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in CD8 T Cell-Mediated Elimination of 3LL-A9 Lewis Lung Carcinoma Cells In Vivo1
*
Institute of Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, Freiburg, Germany; and
Cell Genix Technologie Transfer, GmbH, Freiburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-
in anti-tumor CD8 T
cell immunity was examined in a new tumor model using a CD8 T cell
epitope (GP33) derived from lymphocytic choriomeningitis virus as a
tumor-associated Ag. In contrast with parental 3LL-A9 (A9) Lewis lung
carcinoma cells that progressively grow in C57BL/6 mice, s.c. injection
of GP33-transfected A9GP33 tumor cells induced a protective
GP33-specific CD8 T cell response that led to complete tumor cell
elimination. Tumor regression was dependent on perforin, IFN-, or
TNF-, because A9GP33 tumors developed in mice deficient
in one of these genes. A9GP33 tumors arising in perforin-
and IFN--deficient mice represented GP33 Ag-loss variants,
demonstrating that GP33-specific CD8 T cells from these mice were able
to exert an Ag selection pressure. In contrast, tumor cells growing in
TNF- knock-out mice still expressed the tumor-associated GP33
peptide despite the presence of activated GP33-specific CD8 T cells.
These findings provide evidence for a crucial role of TNF- in A9
tumor cell elimination by CD8 T cells in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which
induce apoptosis in susceptible cells (1). The release of
growth-suppressive soluble factors, such as TNF- or IFN-, is an
alternative way by which CD8 T cells can control tumor growth. Several murine tumor models have been established using genetically modified tumor cells that express defined antigenic CD8 T cell epitopes as a model of TAA (2, 3, 4, 5, 6, 7, 8, 9). Despite expression of the TAA introduced, all of these transfected tumor cells had growth kinetics similar to those of the parental tumor lines. This indicates that expression of CD8 T cell epitopes on tumor cells alone is not sufficient to elicit a protective anti-tumor immune response in general.
The H-2Kb binding peptides MUT1 and MUT2, derived from a mutated connexin 37 gap-junction protein, have been identified as tumor-specific Ags in 3LL Lewis lung carcinoma cells (10). These peptides serve as CD8 T cell epitopes and could also be used as vaccines to protect mice from spontaneous metastasis of 3LL-D122 tumors (11). Despite the expression of these MUT peptides, 3LL parental tumor cells grow in C57BL/6 (B6) mice when injected s.c. In the present study, we have examined the immune response against 3LL-A9 Lewis lung carcinoma cells transfected with a minigene encoding residues 33–41 (GP33 peptide) of the glycoprotein from the lymphocytic choriomeningitis virus (LCMV). In contrast with parental tumor cells, GP33-transfected 3LL-A9GP33 tumor cells induced a protective tumor-specific CD8 T cell response in B6 mice. This new tumor model opens the possibility to examine the role of effector molecules in CD8 T cell-mediated tumor cell elimination in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) mice were obtained from Charles River (Sulzfeld,
Germany). B6.gld and B6 mice deficient in
ß2-microglobulin (ß2m)
(12), I-A (13), perforin (14),
IFN- (15), and TNF- (16) were obtained
from H. Eibel (University Freiburg, Germany), The Jackson Laboratory
(Bar Harbor, ME), T. Brocker (Basel Institute for Immunology,
Switzerland), H. Hengartner (University Hospital Zurich, Switzerland),
M. Kopf (Basel Institute for Immunology), and H. Körner
(University of Erlangen, Erlangen, Germany), respectively. TCR
transgenic (tg) mice (line 318) specific for amino acids 33–41 (GP33
epitope) of the LCMV glycoprotein in association with
H-2Db molecule have been described (17, 18). TCR tg mice deficient in perforin or IFN- were generated
by breeding with perforin- or IFN--deficient B6 mice. The generation
of H8 tg mice expressing the LCMV GP33 epitope sequence under control
of the H-2Kb promoter (H8 mice) (19)
has been described. Mice were bred and kept in a conventional mouse
house facility and were used for experiments 8–16 wk after birth.
