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University of California, Los Angeles-Wadsworth Pulmonary Laboratory and
Jonsson Comprehensive Cancer Center, University of California, Los Angeles, School of Medicine and West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA 90073; and
La Jolla Institute of Allergy and Immunology, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have reported previously that human lung tumor nodules produce significantly greater amounts of IL-10 than does normal lung tissue (3). Although lung tumor cells secrete IL-10, tumor cells also potently induce lymphocyte IL-10 production via a PGE2-mediated pathway, and thus T cell-derived IL-10 appears to be the predominant source of this cytokine in the lung tumor environment (11). To assess the impact of enhanced T cell-derived IL-10 on antitumor immunity in vivo, we utilized a novel transgenic mouse model in which IL-10 is expressed under the control of the IL-2 promoter.
We have shown previously that Lewis lung carcinoma cells (3LL)3 grow more rapidly in IL-10 transgenic mice compared with their control littermates (12). The current studies were undertaken to address the mechanisms responsible for enhanced tumor growth in the IL-10 transgenic mice. Consistent with the in vitro results, we report that IL-10 transgenic mice demonstrate a reduced capacity for Ag presentation, CTL generation, and type 1 cytokine production, reflecting defects in both T cell and APC function. Furthermore, the effects on APC persist in the absence of IL-10-producing transgenic T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine Lewis lung carcinoma cell line (3LL, H2b) was obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were routinely cultured as monolayers in 25-cm2 tissue culture flasks containing RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS (Gemini Bioproducts, Calabasas, CA), penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 2 mM glutamine (JRH Biosciences, Lenexa, KS), and maintained at 37°C in humidified atmosphere containing 5% CO2 in air. The cell line was mycoplasma free, and cells were utilized before the tenth passage for these studies.
Mice
IL-10 transgenic mice were made by standard methods at University of California, Los Angeles, Transgenic Mouse Core Facility (Los Angeles, CA), as previously described (12). Briefly, the human IL-2 promoter-enhancer region from -567 to +54, relative to the transcriptional start site, was cloned upstream of the mouse IL-10 genomic sequence. The mouse IL-10 sequence used in the construct corresponds to positions 1568–6879 of a 7.2-kb BglII fragment, in which position 1568 represents nucleotide 16 of the IL-10 cDNA. The construct was injected into eggs from (C57BL/6 x C3H)F1 females mated to C57BL/6 male, and transgenic founders were backcrossed onto the C57BL/6 background >5 times. Presence of the transgene was confirmed by PCR of mouse tail biopsy. The 5' primer sequence was 5'-TGT CCA CCA CAA TAT GCT ATT CA-3' (positions -372 to -350 of the IL-2 promoter sequence) and the 3' primer sequence was 5'-ATA CTT ACA AAG AAA GTC TTC ACC-3' (positions 1777–1800 of the IL-10 exon 1 of the mouse cDNA). The size of the resulting IL-10 PCR fragment is 800 bp. One microgram of DNA was amplified in a total volume of 50 µl, which contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 200 µM each dNTPs, 0.1 µM primers, 2.5 mM MgCl2, and 2.5 U of Taq polymerase. PCR was performed in a Perkin-Elmer (Norwalk, CT) DNA thermal cycler. The amplification profile for the IL-10 transgene consisted of 40 cycles, with the first cycle denaturation at 94°C for 3 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min, followed by 39 cycles with denaturation at 94°C for 1 min and the same annealing and extension conditions. The extension step in the last cycle was for 10 min. After amplification, the products were visualized against m.w. standards on a 1.5% agarose gel stained with ethidium bromide. IL-10 transgenic mice were bred at the West Los Angeles Veterans Affairs vivarium and maintained in the animal research facility. For all experiments, pathogen-free female transgenic mice or their control littermates (8–12 wk age) were used.
