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CD4-Directed Peptide Vaccination Augments
an Antitumor Response, but Efficacy Is
Limited by the Number of CD8+ T Cell
Precursors
Holly L. Hanson, Silvia S. Kang, Lyse A. Norian, Ken
Matsui, Leigh A. O'Mara and Paul M. Allen
J Immunol 2004; 172:4215-4224; ;
doi: 10.4049/jimmunol.172.7.4215
http://www.jimmunol.org/content/172/7/4215
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Copyright © 2004 by The American Association of
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References
The Journal of Immunology
CD4-Directed Peptide Vaccination Augments an Antitumor
Response, but Efficacy Is Limited by the Number of CD8ⴙ
T Cell Precursors1
Holly L. Hanson,2,3 Silvia S. Kang,2 Lyse A. Norian, Ken Matsui, Leigh A. O’Mara, and
Paul M. Allen4
P
eptide vaccination against defined tumor-associated Ags
(TAAs)5 as a means of treating established tumors or preventing the development of residual tumor growth in highrisk patients is currently an area of intense research. Most peptide
vaccination studies conducted to date have focused on CD8-directed immunization, capitalizing on the identification of MHC
class I-restricted tumor Ags (1–3). Trials in cancer patients have
demonstrated that Ag-specific CD8⫹ T cell expansion and IFN-␥
production can occur in response to peptide vaccination (4 –7).
However, these responses are often short-lived (6) and frequently
do not correspond to protection in terms of objective tumor regression (7–10). The latter observation may be due to a failure of
MHC class I-specific vaccination to induce fully functional CD8⫹
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
Received for publication April 29, 2003. Accepted for publication January 20, 2004.
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 grants from National Institutes of Health, predoctoral
fellowships from the Howard Hughes Medical Institute (to H.L.H. and L.A.O.), and
a Cancer Research Institute postdoctoral fellowship (to L.A.N.).
2
H.L.H. and S.S.K. contributed equally to this work.
3
Current address: Center for the Study of Hepatitis C, The Rockefeller University,
1230 York Avenue, New York, NY 10021.
4
Address correspondence and reprint requests to Dr. Paul M. Allen, Washington
University School of Medicine, Department of Pathology and Immunology, 660
South Euclid Avenue, Campus Box 8118, St. Louis, MO 63110. E-mail address:
[email protected]
5
Abbreviations used in this paper: TAA, tumor-associated Ag; CD40L, CD40 ligand;
ERK, extracellular signal-regulated kinase; KO, knockout; nERK, native ERK peptide; pCTL, cytotoxic T cell precursors; tERK, tumor-derived ERK peptide.
Copyright © 2004 by The American Association of Immunologists, Inc.
T cell effectors (11). Clearly, additional strategies for using the
potential of TAA immunization are needed.
The induction of CTLs as well as maintenance of CD8⫹ T cell
responses have been shown to rely on CD4⫹ T cells. For example,
the efficient generation of CTLs through cross-priming is dependent upon the activation of APCs via CD40-CD40 ligand (CD40L)
interactions between the APC and CD4⫹ T cell (12–17). Additionally, CD4⫹ cells are essential for the secondary expansion of
CD8⫹ T cells that occurs when Ag is re-encountered (18) and for
the generation of CD8⫹ memory T cells (19 –23). In tumor systems, the presence of CD4⫹ T cells has been associated with significant increases in CTL killing capacity; in the numbers of CD8⫹
T cells sustained over time; in recruitment of CD8⫹ T cells, eosinophils, and macrophages to the tumor site; and in long-term
survival and tumor rejection (20, 24 –27). Other mechanisms for
tumor rejection involving IFN-␥ (28) and inducible NO synthase
production (24) have also been found to be dependent on the presence of CD4⫹ T cells. Taken together, these data demonstrate a
critical role for CD4⫹ T cell help in the generation of an optimal
antitumor immune response.
Until recently, however, peptide vaccination directed at boosting antitumor responses in CD4⫹ T cells was not feasible due to a
lack of defined MHC class II-presented TAAs. To overcome this
deficiency, novel molecular protocols have been developed to facilitate identification of tumor cell-derived CD4⫹ T cell epitopes
important in murine models (29, 30) as well as in human disease
(31–34). As a result, a growing number of MHC class II-restricted
epitopes is now known (1, 3), and the efficacy of combined MHC
class I- and II-presented peptide vaccination in cancer patients is
being evaluated (35–37). One study using a peptide composed of
linked CD4- and CD8-directed epitopes in a murine tumor model
0022-1767/04/$02.00
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Peptide vaccination is an immunotherapeutic strategy being pursued as a method of enhancing Ag-specific antitumor responses.
To date, most studies have focused on the use of MHC class I-restricted peptides, and have not shown a correlation between
Ag-specific CD8ⴙ T cell expansion and the generation of protective immune responses. We investigated the effects of CD4-directed
peptide vaccination on the ability of CD8ⴙ T cells to mount protective antitumor responses in the DUC18/CMS5 tumor model
system. To accomplish this, we extended the amino acid sequence of the known MHC class I-restricted DUC18 rejection epitope
from CMS5 to allow binding to MHC class II molecules. Immunization with this peptide (tumor-derived extracellular signalregulated kinase-II (tERK-II)) induced Ag-specific CD4ⴙ T cell effector function, but did not directly prime CD8ⴙ T cells.
