Reversal of CD8+ T Cell Ignorance and Induction of Anti-Tumor Immunity by Peptide-Pulsed APC

Abstract

In the present report, we have studied the potential of naive and activated effector CD8(+) T cells to function as anti-tumor T cells to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Importantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell help.

Full text

Reversal of CD8
ⴙ
T Cell Ignorance and Induction of
Anti-Tumor Immunity by Peptide-Pulsed APC
1
Nava Dalyot-Herman,* Oliver F. Bathe,
†
and Thomas R. Malek
2
*
In the present report, we have studied the potential of naive and activated effector CD8
ⴙ
T cells to function as anti-tumor T cells
to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into
tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in
lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I
CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Impor-
tantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or
CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these
data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the
tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell
help. The Journal of Immunology, 2000, 165: 6731–6737.
I
t is now well-established that some tumors express Ags that
result in induction of anti-tumor immune responses (1, 2).
Although in some cases such a response results in eradication
of the tumor, more often an anti-tumor immune response is inef-
fective, and the tumor ultimately grows. There are a number of
specific mechanisms by which a tumor evades an ongoing immune
response. These vary widely and include: development of tu-
mor-Ag loss variants; down-regulation of surface MHC molecules
or other molecules required for Ag presentation by the tumor cells
(3, 4); induction of anergy of tumor-specific T cells due to lack of
costimulatory molecules on the tumor cells (5–7); and suppression
of the immune response by tumor secretion of inhibitory cytokines
(8, 9). Tumor Ags by nature are usually dysregulated self-proteins
or variants of self-Ags. Therefore, anti-tumor immunity may also
fail due to a weak response to the Ags expressed by the replicating
tumor or because potentially tumor-reactive T cells are tolerant to
the tumor Ags.
Although the fate and function of adoptively transferred tumor-
specific effector T cells have been extensively studied (10–12),
comparatively little is known concerning the initial activation of
tumor-specific T cells in vivo, primarily due to their low frequency
in the peripheral lymphocyte pool. With the introduction of TCR-
transgenic mice, this problem has been overcome. Analysis of an
Ag-specific T cell response to nominal Ags is facilitated by adop-
tive transfer of a relatively low, but detectable, number of TCR-
transgenic T cells to normal mice and then challenging such ani-
mals with the appropriate Ag (13). This approach allows direct
phenotypic and functional characterization of the responding Ag-
specific transgenic T cells during the course of the immune re-
sponse and avoids the complications inherent in direct Ag stimu-
lation of the TCR-transgenic mouse, in which all the T cells are Ag
responsive.
More recently, this method has been adapted to study the in-
duction of anti-tumor immunity in vivo. In this setting, a prede-
termined number of naive TCR-transgenic T cells are adoptively
transferred to mice bearing a tumor that was transfected with an
Ag recognized by the transgenic T cells (14–18). The present
study employed this TCR-transgenic strategy to compare the ca-
pacity of naive and activated effector-transgenic CD8
⫹
T cells to
generate an anti-tumor immune response to a solid tumor. We used
OT-I TCR-transgenic T cells that are specific for OVA
257–264
pep-
tide bound to H-2K
b
(19) as the source of anti-tumor-specific T
cells and the OVA-transfected EL4 cell line, E.G7, as the tumor
cells expressing a tumor-specific Ag, i.e., OVA (20). We demon-
strated that the naive OT-I T cells are functionally blind or igno-
rant of the OVA tumor Ag. This failure of OT-I T cells to respond
to this tumor was overcome by proper Ag presentation, as supplied
by peptide-pulsed professional APC, leading to an effective anti-
tumor immune response. These findings provide a relevant strategy
to overcome tumor Ag ignorance for cancer immunotherapy.
Materials and Methods
Animals
The OT-I TCR-transgenic mice (19) were maintained by breeding het-
erozygous OT-I TCR-transgenic mice to wild-type C57BL/6. The progeny
mice were screened by PCR for the expression of the OVA-TCR gene. Six-
to 10-wk-old female C57BL/6 or CD4-deficient mice on the C57BL/6
background were purchased from The Jackson Laboratory (Bar
Harbor, ME).
