|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4248-4255
Role of Spontaneous and Interleukin-2-Induced Natural Killer Cell
Activity in the Cytotoxicity and Rejection of Fas+
and Fas Tumor Cells
By
Mike Bradley,
Ahmet Zeytun,
Asimah Rafi-Janajreh,
Prakash S. Nagarkatti, and
Mitzi Nagarkatti
From the Department of Biology and the Department of Biomedical
Sciences & Pathobiology, Virginia-Maryland College of Veterinary
Medicine, Virginia Tech, Blacksburg, VA.
 |
ABSTRACT |
In the current study, we investigated whether the naive, poly I:C or
interleukin-2 (IL-2)-induced natural killer (NK)/lymphokine-activated killer (LAK) cells use perforin and/or Fas ligand
(FasL) to mediated cytotoxicity. We correlated these findings with the
ability of mice to reject syngeneic Fas+ and
Fas tumor cells either spontaneously or after IL-2
treatment. The spontaneous NK-cell-mediated cytotoxicity was primarily
perforin based, whereas the poly I:C and IL-2-induced NK/LAK activity
was both FasL and perforin dependent. L1210 Fas+ tumor
targets were more sensitive than L1210 Fas targets to
poly I:C and IL-2-induced cytotoxicity in wild-type, gld/gld,
and perforin knockout mice. When L1210 Fas+ and
Fas- tumor cells were injected subcutaneously (sc) or
intraperitoneally into syngeneic mice, Fas tumor cells
caused mortality earlier than Fas+ tumor cells. Also,
approximately 20% of the mice injected sc with L1210
Fas+ tumor cells survived the challenge(>60 days),
whereas all mice injected similarly with L1210 Fas tumor
cells died. When immunotherapy using IL-2 (10,000 U, three times/d for
a week, followed by once/d for an additional week) was attempted in
mice injected sc with tumor cells, IL-2 treatment was very effective
against mice bearing L1210 Fas+ (40% survival) but not
L1210 Fas (0% survival) tumors. These data correlated
with the finding that the LAK cells from IL-2-injected mice caused
increased cytotoxicity against L1210 Fas+ when compared
with L1210 Fas targets. Also, L1210 Fas+
tumor-bearing mice showed increased tumor-specific cytotoxic T
lymphocyte (CTL) activity when compared with those bearing L1210 Fas tumor cells. Together our studies show for the first
time that expression of Fas on tumor targets makes them more
immunogenic as well as susceptible to CTL- and IL-2-induced LAK
activity. The Fas+ tumor cells are also more responsive
to immunotherapy with IL-2.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
NATURAL KILLER (NK) cells comprise an
important component of the immune system mainly involved in
surveillance against cancer and viral infections.1 NK cells
exhibit spontaneous cytolytic activity against certain tumor cells and
virally infected target cells in a major histocompatability
complex-unrestricted manner. In addition, NK cells can be activated by
binding of monoclonal antibodies (MoAbs) through CD16 receptor to
mediate lysis of target cells called MoAb-dependent cell-mediated
cytotoxicity. The NK cells can also be activated by interleukin-2
(IL-2) and interferon- (IFN-) inducers such as
polyinosonic-polycytdylic acid (poly I:C) to mediate increased lysis of
NK-sensitive targets as well as kill a broader panel of target cells,
including NK-resistant cells.1,2 Such IL-2-induced
cytolytic property shown by NK and T cells has been designated
lymphokine-activated killer (LAK) cell activity.3
Recent studies have shown that cytotoxic T lymphocytes (CTLs) and NK
cells can kill target cells using two distinct lytic pathways. First,
the degranulation pathway which uses perforin possibly in combination
with granzymes,4,5 and secondly, the Fas-based pathway in
which the interaction between Fas-Ligand (FasL) expressed on cytolytic
lymphocytes and Fas on target cells triggers apoptosis and target cell
death.6,7 Moreover, all cytotoxic activity measured in a
4-hour assay can be attributed to perforin and FasL.8,9
Although NK cells have been known to mediate increased lysis of target
cells including those that are NK resistant when activated with IL-2 or
IFN- inducers in vivo, it is not clear whether such lytic activity
results from upregulation of perforin and/or FasL. Inasmuch as
IL-2 is used to activate LAK cells in the immunotherapy of certain
types of cancer,10 it is important to investigate whether
such treatment augments both perforin and FasL-based pathways and
therefore whether it is effective against both Fas+ and
Fas tumors.
Although the role played by FasL in the cytotoxicity mediated by CTL
and NK cells is well established, the outcome of expression of Fas on
the tumor cells and its ability to trigger the antitumor immunity in
the host is not clear. In the current study, we used perforin-deficient
and FasL-defective mice to address the role of perforin and FasL in LAK
cell-mediated cytotoxicity and Fas+ and
Fas L1210 tumor cells to delineate the role played
by Fas in tumor growth and induction of antitumor immunity. The data
showed that IL-2 and poly I:C triggered the induction of both FasL- and
perforin-based cytolytic activity. Also, the survival rate in mice
injected with Fas+ L1210 tumor cells was better than that
observed in mice receiving Fas L1210 cells, due to
the fact that the Fas+ tumor cells induced stronger NK/LAK
and tumor-specific CTL activity when compared with
Fas tumor cells.
| |
MATERIALS AND METHODS |
Mice.
