTL during priming of CD4 T cells against
MUC.1
Because Th-Th cooperation specifically expands CD4 T cells reactive with -VTSA- (8), we assumed that CD4 T cells may be
involved in the mechanism of protection. However, although there
exist only a few studies on CD4 T cell-mediated tumor protection
(21–25), the vast majority of reports emphasized the role of CD8
CTL. Therefore, we considered the possibility that MUC.1-specific
CTL were also induced as a result of the immunization. MUC.1-Tg
mice immunized with ␥1NV2VTSA3-Tg lymphocytes developed
specific CTL that lysed B16-MUC.1, but not B16neo, cells in a 4-h
51
Cr release assay before (Fig. 4, a– c) and after (Fig. 4, d and e)
booster. CTL from MUC.1-Tg mice also lysed EL-4 (H-2b) T lymphoma cells pulsed with the 9-mer peptide TSAPDTRPA, but not
the 11-mer VTSAPDTRPAP, demonstrating that CTL are specific
for a peptide embedded in the 11-mer structure expressed in the
transgene. The protective T cell response was originated in response to immunization and not after contact with tumor cells,
because the induction of CTL preceded tumor implantation (Fig. 4,
upper panels). Therefore, CTL priming probably results from pro-
Next, we investigated the role of CD4 T lymphocytes in tumor cell
lysis as a potential mechanism of protection in vivo. In the first
experiment we purified CD4 T cells from MUC.1-Tg mice immunized with ␥1NV2VTSA3-Tg lymphocytes using anti-CD4 magnetic beads, restimulated them in culture for 5 days, and then tested
them in a 51Cr release assay against B16-MUC.1 target cells. CD4
T cells did not kill even at the highest E:T cell ratio (Fig. 5A, left
panel), whereas whole spleen T cells from the same mice caused
marked lysis (Fig. 5A, right panel). In a second experiment we
generated a VTSA-specific CD4 T cell line again from MUC.1-Tg
mice immunized with ␥1NV2VTSA3-Tg lymphocytes. Cultured
CD4 T cells produced IFN-␥ after in vitro restimulation with the
-VTSA- peptide (Fig. 5B, upper panels), but not ex vivo after
priming (Fig. 5B, lower panels). The CD4 T cell line was unable
to lyse B6-2 cells, a class II⫹ nonsecreting murine B cell hybridoma (H-2d,b), pulsed with the 9-mer -VTSA- or B16-MUC.1 tumor
cells (Fig. 5C), proving conclusively that VTSA-specific CD4 T
lymphocytes lack lytic properties, but secrete IFN-␥.
Table II. Cytokines produced by T cells in response to VTSA
Immunogena
Vehiclea
No. Mice
IL-2b (pg/ml)
IFN-␥b (pg/ml)
IL-4b (pg/ml)
␥1NV2VTSA3
␥1VTSA3
Transgenic B cells
4
4
2550 ⫾ 353
25 ⫾ 15
7358 ⫾ 262
67 ⫾ 12
996 ⫾ 62
12 ⫾ 1
MUC.1 Tg mice were immunized with 2 ⫻ 103 Tg lymphocytes as indicated and sacrificed on day 14.
Tests were performed on culture supernatants collected 40 h after in vitro restimulation with the 11 mer -VTSA- peptide
as detailed in Materials and Methods.
a
b
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a
MUC.1 Tg mice mice were immunized with 1) 5 ⫻ 103 Tg B lymphocytes; 2) 50 ␮g /mouse of -VTSA- peptide emulsified in CFA; 3) a mixture of 50 ␮g of -VTSA- peptide
plus 50 ␮g of -NVDP- peptide emulsified in CFA; or 4) 2 ⫻ 104 B16-MUC.1 tumor cells in saline. SI, Stimulation index.
b
T cell responses were assessed on mice sacrificed 16 days after priming. Tests were performed as described in Materials and Methods.
c
11 mer -VTSA- peptide.
d
9 mer -VTSA- peptide.
The Journal of Immunology
6555
Processing and presentation of VTSA from full-length MUC.1
Discussion
To ensure that the -VTSA- sequence is processed and presented
from full-length MUC.1, an experiment was performed to assess
the ability of DC pulsed with apoptotic B16 MUC.1 cells to restimulate CD4 T cells from MUC.1-Tg mice immunized with
␥1NV2VTSA3-Tg lymphocytes. We reasoned that restimulation of
VTSA-reactive CD4 T cells would constitute proof that the
-VTSA- sequence is processed and presented from full-length
MUC.1. As shown in Fig. 6, DC pulsed with apoptotic B16
MUC.1 cells restimulated CD4 T cells generated in vivo. Both T
cell proliferation (left panel) and production of IFN-␥ (right panel)
were in a range comparable to that in DC pulsed with previously
processed VTSA peptide, used as a positive control.