Tumorigenicity
For tumorigenesis experiments, 3LL tumor cells were inoculated by s.c. injection in the right supra scapular area in transgenic and control littermates, and tumor volume was monitored. Tumor growth was assessed three times per week following tumor implantation. Two bisecting diameters of each tumor were measured with calipers. The volume was calculated using the formula (0.4) (ab2), with "a" as the larger diameter and "b" as the smaller diameter. To determine the dose of tumor-forming established nodules in 50% of mice (TD50) in transgenic and control littermates, a range of 3LL tumor cell inoculums (2.5 x 103 to 5 x 104) was injected s.c., and percentage of tumor take in the animals was recorded. To compare tumor formation in the lungs of transgenic and control littermates, 5 x 105 3LL tumor cells were inoculated i.v. via the lateral tail vein. After 34 days, mice were sacrificed and lungs were isolated for quantitation of tumor surface area. Tumor burden was assessed by microscopic examination of hematoxylin and eosin-stained sections with a calibrated graticule (a 1-cm2 grid subdivided into one hundred 1-mm2 squares). A grid square with tumor occupying >50% of its area was scored as positive, and the total number of positive squares was determined (13).
Lymphocyte transfer
T lymphocytes were isolated from spleens of transgenic mice by Dynal beads using the manufacturer’s protocol (Dynal, Great Neck, NY). IL-10 transgenic T lymphocytes or T lymphocytes from control littermates (3.5 x 107) were transferred to each control littermate 24 h before 3LL tumor cell inoculation (5 x 105 tumor cells) and again 1 wk following tumor inoculation. Tumor volumes were assessed three times per week.
Determination of tumor nodule and splenic T cell IL-10 production
Tumor nodule-associated IL-10 protein was determined in IL-10 transgenic mice and control littermates. IL-10 protein was also determined from splenic T cells. T cells were isolated using Dynal beads coated with Thy-1.1 Ab (PharMingen, San Diego, CA), according to the manufacturer’s instructions. IL-10 production was determined by cytokine-specific ELISA in T cell supernatants after 3 days in culture or directly from homogenized tumor tissue. To address whether factors derived from tumor cells could induce the IL-10 transgene, 1 x 106 splenocytes from IL-10 transgenic mice and negative littermates were cocultured with 105 3LL cells. Following a 3-day coculture, the IL-10 secreted in the culture supernatants was measured by IL-10 ELISA.
Flow cytometry
For flow-cytometric experiments, two or three fluorochromes (PE, FITC, and Tri-color) were used to gate on the CD11c (PharMingen) bright populations of splenic APC or bone marrow-derived dendritic cells (DC) in evaluation of MHC I, MHC II, CD40, B7.1 and B7.2, and F480 (Caltag, San Diego, CA). Flow-cytometric analysis was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) in the University of California, Los Angeles, Jonsson Cancer Center Flow Cytometry Core Facility. Cells were identified as APC by gating based on forward and side scatter profiles. Between 3000 and 5000 gated events were collected and analyzed using CellQuest software (Becton Dickinson).
To determine the source of enhanced IL-10 in the tumor nodules of IL-10 transgenic mice, tumors were harvested, cut into small pieces, and passed through a sieve (Bellco, Vineland, NJ). Intracytoplasmic staining for IL-10 with a PE-labeled anti- mouse IL-10 mAb (PharMingen) and cell surface staining for CD3 with FITC-labeled anti-mouse CD3 were performed, followed by flow cytometry. Tumor cells were identified by gating on in vitro cultured 3LL cells.