C57BL/6+/+ (+/+) and DBA/2 mice were purchased from Charles
River (Boston, MA). C57BL/6 gld/gld (gld) mice were
purchased from Jackson Laboratories (Bar Harbor, ME) and perforin
knockout (KO) mice11 were generously provided
by Dr W.R. Clark (University of California, Los Angeles,
CA). The perforin KO and gld mice were of C57BL/6
origin. The gld and perforin KO mice were bred in our
facilities.12 All mice used in the current study were female and were 3 to 4 weeks old.
Cell lines.
The tumor cell lines used were as follows: YAC-1, a Moloney
virus-induced lymphoma sensitive to NK cells; P815, a mastocytoma resistant to NK cells; L1210 (Fas+ and
Fas), an NK-resistant DBA/2-derived mouse lymphoma
transfected with sense and antisense Fas cDNA.13 All cell
lines were maintained in tissue culture medium, RPMI-1640 (GIBCO, Grand
Island, NY), and supplemented by 10% fetal bovine serum (Atlanta
Biologicals, Norcross, GA), 2 mmol/L glutamine, 50 mmol/L
2-mercaptoethanol, 10 mmol/L HEPES, and 40 mg/mL gentamycin, as
previously described.14
In vitro growth characteristics of L1210 Fas+ and L1210
Fas tumor cells.
L1210 Fas+ and L1210 Fas tumor cells
were cultured in tissue culture flasks at a concetration of 2 × 105 cells/mL. At 12, 24, 36, 48, 60, and 72 hours after in
vitro culture, the viable cell count was performed using trypan blue dye exclusion.
Flow cytometric analysis of Fas.
The tumor cell lines were screened for the expression of Fas by
incubating the cells with normal mouse serum initially to block the Fc
receptors. Next, the cells were washed and incubated with anti-Fas
MoAbs (Jo2; Pharmingen, San Diego, CA) at 4°C for 30 minutes. The
cells were washed and stained with fluorescein isothiocyanate
(FITC)-conjugated anti-hamster IgG [F(ab )2]. The negative controls were stained with normal isotype-matched antibody and the FITC-conjugated secondary antibody. The cells were analyzed using a flow cytometer (Epics V, Model 752; Coulter Corp, Miami, FL).
LAK/NK cells.
NK cells were purified as previously described.15 Briefly,
single-cell suspensions of the spleen were prepared in RPMI-1640 medium
supplemented with 5% fetal calf serum16 using a
homogenizer (Stomacher; Tekmar Co, Cincinnati, OH). Plastic adherence
for 1 hour at 37°C was used to deplete macrophages. The cells were then passed over nylon wool columns and the nonadherent cells used as a
source of spontaneous NK activity. To study the inducible NK activity,
the nonadherent cells were cultured for approximately 48 hours with
1,000 U/mL of IL-2 (kindly provided by Hoffman LaRoche, Nutley, NJ). In
some experiments, NK activity was induced by intraperitoneal (IP)
injection of poly I:C (Sigma Chemical Company, St Louis, MO), at 15 mg/kg suspended in phosphate-buffered saline (PBS).2 In
other experiments, NK cells were activated in vivo by IP injection of
10,000 U of IL-2 three times a day for 5 days.
Isolation of tumor-specific CTL.
DBA/2 mice were injected with 1 × 106 L1210
Fas+ or L1210 Fas live tumor cells in
0.2 mL PBS subcutaneously (sc). On day 5, these mice were injected with
1,3-Bis(2-chloroethyl )-1-nitrosourea, an anticancer drug, at a
concentration of 20 mg/kg body weight as described, so as to enhance
the tumor-specific immune response.17,18 Five days later,
the mice were killed, T cells were purified,18 and 5 × 106 cells were cultured in 24-well tissue culture
plates with irradiated (2,000 R) L1210 Fas+ or L1210
Fas tumor cells (5 × 103) in 2 mL
of medium. After 5 days, the cells were obtained and viable cells were
purified on Ficoll-Hypaque (Sigma Diagnostics, St Louis, MO)
density gradient centrifugation. The cells were tested for
cytotoxicity against 51Cr-labeled L1210 Fas+ or
L1210 Fas tumor cells. The specificity of the CTL
was confirmed by testing the cytotoxicity against YAC-1 and P815
targets.
51Cr-release assay to measure cytotoxicity.