Approximately 31% of BALB/c mice immunized with tERK-II were protected from subsequent tumor challenge in a CD40dependent manner. Priming of endogenous CD8ⴙ T cells in immunized mice was detected only after CMS5 challenge. Heightened
CD4ⴙ Th cell function in response to tERK II vaccination allowed a 12-fold reduction in the number of adoptively transferred
CD8ⴙ DUC18 T cells needed to protect recipients against tumor challenge as compared with previous studies using unimmunized
mice. Furthermore, tERK-II immunization led to a more rapid and transient expansion of transferred DUC18 T cells than was
seen in unimmunized mice. These findings illustrate that CD4-directed peptide vaccination augments antitumor immunity, but
that the number of tumor-specific precursor CD8ⴙ T cells will ultimately dictate the success of immunotherapy. The Journal of
Immunology, 2004, 172: 4215– 4224.
4216
CD4 T CELL PRIMING AUGMENTS CD8 ANTITUMOR RESPONSE
Materials and Methods
Tumor cells and in vivo tumor challenge
CMS5 fibrosarcoma (40) cells were propagated in vitro in RPMI 1640
supplemented with 10% FCS (HyClone Laboratories, Logan, UT), 1 mM
Glutamax (Life Technologies, Gaithersburg, MD), and 50 ␮g/ml gentamicin. For in vivo tumor challenges, tumor cells were removed from culture
flasks by trypsinization, washed twice in PBS, and injected s.c. Tumor
growth was measured every 48 h using calipers and was recorded as the
product of two orthogonal diameters (a ⫻ b). The first diameter was identified as the longest surface length (a), with the second diameter being the
subsequent orthogonal width (b).
Mice
DUC18 TCR transgenic mice on the BALB/c background have been described previously (39). BALB/c mice were obtained from National Cancer
Institute, and CD40 knockout (KO) mice on the BALB/c background
were obtained from The Jackson Laboratory (Bar Harbor, ME). All
experimental mice were age-matched females (8 –12 wk). All mice were
housed in a specific pathogen-free barrier facility at Washington
University.
Peptides
The peptides used in this study were synthesized by standard F-moc chemistry using either a Synergy Peptide Synthesizer (model 432A; Applied
Biosystems, Foster City, CA) or a multiple peptide synthesizer (Symphony/Multiplex; Protein Technologies, Tucson, AZ). All peptides were HPLC
purified, and analyzed by mass spectrometry (Washington University Mass
Spectrometry Resource) for purity. Peptide concentration was determined
by amino acid analysis (model 6300; Beckman, Fullerton, CA). The amino
acid sequences for the peptides used in this study are: tERK-I (136 –142),
QYIHSANVL; tERK-II (133–147), RGLQYIHSANVLHRDLK; native
ERK-I (nERK-I) (136 –142), KYIHSANVL; nERK-II (133–147),
RGLKYIHSANVLHRDLK; OVA (323–339), ISQAVHAAHAEINEAGR
(41). tERK-I and -II denote peptides derived from the mutated tumor form
of ERK, while nERK-I and -II denote the corresponding peptides containing native ERK sequences present in normal tissues. The tERK-I peptide is
aimed at eliciting a CD8⫹ CMS5-specific response, whereas tERK-II is
aimed at generating a CD4⫹ T cell response. The nERK-I and nERK-II
peptides are used as controls for these tumor-specific peptides. The tumorderived peptides (tERK-I and -II) differ from their respective native controls (nERK-I and -II) only at position 136, in which there is an amino acid
substitution of a Q for a K.
Intracellular cytokine staining
Single cell suspensions were made from popliteal and superficial inguinal
lymph nodes from immunized mice, and 3 ⫻ 106 lymph node cells were
stimulated for 18 h in the presence of 3 ⫻ 106 APCs lacking T and B cells
from CB17 SCID mice (kind gift of E. Unanue (Washington University, St.
Louis, MO)) and with no Ag, 10 ␮M tERK-II, 10 ␮M OVA, or 10 ␮M
tERK-I. During the last 4 h of stimulation, 10 ␮g of brefeldin A was added
to the cultures. Cells were stained with CyChrome-conjugated anti-CD8
(BD PharMingen, San Diego, CA) and PE-conjugated anti-CD4 (BD
PharMingen). Stained cells were fixed in 5% formalin and permeabilized
with 0.05% saponin. Permeabilized cells were then stained with FITCconjugated anti-IFN-␥ and immediately analyzed by flow cytometry on a
BD Biosciences FACSCalibur (San Diego, CA) using CellQuest analysis
software. For each sample, 50,000 events falling in the live lymphocyte
gate were collected for analysis.
Immunization and lymph node proliferation assay
BALB/c and CD40 KO mice were immunized in the hind footpad with 20
nmol of peptide emulsified in CFA (Fisher Scientific, St. Louis, MO). After
7 days, popliteal and superficial inguinal lymph nodes were removed from
immunized mice. A total of 5 ⫻ 105 cells/well was cultured in a total
volume of 200 ␮l of RPMI 1640 supplemented with 1% normal mouse
serum, 5 ⫻ 10⫺5 M 2-ME, 1 mM Glutamax, 1% nonessential amino acids,
1% sodium pyruvate, 1% HEPES, and 50 ␮g/ml gentamicin. Assays were
performed in a 96-well flat-bottom tissue culture plate with the indicated
concentrations of each peptide. The cultures were incubated at 37°C for
72 h and then pulsed with 0.4 ␮Ci of [3H]thymidine and harvested 18 –24
h later.