We demonstrate that Th-Th cooperation is an effective way to
elicit protective T cell immunity against a bona fide tumor self-Ag.
There are two aspects to the significance of these findings. One is
that CD4 T cell immunity against a tumor self-Ag and tumor protection were induced strictly by active immunization in vivo. The
second is that this was accomplished by enlisting the response of
tumor (self)-specific CD4 T cells through the help provided by
CD4 T cells specific for a nonself-determinant. This mechanism is
consistent with predictions based on associative recognition of
linked T cell determinants (29) and validates the basic tenet of the
two-signal model for T cell activation (9, 30). We had previously
demonstrated that the simultaneous injection of plasmids coding
FIGURE 4. Induction of MUC.1specific CTL responses after Th-Th cooperation. a– c, MUC.1-Tg mice were
immunized once with 5 ⫻ 103 B lymphocytes Tg for ␥1NV2VTSA3 (a),
␥1VTSA3 (b), or pSVneo (c). The data
are presented as the percentage of specific lysis ⫾ SD of four mice per group.
Similar results were obtained in two additional experiments. d and e,
MUC.1-Tg mice were immunized
twice with 5 ⫻ 103 B lymphocytes Tg
for ␥1NV2VTSA3 (d) or ␥1VTSA3 (e).
Spleen cells were isolated on day 4 after
the second immunization and tested as
described above. The data are presented
as the percentage of specific lysis ⫾ SD
of four mice per group. Similar results
were obtained in two additional experiments. In both priming and booster experiments, four different target cells
were used in each instance as indicated.
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FIGURE 3. Development of protective, tumor-specific immunity in vivo in MUC.1-Tg mice after Th-Th
cooperation. Top panel, MUC.1-Tg mice were immunized on days 0 and 21 with 5 ⫻ 103 Tg B lymphocytes
as indicated. Four days after the booster injection, mice
were challenged with either 2 ⫻ 104 B16-MUC.1 or
B16neo tumor cells, and tumor growth was evaluated
over time. The number of mice is indicated in parentheses. Survival curves were constructed using the
Kaplan-Meier method. Data are cumulative for three
independent experiments. Significance (p ⬍ 0.00005)
was calculated using Fisher’s exact test, based on binomial distribution, where ␣ is reduced according to the
Bonferroni criterion (0.05/number of tests). Bottom
panel, Rechallenge experiment. Mice that survived the
first tumor challenge were subject to a second challenge
with either 2 ⫻ 104 B16-MUC.1 or B16neo tumor cells
70 days later. Survival was calculated as described
above. The number of mice used is indicated in parentheses. The procedures used are in accordance with National Institutes of Health regulations on laboratory animal welfare and approved institutional protocols.
6556
TUMOR PROTECTION BY Th-Th COOPERATION
for -VTSA- and -NVDP- in different sites of the same organ did
not generate immunity to MUC.1 (8), ruling out that -NVDP- may
serve merely as adjuvant. In this study we demonstrate that immunization of MUC.1-Tg mice with 1) the synthetic peptide
-VTSA- in immunological adjuvant with or without helper peptide
-NVDP-, 2) MUC.1⫹ B16 tumor cells, or 3) B lymphocytes Tg for
␥1VTSA3 all failed to induce a detectable CD4 or CD8 T cell
response against -VTSA- (Table I).
CD4 T cells are at the center stage of immune regulation and
play important roles in providing help to B cells (31), in CD8 T
cell priming (32), and in the generation of durable memory CD8 T
cell responses (33–35) with enhanced protection against viruses
(15) and tumors (36). CD4 help can also prevent CD8 T cell tolerance induction in a system in which both CD4 and CD8 T cells
express Tg TCR (37). In this study we show that CD4 T cells
regulate the induction of a CD4 T cell response against a tumor
self-Ag. To the best of our knowledge this is the first direct dem-
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FIGURE 5. Functional characteristics of anti-VTSA CD4 T cells. A, Purified CD4 T cells from MUC.1-Tg
mice immunized once with B lymphocytes Tg for ␥1NV2VTSA3 were tested
in 51Cr release assay against B16
MUC.1 target cells (left panel). Results
refer to the percentage of lysis by CD4
T cells from three individual mice
tested at two E:T cell ratios. Lysis by
whole spleen lymphocytes from the
same mice (right panel) was used as a
control. Each column corresponds to a
single mouse. B, Specific activation of
CD4 T cells. IFN-␥-specific CD4 T
cells were detected after 2-wk culture in
the case of the CD4 T cell line (upper
panels) or ex vivo in the case of spleen
lymphocytes 21 days after priming
(lower panels). In both instances, T
cells were restimulated for 6 h in vitro
with the -VTSA- peptide (right panels)
or the OVA323–339 peptide, used as control (left panels). Results are expressed
as the percentage of IFN-␥ -producing
CD4 T cells. C, Cytotoxic assay using
CD4 T cells. The cytotoxic activity
of CD4 T cell line was tested in 5-wk
cultures in a conventional 51Cr release
assay. Cultured cells were incubated at
the indicated E:T cell ratios with B16MUC.1 cells, B16neo cells, B6-2 cells
pulsed with the 9-mer -VTSA- peptide,
or B6-2 cells pulsed with control
OVA323–339 peptide, as target cells. Results are expressed as the percentage of
specific lysis.
