| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Immunology |
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [T. D. S., S. S. T.], and The Jackson Laboratory, Bar Harbor, Maine 04609 [B. B. K.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-amylase promoter, which leads to the
development of osteogenic osteosarcomas late in life and eventual death
between 12 and 17 months of age. We determined the ability of 501 mice
to respond to the four H2b-restricted T ag CTL epitopes,
which include epitope I (T ag 206–215), epitope II/III (T ag
223–231), the immunorecessive epitope V (T ag 489–497), restricted by
H2-Db, and epitope IV (T ag 404–411), restricted by
H2-Kb. We demonstrate that 501 mice are partially tolerant
to the H2b-restricted T ag epitopes. Immunization of
4-month-old 501 mice with T ag-transformed syngeneic cell lines or a
recombinant vaccinia virus expressing full-length T ag elicited CTL
responses against the H2-Kb-restricted T ag epitope IV
only. In contrast, immunization of 4-month-old 501 mice with
recombinant vaccinia viruses expressing individual T ag epitopes as
minigenes elicited CTLs against epitopes I, IV, and V, but not against
epitope II/III. Complete tolerance to epitopes I, IV, and V developed
in 501 mice, but the age when tolerance was detected varied for each
epitope. Tolerance to epitope I occurred by 6 months of age and was
accelerated in the absence of CD4+ T cells. Tolerance to
the immunorecessive epitope V was observed in 12-month-old 501 mice but
was independent of the presence of osteosarcomas. In contrast, CTLs
specific for epitope IV were detected in mice from 3 to 14 months of
age but not in mice that had developed osteosarcomas. Analysis of
epitope IV-specific CD8+ cells derived from 3-month-old 501
mice with H2-Kb/epitope IV tetramers revealed decreased
numbers of epitope IV-specific CD8+ cells in 501 mice
relative to C57BL/6 mice, with a further decrease in older 501 mice.
Tumor progression resulted in loss of H2-Kb/epitope IV
tetramer staining CD8+ cells. Thus, progression to
tolerance to individual T ag CTL epitopes in 501 mice is epitope
dependent. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The cellular immune response in the C57BL/6 high-responder strain of mice is characterized by the generation of CTLs against three immunodominant epitopes, designated epitope I (amino acid residues 206–215), epitope II/III (amino acid residues 223–231), and epitope IV (amino acid residues 404–411; Refs. 18, 19, 20, 21 ). Epitopes I and II/III are H2-Db restricted, whereas epitope IV is H2-Kb restricted. In addition, CTLs against the immunorecessive H2-Db-restricted epitope V (amino acid residues 489–497) are generated after immunization with T ag variants that lack the three immunodominant T ag epitopes (22) . We have demonstrated recently that induction of CTLs specific for the H2-Kb-restricted epitope IV leads to control of spontaneous T ag-induced choroid plexus tumors in T ag transgenic mice upon X-irradiation and reconstitution with spleen cells from normal C57BL/6 mice (10) , indicating a role for this H2-Kb-restricted T ag epitope in the control of endogenous tumor progression.
Mice of the 501 (H2b) lineage express
SV40 T ag as a transgene from the -amylase promoter, which leads to
T ag expression in salivary glands as well as bone (5)
. T
ag expression in osteoblasts results in the development of osteogenic
osteosarcomas in bones of the axial skeleton as well as the femur and
humerus. Metastases to the liver are detected frequently and are also
found occasionally in the lungs. Expression of T ag in the salivary
glands is detected by 3 months of age with increased expression by 6
months of age, although no neoplasia develops in the salivary
glands.4
Osteosarcomas are first detected by 8 months of age in 501 mice, which
have an average life span of 13 months of age. The CTL response to
individual H2b T ag epitopes as well as the
kinetics of tolerance onset to these CTL epitopes in 501 mice has not
been investigated previously.
In this report, we determined whether 501 mice develop CTL precursors that can be activated against the four defined H2b T ag epitopes both early and late after the onset of T ag expression in the periphery. The results indicate that although 4-month-old 501 mice maintained CTL precursors capable of responding to epitopes I, IV, and V, older mice developed increased tolerance to the T ag CTL epitopes. Loss of CTL responsiveness, however, varied for each epitope. In particular, loss of responsiveness to epitope IV correlated directly with the appearance of T ag-expressing osteosarcomas.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-amylase promoter and have been described previously
(5)
. 501, 501/CD4-/- knockout, and
B6/CD4-/- (23)
mice were bred and
maintained at The Jackson Laboratory and at the animal research
facility of the Milton S. Hershey Medical Center.
Cell Lines and Media.
B6/WT-19 is an SV40 transformed C57BL/6 mouse embryo fibroblast line
that expresses wild-type T ag (24)
. B6/K-1,4,5 was derived
from a T ag-transformed C57BL/6 kidney cell line by sequential
immunoselection with T ag-specific CTL clones and lacks the T ag
epitopes I, II/III, IV, and V (18
, 21
, 25
, 26)
. 501/2484
col 3 was derived from an osteosarcoma of a 501 mouse and expresses
full-length T ag (5)
. All T ag-transformed cell lines were
maintained in DMEM supplemented with 100 units of penicillin/ml, 100
µg of streptomycin/ml, 100 µg of kanamycin/ml, 2 mM
L-glutamine, 10 mM HEPES buffer, 0.075% (w/v)
NaHCO3, and 5–10% FBS. RMA
(H2b) cells (27)
were
maintained in suspension using RPMI 1640 supplemented with 10% FBS,
100 units of penicillin/ml, 100 µg of streptomycin/ml, 2
mM L-glutamine, and 50
µM 2-mercaptoethanol.