Adoptive transfer of DUC18 T cells
Single cell suspensions of DUC18 splenocytes from homozygous transgenic mice were prepared in HBSS, washed twice, and counted. Transferred populations were normalized for V␤8.3 and CD8 dual positivity, as
determined by flow cytometry. Transferred cells were injected i.v. via tail
vein injection.
Enumeration of expanded Thy-1.1 DUC18 T cells
Thy-1.2 BALB/c mice were immunized with 20 nmol of tERK-II peptide
emulsified in CFA or were left unimmunized on day ⫺7. Single cell suspensions of Thy-1.1 DUC18 splenocytes were prepared in HBSS, RBC
lysed, washed, and counted. Transferred populations were normalized for
the number of Thy-1.1 DUC18 T cells based on V␤8.3 and CD8 staining
determined by flow cytometry. Cells were resuspended to contain 1000
naive Thy-1.1 DUC18 T cells in a 200 ␮l vol of HBSS and injected i.v. via
the tail vein into primed or unprimed Thy-1.2 BALB/c recipients on day
⫺1. On day 0, all mice were challenged s.c. with 3 ⫻ 106 CMS5 cells.
Tumor-draining lymph nodes and spleens from individual mice were harvested on days 4, 6, and 8 posttumor challenge. Cells were counted; stained
with V␤8.3 FITC, Thy-1.1 PE, and CD8 CyChrome (BD PharMingen);
and read on the FACSCalibur. The number of transferred Thy-1.1⫹
DUC18 T cells present in each organ was enumerated by multiplying the
percentage of cells that were V␤8.3⫹, Thy-1.1⫹, and CD8⫹ by the total
number of cells.
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illustrated that both Ag-specific CD4⫹ and CD8⫹ T cells were
induced in response to vaccination, and that protective antitumor
immunity could be generated as a result (38). Two studies that
identified unique MHC class II-presented murine TAAs provided
evidence that the CD4⫹ tumor-specific T cell response stimulated
by these Ags can be tumor protective (29, 30). At this time, however, far fewer MHC class II-restricted TAAs have been described
as compared with their MHC class I-restricted counterparts, and
consequently, development of CD4-specific vaccination strategies
remains difficult.
We sought to examine the effects of a CD4-directed vaccination
strategy on CD8-mediated tumor rejection, by evaluating effector
function of both endogenous CD8⫹ T cells and limited numbers of
transferred TCR transgenic tumor-specific precursor CTLs. To accomplish this, we generated a synthetic 17-aa peptide by extending
the sequence of a defined class I-restricted epitope to allow for
binding to the MHC class II molecules present in our previously
described DUC18/CMS5 tumor model system (39). CMS5 is a
MHC class I⫹/class II⫺ fibrosarcoma line derived from a BALB/c
tumor. CD8⫹ T cells from DUC18 TCR transgenic mice express
an ␣␤ TCR specific for a CMS5 tumor rejection Ag that arises
from a point mutation in extracellular signal-regulated kinase 2
(ERK2). The specific CTL epitope comprises aa 136 –144 of the
mutated tumor ERK2 (tERK) bound to H-2Kd and is designated
tERK-I. Transfer of as few as 12,000 naive DUC18 T cells is
sufficient to protect BALB/c mice from subsequent challenge with
CMS5 (39).
In this study, we find that prophylactic vaccination of BALB/c
mice with a CD4-directed peptide, designated tERK-II, induces an
Ag-specific CD4⫹ T cell response as evidenced by proliferation
and IFN-␥ production upon peptide-specific restimulation. Vaccination with tERK-II is sufficient to protect 31% of BALB/c mice
from subsequent challenge with CMS5. In contrast, vaccination
with the 9-aa class I-restricted tERK-I peptide has no protective
effects in vivo. Protection mediated by tERK-II and the resultant
Ag-specific CD4⫹ T cell response is due to enhanced priming of
endogenous CD8⫹ T cells, and relies upon CD40/CD40L interactions in the host. Increased CD4⫹ Th cell function following
tERK-II vaccination allowed transfer of fewer tumor-specific precursor CD8⫹ T cells to prevent tumor growth in vaccinated recipient mice. Our results suggest that this simple strategy of extending
known MHC class I-restricted tumor Ag epitopes to allow presentation by MHC class II molecules is an effective method for generating CD4-directed peptide vaccines that are able to boost tumorspecific CTL function in vivo.