Mixed lymphocyte reaction
To compare the T cell stimulatory capacity of APC from transgenic mice and the control littermates, MLR were performed utilizing BALB/c (H-2d) T cells as the responder cells. Splenic APC (H-2b) from transgenic and control littermates were purified utilizing Ab-mediated complement lysis of T and B cell populations. The following Abs, all obtained from ATCC, were utilized: TIB 207 (anti-CD4), TIB 150 (anti-CD8), TIB 146 (anti-B lymphocyte). After RBC depletion, splenocytes were incubated with a mixture of mAbs and rabbit complement for 60 min at 37°C. Following Ab-mediated depletion, APC were washed twice in complete medium. Subsequently, T cells were purified from BALB/c spleens utilizing Ab-mediated complement lysis of APC and B lymphocytes (Abs TIB 229 and TIB 146; ATCC). APC from transgenic mice and control littermates were co-cultured with splenic BALB/c T cells at varying APC:T cell ratios in 96-well tissue culture plates for 5 days. The responder cell numbers were kept constant and APC numbers were varied. On day 4, the cultures were pulsed with 1 µCi of tritiated thymidine (Amersham, Arlington, IL; sp. act., 62 Ci/mmol/L) for 18 h, and the cells were harvested onto filter mats using a Skatron Cell Harvester. The disks were air dried, placed in scintillation vials with 1 ml of scintillation fluid, and counted in a Beckman scintillation counter (Fullerton, CA). To assess T cell reactivity in IL-10 transgenic and control littermates, MLR were performed with APC from BALB/c (H-2d) mice as stimulators. MLR were also set up with day 8 matured bone marrow-derived DC from IL-10 transgenic mice and control littermates as stimulators with T cells from BALB/c mice as responders.
APC IL-12 production
APC were purified from total splenocyte suspension by Ab-mediated complement lysis of T and B cells, as described above. APC (5 x 106 cells/ml) from tumor-bearing transgenic mice and control littermates were stimulated with or without anti-CD40 (5 µg/ml). Following a 72-h culture, IL-12 secreted in culture supernatants was determined by ELISA.
Cytokine ELISA
IL-10 protein concentrations from mouse splenocytes cultured in
complete medium or 3LL cell supernatants were determined by
IL-10-specific ELISA, as previously described (3).
Briefly, 96-well Costar (Cambridge, MA) plates were coated overnight
with 4 µg/ml of anti-mouse IL-10 mAb JES5-2A5 (PharMingen). The
wells of the plate were blocked with 10% FBS (Gemini Bioproducts,
Calabasas, CA) in PBS for 30 min. The plate was then incubated with the
Ag for 1 h and excess Ag was washed off with PBS/Tween. The plate
was incubated with 1 µg/ml of biotinylated mAb to IL-10 (PharMingen)
for 30 min, and excess Ab was washed off with PBS/Tween. The plates
were incubated with avidin peroxidase, and after incubation in
o-phenylenediamine substrate, the subsequent change in color
was read at 490 nm with a Dynatech MR5000 spectrophotometer (Chantilly,
VA). The rIL-10 used in the assay as a standard was obtained from
PharMingen. For IL-2 and IFN-
determinations, the above procedure
was repeated, but with either IL-2 or IFN- Ab pairs and standards
from PharMingen. The sensitivity of the IL-10, IL-2, and IFN- ELISA
was 15 pg/ml. For IL-12 measurements, an IL-12 ELISA kit was utilized
(Biosource International, Camarillo, CA) and measurements were
performed according to the manufacturer’s instructions. The
sensitivity of the IL-12 ELISA was 5 pg/ml.
Assessment of type 1 cytokine production in vivo
To evaluate Th1 cytokine production in vivo, IL-10 transgenic
mice and control littermates were injected i.v. with staphylococcal
enterotoxin B (SEB; 50 µg) dissolved in pyrogen-free NaCl (0.9%).
The mice were bled 2 h later, and the sera were assayed for IL-2
and IFN- production by ELISA.