Viruses and Synthetic Peptides.
rVVs used in this study have been described previously (28
, 29) and include rVV-941T, which encodes full-length SV40 T ag, a
series of rVVs encoding T ag epitope minigenes designated rVV-I (T ag
sequence 206–215), rVV-II/III (T ag 223–231), rVV-IV (T ag sequence
404–411), and rVV-VAA (T ag 489–497 plus 2 Ala at the COOH terminus)
and a corresponding series of minigenes with a preceding ES and
designated as rVV-ES I, rVV-ES II/III, rVV-ES IV, and rVV-ES V. The
wild-type vaccinia virus strain WR (VV-WR; ATCC VR-119), from which all
of the rVVs were derived, was used in these experiments. All peptides
used were synthesized at the Macromolecular Core Facility of the Milton
S. Hershey Medical Center by FMoc chemistry using an automated peptide
synthesizer (9050 MilliGen PepSynthesizer). Peptides were solubilized
in DMSO and diluted to the appropriate concentration with RPMI 1640.
Peptides used in these experiments correspond to SV40 T ag epitopes I
(SAINNYAQKL), II/III (CKGVNKEYL), IV (VVYDFLKC), and V (QGINNLDNL) as
well as the optimized H2-Db binding peptide
DbN5 (SMIKNLEYM; Refs. 30
and
31
) and the H2-Kb-restricted peptide
gB498–505 (SSIEFARL; Ref. 32
) from HSV.
Immunization of Mice and in Vitro Restimulation of
Bulk CTLs.
Mice were immunized at the indicated ages by i.p. injection of
2–5 x 107 T ag-transformed cells
or by i.v. injection of 1 x 107
pfu rVVs. Mice were sacrificed at 2–4 weeks after immunization, and
single-cell suspensions of RBC-depleted spleen cells were restimulated
in vitro with gamma-irradiated B6/WT-19 cells as described
(30)
. Briefly, 1 x 107 spleen cells from immunized animals were
mixed with 5 x 105
gamma-irradiated (10,000 rads) B6/WT-19 cells in 4 ml of complete RPMI
1640 supplemented with 10% FBS/well of a 12-well tissue culture plate.
To verify vaccinia virus infection, 1 x 107 spleen cells from each vaccinia
virus-infected mouse were restimulated in vitro with
5 x 105
irradiated VV-WR-infected
C57BL/6 spleen cells in 4 ml of complete RPMI 1640 supplemented with
10% FBS/well of a 12-well plate. VV-WR-infected C57BL/6 spleen cell
stimulators were prepared by infection of naive C57BL/6 spleen cells
(1 x 107 per ml PBS/BSA) with
VV-WR (multiplicity of infection, 10) for 1 h at 37°C with
occasional agitation, followed by a 3-h incubation in complete RPMI
1640 at 37°C, 5% CO2. Infected cells were
gamma-irradiated (60,000 rads) and washed free of virus before use.
Maintenance of CTL Clones.
SV40 T ag-specific CTL clones used in this study were maintained by
in vitro passage as described previously (20)
and include clones K-11, K-19 (33
, 34)
, Y-4
(18)
, and H-1 (30)
, which recognize T ag
epitopes I (residues 206–215), II/III (residues 223–231), IV
(residues 404–411), and V (residues 489–497), respectively.
Cytotoxicity Assays.
Assays for CTL lysis were performed on day 6 after in vitro
restimulation as described previously (10)
. Statistical
analysis of responders versus nonresponders in development
of CTLs in tumor-bearing and tumor-free 501 mice was performed using
Fisher’s exact test with a two-sided P < 0.01 considered significant using Instat (GraphPad Software).
Histology and Immunohistochemistry.
For histology and immunohistochemistry, mice were perfused with 10%
formalin, and tissues were fixed with 10% formalin for an additional
24 h. Tissues were decalcified for 8 h prior to embedding in
paraffin blocks. Seven-µm blocks were cut on a microtome and
collected onto positively charged slides. Parallel sections were
stained by H&E or immunohistochemistry for T ag as described previously
(10)
.
X-Rays.
X-rays of mice were performed by the trained veterinary staff of the
Department of Comparative Medicine at the Milton S. Hershey Medical
Center. Mice were anesthetized immediately prior to X-ray and were
placed in a dorsoventral position on a Kodak MIN-R2 cassette (Eastman
Kodak, Rochester, NY) containing Kodak MIN-R film. X-rays were
performed using 400 mAmps and 58 kV for 0.015 s.
Preparation of Class I MHC Tetramers, Staining of
Epitope-specific T Cells, and Flow Cytometry.
Production and characterization of the H2-Kb/T ag
epitope IV (Kb/IV Tet) and
H2-Kb/HSV gB498–505 (Kb/gB
Tet) tetramers were achieved essentially as described (35)
and will be described in detail
elsewhere.5
PE-labeled Kb/IV Tet used in this study was
prepared using an analogue of the native epitope IV peptide containing
a C411L substitution (VVYDFLKL), which enhances the stability of
this peptide with H2-Kb without disrupting T-cell
recognition (data not shown). For staining of lymphocyte populations,
RBC-depleted splenocytes were incubated with rat antimouse CD16/CD32
(PharMingen) and 50 µg/ml streptavidin (Molecular Probes) for 30 min
on ice to block Fc receptors and nonspecific binding of streptavidin
conjugated tetramers, respectively. After a single wash, spleen cells
were incubated with phycoerythrin-labeled tetramers and FITC-labeled
rat antimouse CD8a (53-6.7; PharMingen) for 1 h on ice. Cells were
fixed with 2% paraformaldehyde and analyzed using a FACScan (Becton
Dickinson, San Jose, CA) flow cytometer, and data were analyzed and
prepared using CELLQuest software (Becton Dickinson). The percentage of
CD8+ cells that stained specifically with
Kb/IV Tet was determined by subtracting the
percentage of CD8+, Kb/gB
Tet+ cells from the percentage of
CD8+, Kb/IV
Tet+ cells within the same population.