The Journal of Immunology
Results
Peptide vaccination with tERK-II primes Ag-specific
proliferation
In vivo priming with tERK-II/adjuvant protects BALB/c mice
from subsequent tumor challenge
To determine the protective effects of vaccination using the
tERK-II peptide, mice were immunized with peptide and then
challenged s.c. with CMS5 7 days later. A significant percentage of
mice immunized with tERK-II peptide was protected from subsequent tumor challenge. Vaccination with tERK-I, nERK-II, or
OVA offered no protection (Fig. 2). In five independent experiments, we consistently observed some degree of protection in
tERK-II-vaccinated mice. Collectively, 31% of the mice immunized with tERK-II were fully protected from CMS5 challenge,
whereas none of the mice immunized with OVA, tERK-I, or
nERK-II showed any protection (Fig. 2B). Additionally, tumors
that did grow out in tERK-II-immunized mice were substantially
smaller than tumors growing in untreated mice (Fig. 2A). Interestingly, although immunization with the control peptide OVA (323–
339) elicited a strong CD4⫹ T cell proliferative response (Fig. 1C),
this nonspecific T cell help was not sufficient to inhibit tumor
growth. This observation illustrates that, in agreement with previous reports (46), tumor-specific CD4⫹ T cell proliferation and activation are required for antitumor effects of the immunization, and
that nonspecific local production of cytokines or stimulation of
nonspecific CD4⫹ T cells is not sufficient to help the antitumor
effector response. Therefore, these results demonstrate that CD4directed vaccination of mice with tERK-II was partially efficacious
in protecting mice from CMS5 tumor challenge.
Vaccination with tERK-II/adjuvant does not directly prime a
tERK-I-specific response
Because the tERK-II epitope contains the entire tERK-I epitope, it
was necessary to determine whether the protective effect of the
tERK-II immunization was due to the direct priming of CD8⫹
tERK-I-specific T cells. Mice immunized with the tERK-II peptide
did not have an in vitro recall response to the tERK-I peptide
before tumor challenge, indicating that the tERK-II immunization
did not directly prime a tERK-I-reactive T cell population (Fig.
1B). Additionally, priming BALB/c mice directly with the tERK-I
peptide in adjuvant was not protective against subsequent CMS5
challenge (Fig. 2A), confirming that the efficacy of the tERK-II
peptide immunization was not due to inadvertent priming of a
CD8⫹ tERK-I-specific effector cell population. Therefore, neither
CD4-directed tERK-II immunization nor CD8-directed tERK-I
immunization appears to directly prime endogenous tumor-specific
CD8⫹ T cells.
The protective effect of tERK-II immunization is CD40
dependent
CD40/CD40L interactions are known to be critical for the initiation of effective tumor-specific immune responses through activation of CD4⫹ Th cells (47) and through activation of APCs (48).
We therefore investigated the CD40 dependence of tERK-II-elicited
protection in our system. Both CD40⫺/⫺ and BALB/c mice succumb
to rapid tumor growth following CMS5 challenge (Fig. 3A).
The CD4⫹ T cells stimulated by tERK-II immunization could
help the final CD8⫹ T cell effectors in a manner that is either CD40
independent (49) or depends on the interaction between CD40L on
the activated CD4⫹ T cell and CD40 on APCs. In marked contrast
to the BALB/c mice, in which tERK-II vaccination reproducibly
induced partial protection from subsequent CMS5 tumor growth,
none of the CD40 KO mice were protected from subsequent CMS5
challenge (Fig. 3B). This indicates that the mechanism of protection mediated by tERK-II immunization depends upon the presence of CD40.
CD40 is necessary for tERK-II immunization to elicit tERK-Ispecific CD8⫹ T cell effectors
Because tERK-II immunization did not protect the CD40 KO mice
from CMS5 tumor growth, we investigated whether tERK-II-immunized CD40⫺/⫺ mice were capable of priming any tERK-Ispecific CD8⫹ T cells. We primed BALB/c and CD40-deficient
mice with either tERK-II or OVA control peptide. Seven days after
immunization, mice were either challenged with CMS5 or sacrificed so that the specificity of the immunization could be assayed
by measuring intracellular IFN-␥ production ex vivo. Popliteal and
superficial inguinal lymph node cells were cultured overnight in
the presence or absence of Ag before being stained for CD4, CD8,
and intracellular IFN-␥ (Fig. 4A). CD4⫹ T cells from wild-type
BALB/c mice immunized with tERK-II responded minimally to
tERK-II stimulation, but not at all to the control peptide OVA (Fig.
4B). Importantly, there was no significant population of CD8⫹
IFN-␥-producing cells following tERK-II immunization, even
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We sought to develop a technique to protect mice from tumor
challenge through peptide vaccination. Initial attempts to vaccinate
using the tERK-I epitope showed no therapeutic benefit, and this
approach was abandoned. The failure of MHC class I-restricted
vaccination in other systems has at times been attributed to the lack
of CD4⫹ T cell help (2, 3). To develop a technique to stimulate
tumor-specific CD4⫹ T cell help and examine its effects on the
endogenous tumor-specific CD8⫹ T cell response, we exploited
the identified K136Q mutation in ERK-2, which generates the
CMS5-specific epitope recognized by DUC18 CD8⫹ T cells. The
tERK-I peptide (aa 136 –142) was extended to a total of 17 aa (aa
133–147) to make it an appropriate length to bind class II MHC.
Immunization with this length of peptide in CFA is a well-established technique of generating class II-restricted, CD4⫹ Th cell
responses (42– 44), and not class I-restricted responses (45). This
extended peptide is referred to as tERK-II, the original Kd-restricted epitope as tERK-I, and their normal tissue counterparts as
nERK-II and nERK-I, respectively (Fig. 1A). We then wanted to
ascertain whether tERK-II could induce an immune response.
BALB/c mice were primed s.c. with tERK-II, nERK-II, or a
control peptide (OVA), and the lymph node proliferative responses
were assayed in vitro 7 days after immunization. A strong response
specific for tERK-II was observed, consistent with this being presented by MHC class II. This response did not cross-react with
OVA, and was only slightly cross-reactive with the nERK-II peptide at very high concentrations of peptide stimulation (Fig. 1B).