Cell lines
EL-4, a thymoma derived from the C57BL/6 mouse (H-2
b
), was obtained
from American Type Culture Collection (ATCC, Manassas, VA). OVA-
transfected EL-4, designated as E.G7 (20), was a gift from Dr. M. Bevan
(University of Washington, Seattle, WA). These cell lines were maintained
in RPMI 1640 containing 5% FCS, glutamine (30
␮
g/ml), penicillin (100
U/ml), streptomycin (100
␮
g/ml), and 2-ME (5 ⫻ 10
⫺5
M) (complete
medium).
*Department of Microbiology and Immunology and
†
Department of Surgery, Divi-
sion of Surgical Oncology, University of Miami School of Medicine, Miami,
FL 33101
Received for publication March 24, 2000. Accepted for publication September
13, 2000.
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 Grant DAMD17-98-1-8208 from the Department of
Defense.
2
Address correspondence and reprint requests to Dr. Thomas Malek, Department of
Microbiology and Immunology, University of Miami School of Medicine, 1600 NW
10th Avenue, Miami, FL 33136. E-mail address: tmalek@med.miami.edu
Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

Abs and other reagents
OVA peptide (SIINFEKL) (21) was synthesized by Research Genetics
(Huntsville, AL). Directly conjugated mAbs, including CyChrome-conju-
gated anti-CD8
␣
, PE-conjugated anti-mouse V
␣
2 TCR, and FITC-conju-
gated anti-mouse V
␤
5.1, 5.2 TCR, were purchased from PharMingen (San
Diego, CA). CFSE was purchased from Molecular Probes (Eugene, OR).
Cells were labeled with CFSE as previously described (22). Briefly, cells
(2 ⫻ 10
7
/ml) were incubated with 5
␮
M CFSE (froma5mMstock in
DMSO) in serum-free medium for 10 min at 37°C and washed twice with
cold complete medium and twice with HBSS.
Tumor challenge and adoptive transfer of transgenic T cells
Normal C57BL/6 or CD4
⫺/⫺
mice were injected with 1 ⫻ 10
6
E.G7 cells
in 0.2 ml HBSS s.c. into the midline of the abdomen. The tumor cells were
freshly thawed and grown in culture for 5–10 days before each injection.
Spleen cell suspensions from heterozygous OT-I mice (⬎6 wk of age) were
prepared as previously described (15). Splenocytes containing the indicated
number of transgenic OT-I T cells were injected i.v. in 0.5 ml HBSS 5–7
days after tumor challenge.
FACS analysis
Spleens and draining lymph nodes (LN)
3
(inguinal, brachial) were col-
lected and subjected to FACS analysis as previously described (15). Be-
tween 50,000 and 100,000 events per sample were collected on a FACScan
flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using
CellQuest software (Becton Dickinson). OT-I-transgenic T cells were enu-
merated either by three-color staining for CD8, V
␣
2-TCR, and V
␤
5.1,
5.2-TCR or by the fraction of CSFE-labeled (FITC) OT-I spleen cells that
also stained for CD8. The latter approach is valid because essentially all
CD8
⫹
cells in OT-I mice express the transgenic TCR.
Purification of CD8
⫹
OT-I cells
OT-I T cells were purified by a combination of negative and positive se-
lection. First, B cells were depleted on anti-Ig-coated plates, followed by
further depletion by incubation of the nonadherent cells with anti-CD24
(JIID), anti-NK1.1, and anti-MHC class II and C⬘ for 45 min at 37°C. The
OT-I T cells were then subjected to positive selection using magnetic beads
containing anti-CD8 (Miltenyi Biotec, Auburn, CA). The resulting cells
were ⱖ95% pure as judged by FACS analysis.
T cell proliferation assay
T cells (2 ⫻ 10
5
/well) were cultured in flat-bottom 96-well plates in com-
plete medium containing the indicated stimuli for 3–4 days. EL4 or E.G7
cells were always irradiated (20,000 rad). Then 1
␮
Ci [
3
H]thymidine was
added to the cultures for the last 5–6 h. The cells were harvested with an
automated cell harvester, and the radioactivity incorporated in DNA was
measured in a scintillation counter. Data of triplicate values that varied by
⬍10% of the mean are displayed as ⌬cpm, i.e., cpm from experimental
culture minus cpm from cultures containing only medium.