Intracellular Cytokine Assay.
For staining of intracellular IFN-
, spleen cells were harvested from
rVV-ES IV immunized mice at 14 days after immunization. RBC-depleted
spleen cell suspensions were prepared as above, and 5 x 106 spleen cells were incubated with 1
µM of the indicated synthetic peptides representing T ag
or control epitopes and 1 µg per ml brefeldin A in 2 ml of complete
RPMI 1640 containing 10% FBS/well of a 24-well plate for 6 h at
37°C, 5% CO2. Cells were stained for
intracellular IFN- using the Cytofix/Cytoperm kit (PharMingen)
according to the manufacturer’s specifications. Briefly, stimulated
cells were washed twice, and then Fc receptors were blocked by
incubation with rat antimouse CD16/CD32 (PharMingen) for 20 min,
followed by staining with phycoerythrin-labeled rat antimouse CD8
(PharMingen) for 30 min. After fixation and permeabilization for 20
min, cells were stained with FITC-labeled rat antimouse IFN-
(PharMingen) or an isotype control antibody for 30 min and then
analyzed by flow cytometry as described above. The percentage of
CD8+ cells that express intracellular IFN- was
calculated by subtracting the percentage of cells that stained
nonspecifically with the isotype control antibody from those that
stained specifically for IFN- after stimulation with the specific T
ag epitope, and by further subtracting those cells that stained
positive for IFN- after incubation with an unrelated peptide.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A, panel a; Fig. 1
B, panel c) and are associated
with osteoid and fibrous connective tissue production (Fig. 1
A,
panel c; Fig. 1
B, panel a). Staining for T ag
expression within tumors from distinct anatomical sites and from
multiple animals revealed the presence of T ag-expressing cells
throughout these tumors (Fig. 1, A and B
, panels
b and d). Because expression of SV40 T ag in the
periphery can lead to the induction of tolerance or anergy to T ag CTL
epitopes, we determined whether the four defined
H2b T ag epitopes were expressed by 501 tumors.
The osteoblast line 501-2484 col 3, derived from a 501 osteosarcoma,
was tested for susceptibility to lysis by a panel of CTL clones
specific for each of the four T ag epitopes in a
51Cr-release assay. 501-2484 col 3 cells were
lysed as efficiently as the T ag transformed cell line B6/WT-19 by each
of the CTL clones (Fig. 2
) indicating that epitopes I, II/III, IV, and V are processed and
presented by class I MHC molecules of this 501-derived tumor cell line.
In addition, C57BL/6 mice immunized with 501-2484 col 3 cells developed
CTLs specific for the immunodominant epitopes I, II/III, and IV after
in vitro stimulation of spleen cells (data not shown). These
results indicate that each of the four previously defined
H2b-restricted T ag epitopes are presented by
osteosarcomas from 501 mice.
|
|
A). In contrast, immunization of 501 mice with B6/WT-19
induced CTLs against epitope IV only (Fig. 3B
). These
epitope IV-specific CTLs retained the ability to lyse the T
ag-transformed cell line B6/WT-19, indicating that the 501-derived CTLs
were capable of lysing cells that express physiological levels of the
target epitope. Similar results were obtained from mice immunized with
rVV-941T. Immunization of B6 mice with rVV-941T typically results in
the induction of a strong epitope IV-specific CTL response and weaker
CTL responses to epitopes I and II/III (Fig. 3C
). 501 mice
also developed CTLs against T ag epitope IV but not against epitopes I
and II/III, although the response against epitope IV was less efficient
than in C57BL/6 mice (Fig. 3D
). Thus, our results using
immunization with full-length T ag indicated that 501 mice display
partial tolerance against T ag epitopes in which the ability to respond
to the immunodominant epitopes I and II/III was not detectable and the
response to epitope IV was diminished.
|
Groups of C57BL/6 and 501 mice of 
4 months of age were immunized
with 1 x 107 pfu of rVVs encoding
each of the T ag epitopes as a minigene, with or without a preceding
ES. In the case of epitope V, a minigene construct encoding epitope V
followed by two alanine residues was used instead of the epitope V
sequence alone, because in the absence of these additional residues,
epitope V fails to be immunogenic in C57BL/6 mice when expressed as a
minigene (28)
. After 4 weeks, spleen cells were
restimulated in vitro with B6/WT-19 cells and then tested
for their ability to lyse peptide pulsed RMA cells in a
51Cr-release assay. Immunization of C57BL/6 mice
with each rVV resulted in the induction of CTLs specific for the
appropriate T ag epitope (Fig. 4, I–P
). In contrast, only immunization with rVV-ES I,
rVV-IV, rVV-ES IV, and rVV-ES V induced detectable CTL responses in 501
mice (Fig. 4, A–H
). Thus, 501 mice retain the ability to
respond to T ag epitope I, but this response is only detected if the
mice are immunized with rVV-ES I (Fig. 4B
). This suggests
that a limited number of epitope I-specific CTL precursors are
available to respond or that they are anergic and only respond to a
strong immunization. Immunization of 501 mice with rVV-II/III or rVV-ES
II/III failed to induce epitope II/III-specific CTLs in multiple
experiments (Fig. 4, C and D
), suggesting that
CTLs specific for this epitope were absent from 501 mice or were unable
to respond, even to this strong stimulus. Immunization of 501 mice with
rVVs expressing epitope IV resulted in strong epitope IV-specific CTL
responses (Fig. 4, E and F
), demonstrating that
the relatively decreased responses to epitope IV observed using
immunization with full-length T ag (Fig. 2, B and D
) could be enhanced using this approach. Immunization with
the rVV-V-AA construct failed to induce a significant response in 501
(Fig. 4G
) compared with C57BL/6 (Fig. 4O
) mice,
indicating that the ability of 501 mice to respond to epitope V is also
reduced compared with C57BL/6 mice. Immunization of 501 mice with
rVV-ES V, however, resulted in the induction of efficient epitope
V-specific CTLs (Fig. 4H
). All mice immunized with rVVs
developed vaccinia virus-specific CTLs after in vitro
stimulation of spleen cells with VV-WR-infected C57BL/6 spleen cells,
indicating that vaccinia virus infection of mice had occurred (data not
shown). Thus, the limited response of 501 mice to T ag CTL epitopes I,
IV, and V can be substantially enhanced by immunization with rVVs
expressing T ag epitopes preceded by ES.