Cell lines
3LL-A9 (A9) is an immunogenic and low metastatic cloned line
derived from the 3LL carcinoma and was kindly provided by Lea Eisenbach
(Weizmann Institute of Science, Rehovot, Israel) (20).
A9GP33 cells were derived from parental A9 tumor
cells by gene transfection. The LCMV GP33 minigene was generated as
previously described (21). The resulting transfectants
were cloned and screened for GP33 expression by CTL assays. The
experiments reported in this study were performed with
A9GP33 clone 3. Parental A9 cells were cultured
in DMEM high glucose, supplemented with 10% FCS, glutamine,
streptomycin, and penicillin (all from Life Technologies, Gaithersburg,
MD). The A9GP33 tumor cells were kept in culture
under G418 (800 µg/ml) (Life Technologies) selection. L929 fibroblast
cells and B16.F10 melanoma cells were used as controls for testing
TNF--mediated cytotoxicity.
Tumor cell inoculation
Mice were injected s.c. into the right flank with 107 A9 or A9GP33 tumor cells in 100 µl PBS. Mice were checked for the presence of a palpable tumor and tumor growth was measured with a caliper. Tumor size was calculated as the product of bisecting tumor diameters. Mice bearing a tumor with a diameter >15 mm were killed according to animal care regulations.
Virus
The LCMV-WE isolate was originally obtained from R. Zinkernagel (University Hospital Zurich). It was propagated on L929 fibroblast cells. Mice were infected i.v. with 200 pfu.
Immunohistochemistry
Tumor sections (5 µm) were cut on a cryostat microtome, air dried, fixed in acetone, and blocked with TBS containing 5% mouse serum. Anti-CD8-biotin, anti-CD4-biotin, and anti-CD11b-biotin (all from PharMingen, San Diego, CA) were used as primary mAb followed by avidin-conjugated alkaline phosphatase (StreptAB Complex/AP; DAKO, Hamburg, Germany) and alkaline phosphatase substrate kit I (Vector Laboratories, Burlingame, CA). Sections were counterstained with Mayer’s hemalum.
Cytotoxicity assay
Cytolytic activity of spleen cells after a secondary in vitro
stimulation was assayed in a standard 51Cr
release assay. A9 cells coated with the GP33 (KAVYNFATM) or the control
adenovirus E1A 234-243 peptide (SGPSNTPPEI) at a concentration of
10-6 M were used as target cells. Tumor cells
growing in mice were isolated, cultured in vitro for 1 wk, and then
used as target cells. Spleen cells from day 8 LCMV-immune B6 mice were
used as effector cells. A9 and A9GP33 cells
cultured only in vitro were used as controls. The sensitivity of
A9GP33 tumor cells to TNF- cytotoxicity was
assessed by 51Cr release assay. L929, B16.F10,
A9, and A9GP33 target cells (5 x
103) were labeled with 250 µCi
51Cr and then incubated with serially diluted
recombinant mouse TNF- (20–0.0002 ng/ml) (PharMingen) for 14
h. Results were expressed as percentage of specific lysis =
{[experimental release (cpm) - spontaneous release
(cpm)]/[maximum release (cpm) - spontaneous release
(cpm)]} x 100.
In vitro stimulation
Eight days after s.c. injection of 107 A9GP33 into B6 mice, spleen cells (4 x 106) were cultured with B6 spleen cells (2 x 106) coated with 10-6M GP33 peptide in 1.5 ml of IMDM (Life Technologies) containing 10% FCS supplemented with antibiotics and 2-ME. Spleen cells isolated from LCMV-immune mice 10 wk after infection were cultured under the same conditions. After 5 days, cells were harvested and CD8 T cell activity was determined. Spleen cells (4 x 106) from TCR tg mice were cultured in 1.1 ml IMDM supplemented with 10% FCS, penicillin/streptomycin, 2-ME, and 10-7 M GP33 peptide. After 3 days, cells were harvested (60–70% of tg TCR+ T cells) and CTL activity was determined in a 5-h and 14-h 51Cr release assay.
