| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,,§
*
Laboratory of Molecular and Tumor Immunology, Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, OR, 97213;
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute, Portland, OR 97291; and
Oregon Cancer Center and
§
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, but not IL-4. In these models, T
cell-mediated tumor regression and long-term antitumor immunity are
perforin and FasL independent. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Perforin-dependent cytotoxicity and Fas/Fas ligand (FasL) interactions are the major known cytotoxic effector functions of CD8+ MHC class I-restricted T cells (14, 15, 16). Perforin is a membranolytic pore-forming protein that is stored together with granzymes in cytoplasmic granules of cytotoxic T cells and NK cells (17). After engagement of the TCR, perforin monomers are released from the cytoplasmic granules in a Ca2+-dependent fashion and assembled in the target cell membrane into polymeric pore structures that enable granzymes to invade the target cell and induce apoptosis (15, 18).
Alternatively, Ag-specific activation of effector T cells induces the expression and up-regulation of membrane-bound FasL, which induces apoptosis through Fas ligation on target cells (19, 20). Cross-linking of Fas through ligation of FasL activates the Fas-associated death domain, which triggers the activation of intracellular caspases, leading to apoptosis (21).
In this report we demonstrate that neither perforin nor FasL is required for the therapeutic antitumor effects mediated by adoptively transferred T cells from D5 TVDLN. Therapeutic effects were also observed in a sarcoma tumor model from MCA-310 TVDLN generated in perforin knockout (PKO) mice. However, effector T cells from wild-type (wt), PKO, or generalized proliferative disease (gld) all exhibit a tumor-specific type 1 cytokine profile. These results support the hypothesis that a type-1 cytokine response is associated with and may be critical for T cell-mediated tumor regression.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female C57BL/6J (wt) mice, female C57BL/6 -PFPtm1Sdz (PKO), and B6Smn.C3H-Faslgld (gld) were purchased from The Jackson Laboratory (Bar Harbor, ME). Additional C57BL/6 x 129S6/SvEv (KO) Pfp mice (PKO) and C57BL/6NTac (wt) were purchased from Taconic Laboratories (Germantown, NY). Mice used for experiments were generally 8 to 12 wk old and were maintained in a specific pathogen-free environment. Recognized principles of laboratory animal care were followed (Guide for the Care and Use of Laboratory Animals, National Research Council, 1996), and all animal protocols were approved by the Earle A. Chiles Research Institute Animal Care and Use Committee.
Tumor cell lines
D5 is a poorly immunogenic subclone of the spontaneously arising
B16BL6 melanoma (22, 23). An early passage of the original
BL6 tumor was provided by Dr. E. Gorelick (Pittsburgh Cancer Institute,
Pittsburgh, PA) and was subcloned by limiting dilution culture in Dr.
S. Shu’s laboratory (Cleveland Clinic Foundation, Cleveland, OH). D5
exhibits low to undetectable class I (H-2 Db and
Kb) and Fas (CD95) expression and no class II
expression (provided by Dr. S. Shu). D5-G6 is a stable clone of D5 that
was transduced with a murine GM-CSF retroviral MFG vector (provided by
Dr. M. Arca, University of Michigan, Ann Arbor, MI, Ref.
24). D5-G6 cells secrete 
200
ng/ml/106 cells/24 h GM-CSF. MPR-4, which is a
transformed prostate tumor cell line (generously provided by Dr.
Thompson, Baylor College of Medicine, Houston, TX) from a C57BL/6 mouse
(25), has very low MHC class I expression and no
detectable MHC class II expression as determined by flow cytometry.
MCA-101 (H-2b) is a methyl-cholanthrene-induced
sarcoma that exhibits low to undetectable levels of MHC class I and
class II, and low levels of Fas (kindly provided by Dr. Nick Restifo,
Surgery Branch, National Cancer Institute, National Institutes of
Health). MCA-310 (H-2b) is a
methyl-cholanthrene-induced sarcoma recently developed at the Earle A.
Chiles Research Institute that expresses low levels of MHC class I and
undetectable levels of MHC class II and Fas.
Reagents
The 145-2c11 hybridoma (anti-CD3) was a gift of Dr. J. A. Bluestone (University of Chicago, Chicago, IL). Recombinant human IL-2 was generously provided by the Chiron Corporation (Dr. M. Giedlin, Emeryville, CA). The anti-CD4 GK1.5 (TIB-207) and anti-CD8 2.43 (TIB-210) hybridomas were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Ascites was prepared in DBA/2 mice primed with pristane and immunosuppressed by injection with 200 mg/kg cyclophosphamide. FITC- and PE-labeled isotype control rat IgG, hamster IgG, and mAbs against CD3, CD4, CD8, Fas, FasL, and annexin were purchased from PharMingen (San Diego, CA). Freshly isolated TVDLN cells were blocked with anti-mouse Fc receptor hybridoma 2.4G2 (HB-197, ATCC) culture supernatant before incubation with directly labeled specific Abs. The apoptosis-inducing Fas Ab (clone Jo2) was purchased from PharMingen.
Culture conditions
Lymphocytes and tumor cells were cultured in complete medium (CM), which consisted of RPMI 1640 (BioWhittaker, Walkersville, MD.) containing 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 µg/ml of gentamicin sulfate. This was further supplemented with 50 µM 2-ME (Aldrich, Milwaukee, WI.) and 10% FBS (Life Technologies, Grand Island, NY). Tumor cells were harvested 2 to 3 times per week by brief trypsinization (BioWhittaker) and maintained in T-75 or T-150 culture flasks.
