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T Cells by Administering a Daunomycin-Conjugated Specific Monoclonal Antibody in Early Tumor Lesions Augments the Activity of CTLs and NK Cells1
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Department of Tumor Biology, Institute of Medical Science, University of Tokyo, Minatoku, Tokyo, Japan; and
Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells accumulating in early
tumor lesions and those purified from spleen cells of tumor-bearing
mice attenuate the activity of CTLs and NK cells. We, therefore,
investigated whether depletion of T cells from early lesions of
tumors results in restoration of CTL and NK cell activities and
subsequent regression of tumors. A daunomycin-conjugated
anti-TCR mAb UC7-13D5 (Dau-UC7) was prepared to efficiently
deplete T cells. An in vitro study revealed that Dau-UC7
specifically lysed TCR+ cells and effectively
inhibited splenic T cells from tumor-bearing mice to produce
cytotoxic cell-suppressive factors. Furthermore, intralesional
injections of Dau-UC7 at an early stage of tumor development led to
augmentation of tumor-specific CTL as well as NK cell activities and to
the resultant regression or growth inhibition of the tumors. On
analysis of cytokine profile, T cells transcribed mRNAs for
IL-10 and TGF-ß, but not IL-4 or IFN-, suggesting the T regulatory
1-like phenotype. Finally, a blocking study with mAbs showed that the
inhibitory action of T cells on CTLs and NK cells was at least
partly mediated by IL-10 and TGF-ß. These results clearly
demonstrated the novel mechanism by which T regulatory 1-like T
cells suppress anti-tumor CTL and NK activities by their regulatory
cytokines in early tumor formation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the one hand, mAbs specific for 
ßTCR or TCR are used in
vivo for abrogation of the target recognition by T cells because of
their ability to render TCR internalized or for specific depletion of T
cell subpopulations by apoptosis or Ab-dependent cell-mediated
cytotoxicity (8, 9, 10, 11, 12). On the other hand, however, some TCR-specific
mAbs have been used in vitro as stimulators of T cells. In normal mice
administered a TCR-specific mAb, some TCR-null T cells survive and
remain constant in number after a certain period (13). In tumor-bearing
mice, Ts cells rather than CTLs or Th cells are predominantly primed
and accumulate in the tumor lesions (14, 15). It is possible that
treatment of tumor-bearing hosts with intact TCR-specific mAbs,
originally intended to eliminate Ts cells, instead enhances Ts cell
functions. Therefore, the effect of treatment with anti-TCR mAbs is
thought to be ambivalent and thus may lead to misinterpretation of the
results. Several studies have demonstrated the therapeutic
effectiveness of daunomycin-conjugated Abs on the direct killing of
tumor cells (16, 17). Daunomycin is internalized by cells following
binding of Abs to the cell surface. Thus, daunomycin conjugates with
mAbs specific for TCR on Ts cells may be an efficacious and reliable
tool for complete depletion of Ts cells.
T cells infiltrating at an early stage of tumor development of
MM2 (mammary tumor cell line), MH124 (hepatoma cell line), and B16
(melanoma cell line) suppress CTL and NK cell activities not only by
cell-cell interaction but also by locally producing suppressive factors
(18, 19). The percentage of these T cells in tumor cell
suspensions peaks on days 5–7 after tumor inoculation and gradually
decreases thereafter (19). Therefore, elimination of these cells at the
tumor sites may result in tumor regression by relaxing CTLs and NK
cells. The purpose of this study was to investigate whether
daunomycin-conjugated anti-TCR mAb can effectively damage the
suppressor functions of T cells, whether intralesional treatment
with this conjugates at an early stage can lead to tumor regression by
restoring tumor-specific CTL and NK cell functions, and whether these
T cells produce regulatory cytokines such as IL-10 and TGF-ß.