Culture for APCs
A single-cell suspension of bone marrow cells from normal C57BL/6 mice
was cultured at 0.5 ⫻ 10
6
/ml in complete medium containing 2 ng/ml
murine GM-CSF (PeproTech, Rocky Hill, NJ). Four to 5 days later, ad-
herent cells were collected after incubation with PBS containing 5 mM
EDTA at 37°C for 15 min. The cells were washed with HBSS, incubated
with 1
␮
M OVA peptide for1hat37°C, and washed three times with
HBSS. Mice were injected i.v. with the OVA-pulsed APC in 0.5 ml
of HBSS.
Generation and assay of CTL
Splenocytes (1 ⫻ 10
6
) from OT-I-transgenic mice were cultured in com-
plete medium containing 1 nM OVA peptide, 20 U/ml IL-2, and 40 U/ml
IL-4. After 3 days, the cells were collected, washed, and re-cultured at 1 ⫻
10
5
cells/ml in complete medium containing 20 U/ml IL-2 and 40 U/ml
IL-4 for 2 days. The CTL activity of the cells was measured by a standard
51
Cr release assay as previously described (23) using
51
Cr-labeled EL4 or
E.G7 cells as the targets.
Results
Activation of OT-I in vitro
In this study, the OVA-specific MHC class I-restricted (H2
b
) OT-I
TCR-transgenic CD8
⫹
T cells were used as tumor-specific T cells
by adoptive transfer to mice bearing the E.G7 tumor (OVA-trans-
fected EL4) as a solid tumor. The presence of the OT-I cells in
vivo was assessed by the coexpression of V
␤
5 and V
␣
2ofthe
transgenic TCR on CD8
⫹
T cells. In our initial studies, we deter-
mined the potential to functionally measure low numbers of OT-I
T cells in peripheral lymphoid tissue by evaluating the prolifera-
tive response to OVA peptide or E.G7 in vitro. Dose-response
studies demonstrated that OT-I T cells developed strong prolifer-
ative responses to 10
⫺7
–10
⫺9
M OVA peptide, with detectable
responses in cultures containing as little as 10
⫺12
M OVA peptide
(Fig. 1B). Furthermore, by mixing OT-I spleen cells with normal
C57BL/6 spleen cells in such a manner that the fraction of trans-
genic OT-I cells was predetermined, readily detectable prolifera-
tive responses were routinely generated by a relatively high dose
of OVA peptide (10
⫺9
M) when the cultures contained as few as
1 ⫻ 10
3
OT-I T cells, which is only 0.5% of the total number of
spleen cells in culture (Fig. 1A). Thus, OT-I cells are exquisitely
sensitive to OVA peptide, and OT-I T cells were functionally de-
tected in lymphoid tissues when present at a frequency of 1 in 200.
The E.G7 cells used in this study secreted 560 pg of OVA/ml/
1 ⫻ 10
6
cells after a 24-h culture as determined by ELISA (data
not shown). The addition of as much as 1.0 mg/ml of native sol-
uble OVA protein induced only minimal proliferation of OT-I T
cells in vitro (data not shown). Despite this relatively low level of
3
Abbreviation used in this paper: LN, lymph node.
FIGURE 1. Response of OT-I cells in vitro. A, Response by limiting
number of OT-I cells. B, Dose response to OVA peptide. C, Response to
cell associated-OVA. D, Response by purified OT-I cells. C57BL/6 spleen
cells (2 ⫻ 10
5
/well) containing the indicated number of OT-I T cells (A)or
2 ⫻ 10
3
/well OT-I T cells (B and C) were stimulated for 3 days with the
indicated concentration of OVA peptide, anti-CD3 (5% supernatant), or the
indicated number of irradiated EL4, E.G7, or OVA-pulsed APC. D, A total
of 2.5 ⫻ 10
4
purified OT-I cells were stimulated for 3 days with the in-
dicated concentration of OVA peptide or the indicated number of irradiated
E.G7 in the presence or absence of 1 ⫻ 10
5
accessory cells. Data are
representative of two to five experiments.