Tumor vaccination
D5-G6 tumor cells were harvested by trypsinization, washed twice with HBSS, and subsequently resuspended at 2 x 107 cells per milliliter. One million D5-G6 (GM-CSF-transduced) tumor cells were injected s.c. into both hind and fore flanks of wt, PKO, or gld mice. Eight days following tumor vaccine inoculation, the draining superficial inguinal and axillary LN were harvested. MCA-310 tumor was passaged in wt and PKO mice and harvested before vaccination. The tumor was harvested, cut into small pieces using a pair of scissors and subsequently digested for 2 h using a triple enzyme mixture containing a final concentration of 0.1% collagenase type IV (No. C-5138; Sigma, St. Louis, MO), 0.002% deoxyribonuclease type IV (Sigma No. D-5025), and 0.01% hyaluronidase type V (Sigma No. H-6254) as previously described (26). The tumor digest was filtered, washed twice with HBSS, and subsequently resuspended at 2 x 107 cells per milliliter. One million MCA-310 tumor cells were injected s.c. into both hind and fore flanks of wt and PKO mice. Thirteen days following tumor vaccine inoculation, the draining superficial inguinal and axillary LN were harvested. TVDLN were resuspended at 2 x 106 cells per milliliter in CM and cultured in 24-well plates with 50 µl of 1:40 dilution of 2C11 ascites (anti-CD3). This dilution was determined previously to be optimal for T cell activation. After 2 days of activation, the T cells were harvested and expanded in CM containing 60 IU rhIL-2/ml for 3 additional days. T cells were then harvested, washed twice in HBSS, counted, and used in adoptive transfer, cytotoxicity, and cytokine release assays.
Adoptive immunotherapy
Experimental pulmonary metastases were established by i.v. inoculation of 2 x 105 D5 or of 2 x 105 or 4 x 105 MCA-310 tumor cells into specified mice. Three days later, when metastases were established, T cells were adoptively transferred i.v. Starting on the day of T cell infusion, mice received 90,000 IU IL-2 i.p. once per day for 4 days. Where noted, in vivo depletion of T cell subsets was performed by i.v. administration of anti-CD4 (GK1.5) or anti-CD8 (Lyt-2) ascites (0.5 ml of a 1:5 dilution). These doses of mAb were shown to deplete effectively the appropriate T cell population for at least 13 days (data not shown). Rat Ig (Sigma; No. I-4131, Lot 086H8910) was administered as a control. Animals were sacrificed 13 to 16 days following tumor inoculation by CO2 narcosis. Lungs were resected and fixed in Fekete’s solution. Lungs of mice bearing MCA-310 metastasis were infused with India ink to visualize the metastases. The number of pulmonary metastases was counted in a blinded fashion. Metastases that were too numerous to count accurately were assigned an arbitrary value of 250.
Survival experiments
After establishment of 3-day experimental pulmonary metastases by i.v. inoculation of 2 x 105 D5 cells into wt or PKO mice, 70 x 106 effector T cells generated in either wt or PKO mice were adoptively transferred i.v. Starting on the day of T cell infusion, mice received 90,000 IU IL-2 i.p. once per day for 4 days. The animals were followed for at least 100 days.
Rechallenge experiments
Wild type and PKO mice that survived for longer than 100 days after adoptive transfer of effector T cells were rechallenged with 2 x 104 tumor cells s.c. (about 10x TD50 for D5 tumor; the TD50 is the dose at which 50% of the injected animals will develop tumor). As a control, naive wt or PKO mice were challenged s.c. with the same tumor dose. The size of the developing tumors was determined three times a week by measurement of two perpendicular diameters of the developing tumor using a caliper.
Measurement of cytokines
After activation and expansion, TVDLN were washed, resuspended
in CM + IL-2 (60 IU/ml), and seeded at 4 x
106/2 ml/well in a 24-well plate. The cells were
either cultured without further stimulation or stimulated with either
2 x 105 D5, MPR-4 or MCA-310 tumor cells,
or immobilized anti-CD3. Supernatants were harvested after 24
h, and the release of IFN-, IL-10, and IL-4 was determined in
duplicate by ELISA using commercially available reagents (IFN-,
PharMingen or Genzyme; IL-4, Genzyme; IL-10, PharMingen). The
concentration of the cytokines in the supernatant was determined by
regression analysis.
Cytotoxicity assay
Target cell lysis was assessed by 6-h 51Cr-release assay. Tumor cells were incubated with 100 µCi Na251CrO4 (NEN, Boston, MA) for 1 h, washed twice, and plated into round bottom 96-well plates with 1 x 104 target cells/well in triplicate. The target cells were incubated with LAK or effector T cells at the indicated E:T ratios in a total volume of 200 µl CM at 37°C in a CO2 incubator. The supernatant was harvested and counted, and the percentage specific lysis was calculated as previously described (27). Maximum lysis was achieved by incubating target cells with 2% Triton X-100 detergent. LAK cells, which were used as positive controls for cytotoxicity assays, were generated by culturing wt splenocytes with 6000 IU/ml IL-2 for 3 days.