Assessment of CTL activity
To evaluate the function of CTL from IL-10 transgenic and control littermates, mice were vaccinated three times at weekly intervals with irradiated 5 x 106 3LL cells. Splenic lymphocyte cytotoxicity was determined against 3LL tumors, as previously described. Briefly, 5 x 106/ml (5 ml total) RBC-depleted splenocytes from IL-10 transgenic and control littermates were stimulated in the presence of 5 x 105 mitomycin C-treated 3LL cells and IL-2 (100 U/ml) for 10 days with one medium change on day 5. CTL cytotoxicity was determined against chromium-labeled (51Cr; sp. act., 250–500 mCi/mg; Amersham) 3LL targets at varying E:T ratios for 4 h in 96-well plates. Spontaneous release and maximum release with 5% Triton X also were assessed. Following the 4-h incubation, supernatants were removed and activity was determined using a gamma counter (Beckman). The percent specific lysis was calculated using the formula: percentage lysis = 100 x [(experimental cpm - spontaneous release)/(maximum release - spontaneous release)].
Isolation, in vitro propagation, and peptide loading of DC
Lymphocyte-depleted mouse bone marrow cells were cultured with murine GM-CSF (2 ng/ml) and IL-4 (20 ng/ml) (R&D Systems, Minneapolis, MN) for 8 days, as previously reported (14). Medium was replenished every other day. On day 8, nonadherent DC were isolated by pipetting. The DC were counted and washed twice in PBS, and 1 x 106 DC were prepared for loading with peptides. The MUT 1 and MUT 2 peptides, synthesized by Research Genetics (Huntsville, AL), consist of the 52–59 aa positions of the mutated connexin 37 protein present in the 3LL cell line and have the sequence FEQNTAQP and FEQNTAQA, respectively. Bone marrow-derived DC were incubated in 1 ml of RPMI with 10 µM peptide for 2 h at 37°C. Following incubation, the peptide-loaded DC were washed twice in PBS, and 1 x 106 peptide-loaded DC were administered by intratumoral injection at weekly intervals for 3 weeks in mice bearing 5-day-old established s.c. tumors that were generated previously by implanting 105 tumor cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies documented that 3LL tumors cells grew more
rapidly in IL-10 transgenic mice than did tumors in control mice. This
effect was reversed by in vivo administration of anti-IL-10 mAb
(12). To further assess tumorigenicity of 3LL cells in
IL-10 transgenic mice, mice were challenged with a range of tumor cell
inoculums (2.5 x 103 to 5 x
104 cells) and the percentage of mice with tumors
and tumor volumes were monitored. When compared with littermate
controls, the 3LL tumor cells were significantly more tumorigenic in
IL-10 transgenic mice up to inoculums of 2.5 x
104 tumor cells. The TD50 in IL-10 transgenic
mice was significantly less than the TD50 in controls (7.5 x
103 vs 2.2 x 104
cells, respectively, p < 0.01, Fig. 1
A). Because different organ
sites may have varying capacities to support or limit tumor propagation
(15), the growth of 3LL was assessed in the lungs of IL-10
transgenic mice following i.v. tumor challenge. Thirty-four days
following tumor challenge, IL-10 transgenic mice had a significantly
greater tumor burden within the lungs than did controls
(p < 0.05, Fig. 1B). Thus, the
IL-10 transgenic mice demonstrated a diminished capacity to limit tumor
growth in both s.c. and pulmonary sites.
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Based on the findings noted above, we speculated that IL-10
production would be elevated in IL-10 transgenic mice both locally, at
the tumor site, and systemically, within the spleen. To confirm that
the IL-10 transgene was functional in the IL-10 transgenic mice,
splenocyte IL-10 production was assessed in cells from transgenic and
control mice using in vitro stimulation. There were no significant
differences in constitutive IL-10 production between the groups. In
agreement with previous results (12), stimulation of
freshly isolated spleen cells with one of several stimuli, including
anti-CD3 mAb, IL-2, or Con A, led to significantly more IL-10
production in cells from IL-10 transgenic mice (data not shown). To
determine the extent of splenic IL-10 production in vivo, within the
context of the tumor model, IL-10 production by splenic T cells from
IL-10 transgenic mice and control littermates bearing 3LL tumors was
determined following a 3-day culture (Table I). In nontumor bearers, unstimulated
splenic T cells from IL-10 transgenic mice produced 354 ± 40
pg/ml of IL-10 following in vitro culture, whereas unstimulated splenic
T cells from control mice did not produce detectable IL-10. In 3LL
tumor bearers, splenic T cells from the control mice produced
detectable IL-10, but splenic T cells from IL-10 transgenic mice
produced significantly more of this cytokine than did splenic T cells
from the control mice (Table I). Following culture, unstimulated
splenic T cells from transgenic mice made significantly more IL-10
whether or not mice had been injected with tumor. The relative increase
in IL-10 production in the tumor-injected animals vs the
nontumor-bearing mice is similar for both nontransgenic and IL-10
transgenic mice. To address whether factors derived from tumor cells
could induce the IL-10 transgene, splenocytes from IL-10 transgenic
mice and negative littermates were cocultured with 3LL cells. The IL-10
secreted in the culture supernatants was measured by IL-10 ELISA.
