| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Laboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute, and
Intramural Research Support Program, Science Applications International Corporation, Frederick, MD 21702; and
Cancer Immunology, Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, TNF-
,
and Fas ligand (FasL) in response to Renca. CTL used both granule- and
FasL-mediated mechanisms to lyse Renca, although granule-mediated
killing was the predominant lytic mechanism in vitro. The cytokines
IFN- and TNF- increased the sensitivity of Renca cells to CTL
lysis by both granule- and FasL-mediated death pathways. Adoptive
transfer of these anti-Renca CTL into tumor-bearing mice cured most
mice of established experimental pulmonary metastases, and successfully
treated mice were immune to tumor rechallenge. Interestingly, we were
able to establish Renca-specific CTL from mice gene targeted for
perforin (pfp-/-) mice. Although these
pfp-/- CTL showed reduced cytotoxic activity against
Renca, their IFN- production in the presence of Renca targets was
equivalent to that of wild-type CTL, and adoptive transfer of
pfp-/- CTL was as efficient as wild-type CTL in causing
regression of established Renca pulmonary metastases. Therefore,
although granule-mediated killing is of paramount importance for
CTL-mediated lysis in vitro, some major in vivo effector mechanisms
clearly are independent of perforin. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
was also found to be essential for the success of immunotherapy
(4). Perforin-mediated cytotoxicity and Fas/Fas ligand (FasL)-interaction are major mechanisms for CD8+ T cell-mediated effector function (5, 6). After the TCR engages specific antigenic peptides presented by the MHC of target cells, perforin is released and causes damage to target cell membranes. Various granzymes, and possibly other granule constituents cosecreted with perforin, enter the target cells and induce apoptosis (7). The expression of a variety of cytokines, including FasL, is also enhanced in the T cells after Ag-specific activation. FasL cross-links Fas on the target cell, and in many cells this interaction triggers apoptosis and cell death (6). It has been reported that other members of the TNF family can also be expressed by antitumor effector cells. For example, TNF-related apoptosis-inducing ligand (TRAIL) can interact with its receptors on tumor cells and cause tumor cell death (8). However, the extent to which these cytotoxic mechanisms are required for CTL-mediated tumor destruction in vivo remains controversial, and the relative importance of granule vs FasL-mediated lytic activities are not well understood.
Studies on tumor surveillance in perforin-deficient mice have
demonstrated that perforin-dependent cytotoxicity was crucial for
resistance against injected tumor cell lines, viral and chemical
carcinogenesis as well as spontaneous leukemogenesis
(9, 10, 11), while FasL was reported to play only a minor role
in this setting. Similarly, studies in tumor-bearing,
perforin-deficient mice have also shown a major contribution of
perforin to regression of established tumors (12, 13).
However, many of the tumors used in these previous studies
were intrinsically resistant to Fas-mediated killing (9),
and some of these experimental models used xenotransplantation of human
tumors into mice (12, 13). In such experimental systems,
the species-specific activities of some cytokines and the strength of
xenogeneic immune responses may mask relevant biological roles for
cytokines and FasL-mediated effects. Relatively strong antigenic
stimuli such as xenoantigens may induce antitumor responses
qualitatively and quantitatively different from spontaneously arising
tumors. More recent studies have shown that Fas-associated death
domain-like IL-1
-converting enzyme inhibitory
protein-transfected tumors grew more rapidly than control
transfectants in wild-type (WT) mice, whereas growth rates in SCID mice
were equivalent (14, 15). Control and FLIP transfectants
were lysed equally well in vitro by CTL using granule-mediated lytic
pathways, whereas the FLIP transfectants were much more resistant to
FasL-mediated lysis. These studies suggested that Fas-dependent
apoptosis constituted a more prominent mechanism for tumor clearance in
vivo than had previously been anticipated. Furthermore, Lee et al.
(16) demonstrated that Renca cells overexpressing Fas grew
at much slower rates than control transfectants in vivo, further
indicating that FasL-mediated lysis could be an important effector
mechanism for tumor surveillance by T cells against some tumors.
A number of experimental murine tumors are resistant to FasL-mediated lysis in vitro, making it unlikely that this lytic mechanism constitutes an important antitumor effector pathway for destruction of these tumors in vivo (9). However, some studies have reported that Fas expression can be enhanced on certain murine tumors by in vivo tumor passage (17). In this situation, it is possible that in the in vivo milieu FasL-mediated antitumor effects may be more important than would be predicted from in vitro assays. In this study, we established CTL specific for in vitro reactivity to Renca from mice successfully treated with flavone acetic acid plus IL-2 or IL-12 pulse IL-2. The molecular mechanisms used by these CTL to lyse Renca tumor cells in vitro and cause regression of Renca metastases in vivo were then analyzed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Specific pathogen-free BALB/c mice were obtained from the Animal Production Area, National Cancer Institute (Frederick, MD). Perforin-deficient (BALB/c pfp-/-) mice were kindly provided by Dr. M. Smyth (Peter MacCallum Cancer Institute, Melbourne, Australia) and bred in our specific pathogen-free facility. Animal care was provided in accordance with the procedures outlined in A Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23; 1985).