Fas (CD95)-mediated apoptosis as determined by cytotoxicity assay
Susceptibility of D5 or MCA-101 tumor cells to Fas-induced
apoptosis was assessed by 6-h 51Cr release assay.
Tumor cells were incubated for 24 h in the presence or absence of
10 ng/ml IFN-, which up-regulates Fas (CD95) expression (see Fig. 4
). After incubation with 100 µCi
Na251CrO4
for 1 h, the cells were washed twice and resuspended in CM (200
µl/well) and plated in triplicate into flat-bottom 96-well plates
with 1 x 104 target cells/well. The tumor
cells were incubated at 37°C with or without Fas Ab (Jo2, 10 µg/ml
or 3.3 µg/ml) or anti-CD3 (1:40 or 1:120 of 2C11 ascites).
Protein G (1 µg/ml or 0.33 µg/ml) was added (as recommended by the
supplier, PharMingen: Apoptosis Applied Reagents and Technologies
Instruction Manual, 1st edition, 4/98, p. 4) to enhance
cross-linking of the Abs. The supernatant was harvested from cells kept
at 37°C in a CO2 incubator and counted, and the
percentage specific lysis was calculated as previously described
(27). Maximum lysis was achieved by incubating target
cells with 2% Triton X-100 detergent.
|
The significance of differences in number of metastases between experimental groups was determined by the Wilcoxon rank sum test. Two-sided p values of < 0.05 are considered significant. Each treatment group consisted of at least five mice, and no animal was excluded from the statistical evaluations. The significance of differences in cytokine secretion was determined using the Student paired t test. Two-sided p values of 0.05 are considered significant. Kaplan-Meier analyses and log rank tests were used to determine the significance of differences in survival studies.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To prove that mice with a targeted mutation in the perforin gene
were unable to mediate cytolytic activity, we generated LAK cells from
wt and PKO mice and tested their cytotoxic capability in a 6-h
51Cr release assay. LAK cells generated in wt,
but not PKO, mice lysed D5, MCA-101, and IFN--treated D5 tumor cells
in a dose-dependent manner (Fig. 1, a–c). These data confirm
that PKO mice are deficient in their ability to generate cytolytic
effector cells. As expected, D5 treated with IFN- to increase the
normally low/undetectable level of MHC Class I expression were less
susceptible to LAK lysis. Next, effector T cells generated by
anti-CD3 activation of TVDLN of wt and PKO mice were tested for
cytolytic activity against the same targets. It has previously been
shown that this in vitro activation method induces the maturation of T
cells with therapeutic activity specific for the tumor they were primed
against by vaccination (9, 10). Although the total LN
population of effector T cells were not specifically cytotoxic (data
not shown), TVDLN from wt mice that were enriched for
CD8+ T cells did exhibit specific killing of D5
tumor if they were pretreated with IFN- to up-regulate MHC class I,
but not the unrelated MCA-101 target (Fig. 1, d–f).
However, CD8+ TVDLN from PKO mice were not
cytolytic for D5 or IFN--treated D5 in the same assay. This
demonstrates that effector T cells generated from TVDLN or LAK cells of
PKO mice were unable to lyse specific tumor in short-term cytotoxicity
assays.
|
Eight days after the vaccination of wt and PKO mice with D5-G6,
TVDLN were harvested, and the phenotype of the cells was determined by
flow cytometric analysis. No differences in the phenotype, or the
number of cells recovered, were observed between TVDLN of wt or PKO
mice. T cells from both wt and PKO mice expanded similarly following
stimulation with anti-CD3 and expansion in IL-2, and similar
percentages of CD4 (10% wt vs 21% PKO) and CD8 (82% wt vs 76% PKO)
positive cells were present in the expanded population. To determine
whether effector T cells generated in PKO mice could mediate
therapeutic activity, in vitro activated TVDLN cells were adoptively
transferred into wt mice bearing 3-day established D5 metastases.
Control mice that received only IL-2 developed more than 250
metastases. In contrast, adoptive transfer of 35 or 70 x
106 effector T cells generated from both wt and
PKO mice mediated significant (p < 0.05) tumor
regression (Table I). No difference was
observed between groups treated with wt or PKO T cells. To exclude the
possibility that effector T cells generated in PKO mice indirectly
induce CTL or NK cells in the wt recipient mouse by secretion of
cytokines (e.g., IFN-) at the tumor site that could induce tumor
regression via a perforin-dependent mechanism, we adoptively
transferred effector T cells generated in PKO mice into PKO recipient
animals with established D5 tumor. The growth characteristics of D5
tumor in PKO mice did not differ from the growth characteristics
observed in wt mice. The control PKO mice that were treated with IL-2
alone developed more than 250 pulmonary metastases (Table II). Adoptive transfer of 70 x
106 PKO effector T cells mediated complete tumor
regression in these mice as well (p < 0.05),
confirming that T cell-mediated tumor regression is not a
perforin-dependent process. We also generated MCA-310 TVDLN cells in
PKO mice to investigate the role of perforin in a different tumor
model. Adoptive transfer of 20 x 106 and
10 x 106 effector T cells from both wt and
PKO mice induced complete tumor regression of wt and PKO mice bearing
3-day established MCA-310 metastases (Table III). This confirms that
perforin-mediated cytotoxicity is not crucial for therapeutic efficacy
of adoptively transferred T cells in these two tumor models.