Adoptive transfer and fluorescent cell labeling
Spleen cells of H8 mice were incubated at 5 x 106 cells/ml in ice-cold PBS containing 0.5 µM 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, Oregon) for 10 min at 37°C. The cells were washed once in PBS containing 1% FCS, and 0.5–1.108 cells were injected i.v. into recipient mice. The percentage of CFSE+ donor cells among recipient PBL was determined by flow cytometry on a FACSort flow cytometer (Becton Dickinson, San Jose, CA).
Anti-viral protection
B6 mice were injected s.c. with 107 A9GP33 tumor cells. Seventeen days later, mice were infected i.v. with 200 pfu of LCMV-WE. After 4 days, viral titers in the spleens were determined in a virus plaque assay as described (22).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used a CD8 T cell epitope of the LCMV glycoprotein aa
33–41(GP33) as a TAA. 3LL-A9 Lewis lung carcinoma
(H-2b) cells (designated A9 in this study) were
transfected with a minigene encoding the 9 aa of the GP33 epitope, and
GP33 expression was assessed by 51Cr release
assays using GP33-specific CTL. As shown in Fig. 1
, GP33-transfected A9 cells
(A9GP33) were efficiently lysed by effector cells
from day 8 LCMV-immune B6 mice. Specific lysis of
A9GP33 cells was comparable to the lysis of A9
cells coated with the GP33 peptide. The CTL activity of effector cells
was GP33 specific, because the target cells coated with an irrelevant
Db-binding peptide from adenovirus were not lysed
(Fig. 1).
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To determine the effect of GP33 expression on tumor growth,
parental A9 and transfected A9GP33 cells were
inoculated s.c. into B6 mice. As shown in Fig. 2A, the injection of parental
tumor cells resulted in progressive tumor growth and the mice had to be
killed within 2–3 wk. In striking contrast,
A9GP33 cells only developed small tumors (3–4 mm
diameter) 5–6 days after inoculation, which completely regressed after
10–12 days in B6 mice (Fig. 2B). To assess the role of CD4
and CD8 T cells in A9GP33 tumor rejection, MHC
class I- and class II-deficient mice were used. As shown in Fig. 2, C and D, growth and rejection of parental and
transfected A9 tumor cells were similar in B6 and in
B6.I-A-/- mice. Thus, CD4 T cells were not
crucial in controlling tumor growth in this model. In contrast, a lack
of CD8 T cells in
ß2m-/- mice resulted in
the progressive growth of the transfected A9GP33
tumor cells (Fig. 2F), comparable to parental A9 cells (Fig. 2E). Similar growth of parental and transfected tumor cells
was also observed in H8 transgenic mice, which ubiquitously express the
LCMV GP33 epitope as a transgene (Fig. 2, G and
H). Due to central tolerance induction, H8 mice lack
GP33-specific CD8 T cells (19). Taken together, these data
indicated that the CD8 T cells specific for GP33 were required for
A9GP33 tumor regression.
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T cell priming usually occurs in secondary lymphoid organs.
Therefore, we examined whether A9GP33 tumor cells
could be detected in the lymph nodes draining the tumor. Despite the
sensitivity of our PCR assay (detection of 1 tumor cell in
106 somatic cells), 1, 3, and 14 days after tumor
cell injection, A9GP33 tumor cells were neither
found in draining inguinal lymph nodes nor in mesenterial lymph nodes
nor in the spleen (not shown). However, immunohistology revealed a
massive infiltration of CD11b+ macrophages (red)
at the injection site 6 days after tumor cell inoculation (Fig. 3, upper left). Later on,
tumor cells (blue) became more abundant and
CD11b+ macrophages were less numerous in the
infiltrates (Fig. 3, upper right). This finding points to a
strong inflammatory response early after tumor cell injection, which
may favor the induction of a tumor-specific T cell response at the
tumor site. Indeed, as shown in the bottom panel of Fig. 3, CD4 and CD8 T cells infiltrating the tumor mass were detected.