Cell lines
The Renca tumor cell line is of BALB/c origin. The following cell lines were used as target cells for CTL assays: stretoxotocin-induced renal cell carcinoma-1, a recently derived, streptozotocin-derived RCC of BALB/c origin (18); A20 B cell lymphoma; the C26, a colon cancer of BALB/c origin; the MethA fibrosarcoma of BALB/c origin; L10A leukemia of BALB/c origin; the Harvey BALB/c fibroblast cell line transformed with the Harvey Ras oncogene (kindly provided by Dr. R. Hornung, Science Applications International Corporation, Frederick, MD); P815 mastocytoma (DBA/2); the B16 melanoma (C57BL/6); and the L929 fibrosarcoma (C3H/An). All the lines were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1x nonessential amino acids, 1 mM sodium pyruvate, penicillin (100 U/ml) and streptomycin (100 µg/ml), 10 mM HEPES, and 5 x 10-5 M 2-ME, pH 7.4 (complete medium). Con A blasts (19) and fibroblasts were prepared from spleen cells as previously described.
Reagents
Recombinant human IL-2, recombinant murine IL-12, and
murine rIFN- were generously provided by Chiron (Emeryville,
CA), Genetics Institute (Cambridge, MA), and Genentech (South San
Francisco, CA), respectively. Murine rTNF- was purchased from BD
PharMingen (San Diego, CA). Human rIFN- A/D was kindly provided by
Dr. M. Brunda (Hoffmann-LaRoche, Nutley, NJ). Human rIL-15 and murine
rIL-18 were obtained from PeproTech (Rocky Hill, NJ). The following Abs
were purchased from BD PharMingen: mAbs against mouse
H-2Kd (SF1-1.1), H-2Dd
(34-5-8AS), H-2Ld (28-14-8), CD4 (GK1.5), CD8
(53-6.7), DX-5, TNF- (MP6-XT22), IFN- (XMG1.2), and LFA-1
(M17/4). An anti-mouse ICAM-1 (KAT-1) was purchased from R&D
Systems (Minneapolis, MN). Anti-mouse FasL (MFL-1) and
anti-mouse TRAIL (N2B2) mAb were kindly provided by Dr. H. Yagita
(Juntendo University, Tokyo, Japan; Ref. 20). PMA and
ionomycin were purchased from Sigma-Aldrich (St. Louis, MO).
Establishment and maintenance of CTL line
Renca tumor cells (105) were injected
under the kidney capsule of BALB/c mice. Mice were then treated on days
7–9 after tumor inoculation with FAA and IL-2, as previously described
(2). In some experiments, BALB/c tumor-bearing mice
treated with a combination of IL-2 and IL-12 (3) were used
as a source of CTL. Mice surviving >30 days after therapy were
rechallenged i.v. with 105 Renca cells.
Splenocytes isolated 1 wk later were stimulated in vitro with
IFN--treated (200 U/ml) and irradiated (100 Gy) Renca cells at a
splenocyte:tumor cell ratio of 20:1 in the presence of IL-2 (10 U/ml).
CTL were established and maintained in complete media supplemented with
IL-2 (10 U/ml). Viable cells (5 x 105) were
maintained as bulk CTL lines by weekly restimulation with irradiated
Renca cells (5 x 104) and irradiated (20
Gy) splenic feeder cells of BALB/c mice (4–5 x
106) in 24-well culture plates for the following
experiments. CTL were also established from BALB/c
pfp-/- mice in an identical manner.
Flow cytometric analysis
Flow cytometry analysis was performed on a FACScan (BD
Biosciences, Mountain View, CA) using CellQuest software. For the
detection of FasL or TRAIL on CTL, CTL (2 x
106 cells/ml) were cultured at 37°C for 6, 12,
or 20 h with the following agents in media supplemented with a
matrix metalloproteinase inhibitor (10 µg/ml, KB8301; BD PharMingen):
IFN- (200 U/ml), IFN- (500 U/ml), TNF- (100 ng/ml), IL-2 (500
U/ml), IL-12 (20 ng/ml), IL-15 (150 ng/ml), IL-18 (500 ng/ml),
plate-coated anti-CD3 mAb (10 µg/ml), or PMA (20 ng/ml) plus
ionomycin (1 µg/ml) (P/I). Alternatively, CTL were cultured with
105 Renca cells or Harvey BALB fibroblasts. These
CTL (1 x 106) were incubated with l µg of
biotinylated anti-FasL, anti-TRAIL mAb, or isotype control
(hamster IgG for FasL and rat IgG2a for TRAIL; BD PharMingen) for 30
min at 4°C followed by PE-labeled avidin. After washing with PBS, the
cells were analyzed on a FACScan.
Cell surface markers of Renca were analyzed after overnight treatment
with IFN- (500 U/ml) and/or TNF- (400 U/ml) or media alone by
flow cytometry using FITC-labeled mAbs reactive to
H-2Kd (SF1-1.1), ICAM-1 (3E2), LFA-1(M17/4),
CD48(HM48-1), CD80 (16-10A1), CD86 (GL-1), ICAM-2 (3C4), LFA-1 (M17/4),
CD48 (HM48-1), CD80 (16-10A1), CD86 (GL-1), and PE-labeled mAb reactive
with Fas (Jo2), or isotype-matched mAbs. All mAbs were purchased from
BD PharMingen.