|
|
|
To investigate whether the therapeutic efficacy of effector T
cells generated in PKO mice was mediated by CD4+
or CD8+ cells, we depleted each population in
vivo by i.v. injection of the appropriate Ab immediately following the
adoptive transfer of effector T cells. A dose of Ab was used that was
known to be highly effective at depleting the corresponding T cell
subsets. The T cells remained depleted for the course of the experiment
(data not shown). As shown in Table IV,
CD8+ T cells (CD4-depleted) from both wt and PKO
mice mediated significant (p < 0.05) reduction
in pulmonary metastases, while CD4+ T cells
(CD8-depleted) failed to exhibit therapeutic activity.
|
To investigate whether the adoptive transfer of effector T cells
from PKO mice affects long-term survival, we treated tumor-bearing PKO
mice with effector T cells generated from PKO mice. In three
independent experiments, all IL-2-treated control mice had to be
sacrificed by 20 days. However, animals treated with PKO effector T
cells showed significant survival benefit; all mice demonstrated an
increase in survival compared with IL-2 alone, and 56% (14/25)
survived greater than 100 days (p < 0.01;
summary of three experiments is presented in Fig. 2a). The efficacy of T cells
from PKO mice was not different from wt effector T cells
(p = 0.26). Interestingly, apparent solitary
s.c. tumor metastases that occurred after 60 days were often the reason
that animals treated with T cells were sacrificed.
|
To determine whether surviving mice developed protective immunity,
animals that survived longer than 100 days were rechallenged with a
s.c. injection of 2 x 104 D5 tumor cells.
As a control, 10 naive wt and PKO mice were challenged with the same
tumor dose. As expected, all of the naive mice, regardless of the
status of their perforin gene, developed rapid and progressive tumors
and required sacrifice by 21 days (Fig. 3, a and b). In
contrast, only two of the four PKO mice from the first experiment and
four of the seven mice from the second experiment developed a tumor,
and the onset of tumor development was significantly delayed compared
with naive mice. Two rechallenged animals from experiment 1 remain
tumor-free 140 days following rechallenge and three mice from
experiment 2 remain tumor-free 120 days following tumor challenge.
|
Although tumor destruction in PKO mice treated with PKO T cells
could not be mediated by a perforin-dependent mechanism, other lytic
molecules could be responsible for tumor regression. For example,
expression of FasL on effector T cells affords one alternative method
for tumor destruction (28). Flow cytometric analysis
confirmed that D5 expressed low levels of Fas (Fig. 4
a). Since effector T cells
can express FasL, we postulated that D5-TVDLN may mediate tumor
regression via induction of apoptosis by triggering Fas on D5. To
determine whether D5 was susceptible to Fas-mediated killing, we used
the anti-mouse Fas mAb Jo2. Although we could demonstrate the
apoptotic activity of the Jo2 Ab against wt thymocytes (data not
shown), the Jo2 Ab did not induce apoptosis of D5 tumor cells (Fig. 5). This was confirmed by measuring the
viability of D5 tumor cells in a colony-forming assay, where treatment
with anti-Fas again failed to reduce the viability of D5 tumor
cells as determined by their ability to form colonies (data not shown).
D5-induced IFN- secretion by T cells could up-regulate the
expression of Fas as well as MHC class I and class II molecules (data
not shown). Therefore we evaluated whether in vitro culture with
IFN- would up-regulate Fas expression on D5 which could increase
their susceptibility to anti-Fas-induced apoptosis
(29). D5 and MCA-101 tumor cells were cultured in CM or CM
containing 10 ng/ml IFN-, and Fas expression was evaluated by flow
cytometry 24 h later. Both D5 and MCA-101 tumor cells exhibited
substantially higher levels of Fas following exposure to IFN- (Fig. 4, b and d). Following IFN- treatment, MCA-101
became more susceptible to anti-Fas-mediated killing, but D5
remained resistant to anti-Fas-induced apoptotic death (Fig. 5).
Thus, D5 does not appear to be susceptible to apoptosis upon ligation
of Fas with the anti-Fas Ab (Jo2), which suggests that Fas/FasL
interactions in vivo may not be a primary mechanism of T cell-mediated
tumor regression in our model. To test this hypothesis in vivo, we
generated D5-specific effector T cells from TVDLN of gld
mice and examined their therapeutic activity in the D5 tumor model.
|
Eight days after vaccination of wt and gld mice TVDLN
were harvested, and the phenotype of the cells was determined by flow
cytometric analysis. TVDLN from gld mice contained 11%
double-negative CD4 and CD8 T cells. This was different from wt and PKO
mice. After activation of T cells with anti-CD3 and IL-2, no
double-negative T cells were observed, and the percentage of
CD4+ and CD8+ T cells was
the same as in wt T cells. The percentage of CD4+
cells from wt and gld mice was 20% and 23%, respectively,
and the percentage of CD8+ cells from wt and
gld mice was 82% and 75% respectively. To determine
whether effector T cells generated in gld mice were
therapeutic, in vitro-activated TVDLN cells were adoptively transferred
into wt mice bearing 3-day established D5 metastases. Adoptive transfer
of 35 or 70 x 106 effector T cells
generated from gld mice mediated significant
(p < 0.05) tumor regression (Table V). To exclude the possibility that
effector T cells generated in gld mice indirectly induce CTL
or NK cells in the wt recipient mouse at the tumor site, which can
induce tumor regression via a FasL-dependent mechanism, we adoptively
transferred effector T cells generated in gld mice into
gld recipient animals with established D5 tumor (Table VI). Adoptive transfer of 35 x
106 gld effector T cells mediated complete
tumor regression in gld mice as well, indicating that T
cell-mediated tumor regression is independent of FasL-dependent
mechanisms.