Following a coculture with 3LL cells, there was an enhanced IL-10
production from IL-10 transgenic splenocytes (14,210 ± 36 pg/ml)
compared with splenocytes from negative littermates (3,461 ± 120
pg/ml). The 3LL tumor cells and unstimulated splenocytes from
transgenic and negative littermates produced only modest amounts (160
± 14 pg/ml, 160 ± 10 pg/ml, 173 ± 9 pg/ml, respectively). The
enhanced production of lymphocyte-derived IL-10 in normal tumor-bearing
mice has been previously described (16) and may be related
to PGE2-mediated stimulation of lymphocyte IL-10
production (17). The effect of the IL-10 transgene on
local IL-10 production was even more striking. Tumor nodules from IL-10
transgenic and control mice were analyzed directly for IL-10 following
homogenization. Tumor nodules from IL-10 transgenic mice produced
significantly more IL-10 than did tumor nodules from control mice
(Table II). To determine the source of
the enhanced IL-10 in the tumor nodules of IL-10 transgenic mice
compared with tumor nodules from negative littermates, intracytoplasmic
staining for IL-10 and cell surface staining for CD3 followed by flow
cytometry were performed. The results of these experiments show that
the predominant source of IL-10 in the tumor nodules from IL-10
transgenic mice are T cells with a very small contribution from tumor
cells. Within the gated lymphocyte population, 50% of cells stained
positively for both CD3 and intracytoplasmic IL-10 in single cell
suspensions of the tumor nodule from IL-10 transgenic mice. In
contrast, only 0.4% of 3LL tumor cells stained positively for
intracytoplasmic IL-10. Within the gated lymphocyte population, CD3
cell surface staining for T cells showed that the tumor nodules in
IL-10 transgenic mice had 3 times more T lymphocytes than tumor nodules
of negative littermates (60 vs 20%, respectively, p <
0.01).
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In both patients and murine tumor models, progressive tumor growth
has often been associated with a marked limitation in lymphocyte Th1
cytokine production along with up-regulated IL-10 and Th2 cytokines
(16, 18, 19, 20, 21). We hypothesized that, similar to tumor
bearers, lymphocytes from IL-10 transgenic mice would have a decrement
in their capacity to elaborate IL-2 and IFN- in vivo
(22). Administration of the superantigen SEB to normal
mice rapidly elicits the production of the Th1 cytokines IL-2 and
IFN- (23). This is a potent and consistent stimulus for
Th1 cytokine production in vitro and in vivo, with the early cytokine
production derived from memory and NK T cells. To assess the level of
induction of IL-2 and IFN- in vivo in IL-10 transgenic and control
mice, SEB was administered i.v. in mice with or without 3LL tumors.
After 2 h, the mice were bled and the sera assayed for IL-2 and
IFN- by ELISA. Following SEB injection, IL-10 transgenic mice
produced significantly less IFN- (120 ± 15 vs 1250 ± 50
pg/ml, p < 0.05) and IL-2 (265 ± 20 vs 670
± 100 pg/ml, p < 0.05) than the control SEB-injected
mice (Fig. 2, A and
B). Control mice bearing 3LL tumors had a reduction in IL-2
and IFN- production in response to SEB. In response to SEB,
nontumor-bearing IL-10 transgenic mice produced similar levels of IL-2
and IFN- as did control mice bearing tumors (Fig. 2, A
and B). Tumor-bearing IL-10 mice produced even lower levels
of IFN- and IL-2 than did their nontumor-bearing, IL-10 transgenic
counterparts.