Cytotoxicity assays
Renca cells that had been incubated overnight in the presence or
absence of IFN- (500 U/ml) and TNF- (400 U/ml) were labeled with
111In-labeled oxine
([111In]Ox; Medi-Physics, Silver Spring, MD) as
previously described (19). Other target cells were labeled
with [111In]Ox in the same manner. Briefly,
1 x 106 target cells were incubated with 10
µCi of [111In]Ox for 30 min at room
temperature. Cells were then washed twice in complete medium and
labeled cells (1 x 104) were then incubated
with effector CTL at various E:T ratios for 6–8 or 18 h at 37°C
in a final volume of 200 µl. Short-term cytotoxicity assays of 6–8 h
were used in preference to standard 4-h assays which, although adequate
for granule-mediated killing, tend to underestimate death
receptor-mediated lysis. For inhibition experiments, various mAbs were
added to the culture at a final concentration of 10 µg/ml. For the
inhibition of FasL- or TRAIL-dependent cytotoxicity, CTL activity was
tested in the presence of a neutralizing anti-FasL mAb (MFL-1) or
anti-TRAIL mAb (N2B2) at 10 µg/ml, respectively. For inhibition
of perforin-dependent cytotoxicity, CTL were pretreated with
Concanamycin A (CMA; Sigma-Aldrich) for 2 h at 100 nM, and then
cytotoxic assays were performed in the continuous presence of CMA.
After incubation, supernatants were harvested and counted on a gamma
counter. Specific killing (percentage of cytotoxicity) was calculated
as: [(experimental release - spontaneous release)/(maximal
release - spontaneous release)] x 100. All groups were run in
triplicate, and all experiments were performed three or more times with
similar findings.
Cytokine ELISA
CTL (4 x 104 cells) were incubated
with 104 stimulator cells (E:T = 4:1) or
media alone in 96-well plates at 37°C for 16 h. For the blocking
experiments, mAb was added to the culture at a final concentration of
10 µg/ml. Cell-free supernatant was harvested and subjected to
IFN-, TNF-, or IL-4 ELISA (R&D Systems; sensitivity limits: 2
pg/ml).
N-a-benzyloxycarbonyl-L-lysine thiobenzoyl ester (BLT) esterase release assay
CTL (1 x 105) were incubated with
Renca or IFN-/TNF--treated Renca (1 x
104) for 4 h in 96-well tissue culture
plates with or without anti-LFA-1 mAb. The BLT activity of the
supernatants was measured as previously described
(21).
Adoptive transfer of CTL in vivo
Treatment of pulmonary metastasis model was initiated 3 days after the i.v. injection of 1.5 x 105 Renca cells into syngeneic BALB/c WT mice or BALB/c pfp-/- mice. CTL derived from BALB/c WT or BALB/c pfp-/- mice were transferred i.v on day 3. In initial experiments, CTL or Con A blast T cells as a control were transferred i.v. at 107 cells per day on days 3, 4, and 5 together with IL-2 (10,000 U/day). In later experiments, CTL were simply transferred on day 3 in the absence of IL-2 and survival was monitored, or cohorts of mice were euthanized on day 17, and numbers of lung metastasis were counted.
Statistical analysis
The significance of difference in number of metastasis between experimental groups was determined by the Mann-Whitney U test. Two-sided p values of <0.05 are considered significant. Survival data was analyzed by the log rank test, and p values of <0.01 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Renca cells were injected under the renal capsule of BALB/c mice,
followed by immunotherapy with FAA plus IL-2. The majority (>80%) of
mice undergoing this therapy survived and were rendered tumor-free,
whereas all untreated mice died between 20 and 27 days from extensive
tumor development. Splenocytes from surviving mice were isolated 1 wk
after i.v. rechallenge with tumor, and CTL were generated by mixed
lymphocyte tumor culture in vitro. Seven days after the in vitro
culture was started, cells were >95% CD3+,
CD8+, CD4-,
TCR+, and DX5- T
cells by FACS analysis (data not shown). These enriched CD8 T cells
were maintained as a CTL line by weekly restimulation with Renca cells
and splenic feeder cells, and used in subsequent experiments.
Established CTL were examined for cytotoxicity and cytokine production
in vitro in response to various cell lines (Table I
). CTL showed specific lysis of Renca
but not other syngeneic (BALB/c) or allogenic cell lines. In addition,
CTL showed no lytic activity against autologous fibroblasts or Con A
splenic T cell blasts. IFN- and TNF- were also produced only in
response to Renca. In contrast, CTL showed no production of IL-4 or
IL-12 in response to any stimulator cells (data not shown). These
specific responses against Renca were inhibited with anti-CD8 or
anti-H2Kd blocking mAb, but not by either
anti-CD4 or anti-H-2Dd mAb, clearly
demonstrating the Renca specificity and H-2Kd
restriction of these CTL.
|
and TNF- before the cytotoxicity assay, these cells became
very susceptible to CTL lysis even in short-term assays (Fig. 1A). This indicates that
cytokines routinely produced during the coincubation of CTL and tumor
cells could play an important role in sensitizing Renca to CTL lysis.
This hypothesis was confirmed by inhibition of CTL lysis by
neutralizing mAbs to IFN- and/or TNF- (Fig. 1B).
CTL-mediated lysis of Renca cells was partially inhibited with
anti-TNF mAb in the long term assays, while the lysis was almost
completely inhibited with either anti-IFN- mAb or the
combination of anti-IFN- and anti-TNF- mAb. In contrast,
these neutralizing mAb did not inhibit CTL lysis against Renca cells
that were pretreated with IFN- and TNF- before the assay. These
results suggest that cytokines produced by the CTL on contact with
Renca cells can ultimately sensitize Renca cells to lysis by these CTL.