The results show that successful elimination of T cells
producing Tr1-type cytokines results in tumor regression by the actions
of CTLs and NK cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seven to nine-week-old male C3H/He and C57BL/6 (B6) mice were obtained from Japan SLC (Hamamatsu, Japan). MM2, MH134, B16, and YAC-1 tumor cells were used in this study. MM2, MH134, and B16 were mammary tumor cell lines of C3H/He, a hepatoma cell line of C3H/He, and a melanoma cell line of B6, respectively. MM2 cells were maintained i.p. in C3H/He. MH134 and B16 cells were maintained by culturing them in DMEM (Nissui, Tokyo, Japan) supplemented with 10% FCS. In in vivo experiments, MM2 and MH134 cells (2 x 105 cells/mouse) were inoculated into C3H/He mice i.p. and s.c., respectively. The same number of B16 cells were injected s.c. into B6 mice. YAC-1 cells are an NK-sensitive cell line that was cultured in DMEM supplemented with 10% FCS and used in in vitro assays.
mAbs and chemical substances
Anti-TCR mAb (UC7-13D5)-producing hybridoma was a gift
from Dr. Bluestone (Chicago University, Chicago, IL). UC7-13D5 and
anti-ßTCR mAb (H57-597) were purified from hybridoma culture
supernatants by affinity column chromatography with anti-hamster
IgG-Sepharose after ammonium sulfate precipitation. Anti-hamster
IgG-Sepharose was prepared by covalent coupling of anti-hamster IgG
sheep polyclonal Abs (Organon Teknika, West Chester, PA) with cyanogen
bromide-activated Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden).
Purified forms of anti-CD4 mAb (GK1.5), anti-CD8 mAb (53-6.7),
and hamster IgG were obtained from PharMingen (San Diego, CA).
Anti-IL-10 and anti-TGF-ß1, -2, and -3 neutralizing mAbs, rIL-10,
and rTGF-ß1 were purchased from Genzyme (Cambridge, MA). Daunomycin
was purchased from Calbiochem-Novabiochem (La Jolla, CA). Sodium
periodate and sodium borohydride were purchased from Sigma (St.
Louis, MO).
Ab-daunomycin conjugation
Daunomycin (40 mg/ml) was oxidized with 0.1 M sodium periodate (in PBS) at 20°C for 20 min in a dark room (final 1-ml volume). Immediately, 100 µl of 1 M glycerol was added to the mixture to stop the reaction, and incubation proceeded at 20°C for 30 min. Supernatant obtained after centrifugation of the mixture was used as an oxidized daunomycin solution. One milliliter of 5 mg/ml UC7-13D5 or H57-597 mAb or hamster IgG (in 0.15 M potassium carbonate buffer, pH 9.5) and the oxidized daunomycin solution were mixed and incubated at 20°C for 2 h. After centrifugation, the supernatant (2 ml) containing daunomycin-conjugated Ab was reduced by the addition of 0.6 mg of sodium borohydride and incubation at 4°C for 2 h and was subjected to gel filtration (Bio-Gel P-100, Bio-Rad, Hercules, CA) to separate daunomycin-bound Ab from free daunomycin. Conjugate fractions were collected and used in in vitro and in vivo assays. By assessment of 495 nm (daunomycin) and 280 nm (Ab) absorbances, 50 µg of daunomycin-conjugated UC7-13D5 (Dau-UC7), H57-597 (Dau-H57), and hamster IgG (Dau-IgG) were bound with 2 µg of daunomycin.
Preparation of lymphocytes
T cell-enriched fractions of splenocytes from normal and
MM2-bearing C3H/He mouse splenocytes were prepared using a generally
established method. Spleen cells were hemolyzed with 0.17 M ammonium
chloride. After washing three times, the cells were incubated on a
plastic dish in RPMI 1640 medium (Nissui) supplemented with 10% FCS at
37°C for 1 h to remove dish adherent cells. The dish-nonadherent
cells were collected by gentle shaking and subjected to nylon wool
column. The T cell-enriched fraction passed through a nylon wool column
was used for ß or T cell preparation. The enriched T cells
were incubated with ßTCR (H57-597)- or TCR
(UC7-13D5)-specific mAb at 4°C for 30 min. After washing three times,
Ab-bound cells were mixed with anti-hamster IgG-conjugated magnetic
beads (Dynal, Oslo, Norway) at a ratio of three beads per cell at 4°C
for 1 h on a rocking shaker. Anti-hamster IgG-conjugated beads
were prepared by coupling the anti-hamster IgG (Organon Teknika)
with tosyl-activated magnetic beads (Dynal) according to the Dynal
manual. Cells bound with beads were collected with a magnet and
cultured overnight in RPMI 1640 medium supplemented with 10% FCS to
separate cells from beads. ß and T cells purified by this
manipulation were confirmed to be >96% pure by flow cytometric
analysis using FITC-conjugated ßTCR- or TCR-specific mAb
(PharMingen, San Diego, CA).