|
|
To investigate long-term survival after adoptive transfer of
effector T cells from gld mice, we treated tumor-bearing wt
mice with 70 x 106 effector T cells
generated from gld mice. The IL-2 treated wt mice that did
not receive adoptively transferred T cells were sacrificed by 21 days
(Fig. 2b). However, animals treated with gld and
wt effector T cells showed significant survival benefit compared with
IL-2. All animals treated with T cells from FasL-deficient mice
survived greater than 50 days (p < 0.01). The
efficacy of T cells from gld mice was not different compared
with effector T cells from wt mice (p <
0.01).
Effector T cells generated from PKO mice exhibit a tumor-specific T1 cytokine profile
Recently, we and others have shown a correlation between
tumor-specific cytokine secretion and therapeutic efficacy of
adoptively transferred T cells (12, 23, 30, 31, 32). To
characterize the effector T cells generated in either wt or PKO mice we
examined their ability to secrete IFN-, IL-4, or IL-10 upon exposure
to D5 tumor cells. These analyses were performed on T cells used for
adoptive immunotherapy studies (Tables I and II). Stimulation with
anti-CD3 induced secretion of similar levels of all three cytokines
by effector T cells from wt or PKO mice (Fig. 6). Effector populations from wt and PKO
mice secreted substantial IFN-, specifically in response to D5 tumor
stimulation (p < 0.05). IFN- was not
produced when T cells were cultured alone or in the presence of
syngeneic prostate cancer cells, MPR-4. Thus, tumor-specific release of
IFN- was exhibited by effector T cells generated from PKO mice. A
similar T1 cytokine profile was seen from CD4-depleted effector T cells
but was absent from CD8-depleted effector populations (data not shown).
We next evaluated IL-4 and IL-10 production by the same T cells. In
contrast to IFN-, effector T cells generated from wt or PKO mice did
not secrete IL-4, the prototypical type 2 cytokine, in response to
stimulation with D5 or MPR-4 tumor cells. However, tumor-specific IL-10
secretion was observed for both PKO and wt mice.
|
Effector T cells from FasL-deficient gld mice exhibit a tumor-specific T1 cytokine profile
Next we characterized the cytokine profile of effector T cells
generated from gld mice. Although stimulation with
anti-CD3 induced secretion of similar levels of IFN-, IL-4, or
IL-10 by effector T cells generated from wt or gld mice, the
tumor-specific cytokine response was polarized to a T1 profile (Fig. 7). Effector T cells from gld
mice secreted substantial IFN-, specifically in response to D5 tumor
stimulation (p < 0.05). IFN- was not
produced when T cells were cultured alone or in the presence of the
syngeneic sarcoma MCA-310. As observed for effector T cells from wt and
PKO mice, gld effector T cells did not secrete
tumor-specific IL-4. These results demonstrate that effector cells from
wt, gld, and PKO mice exhibit a dominant tumor-specific type
1 cytokine profile.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Kaegi et al. demonstrated that activation and proliferation of T cells from PKO mice are not impaired and that the number and ratio of CD8+ and NK cells in these mice are not different to wt mice (39). Consistent with these data, no phenotypic difference in the T cell or NK cell populations was observed for wt or PKO mice, either after vaccination in the TVDLN, or after in vitro stimulation and expansion. These results strongly suggest that other PKO-independent effector mechanisms are operational in tumor regression. Nevertheless, a possible contribution of perforin-mediated cytotoxicity in TVDLN cells generated in wt mice cannot be excluded by these experiments.
Although tumor rejection was not impaired by adoptive transfer of T cells lacking perforin, we observed a significant difference in the control of tumor growth in surviving PKO and wt mice that were rechallenged s.c. with live tumor cells. This observation is consistent with previous reports by M. van den Broeck et al., who investigated the role of perforin in tumor surveillance and the control of s.c. tumor growth in mice (40). They showed that, after a s.c. challenge with MC57G fibrosarcoma cells and the lymphoid tumors EL-4 and MBL-2, wt mice exhibited better growth control of the tumor than PKO mice.
The discrepancy between the efficacy of adoptively transferred PKO T cells in the eradication of established 3-day pulmonary metastases and the impaired ability of immune long-term surviving PKO mice to reject or prevent the outgrowth of a s.c. tumor burden, which is accomplished by wt-immune mice, is still uncertain and the subject of further investigation. However, it may be related to the properties of the immune response against a s.c. tumor vs a visceral tumor.