Most of the CTL lysis of Renca cells in vitro was blocked by the
perforin inhibitor CMA (22) in both short- (66.5%
inhibition) and long-term assays (78.2%), suggesting a major effector
role for granule-mediated lysis in vitro (Fig. 1A). However,
some inhibition of lysis was also observed when Abs to FasL were
present. Furthermore, almost all lysis was inhibited with the
combination of CMA and anti-FasL-neutralizing mAb. We also observed
that Renca was resistant to any cytotoxic effects of TNF- in vitro
even at doses of up to 10,000 U/ml (data not shown). In addition,
anti-TRAIL mAb did not inhibit CTL lysis of Renca, even though
Renca is known to be susceptible to TRAIL-mediated killing
(19). These findings suggest that the vast majority of
CTL-mediated lysis of Renca cells in vitro could be accounted for by
either granule- or FasL-mediated mechanisms. Because cytokine
pretreatment sensitized Renca cells to the lytic effects of the CTL,
further studies were performed to determine the molecular basis of this
increased sensitivity to cytolysis.
|
IFN- and TNF- enhanced susceptibility of Renca to CTL lysis
as shown in Fig. 1
. Therefore, the effects of IFN- and
TNF- on Renca target cells were examined in more detail in Fig. 2. Renca cells constitutively express
very low levels of Fas; however, treatment with either IFN- or
TNF- up-regulated Fas expression, and the combination of these
cytokines synergistically enhanced surface Fas expression on Renca. The
enhanced Fas induction by this cytokine combination sensitized of Renca
to FasL mediated killing by CTL, as previously reported
(23). In addition, ICAM-1 is only expressed at low levels
on Renca, but IFN- pretreatment induced high levels of surface
expression. TNF- induced only a slight increase in ICAM-1
expression, and the combination of IFN- with TNF- enhanced ICAM-1
induction more than either cytokine alone. Renca cells express high
endogenous levels of MHC class I, and IFN- and the combination of
IFN- plus TNF- further enhanced expression of the
H-2Kd molecule on Renca cells. CD11a (LFA-1),
CD48 (CD2 ligand), CD80 (B7.1) CD86 (B7.2), and CD102 (ICAM-2) were
neither constitutively expressed nor induced after the cytokine
treatment (data not shown). In short-term cytotoxicity assays, most of
the CTL killing of Renca cells that were pretreated with these
cytokines was inhibited with anti-ICAM-1 mAb and anti-LFA-1 mAb
(Fig. 3A), suggesting that
interaction of ICAM-1 on Renca and LFA-1 on CTL may be a crucial
requirement for granule-mediated killing of Renca cells. Lysis in this
short-term assay was predominantly granule-mediated, since
anti-FasL had little effect. Degranulation of CTL was also
estimated by the release of BLT esterase activity (Fig. 3B).
BLT esterase is stored in the granules together with
perforin/granzymes, and its secretion correlates with the exocytosis of
lytic granules. Supernatants from the culture of CTL and Renca cells
that had been pretreated with IFN- and TNF- showed higher levels
of BLT esterase activity than those from CTL cultured with untreated
Renca. A large part (80%) of this released BLT esterase activity was
inhibited when anti-LFA-1 mAb was present during the CTL and Renca
incubation (Fig. 3B). Anti-ICAM-1 could also block
degranulation, but was usually less efficient than anti-LFA-1 (data
not shown). Binding assays using isotope-labeled effector cells also
showed that specific binding between cytokine-treated Renca and CTL was
4–5 times stronger than binding between untreated Renca and CTL. This
increased binding capacity was also inhibited with anti-LFA-1 mAb
(data not shown). Collectively, this suggests that ICAM-1 and LFA-1
interactions enhanced the binding between CTL and Renca cells. This
resulted in an amplification of signals though the TCR, such that
degranulation was increased. Therefore, the cytokines IFN- and
TNF- sensitize Renca to CTL lysis by at least two mechanisms: the
increase of Fas expression on Renca is crucial for the optimal lytic
effects of FasL, and the induction of ICAM on Renca enhances the
binding between CTL and Renca cells, which increases degranulation
of CTL.
|
|
Because both FasL and TRAIL have been reported to lyse Renca cells
(19, 23, 24, 25), we investigated the stimuli that control
FasL and TRAIL expression on anti-Renca CTL. FasL expression was
up-regulated on CTL after 6 h specific interaction with Renca
cells, but not with the syngeneic Harvey BALB cell line (Fig. 4). CTL also expressed high levels of
FasL after stimulation with P/I or with immobilized anti-CD3 mAb
(data not shown). In contrast, cytokine treatment of CTL with IL-2,
IL-12, and IFN- had minimal effects on CTL expression of FasL (data
not shown). TRAIL was only weakly induced on CTL after incubation with
Renca tumor cells or P/I, indicating FasL and TRAIL are differentially
regulated in these CTL. Interestingly, only IFN- could significantly
induce TRAIL on CTL, while other cytokines including IL-2 and IL-12
only had modest effects. The cytokines IL-15, IL-18, IFN-, and
TNF- had minimal effects on either FasL or TRAIL expression by CTL
(data not shown). These patterns of FasL and TRAIL expression remained
consistent when the same stimuli were used for 12 or 20 h (data
not shown).
|
Because Renca-specific CTL produced high levels of cytokines and
were highly lytic in response to Renca cells in vitro, we next tested
their antitumor efficacy after transfer to tumor-bearing BALB/c mice.