|
). B6/CD4-/- mice responded efficiently to
rVV-ES I in the same experiment, although the response was less
vigorous than in C57BL/6 mice. In contrast,
501/CD4-/- mice developed epitope IV- and
V-specific CTLs after immunization with rVV-ES IV or rVV-ES V,
respectively. Thus, CD4+ T cells were required
for the induction of epitope I-specific CTLs in 501 mice. The CTL
response generated in B6/CD4-/- mice was less
efficient than in C57BL/6 mice, indicating that
CD4+ T-cell help was required for optimal
response but was not necessary to induce CTLs against T ag epitope I in
T ag-negative mice.
|
8 months of
age, T ag expression can be detected in the salivary glands by 3 months
of age, with maximal levels occurring by 6 months of age.4
We asked whether these increased levels of T ag expression in the
periphery altered the response to T ag epitope immunization. 501 mice
were immunized with rVV-ES I, rVV-ES IV, or rVV-ES V at 6 months of
age. Spleen cells from immunized mice were restimulated in
vitro with B6/WT-19 cells prior to assay against peptide-pulsed
RMA cells. 501 mice immunized with rVV-ES I at 6 months of age
developed only weak T ag epitope I-specific CTL responses (Fig. 5A
) compared with control C57BL/6 mice (Fig. 5D
).
This response was reproducibly less efficient than that induced in
younger mice (Fig. 4B
), indicating a decreased ability to
respond to epitope I by 6 months of age. In contrast, immunization of
6-month-old 501 mice with rVV-ES IV and rVV-ES V induced
epitope-specific CTL responses (Fig. 5, B and C
)
comparable with those induced in C57BL/6 mice (Fig. 5, E and F
). Thus, the onset of tolerance to the
H2-Db-restricted epitope I occurs relatively
early in the absence of detectable effects on the CTL response to
epitopes IV and V and correlates with the time when high levels of T ag
were detected in the periphery.
|
respectively) developed efficient
responses to epitope IV. One of two 11-month-old 501 mice immunized
with rVV-ES IV developed a strong epitope IV-specific CTL response,
whereas the other failed to develop a detectable response to epitope IV
(Fig. 6C
). Importantly, mouse 501-34, which failed to
respond, had a palpable tumor of the humerus but was not generally
immunosuppressed because vaccinia virus-specific CTLs were detected
from the same spleen cells after in vitro culture with
vaccinia virus-infected stimulator cells (Fig. 6
C, inset).
Histological analysis of the tumor from mouse 501-34 revealed the
presence of osteogenic osteosarcoma containing T ag-expressing cells
(Fig. 1
B, panels a and b). 501 mice of 12 and 14
months of age retained the ability to respond to epitope IV, although
the response in these older mice was somewhat decreased compared with
the younger mice. The ability of all 501 mice to develop epitope
IV-specific CTLs, except for the 11-month-old tumor-bearing mouse (Fig. 6C
), suggested that tolerance to epitope IV in 501 mice
might not correlate with a specific age but rather with the appearance
of tumors.
|
). Mice were sacrificed, and necropsies were performed to ensure the
presence or absence of tumors. Spleen cells from these animals were
restimulated in vitro with B6/WT-19 cells or VV-WR-infected
C57BL/6 spleen cells. Tumor-free 501 mice developed efficient epitope
IV-specific CTL responses (Fig. 8, A and C
) comparable with that of an age-matched
C57BL/6 mouse (Fig. 8E
). In contrast, both tumor-bearing 501
mice developed only weak epitope IV-specific CTL responses (Fig. 8, B and D
). Spleen cells derived from these same
tumor-bearing mice developed efficient vaccinia virus-specific CTL
responses (Fig. 8, B and D
, insets) comparable
with those from the tumor-free 501 mice (data not shown). Thus,
although tumor-bearing 501 mice are tolerant to T ag epitope IV, they
are not generally immunocompromised because they retain the ability to
develop CTLs against an antigen unrelated to T ag. Histological
analysis of the tumor of the left clavicle from mouse 501-59, shown in
Fig. 7
D, revealed the presence of osteogenic osteosarcoma
that contained T ag-expressing cells throughout the tumor (Fig. 1
B, panels c and d). This indicates that T ag
expression is maintained in the tumors from mice that develop tolerance
to epitope IV. This result is consistent with previous studies that
indicate that T ag expression is required for maintenance of the
transformed phenotype (38
, 39) . In total, seven of seven
tumor-bearing 501 mice over the age of 11 months failed to develop
epitope IV-specific CTLs after immunization with rVV-ES IV, whereas 11
of 11 tumor-free 501 mice over the age of 11 months developed moderate
to strong CTL responses to epitope IV after immunization with rVV-ES IV
(P < 0.01 using Fisher’s exact test; Figs. 6
8
and data not shown). These results suggest that the onset of
tolerance to the H2-Kb-restricted epitope IV in
501 transgenic mice correlates with the appearance of tumors.