6732 REVERSAL OF TUMOR-SPECIFIC T CELL IGNORANCE

secretion of OVA, the E.G7 cells were immunogenic, as the irra-
diated E.G7, but not the parental EL4 thymoma, induced prolifer-
ation by OT-I cells in a fashion comparable to peptide-pulsed APC
(Fig. 1C). Taken together, these data suggest that cell-associated
OVA is much more immunogenic than soluble OVA protein. Fur-
thermore, E.G7 directly stimulated substantial proliferation by
highly purified OT-I T cells in the absence of accessory cells (Fig.
1D). By contrast, the response of these purified T cells to soluble
OVA peptide was dependent upon accessory cells. These results
indicate that the E.G7 can directly present OVA to OT-I.
OT-I T cells lack anti-tumor activity in vivo
Given the potent immunogenicity of E.G7 for OT-I T cells in vitro,
we examined the ability of OT-I T cells to mount an anti-tumor
immune response in vivo. Five days after C57BL/6 mice received
10
6
E.G7 s.c., OT-I T cells were adoptively transferred to these
tumor-bearing mice, and tumor size was measured over time. Each
mouse received 2.5 ⫻ 10
6
OT-I T cells, yielding mice in which
⬃5% of their CD8
⫹
T cells were OT-I. As reported by Jenkins and
coworkers (13), this number of transgenic T cells seeds the pe-
ripheral immune compartment so that the transgenic T cells are
present at a frequency that is still detectable by FACS analysis, but
these cells are not at such a high level as to cause imbalance in the
peripheral immune compartment. When compared with control
mice that did not receive OT-I cells, it is quite apparent that rate of
tumor growth was comparable between both groups of mice (Fig.
2). Transfer of a larger number (4 ⫻ 10
6
) of OT-I T cells still failed
to affect tumor cell growth (data not shown). Thus, OT-I cells did
not generate as obvious an anti-tumor response to E.G7 growing as
did a solid tumor.
To examine whether the adoptively transferred OT-I cells pro-
liferated to E.G7 in vivo, we determined the proportion of OT-I T
cells in the spleen and draining LN of tumor-bearing animals by
three-color FACS analysis by staining for CD8, V
␣
2, and V
␤
5. In
a normal mouse, ⬃0.2% and ⬃0.5% of spleen and LN cells, re-
spectively, express these three surface molecules. After adoptive
transfer of OT-I T cells, the fraction of cells bearing these markers
initially increased to ⬃0.6% for the spleen and ⬃1.3% for the
draining LN (Fig. 3A). Importantly, these numbers decreased over
time for both control and tumor-bearing animals to a level similar
to that seen in normal mice. The total spleen and LN recovery, and
the proportion of CD8
⫹
T cells from mice that were adoptively
transferred with OT-I cells, in the presence or absence of E.G7,
was always comparable (data not shown), further indicating that
there was no obvious lymphoid cell expansion by the tumor. These
findings suggest that OT-I T cells did not proliferate in response to
the E.G7 tumor.
To further evaluate whether OT-I cells responded to E.G7 in
vivo, CFSE-treated OT-I T cells were adoptively transferred to
E.G7-bearing mice. CFSE is an intracellular fluorescent label for
which the fluorescence intensity decreases proportionally upon cell
division (22). When CD8 cells in the draining LN and spleens of
control and tumor-bearing mice were subjected to FACS analysis,
the large majority of cells maintained the original fluorescence
FIGURE 2. Anti-tumor activity of naive OT-I cells. Normal C57BL/6
mice were injected with E.G7 (1 ⫻ 10
6
). Seven days later, mice received
OT-I T cells as indicated, and the tumor size was measured. Detectable
tumor is considered to be above 0.5 cm
2
. The mice were sacrificed when
the tumor reached a size of 2.0 cm
2
. Data shown contain eight mice/group
and are representative of three experiments.
FIGURE 3. OT-I cells do not respond to E.G7 in vivo. A, Proportion of
OT-I T cells in tumor-bearing mice. The percentage of V
␣
2, V
␤
5, CD8
⫹
three-color-positive cells was determined for the spleens and LN of normal
and tumor-bearing mice as described in Materials and Methods. Data
shown contain four mice/group and is representative of three experiments.