CTL specific for Renca cells were tested for therapeutic efficacy
against established experimental pulmonary metastases. In this
experimental metastasis model, CTL were transferred i.v. on days 3–5
together with 10,000 U IL-2. Control mice were treated with an
equivalent number of Con A blasts (>95% CD3+
and CD8+) plus IL-2, or with IL-2 alone. A
portion of the mice were then sacrificed on day 17, and numbers of lung
metastasis were counted (Fig. 5A). Mice treated with IL-2
alone showed a large number of metastasis (>200), whereas mice
receiving specific CTL transfer showed zero or very few metastases
(n = 0–5). In mice receiving Con A blasts, metastases
(n = 125–200) were significantly lower than in mice
receiving IL-2 alone, but were still numerous when compared with the
CTL-treated mice. Furthermore, most mice receiving specific CTL
survived >60 days, while all mice receiving Con A blasts or IL-2 were
dead within 30 days of tumor injection (Fig. 5B). Because
CTL therapy was very effective, we determined the minimal number of CTL
required for adoptive therapy. One injection of 1 x
106 CTL in the absence of IL-2 provided complete
therapeutic benefit, whereas 2 x 105 CTL
could substantially reduce the number of metastases (by >80%), but
did not provide complete therapeutic benefits (data not shown). Mice
that survived adoptive CTL transfer were immune to rechallenge with the
Renca tumor (data not shown).
|
CTL use both granule-mediated and FasL-mediated pathways to lyse
Renca cells in vitro, and the granule-mediated pathway seems to be the
predominant lytic mechanism. To investigate the importance of the
granule-mediated pathway in vivo, CTL were established from
pfp-/- mice. CTL were generated by in vitro
mixed lymphocyte tumor cultures with spleen cells from
pfp-/- mice that had been successfully treated
with FAA plus IL-2, in the same way that CTL were successfully isolated
from WT mice. As expected, pfp-/- CTL showed a
reduced lytic activity against Renca cells in vitro (Fig. 6A), but produced equivalent
amounts of IFN- (Fig. 6B) and TNF- (data not shown)
upon stimulation with Renca, but not with syngeneic A20 cells. Both
lytic activity and cytokine production by
pfp-/- CTL were blocked with anti-CD8.
These results demonstrate the specificity and the expected MHC
restriction of the pfp-/- CTL. Furthermore,
anti-FasL blocked all lysis by pfp-/- CTL,
demonstrating that granule-mediated lysis was absent in these CTL (Fig. 6C). We have previously shown that cytokine-induced ICAM-1
expression on the Renca target cells was important for degranulation of
CTL. Using pfp-/- CTL, we determined whether
ICAM-1 expression enhanced FasL-mediated lysis. An 8 h cytotoxic
assay with IFN- and TNF- pretreated Renca cells was performed
(Fig. 6D), and lytic activity of
pfp-/- CTL was partially inhibited with
anti-ICAM-1 or anti-LFA-1 mAb, and almost completely abrogated
with the combination of anti-LFA-1 and anti-FasL mAbs. These
findings suggest that the interaction of ICAM-1 on Renca cells and
LFA-1 on CTL increases the lytic efficiency of the FasL pathway by
30–50%.
|
).
|
A). No lung metastases were
observed on day 17 in CTL-treated mice using either BALB/c or BALB/c
pfp-/- recipients, while large numbers of
metastases were observed in vehicle-treated mice. Furthermore, BALB/c
or BALB/c pfp-/- mice receiving CTL survived
>60 days after tumor challenge, whereas control mice all died within
30 days (Fig. 8B). These results clearly show that the
granule-mediated pathway is not essential for the regression of Renca
pulmonary metastases after the adoptive transfer of CTL in vivo.
Interestingly, we also consistently observed that
pfp-/- mice developed fewer lung metastases
than WT mice; however, the reason for this remains unclear.