|
|
), and a
10-month-old tumor-bearing 501 mouse also developed an efficient
epitope V-specific CTL response (Fig. 8H
). In contrast,
12-month-old 501 mice failed to generate epitope V-specific CTLs,
regardless of their tumor status (Fig. 8, I and J
). This failure to develop epitope V-specific CTLs was
dependent on the presence of the T ag transgene and not solely
attributable to age because 12-month-old C57BL/6 mice developed strong
epitope V-specific CTLs after immunization with rVV-ES V (Fig. 8K
). In addition, both tumor-bearing and tumor-free 501 mice
developed strong vaccinia virus-specific CTL responses (Fig. 8, I and J
, insets). These results indicate that the
onset of tolerance to the immunorecessive epitope V is not a
direct result of tumor development but might correlate with time of
exposure to endogenous T ag because only 12-month-old mice developed
tolerance.
Decreased Numbers of Epitope IV-specific CTLs Are Detected in 501
Mice.
Our results suggested that decreased responsiveness to T ag epitope IV
correlates with the appearance of osteosarcomas in 501 mice. This loss
of T ag epitope IV-specific CTL reactivity might be explained by loss
of epitope IV-specific CTL precursors through deletion or by the
induction of anergy among epitope IV-specific
CD8+ T cells. To determine whether a quantitative
difference in epitope IV-specific CD8+ T cells
existed between tumor-bearing versus tumor-free 501 mice, we
used class I MHC tetramers (35)
composed of
H2-Kb and T ag epitope IV peptide
(Kb/IV Tet) to determine the number of epitope
IV-specific T cells that developed in rVV-ES IV immunized mice. The
Kb/IV Tet specifically stains CTL clones that
recognize T ag epitope IV as well as T ag epitope IV-specific CTLs from
rVV-ES IV immunized C57BL/6 mice.5
501 siblings were
identified in which one animal had a large osteosarcoma on the cervical
vertebrae, and the other was judged tumor-free by X-ray. These mice
were immunized at 11 months of age, along with 3-month-old 501 mice,
and C57BL/6 mice of 3 and 12 months of age. All mice were sacrificed
after 2 weeks, and their spleen cells were analyzed for the presence of
CD8+, Kb/IV
Tet+ cells. The number of
CD8+ cells that could be induced to secrete
IFN- after a short incubation with epitope IV synthetic peptide was
determined with spleen cells from the same mice in parallel as a
measure of function. C57BL/6 mice of both 3 and 12 months of age
mounted robust epitope IV-specific responses in which 14–16% of
CD8+ cells were stained by
Kb/IV Tet (Fig. 9
A). In contrast, only 3.5% of CD8+
cells were Kb/IV Tet+ in
3-month-old 501 mice, indicating that fewer epitope IV-specific
CD8+ cells develop in 3-month-old 501
versus C57BL/6 mice after immunization with rVV-ES IV. This
is in contrast to the similar levels of lysis detected in bulk CTL
cultures after in vitro restimulation (Fig. 4
, compare
F and N). Staining of spleen cells from an
11-month-old tumor-free 501 mouse immunized with rVV-ES IV revealed a
decrease in Kb/IV Tet+
cells to 0.5% of CD8+ cells (Fig. 9A
), suggesting that although bulk CTL responses against
epitope IV are still detected in older 501 mice, the number of
CD8+ cells that can respond diminishes with age.
No such decrease was noted in C57BL/6 mice, indicating that decreased
epitope IV responsiveness was attributable to endogenous T ag
expression. Analysis of spleen cells from the tumor-bearing 501 sibling
resulted in the inability to detect CD8+,
Kb/IV Tet+ cells above
background. This absence of epitope IV-specific
CD8+ cells corresponds with the inability to
expand epitope IV-specific CTL in vitro from tumor-bearing
501 mice. Analysis of naive spleen cells from either a 4-month-old 501
mouse or a tumor-bearing 10-month-old 501 mouse revealed that
CD8+, Kb/IV
Tet+ cells could not be detected above background
levels (data not shown), indicating that immunization with rVV-ES IV
was required to induce detectable levels of epitope IV-specific
CD8+ T cells.
|
after peptide stimulation with epitope IV correlated well with the
percentage of CD8+, Kb/IV
Tet+ cells from the same mice (Fig. 9B
). This suggests that the majority of
CD8+, Kb/IV
Tet+ cells are functional and do not represent a
population of anergic CD8+ cells expressing TCRs
specific for epitope IV. In addition, spleen cells from the rVV-ES IV
immunized mice from Fig. 9
were restimulated in vitro to
expand epitope IV-specific CTLs. All cultures developed efficient
epitope IV-specific CTLs except for those derived from the 11-month-old
tumor-bearing 501 mouse (data not shown), which is in agreement with
the data in Fig. 8
. Thus, our results indicate that T ag epitope
IV-specific CTL precursors persist in 501 mice beyond the onset of T ag
expression in the periphery but decline in number with age and reach
undetectable levels upon the appearance of osteosarcomas. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previous investigations of peripheral tolerance to SV40 T ag have used transgenic mice that express T ag from a tissue-specific promoter, such as the insulin promoter (3) . Such mice are responsive to T ag CTL epitopes in the absence of T ag expression in the thymus (8 , 11 , 14) , and activation of T ag-specific CTLs in these mice leads to the development of autoimmunity (11 , 13 , 15) . The fate of autoreactive CTLs in these mice, however, differs according to the model used. For example, mice that are transgenic for both full-length T ag and a TCR specific for an H2-Kk-restricted T ag CTL epitope developed spontaneous autoimmunity and failed to show tolerance to T ag (13 , 14) . In contrast, mice with fewer T ag-reactive CTLs showed signs of antigen specific tolerance, similar to our observations, which correlates with the onset of T ag expression in the periphery (11 , 13 , 16 , 17) . Using RIP1-Tag4 mice, which express T ag from the insulin promoter, Ye et al. (11) demonstrated that immunization of RIP1-Tag4 mice with SV40 induces T ag-specific CTLs and leads to a delay in the progression of T ag-induced tumors if mice are immunized prior to the onset of endogenous T ag expression. Immunization after the onset of T ag expression in the periphery failed to control tumor progression, suggesting that expression of the transgene leads to immune tolerance and that a period of time exists prior to T ag expression in the periphery when immunization leads to effective control of tumor progression. The present study, using 501 transgenic mice, reveals that the onset of CTL tolerance to multiple T ag epitopes can occur sequentially after expression of the transgene in the periphery, suggesting that distinct windows of opportunity exist for immunization against the T ag epitopes.