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To demonstrate that A9GP33 tumor cells
induce a GP33-specific CD8 T cell response, three different assays were
used. First, the cytolytic activity of in vitro restimulated spleen
cells from B6 mice inoculated with A9GP33 cells
was determined on A9 target cells pulsed with the GP33 peptide. As
shown in Fig. 4A, spleen cells
from B6 mice injected with the A9GP33 tumor cells
exhibited a GP33-specific lytic activity comparable to that of spleen
cells from LCMV-immune B6 mice. Second, rejection of CFSE-labeled
spleen cells from H8 mice that ubiquitously express GP33 was used as an
in vivo assay for functional GP33-specific T cell activity. As shown in
Fig. 4B, 5 h after the adoptive cell transfer, H8
spleen cells represented 7–10% of PBL of the recipient mice. The
CFSE+ cells decreased within 24 h in
LCMV-immune B6 mice, whereas they persisted in control B6 mice. The
rejection of H8 spleen cells in B6 mice previously injected with
A9GP33 tumor cells demonstrated that
A9GP33 tumor cells were able to induce
GP33-specific T cells in these mice. The slower rejection of H8 spleen
cells in A9GP33-injected B6 mice, when compared
with LCMV-immune B6 mice, further indicated that GP33-specific T cell
priming with tumor cells was less efficient than immunization with live
virus. Third, anti-viral protection induced by
A9GP33 tumor cells was examined. B6 mice injected
with A9GP33 tumor cells were challenged with
LCMV, and viral titers were determined in the spleen 4 days after
infection. As shown in Fig. 4C, the reduction of the virus
titers in mice inoculated with A9GP33 tumor cells
compared with control B6 mice demonstrated that the transfected tumor
cells were able to induce a protective CD8 T cell response. Taken
together, these data show that A9GP33 tumor cells
were able to prime GP33-specific CD8 T cells in B6 mice.
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,
perforin, or IFN-
To investigate the mechanism of A9GP33 tumor
cell elimination by GP33-specific CD8 T cells, tumor cells were
inoculated into mice deficient in either Fas ligand, TNF-, perforin,
or IFN-. As shown in Fig. 5, parental
A9 tumor cells grew in all mice with a similar kinetic. In contrast
with normal B6 mice, which all have rejected
A9GP33 tumor cells, A9GP33
tumor growth was observed in B6 mice deficient in perforin (7/8),
IFN- (9/10), or TNF- (13/16). These data demonstrate that
perforin, IFN-, and TNF- were all required for a fully protective
anti-tumor T cell response in B6 mice. In contrast, Fas-Fas ligand
interaction appeared to play only a minor role in A9 tumor cell
elimination in vivo because only one out of five B6.gld mice developed
an A9GP33 tumor.
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- but not in
TNF--deficient mice
A9GP33 tumor development in the knockout
mice examined could be due to 1) insufficient priming of GP33-specific
CD8 T cells, 2) ineffective tumor cell elimination, or 3) selection of
GP33 Ag-loss variants. To test for GP33 expression,
A9GP33 tumors were isolated, cultured 1 wk in
vitro, and tested in a 51Cr release assay using
spleen cells from LCMV-infected B6 mice as GP33-specific CTL. As shown
in Fig. 6A, all tumors
isolated from ß2m-/- or
H8 transgenic mice still expressed GP33 as a TAA, because the isolated
tumor cells were lysed by GP33-specific CTL as efficiently as the
A9GP33 line kept in in vitro culture. This result
can readily be explained by the fact that these mice lacked
GP33-specific CD8 T cells, either due to the failure of positive
selection in the thymus
(ß2m-/- mice) or due to
central tolerance induction (H8 mice).