onstration of the induction of CD4 T cell immunity against a tumor
self-Ag by active immunization. The data imply that the immunological switch provided by two interacting CD4 T cells in vivo
(one against a nonself and the other against a self Th cell determinant) is powerful enough to initiate a response against a tumor
self-Ag. They also underscore the importance of linked recognition
of endogenously processed Ag as a key requirement in Th-Th
cooperation.
In our model, activation of VTSA-specific CD4 T cells is the
result of Th-Th cooperation, where the response to a self-determinant requires licensing of the Ag-presenting B cell by activated
CD4 T cells specific for a nonself-determinant, a three-cell interaction dependent on CD40 and OX40 (10). In previous studies in
C57BL/6 mice, we demonstrated that this mechanism was sufficient to overcome the poor immunogenicity of the VTSA determinant (10). In this study we show that the same mechanism enables the induction of specific T cell immunity in MUC.1-Tg mice.
The Journal of Immunology
The VTSA peptide is a bona fide subimmunogenic Th cell determinant, because it becomes demonstrably immunogenic by changing the molecular environment (i.e., by creating the conditions for
Th-Th cooperation) while keeping constant the flanking residues,
hence excluding defects in processing of -VTSA- in B lymphocytes harboring the ␥1VTSA3. Together with the fact that the
-VTSA- sequence is processed from full-length MUC.1 (Fig. 6),
we conclude that Th-Th cooperation was key to overcome the
subimmunogenicity of the -VTSA- determinant in both wild-type
mice and MUC.1-Tg mice. Furthermore, the activation of CD8 T
cells against the 9-mer -VTSA- peptide fits within the framework
of a model in which activation of CD8 T cells by Ag-presenting B
lymphocytes is Th dependent (38). Collectively, our data and interpretation of the phenomenon suggest that the immunological
switch that hallmarks functionally Th-Th cooperation is the property of the APC (a B lymphocyte) where inducible costimulation
plays a critical role.
We believe that Th-Th cooperation as an immunological switch
enables immunity against -VTSA- in MUC.1-Tg mice through the
same tempo and requirements for CD40 and OX40 as in wild-type
C57BL/6 mice (8). What remains to be clarified is which barrier
Th-Th cooperation needed to overcome in MUC.1-Tg mice. For
instance, MUC.1-Tg mice possess CD8 T cell precursors for
MUC.1 at comparable frequencies as C57BL/6 mice (12), arguing
that in these mice there exists a residual T cell repertoire as observed in other Tg mice models (39). In the present study
MUC.1-Tg mice were found to respond promptly to immunization
via Th-Th cooperation, suggesting the existence of CD4 T cell
precursors that can be expanded by immunization. Thus, the refractoriness of MUC.1-Tg mice to respond to MUC.1 and to reject
MUC.1⫹ tumors may be due to two concurrent factors: 1) an intrinsic poor immunogenicity of the MUC.1 Ag, an oncofetal glycoprotein; and 2) central or peripheral tolerance. As demonstrated
in this study, Th-Th cooperation, but not immunization with synthetic peptide in immunological adjuvant or with tumor cells, was
per se sufficient to induce MUC.1-specific T cell immunity and
tumor protection in MUC.1-Tg mice. Taken together, the present
findings argue for an effect of Th-Th cooperation in correcting the
subimmunogenicity of the -VTSA- determinant and raise the possibility that it may be effective also in settings where unrespon-
siveness is compounded by a tolerant state. An alternative interpretation would be that the -VTSA- determinant is not processed
from full-length MUC.1, precluding its presentation in thymus
during the establishment of tolerance. In favor of this view is the
fact that the -VTSA- peptide is in all likelihood a poor binder to the
MHC class II cleft because it is relatively short, a fact possibly
reflected in its lack of immunogenicity when emulsified in IFA.
However, as demonstrated in this study, -VTSA- is processed and
presented in Tg B lymphocytes and in DC pulsed with apoptotic
B16-MUC.1 cells (Fig. 6), making this alternative interpretation
unlikely.