Tumor-infiltrating lymphocytes (TILs) were separated from B16 and MH134
lesions. B16 and MH134 tumor cell suspensions were prepared on day 7
after s.c. inoculation (2 x 105/mouse) from
50 lesions. Ten milliliters of B16 and MH134 cell suspensions (1
x 105 cells/ml in PBS supplemented with 10%
FCS) applied on 5 ml of Histopaque 1083 (Sigma) were subjected to
centrifugation at 1000 x g for 30 min at 20°C. The
cells at the interface were collected, washed three times with DMEM,
and used as B16 and MH134 TILs. For separation of T cells from
MM2-infiltrating lymphocytes, 2 x 105 MM2
cells were inoculated i.p. into C3H/He mice. Seven days after
inoculation, ascites containing MM2 and tumor-infiltrating lymphocytes
were collected and then diluted with DMEM containing 10% FCS. After
washing three times, the cells were suspended in DMEM and subjected to
weak centrifugation (500 x g, 10 s, five times).
Approximately 90% of large MM2 cells were precipitated by this
manipulation. The remaining cells in culture supernatants were used as
MM2-tumor infiltrating lymphocytes. T cells were separated from
MM2 tumor-infiltrating lymphocytes using magnetic beads as described
above. Anti-H-2b and anti-MM2 CTLs were
prepared from T cell-enriched splenocytes of C3H/He mice immunized with
B6 lymphocytes and MM2 regressor mice, respectively, as described
previously (18).
Preparation of lymphocyte culture supernatants
Splenic T cells purified from spleens of normal or
MM2-bearing mice and from MM2 tumor-infiltrating lymphocytes were
cultured in RPMI 1640 supplemented with 10% FCS and rIL-2 (5 U/ml) at
37°C for 3 days. The expanded cells were recultured in RPMI 1640
supplemented with 10% FCS in a 24-well plate (Corning, Corning, NY;
1 x 106 cells/well) for 24 h. After
centrifugation, each culture supernatant was collected and added to the
anti-H-2b CTL assays at a 50% volume.
Otherwise, to examine the effect of daunomycin conjugates on the
ability of T cells to produce suppressive factors, T
cells separated from MM2 tumor-infiltrating lymphocytes were cultured
in RPMI 1640 supplemented with 10% FCS and rIL-2 (5 U/ml) for 3 days.
The expanded cells were incubated with Dau-UC7, Dau-H57, Dau-IgG,
UC7-13D5, H57-597, hamster IgG, or daunomycin at varying concentrations
at 4°C for 1 h. After washing three times, these treated cells
were cultured in RPMI 1640 supplemented with 10% FCS in 24-well plates
(1 x 106 cells/well). Culture supernatants
obtained by this manipulation were also added to the
anti-H-2b CTL assays at a 50% volume.
Cytotoxicity assay and cytotoxicity inhibition assay
To test the cytotoxicity of daunomycin-conjugated Ab, purified
ß and T cells (2 x 104
cells/well) were cultured and expanded in an anti-CD3
mAb-immobilized 24-well culture dish with RPMI 1640 supplemented with
10% FCS and 50 U/ml rIL-2. The propagating cells (5 x
106 cells/ml) were radiolabeled with RPMI 1640
containing 10% FCS and 200 µCi Na[51Cr]
(DuPont-New England Nuclear, Boston, MA) for 1 h at 37°C. After
washing four times, 51Cr-labeled ß and
T cells were used as target cells for drug-conjugated Abs.