Although the specific immunological mechanism that leads to the onset of tolerance to T ag CTL epitopes in 501 mice remains to be determined, some possible mechanisms for the induction of tolerance by peripherally expressed tumor antigens include the induction of anergy, deletion of reactive CTLs, or suppression of immunity (40) . The induction of T cell anergy by peripherally expressed antigens has been reported for both CD4+ (41) and CD8+ (42) T cells and may be explained by the recognition of peptide ligands by naive T cells in the absence of costimulation from APCs (43) . Recognition of T-cell epitopes on tumor cells that lack the costimulatory molecules B7-1 (CD80) or B7-2 (CD86) lead to the induction of anergy (44 , 45) , suggesting that their interaction with CD28 on T cells is critical for the induction of tumor-specific immunity. Thus, direct recognition of T ag epitopes on peripheral tissues in 501 mice in the absence of proper costimulation could lead to the induction of CTL anergy in vivo.
Alternatively, naive T lymphocytes might be exposed to T ag epitopes on professional APCs in 501 mice through the mechanism of cross-presentation (46) . Several recent findings support an active role for professional APCs in the induction of T-cell tolerance. For example, engagement of the inhibitory receptor CTLA-4 on T lymphocytes by B7 molecules of activated APCs can induce tolerance directly, suggesting that antigen is transferred to APCs prior to tolerance induction (47) . Multiple studies have now determined that the induction of tolerance among naive T cells is preceded by a period of T-cell proliferation, which is thought to require interaction with professional APCs (41 , 48, 49, 50) . In addition, it is generally believed that naive lymphocytes of the adult do not traffic into peripheral nonlymphoid tissues, where they might be exposed to direct presentation of self epitopes (51 , 52) . Deletion of antigen-specific T cells after cross-presentation of peripherally expressed antigens by bone marrow-derived APCs has been observed for both CD8+ (49 , 53) and CD4+ (54) cells. Thus, the ability of professional APCs to process and present T ag after its expression in the periphery of 501 mice could explain the induction of tolerance to T ag CTL epitopes through cross-presentation. Whether the mechanism of tolerance induction involves deletion of epitope-specific CTL precursors or the induction of anergy remains to be determined.
Recent observations have indicated that the level of antigen expression in the periphery is critical for the induction of tolerance by cross-presentation (55 , 56) . Antigens expressed at low levels in the periphery either were ignored or induced weak proliferative responses in vivo and failed to induce deletion of autoreactive CD8+ T cells. Higher amounts of peripheral antigen, however, resulted in increased proliferation, followed by deletion of autoreactive CD8+ cells. These results indicate that the production of relatively small amounts of antigen in the periphery may not lead to efficient cross-presentation and manifests itself as immunological ignorance. In contrast, higher amounts of antigen production may lead to release of antigen that can be cross-presented by APCs and induce proliferation and subsequent deletion of autoreactive T cells. Thus, changes in the amount of T ag expressed during the life span of 501 mice might lead to the onset of tolerance to T ag CTL epitopes at distinct times.
At least two events in 501 mice might lead to increased levels of T ag in the periphery: (a) increased levels of T ag can be detected in the salivary glands of 501 mice at 6 months of age.4 This time of increased antigen load corresponds with the loss of reactivity to T ag epitope I. Although no direct correlation was established between these two events, our results might be explained by an increase in the availability of H2-Db/epitope I complexes in the periphery, which lead to the induction of tolerance among epitope I-specific CTL precursors; and (b) the progression of T ag-expressing tumors in 501 mice might lead to the release of increased amounts of T ag, particularly as the tumors undergo necrosis or apoptosis. Thus, this second wave of T ag expression might result in increased exposure of epitope IV-specific CTLs to cross-presented antigen. Our results using Kb/IV tetramers indicate that many of the epitope IV-specific CTLs are lost prior to the detection of osteosarcomas, because fewer epitope IV-specific CTLs were detected in 12-month-old, tumor-free 501 mice than in 4-month-old, tumor-free 501 mice. This suggests that tolerance to epitope IV progresses over the life span of 501 mice and is accelerated by the appearance of tumors. Importantly, T ag expression was maintained in the tumors of 501 mice that developed tolerance to epitope IV. These findings are reminiscent of experiments by Kurts et al. (49) in which a steady decline in the number of OVA-specific TCR transgenic T cells was observed after transfer into OVA transgenic mice (RIP-mOVA). Thus, our data support a model in which the pool of T ag epitope-specific CTLs are depleted over time because of increased levels of the transgene in the periphery, finally resulting in the complete loss of T ag epitope-specific CTLs.