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- or
perforin-deficient mice lost GP33 expression, because they were no
longer recognized by GP33-specific CTL. H-2Kb and
Db expression of these ex vivo-isolated tumor
cells were comparable to the A9GP33 line kept in
culture, and the tumor cells were efficiently lysed by GP33-specific
CD8 T cells when pulsed with the GP33 peptide (not shown). These
results indicated that Ag-loss variants arose due to the selection
pressure of the induced GP33-specific CD8 T cells in IFN-- or
perforin-deficient mice. To assess the role of perforin and IFN- in
cell-mediated lysis of A9GP33 tumor cells in
vitro, the CTL activity of effector cells from perforin- or
IFN--deficient TCR tg mice specific for the GP33 peptide were
determined against A9GP33 tumor cells. As shown
in Fig. 6
B, effector cells from TCR tg and TCR tg
IFN--/- mice lysed
A9GP33 target cells in a 5-h standard
51Cr release assay. In contrast, GP33-specific
lytic activity of effector cells from perforin-deficient TCR tg mice
was only detected in a long-term (15 h) assay. The observed lysis was
GP33 specific because parental A9 target cells were not lysed. Taken
together, these data indicate that A9GP33 target
cells can be specifically lysed by perforin- or IFN--deficient
effector cells, which correlates well with the occurrence of A9 GP33
Ag-loss variants in perforin- and IFN--deficient mice.
Surprisingly, all (5/5) A9GP33 tumors isolated
from TNF--deficient mice still expressed the GP33 Ag, because tumor
cells were lysed as efficiently as the A9GP33
line kept in vitro. This indicated that GP33-specific CD8 T cells were
either not induced or were not able to exert a selection pressure on
the growing tumor cells in the absence of TNF-. To test whether
GP33-specific CD8 T cells were present in TNF--deficient mice that
developed A9GP33 tumors, CFSE-labeled spleen
cells from H8 mice were injected into these mice. As shown in Fig. 7, CFSE+ cells
rapidly decreased in A9GP33 tumor-bearing
TNF--deficient mice, whereas they persisted in TNF--deficient
mice not given A9GP33 tumor cells. This result
demonstrated that A9GP33 tumor cells were able to
induce GP33-specific CD8 T cells also in
TNF--/- mice.
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A possible explanation for the impaired ability of
TNF--deficient mice to control tumor growth might be that
A9GP33 tumor cells were susceptible to direct
TNF--mediated cytotoxicity. Therefore, the TNF sensitivity of these
cells was examined in vitro. As shown in Fig. 8, A9 cells were as sensitive as
TNF-sensitive L929 cells to recombinant murine soluble TNF-. This
result could explain the A9GP33 tumor development
in TNF--/- mice despite the presence of
GP33-specific CD8 T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The unique feature of the A9GP33 model could be due to the GP33 epitope used as TAA. The GP33 peptide binds to Db molecules but one amino acid shorter version of this epitope (GP34–43) is also able to bind to the Kb molecules and to be recognized by Kb-restricted CTL from LCMV-infected mice (23, 24). Among several strong CTL epitopes tested, the GP33 peptide was found to be the only one capable of inducing a secondary CTL response in ß2m-/- mice, which have a strongly decreased number of CD8 T cells (25). These data, together with the known immunodominance of the GP33/H-2Db epitope in LCMV infection in H-2b mice, speak for a relatively high frequency of GP33-specific CD8 T cells in the naive T cell repertoire, which could facilitate the induction of a protective immune response against A9GP33 tumor cells. However, due to low frequencies of precursor T cells in normal mice, the actual number of GP33-specific CD8 T cells can neither be determined precisely nor compared with the frequency of T cells specific for other epitopes.