Daunomycin-conjugated H57-597, UC7-13D5, or hamster IgG was added at
varying concentrations to each well containing target cells (1 x
104 cells/200 µl). The mixtures were incubated
at 37°C for 12 h. To examine in vitro CTL induction in TILs,
MM2, MH134, and B16, TILs were treated with daunomycin-conjugated mAbs
(100 ng/ml daunomycin-5 µg/ml Abs) at 37°C for 4 h and washed
three times with DMEM. Dau-Ab-treated TILs were cultured with rIL-2
(100 U/ml)-containing medium at 37°C for 5 days. The expanded cells
were subjected to the CTL assays against
51Cr-labeled tumor target cells. For cytotoxicity
test of CTLs against tumor cells, MM2, MH134, and YAC-1 cells (5
x 106 cells/ml) were radiolabeled with RPMI 1640
containing 10% FCS and 200 µCi of Na[51Cr]
for 1 h at 37°C. Varying numbers of splenic T cells from mice
bearing MM2 or those regressing MM2 were mixed with
51Cr-labeled MM2, MH134, or YAC-1 target cells
(1 x 104 cells) at a final volume of 200
µl and incubated for 12 h at 37°C at varying ratios.
Cytotoxicity inhibition assays were performed to investigate the lytic
mechanisms of cytotoxic effector cells. Varying concentrations of
anti-ßTCR mAb (H57-597), anti-TCR mAb (UC7-13D5),
anti-CD4 mAb (GK1.5), or anti-CD8 mAb (53-6.7) was added in
cytotoxicity assays of splenic T cells from MM2 regressor mice against
MM2 target cells. For cytotoxicity test of
anti-H-2b CTLs, varying numbers of
anti-H-2b CTLs were mixed with
51Cr-labeled B6 lymphoblasts (1 x
104 cells) for 6 h at 37°C. B6
lymphoblasts were prepared by culturing B6 splenocytes with RPMI 1640
supplemented with 10% FCS and 5 µg/ml Con A (Pharmacia Biotech) at
37°C for 3 days. In all cytotoxicity tests, the radio activities of
medium and cells were counted by gamma counter, and the percent
specific lysis was calculated as follows: % specific lysis = (cpm
experimental release - cpm spontaneous release)/(cpm maximum
release - cpm spontaneous release) x 100.
Proliferation assay
H-2b- or MM2-specific CTLs (2 x
105 cells/well) were incubated in triplicate for
24 h in 96-well plates (Corning Glass Works, Corning, NY) in 100
µl of complete medium. Methyl-tritiated thymidine
([3H]TdR; Amersham, Arlington, IL; 1
µCi/well) was added to the culture 8 h before harvest. The cells
were harvested on glass-fiber filters using a cell harvester (Cambridge
Technologies, Watertown, MA), and their radio uptake was measured in a
scintillation counter. Culture supernatants from T cells,
rIL-10, and rTGF-ß1 were added to the H-2b- or
MM2-specific CTL proliferation assay at varying concentrations.
Administration of daunomycin-conjugated Abs in vivo
MM2 or MH134 cells (2 x 105) were inoculated i.p. or s.c. into C3H/He mice, respectively. On either 3 consecutive days, days 4–6 or days 15–17 after tumor inoculation, 100 µg of daunomycin-conjugated Abs containing 5 ng of daunomycin were injected at a tumor site, and subsequent tumor progression was observed. As a negative control, 2 µg of daunomycin alone was injected at a tumor site.
RT-PCR of cytokine mRNAs
Tumor-infiltrating T cells accumulating on day 7 after
MM2 i.p. inoculation and splenocytes from MM2-bearing C3H/He were
prepared as described above. Cells containing PBMC were prepared from
blood of MM2-bearing and regressor mice by treatment with 0.17 M
ammonium chloride. Total RNAs of these cells were extracted with an RNA
extraction kit (RNeasy, Qiagen, Hilden, Germany). First-strand cDNA was
reverse transcribed using each RNA sample and was amplified by PCR with
a RNA PCR kit (GeneAmp RNA PCR Kit, Takara Biomedicals, Osaka, Japan)
according to the manufacturer’s directions. All pairs of primers for
ß-actin, IL-4, IFN- (20), IL-10 (21), and TGF-ß (22) were used
in this PCR study. PCR was run for 35 cycles with a thermal cycler (DNA
amplifier, Sanyo Co., Osaka, Japan) as follows: 1 min at 94°C, 1 min
at 55°C, and 15 s at 72°C. The PCR products and DNA m.w.
marker VI (Boehringer Mannheim, Mannheim, Germany) were loaded in 2%
agarose gels and visualized with UV exposure of 1 µg/ml ethidium
bromide-staining agarose gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells and abrogation of their functions by
Dau-UC7
To prepare the daunomycin-conjugated Abs, the sugar moiety of
daunomycin was cleaved with periodate and then coupled with an
anti-TCR mAb (UC7-13D5), anti-ßTCR mAb (H57-597), or
hamster IgG. Following reduction with sodium borohydride, the
conjugates were separated from the unbound daunomycin by Bio-Gel P-100
column chromatography. Fractionated eluates were collected, and their
absorbances at 280 nm (Abs) and 495 nm (daunomycin) were monitored.