Several possible explanations can be offered regarding the different times for the onset of tolerance to T ag epitopes I, IV, and V. As indicated above, the amount of antigen expressed in the periphery appears to be a determining factor in the onset of CTL tolerance (55 , 56) . Although previous studies examined only the response to a single CTL epitope, we suggest that the levels of individual T ag epitopes presented for CTL recognition in the periphery of 501 mice may vary, subject to the binding properties of the epitopes themselves. We have shown previously that T ag epitope I forms extremely stable complexes with H2-Db molecules (28) . This could lead to the accumulation of a large number of Db/epitope I complexes at the surface of T ag-expressing cells or APCs processing T ag, which might lead to the relatively early onset of tolerance to epitope I. In contrast, T ag epitope V dissociates rapidly from H2-Db and is not expected to accumulate at the cell surface. Thus, the frequency with which epitope V-specific CTL precursors encounter Db/epitope V complexes in 501 mice might be low. T ag epitope IV forms complexes with H2-Kb having an intermediate stability and, therefore, might accumulate an intermediate number of complexes at the cell surface. Thus, varying numbers of T ag epitope/MHC class I complexes may contribute to the development of the hierarchy of tolerance to T ag epitopes observed in 501 mice.
Although immunization with full-length T ag results in the simultaneous induction of CTLs specific for epitopes I and IV, we have shown previously that the frequency of epitope IV-specific CTLs is 4–5-fold higher than epitope I-specific CTLs in C57BL/6 mice using both limiting dilution analysis (30) and tetramer staining of ex vivo lymphocytes.5 Thus, another explanation for the more rapid onset of tolerance to epitope I than to epitope IV in 501 mice is that fewer epitope I-specific CTL precursors than epitope IV-specific CTL precursors need to be affected to result in loss of CTL responsiveness. In support of this scenario, Morgan et al. (56) have shown that the time required to develop peripheral tolerance against a HA CTL epitope in InsHA transgenic mice was directly related to the number of HA-specific TCR transgenic CD8+ cells that were injected. An increase in the number of HA-specific CD8+ T cells transferred into InsHA mice resulted in an increase in the amount of time required to develop peripheral tolerance to HA. Thus, the starting frequency of epitope-specific CTLs in 501 mice, as well as the level of epitope presentation in the periphery might contribute to the hierarchy of tolerance onset observed in our experiments. We are currently addressing these possibilities.
Our results indicate that the ability to develop CTLs against epitope I in 501 mice was dependent on CD4+ T cells, because 4-month-old 501/CD4-/- mice failed to develop epitope I-specific CTLs after immunization with rVV-ES I but were capable of responding to epitopes IV and V after immunization with rVV-ES IV or rVV-ES V, respectively. In contrast, CD4+ cells were not required in T ag-negative animals to develop epitope I-specific CTLs. Thus, epitope I-specific CTL precursors are either absent from the T-cell repertoire of 501/CD4-/- mice or fail to become activated in the absence of CD4+ T-cell help in these mice. Studies on the role of CD4+ T cells in the generation of CTL responses using various systems have revealed that some CTL responses were undetectable (57 , 58) , whereas others apparently were unaffected (59 , 60) by the absence of CD4+ cells. More recent studies, however, have shown that although CTLs are generated in the absence of CD4+ T cells, optimal CTL responses require CD4+ T-cell help (61, 62, 63, 64) . Thus, the generation of epitope I-specific CTLs in 501 mice might be more dependent on CD4+ T cells than in C57BL/6 mice, attributable to tolerogenic mechanisms that reduce the level of CTL responsiveness in 501 mice. The role of CD4+ T-cell help in promoting effective CTL responses recently has been attributed to their ability to "condition" APCs for the subsequent stimulation of CTLs (65, 66, 67) and involves the delivery of a signal through CD40-CD40 ligand.
In addition, the presence of CD4+ T cells may moderate the onset of tolerance in 501 mice. The addition of OVA-specific TCR transgenic CD4+ T cells was shown to prevent the deletion of OVA-specific TCR transgenic CD8+ T cells by the cross-presentation of peripheral OVA in RIP-mOVA transgenic mice (68) , suggesting that CD4+ cells were required for survival of autoreactive CTLs in this model system. Thus, in the absence of CD4+ T-cell help, epitope I specific CTL may be deleted more rapidly in 501 transgenic mice. In support of this idea, we have obtained preliminary evidence that the onset of tolerance to epitopes IV and V is also accelerated in 501/CD4 knockout mice compared with 501 mice (data not shown).
We have shown that an efficient CTL response against three of four T ag epitopes can be induced after immunization of 501 mice with T ag epitope minigenes and that the onset of CTL tolerance is epitope specific. Whether the induction of CTLs against one or more of the T ag epitopes prior to the onset of tolerance in 501 mice will have an effect on tumor progression remains to be determined. These findings, however, indicate that distinct windows of opportunity exist to activate epitope-specific CTLs against endogenous T ag-induced tumors in 501 mice. Thus, in the tumor-bearing host, the effectiveness of immunization against a particular tumor antigen CTL epitope might be determined by the timing of tolerance onset.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by Research Grants
CA25000 (to S. S. T.) and CA37102 (to B. B. K.) from the NIH and
CORE Grant P30 CA34196 from the National Cancer Institute (to
B. B. K.). T. D. S. is supported by the Concern Foundation for
Cancer Research/Cancer Research Institute Fellowship. 
2 To whom requests for reprints should be
addressed, at Department of Microbiology and Immunology, H107, The
Pennsylvania State University College of Medicine, 500 University
Drive, Hershey, PA 17033. Phone: (717) 531-8872; Fax; (717) 531-5578;
E-mail: sst1{at}psu.edu
3 The abbreviations used are: T ag, T antigen;
FBS, fetal bovine serum; rVV, recombinant vaccinia virus; APC, antigen
presenting cell; ES, adenovirus E3/19K endoplasmic reticulum insertion
sequence; HA, hemagglutinin; OVA, ovalbumin; pfu, plaque forming
unit(s); TCR, T-cell receptor; HSV, herpes simplex virus.