The cytotoxicity mediated by NK cells was studied using
51Cr-release assay.14 Target cells (YAC-1,
P815, L1210 Fas+, and L1210 Fas) were
labeled with 100 mCi 51Cr in the form of
Na251CrO4, incubated at 37°C
for 1 hour, washed three times, then seeded in 96-well plates (Costar,
Cambridge, MA) at 5 × 103 cells/well along with
varying numbers of effector cells. The plates were incubated at
37°C for 4 hours. After the incubation, the plates were harvested
with the Titertech collecting system (Skatron Inc, Sterling, VA). The
amount of 51Cr released by the target cells was measured
using a -counter (TmAnalytic, Elk Grove Village, IL). Percent
cytotoxicity was calculated as: (experimental release  control
release)/(total release control release) × 100. In these
experiments, the control release was measured in the presence of target
cells alone, which was usually less than 15%. Total release was
measured by incubating target cells in the presence of 0.1% sodium
dodecyl sulfate.
In blocking studies, the assays were performed in the presence of 2 µg/mL of anti-Fas MoAbs (Jo2; Pharmingen) or 100 nmol of concanamycin
A (ICN Pharmaceuticals Inc, Costa Mesa, CA).19 In these
experiments, the control release was measured both in the presence or
absence of the inhibitors to ensure that the MoAbs or reagents alone
did not alter control release.
In vivo growth characteristic of L1210 tumor cells and
immunotherapy.
Groups of five DBA/2 mice were injected sc or IP with either 1 × 106 L1210 Fas+ or L1210 Fas
tumor cells suspended in 0.2 mL PBS. These mice were injected IP with
10,000 U IL-2 (kindly provided by Hoffman-LaRoche), and suspended in
0.1 mL PBS 3 times/d for 7 days, followed by once a day for an
additional 7 days. The control mice received PBS in a similar fashion.
The mice were observed for tumor growth and survival for 60 days, after
which the experiment was terminated. These experiments were repeated
with consistent results. The mean survival time (MST) was calculated
for each group and compared statistically using a t-test. The
MST for mice that were alive on day 60 was considered to be 60.
| |
RESULTS |
Spontaneous, poly I:C-activated, and IL-2-activated NK/LAK activity in
wild-type, perforin-deficient and FasL-defective mice.
To study the role of perforin and FasL in NK/LAK-cell activity, we used
wild-type, perforin-deficient, or FasL-defective (gld/gld) mice. Also, to investigate the effect of activation, the spontaneous NK-cell activity was compared with that observed after in vitro culture
of cells with IL-2 or after in vivo administration of poly I:C.
The data shown in Fig 1A, B, and C shows
that IL-2-activated NK cells showed maximum cytolytic activity against
NK-sensitive YAC-1 targets in all three groups of mice, followed by
poly I:C-activated and spontaneous lytic activity. Furthermore,
FasL-defective mice showed a similar degree of spontaneous lysis when
compared with the wild-type mice. However, perforin-deficient mice
showed virtually no spontaneous cytolytic activity. It should be noted
that YAC-1 tumor cells were Fas+
(Fig 2). These data demonstrated that the
spontaneous NK activity was primarily perforin based. The fact that
gld/gld mice had similar levels of spontaneous NK activity as
the wild-type mice suggested that FasL was not critical during
spontaneous lysis.
View larger version (15K):
[in this window]
[in a new window]
| Fig 1.
Comparison of spontaneous, poly I:C-, and IL-2-induced
NK/LAK-cell activity in C57BL/6 wild-type, gld/gld, and
perforin KO mice. NK/LAK cells from wild-type (A), gld/gld (B),
and perforin KO (C) mice were tested for cytotoxicity against
NK-sensitive YAC-1 tumor targets. The same symbols were used in (A),
(B), and (C) to depict different types of cytotoxicity. The
cytotoxicity was studied using 51Cr-release assay and the
mean percent cytotoxicity ± SEM of triplicate culture were plotted.
|
|
View larger version (23K):
[in this window]
[in a new window]
| Fig 2.
Flow cytometric analysis of tumor targets for the
expression of Fas. Tumor cell lines were stained with isotype control
(bold histograms) or anti-Fas MoAbs (broken histograms), followed by
FITC-conjugated secondary antibodies and analyzed flow
cytometrically.
|
|
When poly I:C and in vitro IL-2-activated NK/LAK-cell activity were
compared in the three groups of mice, it was noted that the wild-type
mice showed the highest level of cytotoxicity, followed by the
gld/gld mice and perforin-deficient mice. The poly I:C-induced NK activity was comparable between wild-type and gld/gld mice, whereas it was markedly reduced in perforin-deficient mice. These data
suggested that in poly I:C-induced NK activity, perforin played a more
important role than FasL. In contrast, the IL-2-induced NK activity
was based both on FasL and perforin, inasmuch as both perforin-deficient and gld/gld mice showed significant
cytotoxicity and the wild-type mice showed the highest level of
cytotoxicity. However, even in IL-2-induced NK activity, perforin
played a more important role because perforin KO mice had much lower
level of cytotoxicity than gld/gld mice.
Further studies were conducted to investigate the role of perforin and
FasL in cytotoxicity against NK-resistant P815 tumor cells as targets,
which were also found to be Fas+ (Fig 2). The results shown
in Fig 3 suggested that P815 tumor targets
were resistant to spontaneous and poly I:C-induced cytotoxicity. However, in vitro IL-2-activated LAK cells were able to kill P815 cells in all three groups of mice tested. Moreover, in such
IL-2-induced cytolytic activity, both perforin and FasL played a
significant role, inasmuch as both perforin-deficient (Fig 3C) and
FasL-defective (Fig 3B) mice showed cytolytic activity against P815.