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CTL induction may be viewed as a hallmark of effective antitumor
reactivity. To assess CTL activity, IL-10 transgenic mice and
control littermates were vaccinated three times at weekly
intervals with irradiated 3LL cells. Following vaccination, spleens
were isolated and splenic lymphocytes were restimulated with mitomycin
C-treated 3LL cells. CTL from IL-10 transgenic and control mice were
evaluated for cytolytic activity against parental 3LL tumors. CTL from
IL-10 transgenic mice were less effective than were CTL from control
mice in lysing 3LL targets (Fig. 3).
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Previous studies suggest that populations of T cells in the
tumor-bearing host may develop suppressor activities through the
induction of IL-10 gene expression (11, 16). We therefore
predicted that adoptive transfer IL-10 transgenic lymphocytes to
control littermates would diminish the capacity of the recipients to
limit 3LL growth in vivo. To determine whether the immunosuppressive
effect could be transferred, 3.5 x 107
IL-10 transgenic splenic T cells were administered to control mice 1
day before and 1 wk following tumor challenge. Following i.v. transfer
of IL-10 transgenic T cells to control mice, the 3LL tumor growth
revealed a similar pattern to that demonstrated in IL-10 transgenic
mice (Fig. 4), whereas transfer of T
cells from negative littermates to control mice did not alter the
growth pattern of 3LL tumors. Thus, lymphocytes from IL-10 transgenic
mice demonstrated a dominant immunosuppressive effect in control
animals, limiting antitumor responses in normal hosts following
transfer.
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Although IL-10 has been shown to directly inhibit lymphocyte activity (24), studies indicate that the predominant pathway for inhibition of lymphocyte effector functions most often depends upon the capacity of IL-10 to modify APC surface phenotype and cytokine production (5, 10, 25, 26). We therefore evaluated a range of APC activities, including the promotion of MHC alloreactivity, CTL induction, cytokine production, and the induction of antitumor reactivity in vivo.
IL-10 has previously been documented to potently inhibit the
proliferation and cytokine production of alloreactive T cells in MLR
(27). We postulated that exposure to lymphocyte-derived
IL-10 in vivo would lead to a reduction in the capacity of APC to
promote MLR. Utilizing BALB/c T cells as the responder cells, mixed
lymphocyte reactions were performed to compare the T cell stimulatory
capacity of APC from transgenic mice and the control littermates. In
comparison with cells from control mice, both splenic APC and bone
marrow-derived DC from IL-10 transgenic mice revealed a marked
reduction in the capacity to generate an MLR with allogeneic BALB/c
responder T cells (p < 0.01) (Figs. 5A and
6). Consistent with these findings, when
assessed by flow cytometry, splenic APC from IL-10 transgenic mice had
a marked reduction in the mean channel fluorescence (MCF) intensity of
B7.1, B7.2, MHC I, and CD11c expression (p <
0.05) (Table III). In contrast, when MLR
was performed with splenic T lymphocytes from IL-10 transgenic mice
with normal BALB/c APC, there was no significant difference in the
proliferative response compared with the control MLR with normal
lymphocytes (Fig. 5B). The inability to detect a difference
in T cells in this assay may reflect the subtle alteration in cytokine
pattern in naive T cells from IL-10 transgenic mice.
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). Consistent with these findings, DC from IL-10 transgenic mice
evidenced a significant reduction in the expression of B7.1 and MHC II
expression (Table IV).