Mice immunized with tERK-II did not mount a lymph node proliferative response to tERK-I or nERK-I, demonstrating that immunization with the extended peptide did not directly prime a class
I-restricted CD8⫹ T cell response (Fig. 1B). Immunization with
OVA resulted in a strong OVA-specific recall response without
cross-reactivity with nERK-II or tERK-II (Fig. 1C). Immunization
with nERK-II did not break in vivo self-tolerance mechanisms, as
demonstrated by the inability to recall a nERK-II proliferative response in vitro (Fig. 1D). Thus, immunization using this CD4directed peptide tERK-II is able to elicit a robust, specific, endogenous proliferative response.
4217
4218
CD4 T CELL PRIMING AUGMENTS CD8 ANTITUMOR RESPONSE
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FIGURE 1. The tERK-II peptide overlaps the tERK-I epitope and stimulates a lymphoproliferative response. The tERK-II peptide derived from mutated
ERK2 (133–149) contains the tumor-specific alteration from a lysine to a glutamine at position 136 and overlaps the class I-binding CMS5 tumor rejection
Ag (A). Lymph node proliferation assays using 5 ⫻ 105 cells/well were performed 7 days after immunization with 20 nmol of peptide emulsified in adjuvant.
Cultures were incubated for 72 h before addition of [3H]thymidine for the last 18 –24 h. Specific proliferative recall responses from primed mice immunized
with tERK-II (B), OVA (323–339) (C), and nERK-II (D) are shown in response to peptide stimulations with tERK-II (f), nERK-II (E), OVA (Œ), tERK-I
(ƒ), and nERK-I (〫). The data are representative of more than three similar experiments.
upon restimulation with tERK-I, again indicating that the tERK-II
immunization did not directly prime an effector CD8⫹ T cell population (Fig. 4D). The remaining mice were sacrificed 2 wk following CMS5 tumor challenge, and their lymph node cells were
analyzed for cytokine production, as described above. The specificity of the CD4⫹ T cell response remained intact following tumor
challenge. CD4⫹ T cells from both the BALB/c and CD40 KO
animals immunized with tERK-II and challenged with CMS5
made IFN-␥ only in response to tERK-II (Fig. 4C). Although no
CD8⫹ T cells from any of the immunization groups had apprecia-
bly produced IFN-␥ in response to immunization alone, tERK-IIimmunized mice demonstrated a detectable population of tERKI-specific, IFN-␥-producing, CD8⫹ T cells following tumor
challenge. This CD8⫹ T cell response was absent in CD40⫺/⫺
mice (Fig. 4E). The CD4⫹ tERK-II-specific response was diminished in the CD40⫺/⫺ mice; thus, the lack of subsequent
CD8⫹ tERK-I-specific responses could either be due to inadequate generation of CD4⫹ T cell help, or inadequate APC maturation. In either case, the data imply that the protective effect
of tERK-II immunization is associated with the ability to
The Journal of Immunology
generate a tERK-I-responsive CD8⫹ T cell population in a
CD40-dependent manner.
Transfer of 1000 naive DUC18 T cells ablates CMS5 tumor
growth in tERK-II-vaccinated mice
Immunization with tERK-II protects ⬃31% of BALB/c mice from
subsequent CMS5 challenge. The basis for the lack of protection in
the remaining mice could be due to insufficient generation of tumor-specific CD4⫹ or CD8⫹ T cells. We reasoned that a lack of
endogenous tumor-specific CD8⫹ precursors could be corrected
by the transfer of a limited number of transgenic tumor-specific T
cells into immunized mice. Previously, it was determined that at
least 12,000 DUC18 T cells were required to protect BALB/c mice
from a subsequent challenge of 3 ⫻ 106 CMS5 tumor cells (39).
FIGURE 3. CD40 is required for the protective effect of tERK-II immunization. CD40 KO (F) and BALB/c wild-type (WT) (E) mice were
challenged s.c. with 1.5 ⫻ 106 CMS5 tumor cells. Tumor outgrowth was
monitored up to 18 days posttumor challenge. The mean tumor size and SD
of one representative experiment are shown (A). BALB/c WT (filled symbols) or CD40 KO mice (open symbols) were immunized s.c. with 20 nmol
of tERK-II (squares) or OVA (323–339) (triangles) in CFA, and s.c. challenged 7 days later with 1.5 ⫻ 106 CMS5 cells (B).
To determine whether priming with tERK-II would result in protective immunity with a lower number of DUC18 T cells, we immunized mice with tERK-II and transferred 10, 100, or 1000 naive
DUC18 T cells 6 days later. These mice and appropriate controls
were challenged with CMS5 tumors 24 h following T cell transfer,
and tumor growth was monitored. Sixty-four percent of mice that
were immunized with tERK-II peptide alone developed tumors
(Fig. 5 and Table I). Transfer of 1000 naive DUC18 T cells alone
resulted in tumor outgrowth in 81% of the challenged mice, suggesting only partial protection was achieved (Fig. 5 and Table I).