B, Cell division by OT-I cells. OT-I spleen cells were labeled with CFSE
before transferring to normal or tumor-bearing mice (2.5 ⫻ 10
6
). Some
mice received 0.5 ⫻ 10
6
OVA-pulsed APC 1 day later. Three days after
transfer of APC, LN cells were analyzed for cell division by two-color
FACS analysis. Shown are histograms of cells double-stained for CD8 and
CFSE. The percentage of cells that have divided, as seen by a reduction in
CFSE staining intensity, is listed in C. Quantitative analyses of five to six
mice in each group (mean ⫾ SD), based on FACS histograms as shown in B.
6733The Journal of Immunology

intensity, a profile characteristic of cells that have not undergone
cell division. Representative FACS histograms are shown in Fig.
3B, and the results from the analysis of multiple mice are summa-
rized in Fig. 3C. Very few OT-I cells exhibited a decrease in CFSE
staining after adoptive transfer to normal or E.G7-bearing mice. In
contrast, in recipients that were adoptively transferred with CFSE-
treated OT-I cells and subsequently challenged with OVA-pulsed
APC, ⬎50% of the CD8 T cells exhibited a reduced level of CFSE
staining (Fig. 3C), demonstrating a normal and strong response by
OT-I T cells upon encountering Ag in association with a profes-
sional APC. Thus, these data further indicate that naive OT-I T
cells are nonresponsive to E.G7 tumor in vivo.
Because the OT-I T cells were adoptively transferred to mice
with an established tumor, we considered that E.G7 might have
anergized or otherwise suppressed activation of the transgenic T
cells. To test this possibility, the proliferative responses in vitro by
splenic and LN T cells to OVA peptide were assessed 4–7 days
after adoptive transfer to normal or tumor-bearing mice. In both
groups of mice, not only were the OVA-specific proliferative re-
sponses comparable, but the percentage of OT-I cells recovered
from the spleen and LN were also very similar (Table I). This
finding indicates that the failed anti-tumor response by OT-I T
cells was not the result of Ag-specific nonresponsiveness induced
by E.G7 in vivo. Collectively, these data indicate that the failed
anti-tumor response by naive OT-I is largely due to immunological
ignorance of OVA in the context of E.G7.
OT-I effector T cells generate an anti-tumor response
Although E.G7 as a solid tumor routinely failed to activate naive
OT-I T cells in vivo, these cells remained as potential targets for
immune effector cells, if such cells were successfully induced. To
determine whether E.G7 was susceptible to OT-I effector cells, we
tested whether the adoptive transfer of preactivated OT-I T cells
generated an anti-tumor response. Before adoptive transfer, the
OT-I cells were cultured for 3 days with OVA peptide and exog-
enous IL-2 and IL-4, and the effector cells were further expanded
for 2 days by culture with only the cytokines. After this 5-day
culture period, the OT-I cells exhibited potent CTL activity to
E.G7, but not to EL4 (Fig. 4A). These effector CTL were adop-
tively transferred 5 days after s.c. injection of E.G7, and when
compared with control mice, the growth of the tumor was delayed,
especially at a relatively high dose (6 ⫻ 10
6
) of OT-I effector cells
(Fig. 4B). Thus, E.G7 was recognized by, and was accessible to, in
vitro-generated OT-I CD8
⫹
effectors.
Because naive OT-I T cells were readily activated in vivo by
OVA-pulsed APC (see Fig. 3), we next tested whether the in
vivo induction of OT-I effector cells also induced anti-tumor
immunity. Naive OT-I T cells were adoptively transferred to
mice 5 days after s.c. injection of E.G7, and 24 h later the mice
received OVA-pulsed bone marrow-derived APC. As shown
earlier, the tumor grew quickly in mice that received tumor in
the presence of naive OT-1 (Fig. 5A). By contrast, coadminis-
tration of OT-I and OVA-pulsed APC resulted in a substantial
delay in the progression of the tumor. In 40% of the mice, no
tumor was detected 45 days after injection of E.G7, and several
mice that were observed longer remained tumor-free on day 60.
Importantly, there was no inhibition of growth when tumor-
bearing mice received OVA-pulsed APC in the absence of OT-I
T cells. This result indicates that the inhibition of tumor growth
Table I. E.G7 tumor cells do not anergize OT-I cells
a
Expt.