The contribution of perforin-dependent, T cell-mediated cytotoxicity has been intensely investigated in various infectious disease and transplantation models. These studies revealed an important role of CTL-mediated, perforin-dependent cytotoxicity in infections with the noncytopathic virus lymphocytic choriomeningitis virus and in immunity to secondary infections with the intracellular bacterium Listeria monocytogenes (41). In contrast, immune protection against cytopathic viruses such as vaccinia and vesicular stomatitis virus and infections with Semliki Forest virus or hepatitis B virus were independent of perforin-mediated cytotoxicity (15, 42, 43, 44, 45, 46). These infections are controlled by the release of soluble cytokines and neutralizing Abs. The role of perforin in allograft rejection is still controversial. Heart grafts with disparity in the MHC class I gene, but not in the MHC class II gene, were rejected faster in wt mice than in PKO mice (42). In contrast, neither heart grafts that differed in both MHC class I and class II, nor skin grafts that differed in one or in both MHC loci, were rejected faster in wt mice (42). These data show that perforin-mediated cytotoxicity is crucial in some, but not all, cases of CD8+ T cell-dependent immune responses and that other mechanisms, like Ag-specific cytokine release, may be important effector mechanisms of T cells.
Besides perforin-mediated cytotoxicity of CD8+ T cells, FasL expressed on T cells has also been shown to play an important role in CTL-mediated cytotoxicity, especially in the control of peripheral T cell homeostasis (47, 48, 49) and in the control of viral infections (50).
Although T cells from PKO mice lack perforin-mediated cytotoxicity,
they can express FasL and induce apoptosis upon ligation to Fas on
target cells (14, 39). Carter et al. demonstrated that T1
CD8+ effector cells but not T2
CD8+ effector cells generated in wt or PKO mice
exhibit FasL-mediated cytotoxicity (51). Since both PKO
and wt T cells express a T1 cytokine profile, we hypothesized that FasL
might induce apoptosis of the tumor cells. FasL-induced apoptosis is
dependent upon the expression of Fas on the target cell. Although
cultured D5 tumor cells express a very low level of Fas, as determined
by flow cytometry, we were unable to detect Fas on in vivo passaged or
freshly isolated D5 tumor nodules (data not shown). This is consistent
with reports of others (29). Furthermore, treatment of D5
with anti-Fas mAb failed to induce apoptosis. Because IFN-
up-regulates Fas expression on murine melanoma cells (29)
and effector T cells specifically secrete IFN- in response to D5
stimulation, we reasoned that PKO T cells that traffic to the lung
could secrete IFN-, following interaction with D5 or APC, and induce
local expression of Fas on D5. In vitro exposure of D5 to IFN- (10
ng/ml) for 24 h led to marked up-regulation of Fas (Fig. 4) and
MHC class I (Kb, Db) and
class II (IAb) (data not shown). However, IFN-
pretreatment did not render D5 susceptible to killing by PKO or wt
effector T cells in a 6-h 51Cr release assay
(Fig. 1), or as determined by flow cytometric or colony-forming assays
(data not shown).
Another method to determine the impact of Fas/FasL interactions in vivo is to examine our model in FasL-deficient (gld) mice. Since adoptive transfer of T cells generated in gld mice mediated complete tumor regression in 3-day tumor bearing wt and gld mice, and because significant prolongation of survival was noted in the treated animals, it is unlikely that tumor regression is dependent on FasL-mediated apoptosis.
The basis for the resistance of D5 melanoma to FasL-mediated apoptosis is unknown.
Polymerization of Fas through FasL is known to activate Fas-associated
death domain, which then activates caspase 8 (Fas-associated death
domain-like IL-1 converting enzyme (FLICE)) (52). However,
cross-linking of Fas does not necessarily induce apoptosis. Recently,
apoptosis inhibitors like FLICE-inhibiting protein (FLIP) have been
identified (53). Human melanoma cell lines can express
FLIP (54). Since we are not able to induce apoptosis in D5
by Fas mAb despite up-regulation of Fas after incubation with IFN-,
it can be speculated that FLIP or other downstrean inhibitors that are
activated or constitutively expressed inhibit apoptosis induction
in D5.
Recently, Thomas et al. have shown that 10 human melanoma cell lines
were resistant to apoptosis induction after treatment with the FasL,
TNF-
, and CD40L. However 7 of these 10 cell lines tested were
susceptible to TNF-related apoptosis-inducing ligand (TRAIL)-induced
apoptosis (54). Whether TRAIL or other members of the TNF
family are involved in T cell-mediated tumor regression in the D5 model
can only be speculated. In preliminary experiments, we have been unable
to induce apoptosis of D5 with human recombinant TRAIL (our unpublished
observations).
Thus far we have no evidence that direct cell-mediated cytolytic activity is responsible for tumor regression mediated by adoptive transfer of tumor-specific T cells.
If tumor regression is not dependent on direct cell-mediated cytotoxicity, indirect mechanisms through activation of other cells with potential tumoricidal activity are likely to play a major role.
Barth et al. demonstrated a correlation between tumor-specific IFN-
and TNF- release and tumor regression (31). We have
recently shown a direct correlation between tumor-specific IFN-
release and therapeutic efficacy of TVDLN T cells in the D5 tumor model
(23). Others have shown that in vivo administration
of neutralizing mAb to IFN- and GM-CSF in a mouse tumor
model significantly inhibited the therapeutic efficacy of effector
T cells (55). Nagoshi et al. recently
identified that host CD8+ T cells recruited and
activated by IFN- secreted by tumor-specific T cells were crucial
for tumor regression (56).