Renca-specific CTL generated from WT and pfp-/-
mice successfully treated with IL-12 pulse IL-2 therapy gave identical
results for the treatment of established experimental pulmonary
metastases. Overall, these results unequivocally demonstrate a clear
dichotomy between tumor-specific lysis of tumor cells in vitro and
rejection of tumor in vivo. The results implicate non-pfp-dependent
mechanisms that may include Fas/FasL interactions as critical events in
the regression of pulmonary Renca metastases.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and TNF- sensitize the
tumor cells to lysis by both pathways, suggesting these cytokines could
play an important role not only in the induction but also in the
effector phase of the immune response. Sensitization of tumor cells to
CTL-mediated lysis by increased expression of class I (26)
or increased class I and ICAM-1 expression (27) has been
reported in other experimental models. In our study, the induction of
ICAM-1 and enhanced expression of Fas on the Renca target cells in
response IFN- and TNF- was instrumental for sensitizing these
tumor cells to lysis by CTL (Fig. 1). Interaction between ICAM-1 on the
tumor cells and LFA-1 on the CTL could stabilize the TCR interaction
with Ag, thus, amplifying signals to the CTL. Granule-mediated lysis of
Renca by CTL seems particularly dependent on ICAM-1/LFA-1 interactions
(Fig. 3). This would be consistent with previous findings that a
stronger signal was required to trigger degranulation as opposed to
FasL-mediated lysis (28, 29, 30, 31, 32). Nonetheless, ICAM-1
expression on Renca also enhanced FasL-mediated lysis by 30–50%
showing that ICAM-1/LFA-1 interaction can enhance tumor cell lysis via
multiple killing pathways (Fig. 6). The anti-Renca CTL generated in this study showed potent antitumor effects upon adoptive transfer to mice bearing established Renca pulmonary metastases. This finding allowed us to study the relative importance of different cytotoxic effector functions of CTL in vivo. This model allowed us to show that Renca lung metastases could be destroyed efficiently in the complete absence of perforin and granule-mediated cytotoxicity. These findings extend previous reports on clearance of lung metastases by CTL in a variety of murine tumors (33), as well as the murine melanoma model B16BL6-D5 (34), and the murine sarcomas MCA-310 (34) and MCA-205 (35). In the aforementioned studies, tumor-draining lymph node (TDLN) cells that had been expanded in vitro with anti-CD3 and IL-2 were used as a source of effector T cells. In contrast to our studies, relatively high numbers (20–70 x 106) of these TDLN-derived CTL were required for successful adoptive immunotherapy of lung metastases, whereas as few as 1 x 106 anti-Renca CTL could provide complete regression of Renca pulmonary metastases. The reason for this quantitative difference in efficacy could reflect the way in which the respective CTL were generated. The short in vitro expansion of local TDNL cells (derived early in an immune response to tumor) with anti-CD3 and IL-2 could enhance the propagation of many T cells from the lymph node; however, the numbers of T cells in this population with functional antitumor effector capacity could be quite low. By contrast, our CTL were generated from mice which had rejected Renca after biological therapy, and had been rechallenged with tumor. Furthermore, T cells from the spleens of these mice were then expanded in vitro in the presence of irradiated tumor cells and IL-2. It would be anticipated that such a regimen would preferentially select for T cells with a high affinity for tumor. Nonetheless, despite quantitative differences in antitumor efficacy of the various CTL, our results in the Renca pulmonary metastases model are in close agreement to those previously reported using TDLN cells. That is, adoptive transfer of CTL derived from pfp-/- mice into pfp-/- mice bearing Renca pulmonary metastases resulted in complete tumor regression.
In the previous studies of Winter et al. (34, 36) and Peng
et al. (35), it was also noted that TDLN-derived CTL from
gld and IFN- -/- mice also
displayed dramatic therapeutic benefits on administration to mice with
pulmonary metastases, suggesting that neither FasL nor IFN- could
account for the destruction of pulmonary metastases. However, in
contrast to the tumors used in these studies, the Renca tumor we used
is very sensitive to FasL-mediated lysis. We were unable to generate
anti-Renca CTL from BALB/c gld/gld or IFN-
-/- mice using our protocol, because these mice
do not respond to in vivo immunotherapy (4).
Interestingly, the transfer of WT BALB/c anti-Renca CTL plus
neutralizing Ab to FasL did not block the antitumor effects in the
pulmonary metastasis model (data not shown), suggesting FasL may not be
crucial for the destruction of Renca lung metastases. Nonetheless, with
one cytotoxic pathway ablated, it is still possible that the second
pathway could compensate, particularly in the Renca model where the
tumor cells are very sensitive to both CTL lytic pathways. Future
studies using CTL from mice deficient for both FasL and perforin will
be required to determine the importance of these lytic pathways in
antitumor responses to Renca in vivo. We are currently breeding these
mice, and plan to generate CTL from them using various immunization
protocols.
Although we observed no essential role for perforin in eradication of pulmonary Renca metastases by CTL, we cannot rule out the possibility that perforin could play an essential role in CTL-mediated rejection of Renca metastases in other tissue compartments. In the MCA-205 model, differences in perforin dependence were noted in the rejection of pulmonary or intracranial vs s.c. metastases (35). The rejection of s.c. tumor was perforin-dependent, whereas the rejection of pulmonary or intracranial metastases could proceed without an absolute requirement for perforin. Therefore, the relative importance of perforin for the therapeutic effects of CTL may depend on the anatomical location of the tumor. In a recent study on immunotherapy of the B16 melanoma, interesting differences in tumor rejection were observed depending on whether a tumor vaccine was applied in a prophylactic or therapeutic setting (37). In the prophylactic setting (several immunizations before tumor challenge), full protection was conferred in the absence of CD4, CD8, or NK 1.1+ cells at the time of tumor challenge. Therefore, the prophylactic application of the vaccine allowed for the generation of antitumor responses that were not critically dependent on any one particular lymphocyte subset. By contrast, in the therapeutic setting (vaccine administered shortly after tumor challenge) depletion of CD8+ and NK 1.1+ cells hampered the rejection of small established tumors. Other studies in the B16 melanoma model using prophylactic vaccination showed that neither perforin nor FasL were important for the rejection of lung metastases (38). However, it should be noted that in the more clinically relevant therapeutic setting, perforin was essential for the rejection of the s.c. B16 tumors (37). pfp-/- mice did not respond to therapeutic vaccination of B16 cells with either tumor rejection or vitiligo, which can be a surrogate marker for an autoimmune response in this model. Thus, CTL may trigger multiple effector mechanisms to promote tumor destruction, and the dominant pathway(s) may vary depending on the anatomical location of the tumor.