However, expression of the GP33 epitope on tumor cells does not always result in tumor regression. B16.F10GP33 melanoma cells generated with the GP33 expression vector used here for A9 cells grow progressively in syngeneic B6 mice and in LCMV TCR tg mice (21). This shows that both GP33 expression and a high precursor frequency of CD8 T cells specific for the tumor Ag may not always be sufficient to induce tumor regression. Our results in the A9GP33 model are in contrast with a recent report by Hermans and colleagues (26), who also used Lewis lung carcinoma cells and the GP33 peptide as a model Ag. These authors found that parental and GP33-transfected tumor cells grew in B6 mice and in LCMV TCR tg mice with similar kinetics. A difference in GP33 expression levels of the two Lewis lung carcinoma lines used could explain these discrepancies. The features of the A9GP33 tumor model shown here are also distinct from a tumor model first described by Kundig and colleagues using MC57G fibrosarcoma cells and the glycoprotein of LCMV as defined TAA (27). In contrast with the A9 model, both parental and transfected MC57G cells are rejected in syngeneic hosts after s.c. tumor cell injection.
The A9GP33 tumor model shows both similarities and differences to another recently described tumor model that uses HIV gp160 as a model viral tumor Ag (28). As with A9GP33 tumor cells, injection of 15-12-RM-3T3gp160 tumor cells also induced a gp160-specific CTL response, which led to transient regression of the tumors. In contrast with our findings, in the HIV gp160 model recurrent tumors that had lost gp160 expression developed in BALB/c mice but not in mice deficient in CD4 T cells. These differences could be explained by the model Ag (GP33 peptide vs gp160 protein) and/or by the tumor cell types (3LL-A9 vs 15–12-RM-3T3) used.
The site of activation of CD8 T cells recognizing nonmetastatic tumors is a subject of controversy (27, 29, 30). We failed to detect A9GP33 tumor-derived DNA in tumor-draining lymph nodes. This result may suggest that homing of A9GP33 tumor cells into lymphoid tissues is not crucial for the activation of GP33-specific T cells in our model. However, we cannot exclude the possibility that T cell activation by the B7-negative A9GP33 tumor cells occurred via cross-priming, involving host-derived professional APCs migrating from the tumor injection site to regional lymph nodes. Immunohistochemical analysis of the A9GP33 tumor sections revealed a massive macrophage infiltration early after tumor cell injection. The macrophages recruited into the tumor mass may present TAA to CD8 T cells and thereby act as professional APCs.
The fact that A9GP33 tumors were rejected
in wild-type B6 mice, but grew in most of mice deficient in perforin,
IFN-, or TNF- indicates that all of these molecules play a role
in controlling A9GP33 tumor development. Parental
and GP33-transfected A9 cells grew in H8 mice with a similar kinetic.
In contrast, A9GP33 tumor growth was
significantly delayed in all of the knock-out mice mentioned above.
This indicates that initial but not complete immunological control of
A9GP33 tumor growth can be mediated by perforin-,
IFN--, or TNF--independent effector mechanisms. The finding that
A9GP33 tumors regressed in most (4/5) B6.gld mice
argues against a crucial role for Fas ligand in A9 tumor cell
elimination in vivo.
Interestingly, all A9GP33 tumors isolated
from perforin- and IFN--deficient mice represented GP33 Ag-loss
variants. How can the outgrowth of these variants be explained? In
contrast with wild-type mice, which rapidly clear
A9GP33 tumor, tumor cell elimination probably
occurs less efficiently and more slowly in the absence of perforin or
IFN-. This would allow tumor cells to undergo more cell divisions,
which will increase the possibility for spontaneous generation of GP33
"escape" variants. The GP33-specific CD8 T cells induced will
finally eliminate GP33-expressing tumor cells but will not affect the
growth of cells that have lost GP33 expression. An impaired ability of
mice lacking perforin (31, 32, 33, 34, 35) or IFN- receptors
(36) to control the growth of transplanted or induced
tumors has been previously demonstrated. A role of tumor-specific CD8 T
cells in the in vivo selection of Ag-loss variants has been suggested
after analyzing Ags expressed on human melanoma cell lines derived from
metastases appearing at intervals of several years (37).
Our study provides experimental evidence that selection of Ag-loss
tumor variants is favored in the presence of tumor-specific CD8 T cells
that lack perforin or IFN-. The lack of perforin will mainly affect
the lytic potential of effector cells, whereas a deficiency in IFN-
could influence tumor Ag presentation, induction, and the effector
phase of a CD8 T cell response.