As shown here, effector T cells generated in wt, PKO, and
gld mice release tumor-specific IFN- but not IL-4 upon
stimulation with the parental tumor. IFN- is known to up-regulate
MHC class I and II on D5 in vitro and on the parental B16 tumor in vivo
and might enhance tumor recognition by CD4 and CD8 cells in vivo
(57). Furthermore, IFN- has been shown to activate
tumoricidal activity of NK cells as well as their release of IL-12,
which in turn activates macrophages and dendritic cells and potentially
induces more T1 cells. Activated macrophages exhibit increased
tumoricidal activity in vitro and in vivo (58, 59, 60, 61, 62, 63).
By immunohistochemistry, we could show that macrophages accumulate in
the lung around tumor metastasis following adoptive immunotherapy with
PKO or wt T cells. This was most noticeable on day 2 after cell
transfer, and was not seen in control mice. How these macrophages were
attracted to the tumor site, and whether they were involved in tumor
regression is the focus of future investigations. Interestingly, we
observed tumor-specific release of IL-10 by therapeutic effector T
cells. This was more pronounced for effector cells generated in PKO and
gld mice. The tumor-specific IL-10 produced by highly
therapeutic T cells is interesting, in light of recent observation that
IL-10 appeared to inhibit the antitumor activity of transferred T
cells. Aruga et al. reported that distinct nontherapeutic
Vß-subpopulations of TVDLN T cells from LN-draining MCA-207 secreted
tumor-specific IL-10, but not IFN- or GM-CSF, whereas therapeutic
Vß subpopulations secreted tumor-specific IFN- and GM-CSF
(30). Furthermore, in vivo neutralization of IL-10 by i.v.
injection of anti-IL-10 mAb following adoptive transfer of effector
T cells enhanced the antitumor activity of the transferred T cells.
Since IL-10 potentially down-regulates Th1 responses of T cells and has
also been shown to abrogate local T cell responses in established
cutaneous basal and squamous cell carcinomas (64), it is
interesting to speculate whether blocking IL-10 in our model would
further augment the therapeutic efficacy of effector T cells. IL-10 is
also a potent inhibitor of macrophage functions and inhibits their
cytokine synthesis, MHC class II expression, and respiratory burst
(65, 66, 67). Additional experiments neutralizing IL-10 will
address the role of IL-10. Whether other T1 cytokines like TNF- or
TNF-ß are crucial for tumor regression is also under
investigation.
It is important to note that, while we have analyzed both the perforin and FasL effector mechanism separately, it is possible that, in the absence of one lytic mechanism, another lytic mechanism can compensate and mediate antitumor activity. Future studies utilizing animals deficient for both perforin and FasL will be necessary to rule out this possibility. Nonetheless, these studies document that neither perforin- nor FasL-dependent mechanisms are required to mediate tumor regression in this model. These observations raise the possibility that T1 cytokines may indirectly mediate therapeutic activity by the induction and activation of macrophages, which may be the mediators of tumor regression in this model.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Department of Surgery, Ludwig-Maximilians-Universität München, Klinikum Grosshadern, Marchioninistraße 15, 81377 Munich, Germany.
3 Address correspondence and reprint requests to Dr. Bernard A. Fox, Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, 4805 Northeast Glisan, Portland, OR 97213. E-mail address:
4 Abbreviations used in this paper: TVDLN, tumor vaccine-draining lymph node; D5, B16BL6-D5; D5-G6, B16BL6-D5 stably transfected with murine GM-CSF; LN, lymph node; LNC, LN cells; 2C11, hamster anti-mouse CD3 
-chain; PKO, perforin knockout; FasL (CD 95L), Fas ligand; gld, generalized lymphoproliferative disease; wt, wild type; CM, complete medium; FLICE, Fas-associated death domain-like IL-1- converting enzyme; FLIP, FLICE-inhibiting protein; TRAIL, TNF-related apoptosis-inducing ligand.