Days after
Adoptive
Transfer Cells from:
% OT-I
b
cpm ⫻ 10
3c
Spleen LN Spleen LN
1 7 Tumor-bearing mice 0.53 ⫾ 0.03 1.39 ⫾ 0.06 22.0 ⫾ 3.3 88.3 ⫾ 24.0
Normal mice 0.67 ⫾ 0.03 1.23 ⫾ 0.03 24.3 ⫾ 0.4 76.0 ⫾ 16.6
2 4 Tumor-bearing mice 0.56 ⫾ 0.08 1.50 ⫾ 0.30 15.0 ⫾ 4.2 92.0 ⫾ 12.3
Normal mice 0.55 ⫾ 0.12 1.48 ⫾ 0.21 13.5 ⫾ 3.1 101.3 ⫾ 7.4
3 4 Tumor-bearing mice 0.58 ⫾ 0.02 0.94 ⫾ 0.25 23.1 ⫾ 4.0 71.5 ⫾ 21.0
Normal mice 0.60 ⫾ 0.07 1.10 ⫾ 0.15 21.0 ⫾ 1.0 63.5 ⫾ 15.4
a
Normal C57BL/6 mice were injected with E.G7 (1 ⫻ 10
6
). Five day later mice received 4 ⫻ 10
6
OT-I cells (experiment 1) and 2.5 ⫻ 10
6
OT-I cells (experiments 2
and 3). Spleen and LN were harvested at the indicated days, after adoptive transfer, and were subjected to analyses. Data shown contain three to four mice per group in each
experiment.
b
Determined by FACS analysis of three color positive cells for CD8, V
␣
2, and V
␤
5.
c
Determined by proliferation of spleen and LN after culture for 3 days with 1 nM OVA peptide.
FIGURE 4. Activity of effector OT-I CTL cells. A, CTL activity in vitro
as measured by
51
Cr-release assay against E.G7 and EL4. B, Anti-tumor
activity of OT-I effector cells. Normal C57BL/6 mice were injected with
E.G7 (1 ⫻ 10
6
). Five days later, mice received the indicated number of T
cells and tumor size recorded as described in Fig. 2. Effector OT-I CTL
cells were generated as described in Materials and Methods. Data shown
contain five mice/group and are representative of two experiments.
6734 REVERSAL OF TUMOR-SPECIFIC T CELL IGNORANCE

is dependent upon the presence of the adoptively transferred
transgenic T cells. Thus, after appropriate activation either in
vitro or in vivo, E.G7 was susceptible to tumor (OVA)-specific
OT-I effector cells.
To determine whether this anti-tumor response required CD4
⫹
T cells, we transferred OT-I T cells to tumor-bearing CD4-defi-
cient mice (Fig. 5B). Similar to E.G7-bearing C57BL/6 normal
mice, the growth of E.G7 was delayed in the CD4
⫺/⫺
recipient
mice, if they received OVA-pulsed APC, and two of six mice
remained tumor-free after 50 days.
The E.G7 tumor eventually grew in all mice that received in
vitro-induced OT-I effector cells and in some mice that were stim-
ulated with OVA-pulsed APC in vivo. The E.G7 cells were ex-
cised from one such mouse in each of the treatment groups and
grown in culture for at least 7 days. These cells were then used as
targets for OT-I CTL generated by in vitro culture. The E.G7 cells
obtained from the mice treated with ex vivo-induced OT-I effector
cells were nearly as good targets for OVA-specific CTL as the
parental E.G7 (Fig. 6). Thus, the tumor outgrowth from this mouse
appears to be the result of a failure of the adoptively transferred
CTL to completely kill the tumor. However, E.G7 from the in vivo
APC-treated mice were not lysed by the OVA-specific CTL, sug-
gesting that tumor outgrowth in this case was caused by the se-
lection of a tumor variant that escaped the effector OT-I CTL. In
addition, ELISA analysis confirmed that these cells failed to se-
crete a detectable OVA level (data not shown). Collectively, these
data raise the possibility that in vivo-induced effector T cells in-
duced a more potent anti-tumor immune response than adoptive T
cell immunotherapy.