Impressively, tERK-II-primed mice that also received 1000
DUC18 T cells were now completely protected from CMS5 challenge (Fig. 5 and Table I). Titration of the number of transferred
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FIGURE 2. Immunization with tERK-II is protective against subsequent CMS5 challenge. BALB/c mice were immunized s.c. with 20 nmol
of tERK-II (f), tERK-I (䡺), nERK-II (E), nERK-I (F), or OVA (323–
339) (Œ) with adjuvant. Seven days postimmunization, groups of four mice
were s.c. challenged with 1.5 ⫻ 106 CMS5 cells. Mean tumor size in mice
from one representative experiment is shown (A). The collective percentage of tumor-free mice from at least three independent experiments is
shown (B). Numbers over bars indicate the number of tumor-free mice of
the total number of mice per treatment group.
4219
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CD4 T CELL PRIMING AUGMENTS CD8 ANTITUMOR RESPONSE
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FIGURE 4. Peptide immunization primes peptide-specific CD4⫹ T cells, but tERK-I-specific CD8⫹ T cells are not present until after tumor challenge.
Groups of BALB/c or CD40 KO mice were examined for intracellular IFN-␥ production according to the above experimental outline (A). Mice were
immunized s.c. with 20 nmol of OVA (323–339) or tERK-II peptide in adjuvant. Seven days after immunization, mice were either harvested for their lymph
nodes (prechallenge condition) or challenged with 1.5 ⫻ 106 CMS5 tumor cells and harvested for their lymph nodes 2 wk posttumor challenge (postchallenge). A total of 3 ⫻ 106 lymph node cells collected before CMS5 tumor challenge (prechallenge; B and D) was stimulated ex vivo for 18 h with 10
␮M OVA (323–339) (f), tERK-II (o), tERK-I (䡺), or no Ag (u). Cells were stained with IFN-␥ FITC, CD4 PE, and CD8 CyChrome to assess the percentages
of intracellular IFN-␥-positive cells in the CD4⫹ T cell compartment (B) or the CD8⫹ T cell compartment (D). Intracellular IFN-␥ staining in lymph node CD4⫹
and CD8⫹ T cells from immunized mice was also assessed 2 wk after s.c. challenge with CMS5 tumor cells (postchallenge; C and E).
DUC18 T cells demonstrated that a low level of enhanced protection could be obtained by transferring as few as 100 DUC18 T
cells in conjunction with tERK-II immunization (Table I). Interestingly, immunization with OVA peptide before transfer of 1000
naive DUC18 T cells resulted in tumor outgrowth in 100% of the
challenged mice, compared with 81% of mice treated with 1000
DUC18 T cells alone (Table I). Additionally, tumor size was increased in the mice that were treated with OVA and 1000 DUC18
T cells relative to mice receiving 1000 DUC18 T cells alone (data
not shown). Although the underlying mechanism for this effect has
not been elucidated, it could result from the generation of a nontumor-specific response that interferes with DUC18 T cell function
The Journal of Immunology
4221
Table I. Effects of titrated numbers of transferred tumor-specific
DUC18 T cells on subsequent tumor growtha
Group
No. of TumorPositive Mice
PBS
1000 DUC18 cells alone
1000 DUC18 cells plus tERK-II peptide
100 DUC18 cells plus tERK-II peptide
10 DUC 18 cells plus tERK-II peptide
1000 DUC18 cells plus OVA (323–339) peptide
10/10
17/21
1/20
4/10
6/10
19/19
a
Mice were untreated or immunized with peptide plus adjuvant, and 6 days later
received DUC18 T cells or PBS, as shown. Twenty-four hours after T cell transfer,
mice were challenged with CMS5. The numbers of mice bearing measurable tumors
by 3 wk postchallenge are shown. Data represent cumulative totals from at least two
independent experiments.
in vivo. Despite this, the major finding of these experiments remains that tERK-II priming alone was not fully protective against
subsequent CMS5 challenge because sufficient numbers of endogenous CMS5-specific precursor T cells were not present in
BALB/c mice.
To further define the mechanism of tERK-II-mediated protection, we examined the effect of tERK-II priming on the expansion
of DUC18 T cells in tumor-bearing mice. BALB/c Thy-1.2⫹ recipient mice were immunized with tERK-II, or were left unprimed,
on day ⫺7 before the transfer of 1000 naive Thy-1.1⫹ DUC18 T
cells on day ⫺1. The mice were then challenged s.c. with CMS5
tumors on day 0. On days 4, 6, and 8 posttumor transfer, draining
lymph nodes and spleens from the mice were individually harvested, counted, and stained for V␤8.3, CD8, and Thy-1.1. Flow
cytometry was conducted to determine the percentages of
V␤8.3⫹CD8⫹Thy-1.1⫹ cells present in each organ, and this percentage was then used to determine the absolute number of transferred Thy-1.1⫹ DUC18 T cells present at each time point (Fig. 6).
Expansion of the Thy-1.1⫹ DUC18 cells in tERK-II-primed mice
occurred rapidly and transiently in the spleen, with an approximate
20-fold expansion seen at day 4, but only moderate expansion seen
by days 6 and 8. This timing coincided with the tumor rejection
kinetics in which rejection begins between days 6 and 8, suggesting that these Thy-1.1⫹ DUC18 T cells have homed into the tumor
site at these time points. In contrast, minimal numbers of Thy-1.1⫹
DUC18 T cells were detected in the spleen of unimmunized mice
on day 4, and a delayed, but stable increase in Thy-1.1⫹ DUC18
T cell numbers in the spleen was seen by days 6 and 8 posttumor
challenge. Taken together, these results suggest that tERK-II priming resulted in more rapid expansion kinetics and increased trafficking efficiency to the tumor site by the Thy-1.1⫹ DUC18 T cells
compared with unimmunized mice.