The effects of adoptively transferred CD8 T cells in vivo could be
diverse. In addition to direct killing of tumor cells, indirect effects
of locally released cytokines on the tumor environment might be
involved in tumor regression. Cytokines could induce local inflammation
at tumor site and recruit other effector cells including macrophages,
neutrophils, or NK cells. Macrophages and granulocytes have been
observed infiltrating the lungs of B16BL6-D5 tumor-bearing mice after
CTL adoptive immunotherapy (34). Furthermore, the
production of cytokines or chemokines such as Mig or IP-10 at the tumor
site could have antiangiogenic effects on the tumor vasculature
(39, 40, 41). A recent study of IL-12 pulse IL-2 therapy in
Renca model suggested a unique interrelationship between IFN- and
the Fas/FasL pathway in mediating vascular endothelial apoptosis,
inhibition of tumor neovascularization, and overall tumor regression
(4). Furthermore, it was hypothesized that
CD8+ T cells were the most likely source of FasL.
The expression of Fas by endothelial cell lines in vitro is enhanced by
IFN- and TNF- as is their sensitivity to FasL-mediated lysis
(data not shown). Therefore, in a similar manner to direct Renca lysis,
local cytokine production could sensitize endothelial cells to
FasL-mediated "bystander" lysis by anti-Renca CTL. Whether
specific anti-Renca CTL can promote concomitant vascular damage on
infiltration of the tumor bed in vivo is of great interest, and is
currently under investigation.
We have found no role for TRAIL in the direct lysis of Renca by CTL in vitro, despite the fact that Renca cells are known to be sensitive to the lytic effects of TRAIL (19, 25). TRAIL expression on human T cells is increased by type I IFNs, and this has been proposed to be a possible mechanism to explain the antitumor effects of IFNs in human renal cancer (42). In addition, TRAIL has also been implicated in the destruction of Renca liver metastases during IL-12 treatment, where liver NK cells seem to be the major source of TRAIL (24). Whether anti-Renca CTL play a direct or indirect role in the rejection of hepatic as well as peritoneal, lymphoid, or s.c. Renca metastases is unknown, and is currently under investigation. In conclusion, our results rule out a requirement for perforin in CTL-mediated tumor regression of pulmonary metastases in the Renca renal cancer. This begs the question as to exactly how T cells can destroy lung metastases of this tumor. The identification of molecular pathway(s) at different anatomical sites that are important for the CTL-mediated tumor destruction in vivo could provide important insights for the design of improved immunotherapeutic strategies for renal cancer in the future.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Division of Immunobiology, National Institute of Biological Standards and Controls, Potters Bar, Hertfordshire, U.K.
3 Address correspondence and reprint requests to Dr. Thomas J. Sayers, Science Applications International Corporation, National Cancer Institute, Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address: Sayers{at}mail.ncifcrf.gov
4 Abbreviations used in this paper: RCC, renal cell carcinoma; TRAIL, TNF-related apoptosis-inducing ligand; FasL, Fas ligand; P/I, PMA plus ionomycin; [111In]Ox, 111In-labeled oxine; TDLN, tumor-draining lymph node; CMA, Concanamycin A; WT, wild type; BLT, N-a-benzyloxycarbonyl-L-lysinethiobenzoyl ester.
Received for publication October 29, 2001. Accepted for publication January 28, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy. J. Clin. Invest. 108:51.[Medline]
-dependent delay of in vivo tumor progression by Fas overexpression on murine renal cancer cells. J. Immunol. 164:231.-dependent natural killer cell protection from tumor metastasis. J. Exp. Med. 193:661.-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. mechanisms that cannot be predicted from in vitro T cell characteristics. J. Immunol. 165:7116. in vaccine-induced antitumor immunity versus adoptive immunotherapy. J. Immunol. 166:7370.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]
|
|
|
|
 |
|
 W. H. Shingler, P. Chikoti, S. M. Kingsman, and R. Harrop Identification and functional validation of MHC class I epitopes in the tumor-associated antigen 5T4 Int. Immunol., August 1, 2008; 20(8): 1057 - 1066. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Shanker, A. D. Brooks, C. A. Tristan, J. W. Wine, P. J. Elliott, H. Yagita, K. Takeda, M. J. Smyth, W. J. Murphy, and T. J. Sayers Treating Metastatic Solid Tumors With Bortezomib and a Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Agonist Antibody J Natl Cancer Inst, May 7, 2008; 100(9): 649 - 662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. VanOosten and T. S. Griffith Activation of Tumor-Specific CD8+ T Cells after Intratumoral Ad5-TRAIL/CpG Oligodeoxynucleotide Combination Therapy Cancer Res., December 15, 2007; 67(24): 11980 - 11990. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. D. Sloan and K. R. Jerome Herpes Simplex Virus Remodels T-Cell Receptor Signaling, Resulting in p38-Dependent Selective Synthesis of Interleukin-10 J. Virol., November 15, 2007; 81(22): 12504 - 12514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Greeneltch, M. Schneider, S. M. Steinberg, D. J. Liewehr, T. J. Stewart, K. Liu, and S. I. Abrams Host Immunosurveillance Controls Tumor Growth via IFN Regulatory Factor-8 Dependent Mechanisms Cancer Res., November 1, 2007; 67(21): 10406 - 10416. [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]