However, perforin KO mice expressed lower levels of cytotoxicity than
the gld/gld mice, thereby suggesting that P815 cells were more
sensitive to perforin than FasL.
View larger version (24K):
[in this window]
[in a new window]
| Fig 3.
Spontaneous and induced NK/LAK activity against P815
tumor targets. NK cells from wild-type (A and D), gld/gld (B
and E), and perforin KO (C and F) mice were tested for cytotoxicity
against NK-resistant P815 tumor targets as described in Fig 1. In (D),
(E), and (F), in vitro IL-2-activated LAK cells were used as effectors
and cytotoxicity was performed in the presense of medium, concanamycin
A (100 nmol/L), or anti-Fas MoAbs (2 µg/mL). The symbols used in (B)
are common to (A) and (C). Also, symbols shown in (E) are similar to
those used in (D) and (F).
|
|
To further address the role of FasL and perforin in the killing of P815
tumor targets by IL-2-activated LAK cells, the cytotoxicity was
measured in the presence of concanamycin A, which is known to inhibit
perforin-based but not FasL-based cytotoxicity,19,20 and
anti-Fas MoAbs (Jo2) known to inhibit FasL-based
cytotoxicity.21 The data shown in Fig 3D, E, and F
indicated that anti-Fas antibody caused significant inhibition in the
lysis of P815 targets by LAK cells from wild-type and
perforin-deficient mice but failed to inhibit the lysis mediated by LAK
cells from gld/gld mice. Moreover, concanamycin A inhibited the
LAK activity from wild-type and gld/gld mice but not from
perforin-deficient mice. Together, these data corroborated the
observation that the lysis of P815 target cells by LAK cells was both
perforin and FasL based, although perforin played a more important role
than FasL.
To ensure that the IL-2-induced cytolytic activity was primarily
dependent on LAK cells (T cells + NK cells) but not macrophages, the
cells were analyzed for CD3, NK1.1, and Mac-3 in all three strains of
mice. The data shown in Table 1 indicated
that wild-type, gld/gld, and perforin-deficient mice had
similar level of T cells and NK cells before and after IL-2 activation.
Also, such cultures were devoid of macrophages. The data shown in Table
1 also indicated that perforin-deficient and gld/gld mice had
similar proportion of T and NK cells when compared with the wild-type
mice. These data are consistent with recent studies from our lab in
which it was noted that IL-2 treatment in vivo caused identical
phenotypic changes in lymphocytes from wild-type, gld/gld, and
perforin KO mice.22
NK/LAK-cell-mediated cytotoxicity against Fas+ and
Fas tumor targets in wild-type, perforin-deficient,
and FasL-defective mice.
To exclude the possible variations observed in target cell
susceptibility and to address the role of Fas, we used L1210 tumor cells which had been transfected with Fas-sense (Fas+) or
antisense (Fas).13 The expression of Fas
on these cell lines was confirmed by flow cytometric analysis (Fig 2).
As shown in Fig 4A, B, and C,
Fas L1210 tumor targets were resistant to
spontaneous and poly I:C-activated killing by NK cells in all three
groups of mice, similar to the NK-resistant P815 cells (Fig 2).
However, after IL-2 activation, LAK cells from wild-type (Fig 4A) and
gld/gld (Fig 4B) mice mediated significant lysis, whereas
similar cells from perforin KO mice (Fig 4C) failed to show lytic
activity. In fact, LAK cells from gld/gld mice mediated
increased cytotoxicity when compared with the wild-type mice, the
reason for which was not clear.
View larger version (23K):
[in this window]
[in a new window]
| Fig 4.
NK/LAK-cell-mediated cytotoxicity against
Fas+ and Fas tumor targets. NK/LAK cells
from wild-type (A and D), gld/gld (B and E), and perforin-KO (C
and F) mice were tested for cytotoxicity against L1210
Fas (A, B, C) or L1210 Fas+ (D, E, F)
tumor targets. The cytotoxicity was performed as described in Fig 1.
|
|
When L1210 Fas+ targets were similarly tested (Fig 4, lower
panel), it was noted that they were resistant to spontaneous killing by
the NK cells, similar to Fas L1210 targets, in all
three groups of mice. Interestingly, when NK cells activated with poly
I:C were tested, the Fas+ targets became susceptible to
cytotoxicity. Such poly I:C-induced cytotoxicity was both FasL and
perforin based because both gld/gld (Fig 4E) and perforin KO
(Fig 4F) mice showed significant cytotoxicity. However, such
cytotoxicity was less than that observed in wild-type mice (Fig 4D).
Similar results were also seen using LAK cells activated with IL-2 in
vitro (Fig 4D, E, and F). Overall, when L1210 Fas+ and
Fas targets were compared (Fig 4, lower
v upper panel), Fas+ targets were found to be more
susceptible to lysis after poly I:C or IL-2 activation of NK/LAK cells,
thereby suggesting that activation of NK/LAK cells triggers not only
the perforin-based but also the FasL-based pathway.