Surprisingly, all A9GP33 tumors growing in
TNF--deficient mice still expressed GP33, because they were
recognized by GP33-reactive CTL as efficiently as the original cells
used for injection. The rejection of GP33-expressing H8 spleen cells in
A9GP33 tumor-bearing TNF--deficient mice
demonstrated that a GP33-specific T cell response was induced in these
mice. However, GP33-specific T cells were not able to exert selection
pressure in the absence of TNF-. The precise mechanism of how
TNF- exerts its function in this tumor model is not yet fully
understood. It is possible that soluble or membrane-bound TNF-
produced by activated GP33-specific CD8 T cells leads directly to lysis
of A9GP33 tumor cells in vivo. Alternatively,
other TNF--expressing cell types, such as macrophages activated by
GP33-specific T cells, may function as effector cells. The observed
TNF- sensitivity of the A9 tumor cells would fit into such
scenarios. TNF- may also function indirectly by inducing expression
of adhesion molecules and by regulating the migration of macrophages,
dendritic cells, and NK cells into the tumor mass (38, 39). The fact that a GP33-specific T cell response was induced
in A9GP33 tumor-bearing mice argues that tumor Ag
presentation was not severely affected in TNF--deficient mice.
Alternatively, TNF- has been demonstrated to play an essential role
in migration of NK cells into the peritoneum of tumor-bearing mice
(34). Therefore, one may speculate that the traffic of CD8
effector T cells into the tumor mass may be limited in the absence of
TNF-. Such an interpretation would explain tumor outgrowth without
selection of Ag-loss variants in TNF--deficient mice, despite the
presence of primed GP33-specific T cells. However, we failed to observe
significant differences in macrophage and CD8 T cell infiltration into
the A9GP33 tumor mass between wild-type and
TNF--deficient B6 mice (data not shown). The
A9GP33 tumor model described here will be of
great value for analyzing the important role of TNF- in Ag-specific
tumor cell elimination mediated by CD8 T cells in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hanspeter Pircher, Institute of Medical Microbiology and Hygiene, Department of Immunology, Hermann-Herder-Strasse 11, University of Freiburg, D-79104 Freiburg, Germany. E-mail address:
3 Abbreviations used in this paper: TAA, tumor-associated Ag; LCMV, lymphocytic choriomeningitis virus; B6, C57BL/6 mice; CFSE, 5- and 6-carboxyfluorescein diacetate succinimidyl ester; tg, transgenic; ß2m, ß2-microglobulin.
Received for publication September 29, 1999. Accepted for publication January 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A. Lamikanra, Z.-K. Pan, S. N. Isaacs, T.-C. Wu, and Y. Paterson Regression of Established Human Papillomavirus Type 16 (HPV-16) Immortalized Tumors In Vivo by Vaccinia Viruses Expressing Different Forms of HPV-16 E7 Correlates with Enhanced CD8+ T-Cell Responses That Home to the Tumor Site J. Virol., October 15, 2001; 75(20): 9654 - 9664. [Abstract] [Full Text] [PDF]
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B. K. Helmich and R. W. Dutton The Role of Adoptively Transferred CD8 T Cells and Host Cells in the Control of the Growth of the EG7 Thymoma: Factors That Determine the Relative Effectiveness and Homing Properties of Tc1 and Tc2 Effectors J. Immunol., June 1, 2001; 166(11): 6500 - 6508. [Abstract] [Full Text] [PDF]
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) and parental A9 cells
coated with the GP33 (•) or the adenovirus E1A peptides (
) were
used as target cells in a 5-h 51Cr release assay with day 8
LCMV-immune B6 spleen cells as effector cells. Spontaneous release was
<20%. This result is representative of several experiments.
, respectively) or the
adenovirus E1A peptide (
, respectively) in a
51Cr release assay. B, Rejection of
GP33-expressing H8 donor cells in mice previously injected with
A9GP33 tumor cells. Control B6 mice (
) and parental A9 target cells (