Discussion
In the present study, we showed that a tumor-immune response
could not be elicited in tumor-bearing mice after the adoptive
transfer of a relatively high number of naive transgenic tumor-
specific CD8
⫹
T cells. Importantly, as assessed at several different
time points after adoptive transfer, the tumor-specific transgenic T
cells were not activated to proliferate in both the spleen and the
draining LN in the presence of the growing solid tumor. The fail-
ure to activate the T cells was not because the tumor anergized
them, because OT-I T cells derived from tumor-bearing mice
readily proliferated upon Ag challenge in vitro. Therefore, our data
indicate that failed anti-tumor responses to E.G7 were due to im-
munological ignorance, i.e., a failure of the host immune system to
recognize OVA, in this case in the context of a tumor cell, during
the induction phase of the immune response.
It was surprising that the OT-I T cells remained ignorant of
E.G7 when adoptively transferred in vivo considering the robust
proliferative response of the T cells to irradiated E.G7 tumor cells
in vitro. The activation of OT-I T cells in vivo has been shown to
be dependent on Ag presentation by short-lived bone marrow-de-
rived APC within the draining LN (24). Furthermore, when OVA
is cell associated, e.g., in transgenic
␤
-islet cells, activation of
OT-I T cells is dependent upon cross-priming, i.e., the
␤
-islet-
associated OVA is ultimately processed by an exogenous class I
pathway and presented to OT-I T cells by professional APC (24).
Cross-priming is facilitated by either destruction of the Ag-con-
taining cells and/or a high level of expression of the cell-associated
Ag (25). In the case of
␤
-islet cells, when cross-priming was not
facilitated, the OVA-containing
␤
-islets were ignored by OT-I T
cells (26).
The failure of OT-I T cells to recognize E.G7 is not analogous
to that described for OVA-containing
␤
-islets, because E.G7 were
able to directly present OVA to OT-I. It is likely that two factors
promoted ignorance of E.G7. First, ignorance in our model appears
to be at least in part the result of lack of contact between the OT-I
tumor-specific T cells and OVA associated with the E.G7 tumor
cells. Naive OT-I T cells appear not to readily migrate to the site
of the tumor, preventing their direct activation by E.G7. Consistent
with this hypothesis, so far we have not detected OT-I cells within
the tumor site as determined by using CFSE-labeled OT-I cells and
FACS analysis of the excised tumors (data not shown). Similarly,
E.G7 does not obviously traffic to the spleen or the LN following
s.c. or i.v. injections (data not shown). These observations suggest
that OT-I T cells do not encounter E.G7 either at the site of the
tumor or within the draining LN. Second, there is no indication
that the E.G7 was able to cross-prime OT-I T cells by host
APC. E.G7 produced relatively low levels of OVA, and minimal
destruction of the rapidly growing E.G7 tumor is expected, espe-
cially early after injection of the tumor, conditions that would not
favor cross-priming by the APC of the recipient mice. The obser-
vation that professional APC consistently activated OT-I T cells in
the spleen and draining LN of E.G7-bearing mice demonstrates
that ignorance to E.G7 is not due to a failure of the naive OT-I T
cells to ultimately migrate to secondary lymphoid tissue, or to
generalized immune suppression by E.G7, although we cannot ex-
clude other means by which OT-I are ignorant of E.G7.
FIGURE 5. Peptide-pulsed APC induce anti-tumor response by naive
OT-I cells. Normal C57BL/6 mice (A) or CD4-deficient mice (B) were
injected with E.G7 (1 ⫻ 10
6
). Five days later, the mice received naive OT-I
T cells (3 ⫻ 10
6
) and 1 day later peptide-pulsed APC (0.5 ⫻ 10
6
)as
indicated. The size of the tumor was recorded as described in Fig. 2. Data
shown contain five mice per group and are representative of two experi-
ments (A) and six mice per group (B).
FIGURE 6. Lysis of tumor cells from OT-I-treated mice by OT-I ef-
fector cells in vitro. Tumors were excised from treated mice, as designated,
grown in culture for at least 7 days, and labeled with
51
Cr to serve as targets
for OT-I CTL (as described in Materials and Methods).
51
Cr-labeled E.G7
and EL4 served as positive and negative control targets, respectively.