In vivo administration of IL-2 upregulates both perforin- and
FasL-based cytotoxicity.
To further corroborate in vivo that IL-2 activation upregulates both
perforin- and FasL-based cytolytic activity, wild-type, gld/gld, and perforin KO mice were administered with IL-2 for 5 days and the spleen cells were tested for lytic activity against Fas and Fas+ L1210 tumor cells. The data
shown in Fig 5 demonstrated that Fas+ targets were markedly more sensitive to cytotoxicity
by in vivo IL-2-activated LAK cells from wild-type mice when compared
with the Fas L1210 tumor cells (Fig 5A). Also, the
wild-type mice (Fig 5A) showed the highest level of cytotoxicity when
compared with gld/gld (Fig 5B) or perforin-deficient (Fig 5C)
mice, which showed moderate degree of lysis. These data indicated that
in vivo IL-2 administration triggered both perforin- and FasL-based
cytotoxicity and that such cytotoxicity was more effective against
Fas+ tumor targets.
View larger version (13K):
[in this window]
[in a new window]
| Fig 5.
Cytotoxicity mediated by LAK cells activated in vivo with
IL-2. Wild-type (A), gld/gld (B), or perforin KO (C) mice were
injected with 10,000 U of IL-2 twice a day for 4 days and the LAK cells
were tested for cytotoxicity against L1210 Fas and L1210
Fas+ tumor targets. Cytotoxicity was studied as described
in Fig 1.
|
|
Effect of immunotherapy with IL-2 against Fas+ and
Fas L1210 tumor growth.
Inasmuch as the expression of Fas caused increased susceptibility of
tumor cells to lysis by activated LAK cells, we next addressed whether
Fas+ and Fas L1210 tumor cells would
exhibit differential ability to induce tumors in the syngeneic host and
whether IL-2 immunotherapy would have varying effects on the growth of
these tumor cell lines in vivo. To this effect, 1 × 106 Fas+ or Fas L1210 tumor
cells were injected sc and were administered either with PBS as a
control or IL-2 (10,000 U three times/d for 1 week, followed by once/d
for an additional week). The survival of the mice was monitored for
approximately 60 days. As shown in Fig 6,
all L1210 Fas tumor cell + PBS-injected mice died
with an MST = 18.2 ± 6.0 days. In contrast, IL-2-treated
L1210 Fas-bearing mice survived for longer periods
(MST = 27.4 ± 4.6 days). This increase in MST resulting from IL-2
treatment was significant (P < .05) when compared with the
MST in PBS-treated Fas tumor-bearing mice.
Interestingly, mice bearing Fas+ L1210-induced tumor
survived for longer periods (MST = 37.9 ± 6.1 days), with a
significant proportion (approximately 20%) surviving for more than 60 days, at which time the experiment was terminated. Thus, the MST in
mice bearing Fas+ L1210 tumor was greater than those
bearing Fas tumor (P < .05). Moreover,
IL-2 treatment of Fas+ tumor-bearing mice led to increased
survival rate (40%) with an MST of 43.8 ± 15.5. Together, these
data showed that mice bearing L1210 Fas+ tumor cells
survived for longer periods than those bearing Fas
L1210 tumor cells. Furthermore, IL-2 therapy was more effective against
Fas+ than Fas tumor cells. It should be
noted that IP injection of Fas+ and Fas
L1210 tumor cells into DBA/2 mice also led to distinct survival rates.
The Fas L1210 tumor cell-bearing mice all died on
day 12, whereas L1210 Fas+ tumor-bearing mice died with an
MST = 33.2 ± 7.6 (P < .01). However, IL-2 treatment was
not effective against IP-injected tumor because IL-2-treated L1210
Fas+ tumor-bearing mice died with an MST of 31.5 ± 5.68 days, and Fas tumor-bearing mice died with an MST of
18.4 ± 1.68 days.
View larger version (22K):
[in this window]
[in a new window]
| Fig 6.
Effect of immunotherapy with IL-2 against growth of L1210
Fas+ and L1210 Fas tumor cells in
syngeneic host. L1210 Fas+ and Fas tumor
cells (1 × 106) were injected sc into group of five
syngeneic (DBA/2) mice. The mice were injected with PBS (control) or
with IL-2 (10,000 U/mouse, 3 times a day for 1 week followed by once a
day for additional 7 days). The mice were observed for tumor growth and
survival. The experiment was terminated after 60 days.
|
|
To rule out the possibility that the differences in the MST of mice
injected with Fas+ and Fas tumor cells
were caused by alterations in the growth characteristics of the tumor
cells, the in vitro growth pattern of the cells was investigated. The
data shown in Fig 7 indicated that L1210
Fas+ and L1210 Fas tumor cells had
similar growth curves when tested for 12 to 72 hours after in vitro
culture.
View larger version (17K):
[in this window]
[in a new window]
| Fig 7.