Free daunomycin and free hamster IgG were eluted over 20 and in 8–15
fractions, respectively (Fig. 1
D). The daunomycin conjugates
had a dual absorbance derived from daunomycin and Ab and were also
eluted in nearly the same fractions as free Abs (Fig. 1, A–C), indicating successful conjugation. The conjugates of
UC7-13D5, H57-597, and hamster IgG contained daunomycin at a ratio of
approximately six daunomycin moieties to one Ab.
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ßTCR+
and TCR+ cell populations were separated
from normal C3H/He mice and were cultured over a short term under CD3
stimulation. The expanded cells were 51Cr labeled
and incubated with daunomycin-conjugated UC7-13D5 (Dau-UC7), H57-597
(Dau-H57) or hamster IgG (Dau-IgG), unconjugated UC7-13D5 (UC7),
H57-597 (H57) or hamster IgG (IgG), or daunomycin alone. Following 12-h
incubation, ßTCR+ and
TCR+ cells were killed by Dau-H57 and
Dau-UC7, respectively, while neither conjugate exhibited substantial
cytotoxicity against the irrelevant T cell population (Fig. 2A). Dau-IgG, any of the
unconjugated Abs (data not shown), or daunomycin did not lyse
ßTCR+ or TCR+
cell target.
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T cells suppress NK lineage cells and/or
CTLs at least partly by releasing soluble factor(s) (18, 19). To
confirm the functional abolishment of T cells by Dau-UC7, we
used culture supernatants from T cells by testing their
immunosuppressive activity against CTLs. T cells purified from
MM2-bearing mice were treated with Dau-UC7, and their culture
supernatant was added in assays of cytotoxicity and proliferation of
anti-H-2b CTL. T cells separated from
splenocytes or tumor-infiltrating lymphocytes of MM2-bearing mice
produced suppressive factors against
anti-H-2b CTL activity (Fig. 2
, B
and C). The treatment of these T cells with Dau-UC7
totally abrogated their ability to secrete suppressive factors. In
contrast, the T cells exposed to Dau-H57, Dau-IgG, any of the
unconjugated Abs, or daunomycin produced these factors unchangedly.
Interestingly, the T cells treated with UC7 produced these
suppressants more vigorously than untreated cells, suggesting that
intact UC7 mAb transmits activation signals into the T cells.
These results further demonstrated that daunomycin-conjugated mAbs were
able to attenuate the development of specific lymphocytes. Tumor regression by intralesional injections of Dau-UC7 at an early stage of tumor development
The above finding raised the possibility that intralesional
injections of Dau-UC7 restore the function of tumor-specific CTLs and
NK cells by depressing T cells and result in subsequent
inhibition of tumor development. When Dau-UC7 was intralesionally given
on days 4, 5, and 6 after i.p. inoculation of MM2, the MM2 tumor
regressed completely (Figs. 3A
and 4), and the MM2 regressor mice
subsequently survived for over 10 mo. On the other hand, the growth of
MH134 tumor was also suppressed by the administration of Dau-UC7 on
days 4, 5, and 6 after s.c. inoculation of tumor cells, although the
MH134 tumor gradually developed thereafter, and all of the mice died
within 2 mo after the tumor inoculation (Fig. 3B). In
contrast, there were no therapeutic effects when Dau-UC7 was
administered intralesionally on days 15, 16, and 17 after MM2 or MH134
inoculation (data not shown). This is consistent with our previous
finding that T cells function as suppressor cells in an early
tumor formation (19). No attenuation of tumors was found when Dau-IgG
or daunomycin alone was used at the same concentration as Dau-UC7
(Figs. 3 and 4). A weak inhibition of MM2 tumor progression was found
in mice treated with Dau-H57 on days 4, 5, and 6 after i.p. MM2
inoculation, while such an inhibition of tumor growth was not observed
in MH134-bearing mice (Fig. 3B). Subsequently, the mice died
within 25 days following vigorous progression of MM2 (Fig. 3A). These results raise the possibility that a portion of
ß T cell populations modestly inhibit antitumor cytotoxic cells in
early formation of MM2 lesions. This may be in accordance with our
previous finding that early-appearing Th2-ß T cells exhibit
suppressive features against NK activities (19).