Although we are aware of no precedent for immunity against a
defined Th cell determinant of a tumor self-Ag by active immunization alone, there exists evidence that memory CD4 T cells
generated in wild-type C57BL/6 mice adoptively transferred into
MUC.1-Tg mice provide specific tumor immunity and increase
survival (12). In our study, protected MUC.1-Tg mice developed
both Ag-specific CD4 and CD8 T cell responses. We favor a scenario where activation of the first CD4 T cell against the nonselfdeterminant is a prerequisite for the subsequent induction of antiMUC.1 CD4 T cells and thereafter of CTL via intraepitope (11mer 3 9-mer) processing. Anti-MUC.1 CTL have been shown to
be tumor protective in adoptive transfer experiments (40). Thus,
we propose that protection conferred by the antitumor immune
response triggered by Th-Th cooperation may be due to the combined effects of anti-MUC.1 CD4 T cells producing IFN-␥ and
CD8 T cells with conventional cytotoxic activity. The effect of
IFN-␥ may be mediated by the tumoricidal activity of inducible
NO synthase and NO produced by activated macrophages (41– 43)
or through the inhibition of angiogenesis (44). A role for CD4 T
cells and IFN-␥ in rejection of B16-MUC.1 tumors in C57BL/6
mice has been suggested (45). Future studies will need to address
these issues and ascertain the relative importance of CD4 and CD8
T lymphocytes in the mechanism(s) of protection by adoptive
transfer and selective in vivo depletion of T cell populations.
The induction of a tumor-specific CD4 T cell response via
Th-Th cooperation may also be an important source of IL-2 helping the local antitumor effect of specific CD8 T cells concomitantly
induced. It is becoming apparent that CD8 T lymphocytes of subjects immunized with synthetic peptides of tumor Ags fail to cause
tumor regression, being, by and large, in a state of anergy that can
be reversed in vitro by exogenous IL-2 (46). Similarly, tumor regression by adoptive antitumor T cell therapy combined with active vaccination requires the in vivo administration of IL-2 (47).
Therefore, IL-2 produced locally during Th-Th activation may
positively affect the functional status of antitumor CTL.
The results and conclusions of the study presented raise an interesting question about the possible role of Th-Th cooperation as
a mechanism to generate or amplify autoimmunity. Although it has
been known for a long time that immunization with allogeneic or
xenogeneic tissues induces autoreactive B and T cell responses
and, in some instances, organ-specific autoimmune diseases, no
direct molecular evidence exists for an involvement of Th-Th cooperation. A plausible argument would be that if the initiation of
an autoimmune response reflects both the manner in which selfAgs are presented to the immune system and the immune status of
Ag-specific T and B cells, Th-Th cooperation could easily link the
response to a cross-reacting heterologous Ag (seemingly functioning as a nonself-determinant) to that against the a self-determinant.
Thus, Th-Th cooperation could be responsible for the activation of
autoreactive CD4 T cells and together with epitope spreading (48)
could represent the molecular substrate for the de novo generation
or amplification of autoreactive T cell responses in vivo. In this
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FIGURE 6. CD4 T cells induced by immunization against VTSA are
restimulated by processed MUC.1 Ag. CD4 T cells from immunized mice
were cultured with DC pulsed with either apoptotic tumor cells or the
11-mer -VTSA- peptide. Left panel, The specific proliferative response is
shown. Results are expressed as the mean cpm ⫾ SD of triplicate cultures.
Right panel, IFN-␥ production of the same cultures. Supernatants were
collected after 40 h of culture. Experiments were performed using a DC to
CD4 T cell ratio of 1:40.
6557
6558
scenario the rate-limiting factor would be the availability, or accessibility, of a nonself-determinant that must, according to the
postulate of Th-Th cooperation (49), be presented in linked association with a self-determinant. Ultimately, the frequency with
which Th-Th cooperation could lead to autoimmunity will depend
on the accumulation of insertional or deletional mutations in the
genome of the individual that give origin to a nonself-determinant,
the association with predisposing MHC genes (50), and the escape
from control by autoimmune regulatory genes such autoimmune
regulator (51).
In summary, this study demonstrates that CD4 T cell immunity
to a tumor self-Ag can be induced strictly by activating the immunological switch provided by the cooperation between two Th
cells. This finding strengthens the importance of cell-cell cooperation within the dynamics of immune regulation and the ability of
the immune system to generate antiself responses. They also point
to new ways in which the immune response against tumor Ags can
be manipulated and protective antitumor immunity induced.
We thank Drs. M. Cohn and J. Hernandez for critically reading the manuscript and for their helpful suggestions, and we are indebted to Dr. S.
Gendler for originally providing the colony of MUC.1-Tg mice and B16MUC.1 cells.
Disclosures
M. Gerloni is an employee of Cosmo Bioscience, which is developing a
vaccine based on the use of transgenic lymphocytes.
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