In vitro growth characteristics of L1210
Fas+ and Fas tumor cells. The tumor cells
were cultured in vitro as described in Materials and Methods and at
various time intervals the cells were obtained and a viable count was
determined. The data represent mean ± SEM of triplicate cultures.
|
|
Tumor-specific CTLs also show preferential killing of
Fas+ but not Fas target cells.
Because the L1210 tumor cells could evoke CTL responses, T cells were
purified from DBA/2 mice injected with Fas+ or
Fas L1210 tumor cells and cultured in vitro with
respective irradiated tumor cells and tested for cytotoxicity against
Fas+ and Fas targets. The data shown in
Fig 8A suggested that CTLs from
Fas+ tumor bearers mediated stronger lytic activity against
Fas+ as well as Fas tumor targets,
whereas CTLs from Fas tumor-bearing mice showed weak
or no significant lytic activity. These data demonstrated that
Fas+ L1210 tumor cells triggered increased CTL activity
when compared with the Fas L1210 tumor cells, a
finding which may account for increased survival of Fas+
tumor-bearing mice when compared with the Fas
tumor-bearing mice. It should be noted that the CTL obtained from
Fas+ tumor-bearing mice could kill Fas+ or
Fas targets to the same extent. This suggested that
such CTL-mediated cytotoxicity was primarily perforin rather than FasL
mediated. To corroborate that the cytotoxicity in the above study was
mediated by CTL, we tested the lytic activity against a nonspecific
H-2d tumor cell line such as P815. The data shown in Fig 8B
indicated that the CTL raised against L1210 tumors failed to kill P815
target cells, thereby indicating the involvement of L1210
tumor-specific CTL.
View larger version (17K):
[in this window]
[in a new window]
| Fig 8.
Increased tumor-specific CTL activity in mice bearing
L1210 Fas+ but not L1210 Fas cells.
Purified T cells from DBA/2 mice bearing L1210 Fas+ or
L1210 Fas tumor cells were obtained and cultured with
irradiated L1210 Fas+ and Fas tumor cells,
respectively, for 5 days. Next, the harvested cells were tested for
cytotoxicity against L1210 Fas+ or Fas
tumor targets (A) or against P815 (B) as described in Fig 4. In the
symbols, the first column represents source of T cells, the second
shows stimulator cells, and the third depicts target cells tested. For
example, Fas+ / Fas+ / Fas+
represents T cells from mice bearing Fas+ tumor cells
stimulated with Fas+ tumor cells in vitro and tested for
cytotoxicity against Fas+ targets.
|
|
| |
DISCUSSION |
In the current study we showed that poly I:C or IL-2 triggers NK/LAK
activity by enhancing both FasL- and perforin-based cytotoxicity. Fas+ tumor targets were found to be more sensitive to poly
I:C- or IL-2-induced cytotoxicity when compared with the
Fas tumor targets. Also, mice bearing
Fas+ L1210 tumor cells survived for longer periods and a
significant proportion of mice injected sc with the tumor rejected the
tumor, when compared with the mice bearing
Fas L1210 tumor cells, which died from rapid tumor
growth. Moreover, IL-2 administration in vivo was shown to trigger both
FasL- and perforin-based cytolytic activity and was able to increase
the MST of both Fas+ and Fas
tumor-bearing mice, particularly on sc injection. Furthermore, IL-2
therapy was more effective in sc-injected Fas+
tumor-bearing mice in which approximately 40% of the mice survived for
more than 60 days, when compared with Fas
tumor-bearing mice in which 0% of the mice survived.
The role of perforin in NK/LAK-cell-mediated cytotoxicity has been well
established.1 In addition, recent studies have shown the
existence of a perforin-independent pathway in NK/LAK-cell- or
T-cell-mediated cytotoxicity based on the interactions between FasL
expressed on effector cells and Fas receptor on the target cells.23-25 In the current study, we investigated the role
of perforin and FasL during spontaneous and induced NK/LAK activity
using a variety of NK-sensitive and NK-resistant cells, as well as
Fas+ and Fas target cells. Using
NK-sensitive YAC-1 targets, we noted that the spontaneous cytotoxicity
was mainly perforin dependent, inasmuch as perforin-deficient mice
showed no significant levels of cytotoxicity and the gld/gld
mice showed cytotoxicity comparable with or higher than the wild-type
mice. However, on activation with poly I:C in vivo or IL-2 in vitro,
the inducible cytotoxicity against YAC-1 and P815 targets was dependent
on both perforin and FasL. Moreover, in such cytolytic activity,
perforin played a more important role because the perforin KO mice
showed lower levels of inducible cytolytic activity when compared with
the gld/gld mice. This can be explained by the fact that YAC-1
and P815 tumor targets expressed lower levels of Fas, and moreover, not
all cells within the population expressed Fas.