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T cells from
tumor lesions. Therefore, it was suggested that depletion of such
T cells from early tumor lesions results in the vigorous
expansion of lesional CTLs in vitro. Certainly, anti-MM2 and -MH134
cytotoxic cells were much more rapidly induced in Dau-UC7-treated TILs
than in Dau-hamster IgG-treated or untreated cells (Fig. 5, A and B).
Furthermore, similar results were obtained from the study using B16
TILs (Fig. 5C), suggesting that the attenuated cytotoxic
cell activities by T cells are generally seen in early tumor
lesions. We further confirmed activation of tumoricidal cytotoxic cells
in mice treated with Dau-UC7 by an in vitro study. Splenocytes
separated from Dau-UC7-treated MM2 regressor mice lysed MM2, but not
MH134 tumor cells, vigorously (Fig. 6A) in an ßTCR- and
CD8-dependent manner (Fig. 6B), whereas no cytotoxicity was
exhibited by splenocytes isolated from untreated MM2-bearing mice and
control Dau-IgG-treated MM2-unregressing mice (Fig. 6A).
This indicates that the activity of tumor-specific CTLs was powerfully
augmented by removing T cells from tumor-bearing mice. It is
notable that splenocytes from MM2 regressor mice were also modestly
cytotoxic against NK-sensitive YAC-1 tumor cells (Fig. 6A).
This also suggests that NK cell activity was elevated by depleting
T cells. Taken together, these findings indicate that Dau-UC7
exerts its antitumor action by damaging T cells that function as
down-regulators against CTLs and NK cells.
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T cells
To investigate the cytokine expression pattern of T cells
accumulating in tumor lesions, T cells were freshly isolated
from 7-day ascites fluid of i.p. inoculated MM2 using
TCR-specific mAb (UC7-13D5)-conjugated magnetic beads. Their
cytokine profile was examined by PCR of cDNA with primers specific for
IL-4, IL-10, IFN-, and TGF-ß. Fig. 7
shows that freshly isolated MM2-infiltrating T cells transcribed
IL-10 and TGF-ß mRNAs, whereas neither amplified product of IL-4 nor
IFN- was detected. In addition, when cultured over a short term in
the presence of IL-2, these T cells secreted IFN- but not
IL-4 (data not shown), suggesting the IFN--producing capacity of the
T cells. Since it has recently been reported that
CD4+ T lymphocytes producing IL-10, TGF-ß, and
IFN- are a novel population, termed Tr1 cells (5), these results
suggested that T cells accumulating in MM2 tumor lesions are of
the Tr1 type.