|
|
|
|
 |
|
 T. Ramirez-Montagut, A. Chow, A. A. Kochman, O. M. Smith, D. Suh, H. Sindhi, S. Lu, C. Borsotti, J. Grubin, N. Patel, et al. IFN-{gamma} and Fas Ligand Are Required for Graft-versus-Tumor Activity against Renal Cell Carcinoma in the Absence of Lethal Graft-versus-Host Disease J. Immunol., August 1, 2007; 179(3): 1669 - 1680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zamai, C. Ponti, P. Mirandola, G. Gobbi, S. Papa, L. Galeotti, L. Cocco, and M. Vitale NK Cells and Cancer J. Immunol., April 1, 2007; 178(7): 4011 - 4016. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Abdool, E. Cretney, A. D. Brooks, J. M. Kelly, J. Swann, A. Shanker, E. W. Bere Jr., W. M. Yokoyama, J. R. Ortaldo, M. J. Smyth, et al. NK Cells Use NKG2D to Recognize a Mouse Renal Cancer (Renca), yet Require Intercellular Adhesion Molecule-1 Expression on the Tumor Cells for Optimal Perforin-Dependent Effector Function J. Immunol., August 15, 2006; 177(4): 2575 - 2583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. MacGregor, Q. Li, A. E. Chang, T. M. Braun, D. P.M. Hughes, and K. T. McDonagh Ex vivo Culture with Interleukin (IL)-12 Improves CD8+ T-Cell Adoptive Immunotherapy for Murine Leukemia Independent of IL-18 or IFN-{gamma} but Requires Perforin. Cancer Res., May 1, 2006; 66(9): 4913 - 4921. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Balza, L. Mortara, F. Sassi, S. Monteghirfo, B. Carnemolla, P. Castellani, D. Neri, R. S. Accolla, L. Zardi, and L. Borsi Targeted Delivery of Tumor Necrosis Factor-{alpha} to Tumor Vessels Induces a Therapeutic T Cell-Mediated Immune Response that Protects the Host Against Syngeneic Tumors of Different Histologic Origin Clin. Cancer Res., April 15, 2006; 12(8): 2575 - 2582. [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]
|
|
|
|
|
|
K. Liu, S. A. Caldwell, and S. I. Abrams Immune Selection and Emergence of Aggressive Tumor Variants as Negative Consequences of Fas-Mediated Cytotoxicity and Altered IFN-{gamma}-Regulated Gene Expression Cancer Res., May 15, 2005; 65(10): 4376 - 4388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Liu, S. A. Caldwell, and S. I. Abrams Cooperative Disengagement of Fas and Intercellular Adhesion Molecule-1 Function in Neoplastic Cells Confers Enhanced Colonization Efficiency Cancer Res., February 1, 2005; 65(3): 1045 - 1054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Hill, S. J. Youde, S. Man, G. R. Teale, A. J. Baxendale, A. Hislop, C. C. Davies, D. M. Luesley, A. M. Blom, A. B. Rickinson, et al. Activation of CD40 in Cervical Carcinoma Cells Facilitates CTL Responses and Augments Chemotherapy-Induced Apoptosis J. Immunol., January 1, 2005; 174(1): 41 - 50. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Tykodi, E. H. Warren, J. A. Thompson, S. R. Riddell, R. W. Childs, B. E. Otterud, M. F. Leppert, R. Storb, and B. M. Sandmaier Allogeneic Hematopoietic Cell Transplantation for Metastatic Renal Cell Carcinoma after Nonmyeloablative Conditioning: Toxicity, Clinical Response, and Immunological Response to Minor Histocompatibility Antigens Clin. Cancer Res., December 1, 2004; 10(23): 7799 - 7811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Hollenbaugh, J. Reome, M. Dobrzanski, and R. W. Dutton The Rate of the CD8-Dependent Initial Reduction in Tumor Volume Is Not Limited by Contact-Dependent Perforin, Fas Ligand, or TNF-Mediated Cytolysis J. Immunol., August 1, 2004; 173(3): 1738 - 1743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chakraborty, S. I. Abrams, C. N. Coleman, K. Camphausen, J. Schlom, and J. W. Hodge External Beam Radiation of Tumors Alters Phenotype of Tumor Cells to Render Them Susceptible to Vaccine-Mediated T-Cell Killing Cancer Res., June 15, 2004; 64(12): 4328 - 4337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. D. Sloan, G. Zahariadis, C. M. Posavad, N. T. Pate, S. J. Kussick, and K. R. Jerome CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase J. Immunol., December 15, 2003; 171(12): 6733 - 6741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Merritt, A. Mahtabifard, R. E. Yamada, R. G. Crystal, and R. J. Korst Cisplatin augments cytotoxic T-lymphocyte-mediated antitumor immunity in poorly immunogenic murine lung cancer J. Thorac. Cardiovasc. Surg., November 1, 2003; 126(5): 1609 - 1617. [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]
|
|
|
|
|
|
M. Chakraborty, S. I. Abrams, K. Camphausen, K. Liu, T. Scott, C. N. Coleman, and J. W. Hodge Irradiation of Tumor Cells Up-Regulates Fas and Enhances CTL Lytic Activity and CTL Adoptive Immunotherapy J. Immunol., June 15, 2003; 170(12): 6338 - 6347. [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, Y. Hayakawa, A. D. Brooks, J. Wine, R. H. Wiltrout, H. Yagita, J. E. Tanner, M. J. Smyth, and T. J. Sayers Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Apoptosis Is an Important Endogenous Mechanism for Resistance to Liver Metastases in Murine Renal Cancer Cancer Res., January 1, 2003; 63(1): 207 - 213. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