Recently, freshly isolated human and murine NK cells were found to
express FasL and mediate Fas-based cytotoxicity.26,27 However, in the current study we noted that gld/gld mice had
normal levels of spontaneous NK activity against YAC-1 targets. Also, the NK cells failed to mediate spontaneous cytotoxicity against NK-resistant L1210 Fas+ transfectants despite the fact that
such cells expressed high levels of Fas. The reason for the differences
in previous and current studies is not clear. One possibility is that
in the earlier study,26,27 the targets used were different
from those used in the current study. Thus, the use of FasL by NK cells
mediating spontaneous cytotoxicity may depend on the nature of target
cells and not merely on the expression of Fas on target cells. Also, in
the earlier study,27 the investigators used a 12-hour
fluorescent dye assay to study cytotoxicity. During such long-term
cytotoxicity, molecules such as tumor necrosis factor have been shown
to be involved, unlike short-term assays in which the cytotoxicity is mediated exclusively by FasL and perforin.9
In the current study, comparison between Fas+ and
Fas L1210 transfectants yielded interesting results.
The Fas+ L1210 transfectants were more sensitive to poly
I:C- and IL-2-activated NK/LAK-cell-mediated cytotoxicity when
compared with Fas L1210 target cells. These data
together suggested that Fas expression on target cells can increase the
susceptibility of the tumor targets to induced NK/LAK activity. The
fact that perforin-deficient mice completely failed to mediate lysis of
Fas L1210 targets and that they could mediate lysis
of Fas+ L1210 cells to a lower level than the
gld/gld or wild-type mice suggested that perforin may play an
important role in induced NK/LAK activity. It should also be noted that
despite the strong expression of Fas on the L1210 tumor cells,
FasL-based cytotoxicity as observed in perforin KO mice was lower than
that seen in wild-type mice. Similarly, the perforin-based lytic
activity observed in gld/gld mice was also less than that seen
in wild-type mice. Thus, the presence of both perforin- and FasL-based
cytotoxicity was clearly an advantage to efficiently kill
Fas+ tumor targets.
To further corroborate and translate the in vitro results to tumor
rejection in vivo, we compared the ability of Fas+ and
Fas L1210 transfectants to grow and induce tumors in
syngeneic mice and tested whether IL-2 administration would inhibit the
growth of these tumor cells. In these studies, it was striking that
mice injected with Fas+ tumor cells survived for longer
periods than those receiving Fas tumor cells.
Furthermore, a significant proportion of mice injected sc with
Fas+ tumor cells could reject the tumor, and this was
further enhanced after IL-2 administration. It should be noted that the
IL-2 treatment was effective against sc- but not IP-injected tumors.
This may be because IP-injected tumors may metastasize faster and kill the host earlier as evident from a shorter MST, thereby preventing an
effective antitumor immunity to develop and act on the tumor cells. In
contrast, the sc tumors may metastasize slower as observed from longer
MST, because of which IL-2 treatment may be more effective. In the
current study it was also noted that Fas+ tumor-bearing
mice had higher levels of tumor-specific CTL activity when compared
with the Fas tumor-bearing mice. Also, T cells from
Fas tumor-bearing mice failed to mediate cytolytic
activity against Fas+ targets. These data suggested that
the expression of Fas on tumor cells may make them more immunogenic.
Together, the current study shows that IL-2 therapy may be more
effective against Fas+ tumors. Furthermore, transfection of
the Fas gene into Fas tumor cells may offer a novel
approach to trigger antitumor immunity.
| |
FOOTNOTES |
Submitted March 16, 1998;
accepted July 29, 1998.
Supported in part by National Institutes of Health grants (AI01392 and
HL 58641).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mitzi Nagarkatti, PhD, Dept of Biomedical
Sciences & Pathobiology, VA-MD College of Vet Med, Phase II, Duckpond
Dr, Virginia Tech, Blacksburg, VA 24061.
| |
REFERENCES |
1.
Trinchieri G:
Biology of natural killer cells.
Adv Immunol
47:187, 1989[Medline]
[Order article via Infotrieve]
2.
Oehler JR, Herberman RB:
Natural cell-mediated cytotoxicity in rats. Effects of immunopharmacological treatments on natural reactivity augmented by polyinosinic polycytidylic acid.
Int J Cancer
21:221, 1978[Medline]
[Order article via Infotrieve]
3.
Grimm EA, Muzumder A, Zhang HZ, Rosenberg SA:
Lymphokine activated killer cells phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin-2-activated autologous human peripheral blood lymphocytes.
J Exp Med
155:1823, 1983[Abstract/Free Full Text]
4.
Podack ER, Hengartner H, Lichtenheld MG:
A central role of perforin in cytolysis?
Annu Rev Immunol
9:129, 1991[Medline]
[Order article via Infotrieve]
5.
Smyth MJ, Trapani JA:
Granzymes: Exogenous proteinases that induce target cell apoptosis.
Immunol Today
16:202, 1995[Medline]
[Order article via Infotrieve]
6.
Kagi D, Ledermann B, Burki K, Zinkernagel RM, Hengartner H:
Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo.
Annu Rev Immunol
14:207, 1996[Medline]
[Order article via Infotrieve]
7.
Kojima H, Shinohara N, Hanaoka S, Someya-Shirota Y, Takagaki Y, Ohno H, Saito T, Katayama T, Yagita H, Okumura K, Shinkai Y, Alt FW, Matsuzawa A, Yonehara S, Takayama H:
Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes.