Discussion
In this study, we investigated the ability of a CD4-directed peptide
vaccine to elicit protective T helper and CTL responses against a
defined TAA in the murine CMS5 tumor model system. The
tERK-II peptide was generated by extending the amino acid sequence of a known 9-aa CTL epitope, tERK-I. Although this 17-aa
tERK-II peptide encompassed the tERK-I sequence, no direct
CD8⫹ T cell priming was observed as a result of vaccination, and
CD8⫹ T cell effector function was only detectable following tumor
challenge. Vaccination with tERK-I elicited no protective effects
in vivo. In contrast, a low level of protection from subsequent
tumor challenge was observed following tERK-II vaccination
alone. Transfer of minimal numbers of tumor-specific CD8⫹
DUC18 T cells in conjunction with tERK-II vaccination resulted in
protection from tumor challenge in 95% of treated mice. The underlying mechanisms for this combined effect may be due to increased expansion kinetics of transferred DUC18 T cells, and enhanced trafficking to the tumor site. Ag-specific priming of CD4⫹
T cells was required for protection, as the use of an irrelevant OVA
peptide failed to stimulate any protective immune response. Therefore, the tERK-II peptide effectively generated CD8⫹ T cell-mediated antitumor immunity indirectly, via its effects on CD4⫹ Th
cells.
Our current findings highlight the importance of CD4⫹ T cells
in the establishment of antitumor immunity in the CMS5 model.
However, in our initial description of the DUC18/CMS5 tumor
model system, we observed that CD4⫹ T cells were unnecessary
for rejection of CMS5 tumor challenge in DUC18 TCR transgenic
mice (39). In these mice, ⬃35–50% of CD8⫹ T cells are tumor
specific, and therefore the precursor frequency of antitumor CTLs
is high, with millions present per mouse. A titration of naive
DUC18 T cells demonstrated that adoptive transfer of 12,000 –
30,000 tumor-specific cells was needed to protect 90% of recipient
BALB/c mice from subsequent tumor challenge (39). We now find
that in the presence of enhanced CD4⫹ Th cell function posttERK-II vaccination, transfer of only 1000 naive DUC18 T cells is
sufficient to protect 95% of BALB/c mice against CMS5 challenge
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FIGURE 5. Transfer of 1000 naive DUC18 T cells into tERK-II-primed
mice protects from CMS5 challenge. Mice were left unimmunized (f and
Œ) or were immunized with 20 nmol of tERK-II (‚, 䡺) in adjuvant. Six
days later, some mice received 1000 naive DUC18 T cells i.v. (Œ, 䡺).
Twenty-four hours following T cell transfer, mice were s.c. challenged
with 3 ⫻ 106 CMS5 tumor cells. Tumor growth was monitored over the
course of the experiment. Data represent the mean ⫾ SE for groups of 10
mice; results combined from two separate experiments are shown. ⴱ, Indicates that one or more of the mice were sacrificed from the groups (‚, f)
at the indicated time points due to large tumor burdens.
Use of vaccination plus adoptive transfer of tumor-specific
CD8⫹ T cells resulted in a level of efficacy far above what either
treatment showed alone. Thus, as has been observed in other systems, we have demonstrated that CD4⫹ antitumor T cells have a
large capacity to augment CD8⫹ T cell responses. Ultimately, the
number of antitumor CD8⫹ T cells is a determining factor in the
generation of an effective antitumor response, and this threshold
can be lowered by providing adequate CD4⫹ T cell help.
4222
CD4 T CELL PRIMING AUGMENTS CD8 ANTITUMOR RESPONSE
(Fig. 5 and Table I). This suggests that when the precursor frequency of tumor-specific CTLs is low, CD4⫹ T cells become critical for the development of successful antitumor immunity. Recent
work from the Heath laboratory (50) supports this idea, illustrating
that CTL immunity is dependent upon CD4⫹ Th cells at low precursor frequencies (104-105 cytotoxic T cell precursors (pCTL)/
mouse), but proceeds independently of CD4⫹ Th cells at high
precursor frequencies (106 pCTL/mouse) (50).