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T cell
suppression of CTL and NK activities, culture supernatants of
MM2-infiltrating T cells were mixed with IL-10- and
TGF-ß-specific neutralizing mAbs and added to the culture of the
anti-MM2 CTL proliferation assay. The inhibitory effect of the
T cell culture supernatant was reduced by the addition of either
anti-IL-10 or anti-TGF-ß mAb in a dose-dependent manner (Fig. 8A). CTLs proliferated more
vigorously when both IL-10 and TGF-ß were neutralized, suggesting an
additional or synergistic effect of IL-10 and TGF-ß on the
suppression of CTLs. To further examine the inhibitory role of IL-10
and TGF-ß, splenocytes from MM2-bearing mice were cultured in low
dose (10 U/ml)-IL-2-containing medium in the presence of anti-IL-10
and anti-TGF-ß mAbs. This neutralization led to augmentation of
the lytic activity of cultured cells against MM2 as well as YAC-1 (Fig. 8B), consistent with the augmentation of NK cytotoxicity in
mice that regressed MM2 tumors following injection of Dau-UC7 (see Fig. 6A). An RT-PCR study revealed that a reduction in IL-10 and
TGF-ß mRNA transcriptions of PBMC was correlated with regression of
MM2 tumor cells in Dau-UC7-treated MM2-bearing mice (Fig. 8C). The inhibitory effect of TGF-ß on the growth of CTLs
was further confirmed by the finding that rTGF-ß1 suppressed the
proliferation of CTLs in response to MM2 in a dose-dependent manner
(Fig. 8D). However, only weak inhibition was obtained with
rIL-10, and the effect of the mixture of rIL-10 and rTGF-ß was
similar to that of TGF-ß alone. Since IL-10 neutralization of culture
supernatant from T cells abolished its inhibitory activity (see
Fig. 8A), it is possible that another suppressive factor(s)
that synergizes with IL-10 is present in T cell culture
supernatants. These results demonstrated that Tr1 cytokines produced by
T cells participate in the attenuation of CTL and NK
activities.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells inhibit CTL generation and NK activity, and
that elimination of this type of T cells from tumor-bearing mice
with a daunomycin-conjugated specific mAb augments the activity of CTLs
and NK cells and subsequent tumor regression. The in vitro study
further confirmed the accumulation of T cells functioning as
suppressors against tumoricidal lymphocytes in early tumor lesions, as
cytotoxic cells were rapidly induced in T cell-depleted TILs of
melanoma as well as hepatoma and mammary tumors. Our previous study has
shown that Th2-type T cells present at an early stage of B16
melanoma development also exert an inhibitory action on CTLs and NK
cells. Those T cells dominantly infiltrate in early tumor
lesions, and their number subsequently decreases thereafter (19). Thus,
T cells in these two systems function as immunosuppressors
against effector T cells by producing Tr1 or Th2 cytokines in innate
immune responses (23). It is well known that Th2 cytokines
down-regulate Th1 cell functions, resulting in abolishment of effective
induction of CTL and NK cells (24). Likewise, Tr1 cells directly
attenuate Ag-specific immune responses mediated by Th1 cells (5, 25).
In our preliminary study, Th2-type T cells in the B16 system
also secrete a great amount of TGF-ß (unpublished data), suggesting a
similarity between these two types of T cells. Since TGF-ß is an
autocrine and paracrine inhibitor of CTLs (26, 27), this cytokine seems
to be a key factor in the T cell-mediated suppression of
antitumor activities. In fact, blocking of TGF-ß with mAb abrogated
the activity of T cell supernatants, and the function of
T cells was replaced by exogenously added TGF-ß.
On the other hand, the effect of IL-10 on suppression of CTL generation
and NK activities remains controversial. IL-10 suppresses the
cytotoxicity of and IFN- production by NK cells, and the induction
of tumor-specific CTLs (28, 29), whereas IL-10 augments CTL and NK
activities synergistically with IL-2 (30, 31, 32). In our study
neutralization of IL-10 in T cell culture supernatants resulted
in elevation of CTL and NK activities, indicating the down-regulatory
role of IL-10. However, the exogenous addition of IL-10 did not
suppress the proliferative response of CTLs as did TGF-ß. This
suggests that as yet unelucidated factors that synergize with IL-10 are
required for the suppression. Our previous study has demonstrated that
T cells release soluble suppressant(s) acting on the cytolytic
effector phase of CTLs, and this soluble factor seems to be different
from TGF-ß and IL-10 (18). This unidentified factor may participate
in the synergistic inhibition with IL-10. Taken together, Tr1 cytokines
IL-10 and TGF-ß are involved in the mechanism underlying the T
cell-mediated inhibition of CTL and NK activities.
Many reports, however, have provided in vitro evidence for a cytotoxic
effector role for T cells against tumor cells (33, 34, 35). The vast
majority of these observations were obtained from experiments using
culture conditions with high doses of IL-2. Since the amounts of Th2
cytokines, including IL-4 and IL-10, are frequently elevated in
tumor-bearing mice (1, 36), T cells may overt their suppressive
immunoregulatory capacity under such Th2-predominant conditions. In
this situation, cultivation of T cells under artificial
IL-2-rich conditions possibly converts their function to cytotoxic
cells. Our observation that fresh MM2-infiltrating T cells
acquire the ability to secrete IFN- after short term culture with
IL-2 suggests the conversion of immunosuppressive T cells to
cytotoxic cells by Th1 cytokines. Alternatively, it is possible that
some T cells originally distributed in certain organs, such as
skin, liver, intestine, periphery, and reproductive tracts (37) acquire
immunosuppressive activity when they accumulate in tumor lesions.