Immunity
1:357, 1994[Medline]
[Order article via Infotrieve]
8.
Glass A, Walsh CM, Lynch DH, Clark WR:
Regulation of the Fas lytic pathway in cloned CTL.
J Immunol
156:3638, 1996[Abstract]
9.
Lee RK, Spielman J, Zhao DY, Olsen KJ, Podack ER:
Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.
J Immunol
157:1919, 1996[Abstract]
10.
Mule JJ, Shu S, Schwartz SL, Rosenberg SA:
Adoptive immunotherapy of established pulmonary metastasis with LAK cells and recombinant IL-2.
Science
225:1487, 1984[Abstract/Free Full Text]
11.
Walsh CM, Matloubian M, Liu CC, Ueda R, Kurahara CG, Christensen JL, Huang MT, Young JD, Ahmed R, Clark WR:
Immune function in mice lacking the perforin gene.
Proc Natl Acad Sci USA
91:10854, 1994[Abstract/Free Full Text]
12.
Seth A, Pyle RH, Nagarkatti M, Nagarkatti PS:
Expression of the J11d marker on peripheral T lymphocytes of MRL-lpr/lpr mice.
J Immunol
141:1120, 1988[Abstract]
13.
Rouvier E, Luciani MF, Golstein P:
Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity.
J Exp Med
177:195, 1993[Abstract/Free Full Text]
14.
Seth A, Gote L, Nagarkatti M, Nagarkatti PS:
T-cell-receptor-independent activation of cytolytic activity of cytotoxic T lymphocytes mediated through CD44 and gpmel-14.
Proc Natl Acad Sci USA
88:7877, 1991[Abstract/Free Full Text]
15.
Nagarkatti M, Nagarkatti PS, Kaplan AM:
Differential effects of BCNU on T cell, macrophage, natural killer and lymphokine-activated killer cell activities in mice bearing a syngeneic tumor.
Cancer Immunol Immunother
27:38, 1988[Medline]
[Order article via Infotrieve]
16.
Hammond DM, Nagarkatti PS, Gote LR, Seth A, Hassuneh MR, Nagarkatti M:
Double-negative T cells from MRL-lpr/lpr mice mediate cytolytic activity when triggered through adhesion molecules and constitutively express perforin gene.
J Exp Med
178:2225, 1993[Abstract/Free Full Text]
17.
Nagarkatti M, Toney D, Nagarkatti PS:
Immunomodulation by various nitrosoureas and its effect on the survival of the host bearing a syngeneic tumor.
Cancer Res
49:6587, 1989[Abstract/Free Full Text]
18.
Nagarkatti M, Kaplan AM:
The role of suppressor T cells in BCNU-mediated rejection of a syngeneic tumor.
J Immunol
135:1510, 1985[Abstract]
19.
Zeytun A, Hassuneh M, Nagarkatti M, Nagarkatti PS:
Fas-Fas-ligand-based interactions between tumor cells and tumor-specific cytotoxic T lymphocytes: A lethal two-way street.
Blood
90:1952, 1997[Abstract/Free Full Text]
20.
Kataoka T, Shinohara N, Takayama H, Kondo S, Yonehara S, Nagai K:
Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and fas-based lytic pathways in cell-mediated cytotoxicity.
J Immunol
156:3678, 1996[Abstract]
21.
Yang Y, Mercep M, Ware CF, Ashwell JD:
Fas and activation-induced Fas ligand mediate apoptosis of T cell hybridomas: Inhibition of Fas ligand expression by retinoic acid and glucocorticoids.
J Exp Med
181:1673, 1995[Abstract/Free Full Text]
22.
Rafi A, Zeytun A, Bradley M, Sponenberg P, Grayson R, Nagarkatti M, Nagarkatti PS:
Evidence for the involvement of Fas ligand and perforin in the induction of vascular leak syndrome.
J Immunol
161:3077, 1998[Abstract/Free Full Text]
23.
Oshimi Y, Oda S, Hond Y, Nagata S, Miyazaki S:
Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells.
J Immunol
157:2909, 1996[Abstract]
24.
Lee RK, Spielman J, Zhao DY, Olsen KJ, Podack ER:
Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.
J Immunol
157:1919, 1996
25.
Yagita H, Hanabuchi S, Asano Y, Tamura T, Nariuchi H, Okumura K:
Fas-mediated cytotoxicity A new immunoregulatory and pathogenic function of Th1 CD4+ T cells.
Immunol Rev
146:223, 1995[Medline]
[Order article via Infotrieve]
26.
Oshimi Y, Oda S, Honda Y, Nagata S, Miyazaki S, Oshimi Y:
Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells.
J Immunol.
157:2909, 1996
27.
Arase H, Arase N, Saito T:
Fas-mediated cytotoxicity by freshly isolated natural killer cells.
J Exp Med
1811:235, 1995
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
D. Vermijlen, D. Luo, C. J Froelich, J. P. Medema, J. A. Kummer, E. Willems, F. Brae | |
Top
Abstract
Introduction