In clinical settings, the precursor frequency of tumor-reactive
CD8⫹ CTLs is predicted to be low, with the exception of patients
undergoing adoptive immunotherapy with in vitro expanded tumor-infiltrating lymphocytes or CTL clones. A requirement for
CD4⫹ T cell help in mounting effective CTL immunity from low
numbers of precursors may explain the failure of many MHC class
I-restricted peptide vaccination studies to generate objective tumor
regression in cancer patients. Numerous studies have demonstrated
vaccination-induced expansion of Ag-specific CD8⫹ T cells in human trials, without detecting a corresponding objective regression
of tumor load in the majority of these patients (5, 7–9, 51). A
detailed examination of peptide vaccine-induced CD8⫹ T cells in
one study found that 73% of Ag-specific CD8⫹ T cells had an
effector phenotype, as evidenced by being CD27⫺, but fewer than
20% of these effector CD8⫹ T cells expressed perforin (11). These
findings reinforce classic murine studies demonstrating that deficient CD4⫹ T cell help can lead to the presence of CD8⫹ T cells
that proliferate and up-regulate surface activation markers in re-
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FIGURE 6. tERKII priming results in a more rapid and transient expansion of transferred naive Thy-1.1 DUC18 T cells in the spleen. BALB/c
Thy-1.2 mice were immunized with tERK-II (circles, ‚) or left unimmunized (squares, ƒ) on day ⫺7. On day ⫺1, 1000 naive Thy-1.1 DUC18
cells were transferred i.v. to Thy-1.2 BALB/c recipients. The following day
(day 0), both groups were challenged s.c. with 3 ⫻ 106 CMS5 cells. On
days 4, 6, and 8 posttumor challenge, the tumor-draining lymph nodes and
spleens were individually harvested and analyzed from each animal. Cells
were counted and then stained with V␤8.3 FITC/Thy-1.1 PE/CD8 CyChrome to determine the number of transferred Thy-1.1 DUC18 T cells
that had expanded in vivo at each time point. On day 4, there are four mice
per group from two separate experiments; on day 6, there are eight mice per
group from three separate experiments; and on day 8, there are 15 mice in
the unimmunized group and 14 mice in the tERK-II-immunized group
from three separate experiments.
sponse to encounter with cognate Ag, but do not acquire effector
function and cytolytic capabilities (52–54).
CD4⫹ Th cells may contribute to the generation of protective
antitumor immunity via several different mechanisms. The interaction of CD40L on activated CD4⫹ Th cells with CD40 on dendritic cells provides a maturation signal for DCs, increasing their
ability to provide essential costimulatory signals to pCTLs (12–
14). As a result, CD4⫹ Th cell expansion and activation can indirectly lead to the development of functional CD8⫹ effector T
cells. Because vaccination with tERK-II induces CD8⫹ T cellmediated protective immunity only indirectly (Figs. 1B and 4), and
CD40/CD40L interactions were required for the generation of
CTL effector function (Figs. 3 and 4), this mechanism is likely to
be operative in our CD4-directed peptide vaccination study. CD4⫹
cells may also regulate CTL development independently of CD40/
CD40L interactions. Although less well defined, in vitro and in
vivo studies have demonstrated that CD4 cells can trigger DC
maturation through a CD40-independent process, and can stimulate CD8⫹ T cell effector function directly through cytokine production (49). Because the protective effects of tERK-II vaccination
were completely ablated in the absence of CD40 (Fig. 3B), CD40independent routes of CD4⫹ T cell help for CTL generation do not
appear to have a significant role in our model system. We also
observed that CD40/CD40L interactions impact the generation of
CD4⫹ Th cell function, as evidenced by the decreased levels of
IFN-␥-producing CD4⫹ T cells in CD40⫺/⫺ mice (Fig. 4, B and
C). This finding is in agreement with previous data from the Flavell laboratory (47), which indicated that CD40/CD40L interactions were required for optimal priming of CD4⫹ Th cells (47).
The current study focused on the ability of CD4-directed
tERK-II vaccination to augment antitumor immunity through indirect effects on tumor-specific CD8⫹ T cells. However, tERK-IIprimed CD4⫹ Th cells may also function as effector cells to eliminate tumors in a more direct manner. CD4⫹ T cells have been
shown to reject MHC class II-deficient tumor challenges through
IFN-␥ production, which appeared to act on host cells rather than
on the tumor cells themselves (28). Additionally, the existence of
a rare population of MHC class I-restricted CD4⫹ T cells (55) has
been previously reported. These CD4⫹ cytotoxic cells have been
shown to display tumoricidal activity in an MHC class I-restricted
manner (56, 57). Thus, although the CMS5 tumor lacks MHC class
II expression, IFN-␥ produced by CD4⫹ T cells activated in response to tERK-II immunization could have some tumoricidal activity. We did not test these possibilities experimentally, because
we saw optimal tumor rejection only upon transfer of CD8⫹
DUC18 T cells into tERK-II-vaccinated mice (Fig. 5 and Table I).
Therefore, our data support a model in which the primary function
of CD4⫹ T cells is to provide help for tumor-specific CD8⫹ CTLs.
Several recent reports have illustrated the benefits of eliciting a
CD4⫹ T helper function in the context of antitumor immunity. An
experimentally isolated class II-restricted TAA was shown in a
similar murine fibrosarcoma model to have tumor-protective activity when provided in adjuvant (30). Additionally, adoptive
transfer of class II-restricted T cell clones was also recently shown
to have antitumor efficacy in two murine models (29, 30), although
the exact mechanism of such antitumor activity was not clear.
Work done by Melief and colleagues (38) using linked CD4 and
CD8 epitopes in a single long peptide illustrated that vaccination
with this peptide could induce effector function in both cell populations, and could lead to the regression of large, established tumors. A series of studies conducted in patients with Her-2/neuoverexpressing tumors demonstrated that CD8-directed peptide
vaccination resulted in CTL expansion that was short-lived, but not
protective; however, vaccination with 15-aa peptides containing
The Journal of Immunology
Acknowledgments
We thank Darren Kreamalmeyer for his careful management of the mouse
colony, Steve Horvath for peptide synthesis and purification, and Jerri
Smith for manuscript preparation.
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CD4 T CELL PRIMING AUGMENTS CD8 ANTITUMOR RESPONSE