4 I. Marton, S. E. Johnson, I. Damjanov, K. S.
Currier, J. P. Sundberg, and B. B. Knowles. Expression and immune
recognition of SV40 Tag in transgenic mice that develop metastatic
osteosarcomas, submitted for publication.
5 L. M. Mylin, T. D. Schell, M. Epler, D.
Roberts, A. Bosteanu, E. J., Collins, J. A. Frelinger, S. Joyce, and
S. S. Tevethia. Quantitation of CD8+ T lymphocyte responses to
multiple epitopes from SV40 large T antigen in C57BL/6 mice immunized
with SV40, SV40 T antigen-transformed cells, or with vaccinia virus
recombinants expressing full-length T antigen or epitope minigenes,
submitted for publication.
Received 10/21/99. Accepted 3/29/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-amylase-SV40 T antigen hybrid gene. Science (Washington DC), 244: 460-463, 1989.-amylase-SV40 T-antigen hybrid gene. Am. J. Pathol., 137: 259-262, 1990.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  G. Willimsky, M. Czeh, C. Loddenkemper, J. Gellermann, K. Schmidt, P. Wust, H. Stein, and T. Blankenstein Immunogenicity of premalignant lesions is the primary cause of general cytotoxic T lymphocyte unresponsiveness J. Exp. Med., July 7, 2008; 205(7): 1687 - 1700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Degl'Innocenti, M. Grioni, G. Capuano, E. Jachetti, M. Freschi, M. T.S. Bertilaccio, R. Hess-Michelini, C. Doglioni, and M. Bellone Peripheral T-Cell Tolerance Associated with Prostate Cancer Is Independent from CD4+CD25+ Regulatory T Cells Cancer Res., January 1, 2008; 68(1): 292 - 300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Otahal, B. B. Knowles, S. S. Tevethia, and T. D. Schell Anti-CD40 Conditioning Enhances the TCD8 Response to a Highly Tolerogenic Epitope and Subsequent Immunotherapy of Simian Virus 40 T Antigen-Induced Pancreatic Tumors J. Immunol., November 15, 2007; 179(10): 6686 - 6695. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Otahal, T. D. Schell, S. C. Hutchinson, B. B. Knowles, and S. S. Tevethia Early Immunization Induces Persistent Tumor-Infiltrating CD8+ T Cells against an Immunodominant Epitope and Promotes Lifelong Control of Pancreatic Tumor Progression in SV40 Tumor Antigen Transgenic Mice. J. Immunol., September 1, 2006; 177(5): 3089 - 3099. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Ryan and T. D. Schell Accumulation of CD8+ T Cells in Advanced-Stage Tumors and Delay of Disease Progression following Secondary Immunization against an Immunorecessive Epitope J. Immunol., July 1, 2006; 177(1): 255 - 267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Otahal, S. C. Hutchinson, L. M. Mylin, M. J. Tevethia, S. S. Tevethia, and T. D. Schell Inefficient Cross-Presentation Limits the CD8+ T Cell Response to a Subdominant Tumor Antigen Epitope J. Immunol., July 15, 2005; 175(2): 700 - 712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. O'Mara, L. A. Norian, D. Kreamalmeyer, J. M. White, and P. M. Allen T Cell-Mediated Delay of Spontaneous Mammary Tumor Onset: Increased Efficacy with In Vivo versus In Vitro Activation J. Immunol., April 15, 2005; 174(8): 4662 - 4669. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. M. Haeryfar, R. J. DiPaolo, D. C. Tscharke, J. R. Bennink, and J. W. Yewdell Regulatory T Cells Suppress CD8+ T Cell Responses Induced by Direct Priming and Cross-Priming and Moderate Immunodominance Disparities J. Immunol., March 15, 2005; 174(6): 3344 - 3351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. D. Schell In Vivo Expansion of the Residual Tumor Antigen-Specific CD8+ T Lymphocytes That Survive Negative Selection in Simian Virus 40 T-Antigen-Transgenic Mice J. Virol., February 15, 2004; 78(4): 1751 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Staveley-O'Carroll, T. D. Schell, M. Jimenez, L. M. Mylin, M. J. Tevethia, S. P. Schoenberger, and S. S. Tevethia In Vivo Ligation of CD40 Enhances Priming Against the Endogenous Tumor Antigen and Promotes CD8+ T Cell Effector Function in SV40 T Antigen Transgenic Mice J. Immunol., July 15, 2003; 171(2): 697 - 707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zheng, J.-X. Gao, H. Zhang, T. L. Geiger, Y. Liu, and P. Zheng Clonal Deletion of Simian Virus 40 Large T Antigen-Specific T Cells in the Transgenic Adenocarcinoma of Mouse Prostate Mice: An Important Role for Clonal Deletion in Shaping the Repertoire of T Cells Specific for Antigens Overexpressed in Solid Tumors J. Immunol., November 1, 2002; 169(9): 4761 - 4769. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. Schell and S. S. Tevethia Control of Advanced Choroid Plexus Tumors in SV40 T Antigen Transgenic Mice Following Priming of Donor CD8+ T Lymphocytes by the Endogenous Tumor Antigen J. Immunol., December 15, 2001; 167(12): 6947 - 6956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. Schell, J. D. Lippolis, and S. S. Tevethia Cytotoxic T Lymphocytes from HLA-A2.1 Transgenic Mice Define a Potential Human Epitope from Simian Virus 40 Large T Antigen Cancer Res., February 1, 2001; 61(3): 873 - 879. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), II/III (•), IV (
), V (
), or the control
epitope DbN5 (
), and the T ag-transformed cell lines B6/WT-19 (
)
and B6/K-1, 4, 5 (
).