Interestingly, a study from another group has shown that
extrathymically differentiated T cells may negatively regulate
immune reactions, as administration of hepatic T cells leads to
unresponsiveness to skin allograft (38). Our preliminary study showed
that T cells accumulate markedly in early tumor lesions of
athymic nude mice (N. Seo et al., unpublished observation), further
providing evidence for the participation of extrathymic T cells
in the suppression of tumor immunity. In addition, studies from
experimental pregnancy have revealed that the appearance of extrathymic
T cells in early decidua of pregnant mice is a crucial event for
the maintenance of pregnancy (39, 40). These different lines of studies
suggest the role for extrathymic T cells in suppression of
cytotoxic cell-mediated immune reactions.
It should be noted that the intralesional administration of Dau-UC7 at
an early stage of tumor development also augmented NK activity. The
NK-suppressive role of IL-10 and TGF-ß has been demonstrated by
several groups (28, 29, 41). However, it has also been reported that
IL-10 and TGF-ß down-modulate MHC class I expression on target cells
and render cells NK sensitive (42, 43). We have previously demonstrated
the modulation of NK activity by class I molecules on bystander cells
in tumor lesions (19). Thus, the activity of NK cells seems to be
regulated bivalently by Tr1 cytokines. In another line of studies, it
has been reported that NK cells are important in the generation of
tumor-specific CTLs, because NK cell-depleted B16 tumor-bearing mice
fail to induce CTLs at their tumor sites (44). Thus, T cells in
tumor-bearing mice are suggested to suppress both NK cell activity and
subsequent generation of anti-tumor CTLs.
Daunomycin-conjugated TCR-specific mAbs were very useful for the
elimination of T cells. In contrast, unconjugated mAbs specific
for TCR did not inhibit but, rather, enhanced T cell function. The
conjugates were prepared by the classical method in this study. It has
been documented that the daunomycin conjugates prepared by the dextran
bridge method lyse target cells more effectively than those by direct
binding method because of the high efficacies of internalization and
the release of daunomycin in the cytoplasm of target cells (45, 46).
Furthermore, daunomycin bound to dextran without any Abs exhibited more
vigorous antitumor cell cytotoxicity than the free form (47).
Therefore, daunomycin-anti-TCR Ab conjugates synthesized via the
dextran bridge may be a more powerful tool than conjugates constructed
by the classical method. On the other hand, since diphtheria
toxin-conjugated (48, 49) and ricin-conjugated (50, 51) tumor
cell-specific Abs have been used for targeting of tumors, we suggest
that Ts cell-specific Abs bound with diphtheria toxin or ricin may also
be useful as an effective eliminator of T cells compared with
daunomycin conjugates.
In conclusion, elimination of T cells that down-regulate CTLs and/or NK
lineage cells is one strategy for tumor immunotherapy. Depletion of the
down-regulator such as Th2- and Tr1-type T cells, when the
treatment is efficacious but not harmful, may be of clinical importance
for the development of an alternative way to enhance immunity against
tumor cells.
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Acknowledgments |
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Footnotes |
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2 Current address: Tumor Laboratory, The Foundation for Basic Research of Oncology, Kokubunji, Tokyo, Japan.
3 Address correspondence and reprint requests to Dr. Naohiro Seo, Department of Dermatology, Hamamatsu University School of Medicine, Handa-cho 3600, Hamamatsu 431-3192, Japan. E-mail address:
4 Abbreviations used in this paper: Tr, T regulatory; Ts, suppressor T; Dau, daunomycin; TIL, tumor-infiltrating lymphocyte.
Received for publication January 25, 1999. Accepted for publication April 23, 1999.
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), Dau-hamster IgG (
), or daunomycin
(
) at the indicated concentrations of daunomycin or daunomycin in
conjugates. In conjugates, 20, 100, and 200 ng of daunomycin correspond
to 1, 5, and 10 µg of Abs, respectively. Data are expressed as means
of duplicate assays. B, MM2 tumor-infiltrating 