Received for publication April 28, 1999. Accepted for publication July 26, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-facilitated rejection of murine B16 melanomas. J. Immunol. 161:897. and tumor necrosis factor have a role in tumor regressions mediated by murine CD8+ tumor-infiltrating lymphocytes. J. Exp. Med. 173:647. and granulocyte/macrophage colony-stimulating factor) producing donor tumor-infiltrating lymphocytes. J. Immunol. 160:334. interferon induces the priming step in macrophage activation for tumor cell killing. J. Immunol. 130:2011.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  A. Shanker, A. D. Brooks, K. M. Jacobsen, J. W. Wine, R. H. Wiltrout, H. Yagita, and T. J. Sayers Antigen Presented by Tumors In vivo Determines the Nature of CD8+ T-Cell Cytotoxicity Cancer Res., August 15, 2009; 69(16): 6615 - 6623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. S. Umeshappa, H. Huang, Y. Xie, Y. Wei, S. J. Mulligan, Y. Deng, and J. Xiang CD4+ Th-APC with Acquired Peptide/MHC Class I and II Complexes Stimulate Type 1 Helper CD4+ and Central Memory CD8+ T Cell Responses J. Immunol., January 1, 2009; 182(1): 193 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Tatum, L. M. Mylin, S. J. Bender, M. A. Fischer, B. A. Vigliotti, M. J. Tevethia, S. S. Tevethia, and T. D. Schell CD8+ T Cells Targeting a Single Immunodominant Epitope are Sufficient for Elimination of Established SV40 T Antigen-Induced Brain Tumors J. Immunol., September 15, 2008; 181(6): 4406 - 4417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Zhang, A. Barber, and C. L. Sentman Chimeric NKG2D Modified T Cells Inhibit Systemic T-Cell Lymphoma Growth in a Manner Involving Multiple Cytokines and Cytotoxic Pathways Cancer Res., November 15, 2007; 67(22): 11029 - 11036. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Yang, N. ud Din, D. D. Browning, S. I. Abrams, and K. Liu Targeting Lymphotoxin {beta} Receptor with Tumor-Specific T Lymphocytes for Tumor Regression Clin. Cancer Res., September 1, 2007; 13(17): 5202 - 5210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. T. van der Veken, R. S. Hagedoorn, M. M. van Loenen, R. Willemze, J.H. F. Falkenburg, and M. H.M. Heemskerk {alpha}{beta} T-Cell Receptor Engineered {gamma}{delta} T Cells Mediate Effective Antileukemic Reactivity. Cancer Res., March 15, 2006; 66(6): 3331 - 3337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Sussman, R. Parihar, K. Winstead, and F. D. Finkelman Prolonged Culture of Vaccine-Primed Lymphocytes Results in Decreased Antitumor Killing and Change in Cytokine Secretion Cancer Res., December 15, 2004; 64(24): 9124 - 9130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, and R. W. Dutton Tc1 and Tc2 Effector Cell Therapy Elicit Long-Term Tumor Immunity by Contrasting Mechanisms That Result in Complementary Endogenous Type 1 Antitumor Responses J. Immunol., February 1, 2004; 172(3): 1380 - 1390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, J. C. Hylind, and R. W. Dutton Effector Cell-Derived Lymphotoxin {alpha} and Fas Ligand, but not Perforin, Promote Tc1 and Tc2 Effector Cell-Mediated Tumor Therapy in Established Pulmonary Metastases Cancer Res., January 1, 2004; 64(1): 406 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Greil, G. Anether, K. Johrer, and I. Tinhofer Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools J. Leukoc. Biol., September 1, 2003; 74(3): 311 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Caldwell, M. H. Ryan, E. McDuffie, and S. I. Abrams The Fas/Fas Ligand Pathway Is Important for Optimal Tumor Regression in a Mouse Model of CTL Adoptive Immunotherapy of Experimental CMS4 Lung Metastases J. Immunol., September 1, 2003; 171(5): 2402 - 2412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Qin, J. Schwartzkopff, F. Pradera, T. Kammertoens, B. Seliger, H. Pircher, and T. Blankenstein A Critical Requirement of Interferon {gamma}-mediated Angiostasis for Tumor Rejection by CD8+ T Cells Cancer Res., July 15, 2003; 63(14): 4095 - 4100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Poehlein, H.-M. Hu, J. Yamada, I. Assmann, W. G. Alvord, W. J. Urba, and B. A. Fox TNF Plays an Essential Role in Tumor Regression after Adoptive Transfer of Perforin/IFN-{gamma} Double Knockout Effector T Cells J. Immunol., February 15, 2003; 170(4): 2004 - 2013. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Seki, A. D. Brooks, C. R. D. Carter, T. C. Back, E. M. Parsoneault, M. J. Smyth, R. H. Wiltrout, and T. J. Sayers Tumor-Specific CTL Kill Murine Renal Cancer Cells Using Both Perforin and Fas Ligand-Mediated Lysis In Vitro, But Cause Tumor Regression In Vivo in the Absence of Perforin J. Immunol., April 1, 2002; 168(7): 3484 - 3492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Saio, S. Radoja, M. Marino, and A. B. Frey Tumor-Infiltrating Macrophages Induce Apoptosis in Activated CD8+ T Cells by a Mechanism Requiring Cell Contact and Mediated by Both the Cell-Associated Form of TNF and Nitric Oxide J. Immunol., November 15, 2001; 167(10): 5583 - 5593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Bonnotte, N. Larmonier, N. Favre, A. Fromentin, M. Moutet, M. Martin, S. Gurbuxani, E. Solary, B. Chauffert, and F. Martin Identification of Tumor-Infiltrating Macrophages as the Killers of Tumor Cells After Immunization in a Rat Model System J. Immunol., November 1, 2001; 167(9): 5077 - 5083. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Winter, H.-M. Hu, K. McClain, W. J. Urba, and B. A. Fox Immunotherapy of Melanoma: A Dichotomy in the Requirement for IFN-{{gamma}} in Vaccine-Induced Antitumor Immunity Versus Adoptive Immunotherapy J. Immunol., June 15, 2001; 166(12): 7370 - 7380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Peng, J. C. Krauss, G. E. Plautz, S. Mukai, S. Shu, and P. A. Cohen T Cell-Mediated Tumor Rejection Displays Diverse Dependence Upon Perforin and IFN-{gamma} Mechanisms That Cannot Be Predicted From In Vitro T Cell Characteristics J. Immunol., December 15, 2000; 165(12): 7116 - 7124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-M. Hu, H. Winter, W. J. Urba, and B. A. Fox Divergent Roles for CD4+ T Cells in the Priming and Effector/Memory Phases of Adoptive Immunotherapy J. Immunol., October 15, 2000; 165(8): 4246 - 4253. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