6735The Journal of Immunology

The value of using adoptive transfer of TCR- transgenic T cells
to mice bearing tumors transfected with a model Ag is that this
approach provides insight into the strength and duration of an anti-
tumor T cell response. So far, two major outcomes have been
observed in these types of studies. As we have seen for E.G7 as a
solid tumor, tumor-specific TCR-transgenic T cells have been re-
ported to be ignorant of L
d
-transfected AG104A fibrosarcoma (27)
and a glycoprotein of lymphocytic choriomeningitis virus after
transfection into either Lewis lung carcinoma or the MC57G fi-
brosarcoma (17, 18). In the latter case, T cell ignorance required
that the tumor be transplanted as a solid tumor fragment rather than
s.c. injection of a single-cell suspension. These observations and
our results suggest that T cell ignorance represents one important
reason for failed anti-tumor immunity. Our data indicate that im-
munological ignorance may pertain to tumor-specific T cells bear-
ing a high affinity for TCR, as OT-I T cells are extremely sensitive
to OVA-peptide and proliferate to as little as 1 pM of peptide in
vitro, a dose that is 1000-fold lower than that required to activate
the lymphocytic choriomeningitis virus-specific TCR-transgenic T
cells (17).
In other studies of the adoptive transfer of TCR-transgenic T
cells to Ag-transfected tumors, an initial transient anti-tumor re-
sponse was observed (14, 15, 28). In several cases, failed anti-
tumor immunity was shown to be due to anergy of the tumor-
specific T cells. This has been observed for both MHC class I- and
II-restricted TCR-transgenic T cells. Interestingly, in the case of
the MHC class I T cell response, OT-I T cells and E.G7 tumor cells
also served as the model system (15). In that study, the E.G7 cells
were injected into the peritoneal cavity 1 day after adoptive trans-
fer of the OT-I T cells, which led to initial activation and prolif-
eration of the OT-I T cells in vivo. Effective anti-tumor immunity
failed in part due to CTLA4-mediated down-regulation of endog-
enous CD4 helper activity (29). This finding markedly contrasts
with our result in which we found that OT-I T cells were ignorant
of E.G7 as a solid tumor. We have compared the E.G7 subline
maintained in our laboratory with that used by Srikant et al. (15)
and found that our subline failed to activate OT-I after injection
i.p. whereas the E.G7 subline used by Shrikant and coworkers
activated OT-I when present as a solid tumor. This indicates that
the different pattern of results is unlikely to be due to differences
in experimental protocols and/or responses to a systemic vs solid
tumor. Furthermore, both sublines of E.G7 produce similar levels
of OVA. Therefore, it is most likely that these two cell lines ex-
press some intrinsic undefined difference, as the cells were inde-
pendently passaged for a considerable period of time.
Although in our study naive OT-I T cells failed to inhibit the
growth of E.G7, anti-tumor immune responses were elicited by
OT-I effector cells, confirming that the OT-I T cells have sufficient
affinity to specifically attack the developing E.G7 solid tumor. Ef-
fector OT-I T cells were generated either ex vivo in culture or by
in vivo stimulation of the naive adoptively transferred OT-I T cells
with peptide-pulsed bone marrow-derived APC. This illustrates
that T cell ignorance can be overcome simply by proper Ag pre-
sentation of a tumor Ag. Furthermore, by using CD4-deficient
mice, we demonstrated that this anti-tumor activity was indepen-
dent of CD4 T cell help. Although many other studies clearly
indicate a need for CD4
⫹
T cells for anti-tumor responses (30–33),
our data, similar to that reported by Wick et al. (27), demonstrate
that direct activation of CD8 T cells can be sufficient for potent
anti-tumor immunity.
Several other studies have demonstrated that bone marrow-de-
rived APC effectively inhibit tumor growth (17, 34), perhaps by
activation of a population of ignorant tumor-specific T cells. It is
interesting to note that so far we have only “cured” E.G7 with OT-I
cells when the mice were immunized with OVA-pulsed APC.
These findings raise the prospect that effective anti-tumor immu-
nity may be facilitated by approaches that both increase the fre-
quency of tumor-specific T cells and induce activation of such T
cells by vaccination with tumor-Ag-containing APC.
Acknowledgments
We thank Dr. E. Codias for critically reading this manuscript,
P. Scibelli and T. Nguyen for technical assistance, and the Sylvester Com-
prehensive Cancer Center for support of the FACS facilities.
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6737The Journal of Immunology