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To evaluate the capacity of DC to mediate specific in vivo
antitumor effects, the in vitro activated DC were loaded with MUT 1 and
MUT 2 peptides, and their capacity to cure 5-day-old established tumors
in IL-10 transgenic and control mice was assessed. C57BL/6 DC loaded
with MUT 1/MUT 2 peptides mediated complete eradication of established
tumors in all normal control mice, but it mediated eradication in only
two of six animals when treating IL-10 transgenic mice bearing 3LL
tumors. DC from IL-10 transgenic mice loaded with MUT 1/MUT 2 peptides
were ineffective in mediating any significant tumor reduction in
C57BL/6 control mice bearing 3LL, thereby demonstrating APC as well as
T cell defects (Table V). These APC
defects were apparent even after short-term (8-day) culture in the
absence of T cells. To determine whether contaminating lymphocytes
producing IL-10 within our DC cultures were contributing to
modifications in function and phenotype, we repeated these experiments
using anti-IL-10 mAb within the medium throughout the culture
period. No restoration of DC function was observed, suggesting that the
detrimental exposure to IL-10 occurred in vivo (data not shown).
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The capacity for professional APC to produce IL-12 has been
correlated with APC functional maturation (35). To
determine the role of increased lymphocyte-derived IL-10 on splenic APC
IL-12 production, IL-12 released by IL-10 transgenic and control mice
splenic APC was assessed. Unstimulated APC from control mice, prepared
by negative selection of T and B cells, produced significantly more
IL-12 than did APC from IL-10 transgenic mice (110 ± 4.5 vs
25 ± 2.5 pg/ml, respectively; p < 0.01).
Following stimulation with anti-CD40 mAb, APC from control mice
produced a 3.5-fold greater induction of IL-12 than did the APC from
IL-10 transgenic mice (Fig. 7). To
determine whether the reduced levels of IL-12 production from APC in
IL-10 transgenic mice were accompanied by a decrement in CD40 receptor
expression by IL-10 transgenic APC, flow-cytometric analysis was
performed. APC from IL-10 transgenic mice expressed significantly less
cell surface CD40 than did controls (28% (233 MCF) vs 64% (458 MCF),
p < 0.05) (Table III
). Further studies will be
required to determine whether the decreased expression of CD40 is the
cause of limited IL-12 production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the tumor-bearing host, a decrement in type 1 cytokine production
has been suggested to play a significant role in the insufficient
induction of specific cell-mediated antitumor immunity (22, 37). Based on our previous studies (11, 12, 38) and
other recent reports (16, 39, 40), we postulated that
lymphocyte-derived IL-10 could contribute significantly to reduced type
1 cytokine production capacity in vivo. To assess this parameter, we
administered a potent type 1 cytokine inducer to IL-10 transgenic and
control mice. Superantigens, including SEB, have been demonstrated to
bind APC MHC class II molecules and stimulate a large population of
CD4+ and CD8+ lymphocytes
through interactions with specific TCR Vß phenotypes
(23). As a result of these interactions, IL-2 and IFN-,
as well as other cytokines, are potently induced. Our findings of
reduced IL-2 and IFN- release in IL-10 transgenic mice in response
to SEB are consistent with the results of Florquin et al.
(41) and Bean et al. (42). These studies
demonstrated that IL-10 limits the lethal effects of SEB challenge in
vivo. Thus, the readily induced, augmented levels of IL-10 produced by
lymphocytes in IL-10 transgenic mice may directly limit their capacity
for IL-2 and IFN- production.
Although IL-10 has been shown to have the capacity to directly suppress T cell activities (24), the inhibitory effects of IL-10 have most often been found to be mediated indirectly through modulation of APC function (5, 10, 25, 26). IL-10 has the capacity to decrease APC MHC and TAP expression and to down-regulate critical costimulatory molecules including CD54, CD80, and CD86 (26, 43, 44, 45). Based on these previously documented activities of IL-10, a range of immune parameters was evaluated in the IL-10 transgenic mice, including APC functional activities and phenotype, cytokine production, alloreactivity, CTL generation, and the capacity for DC to mediate therapeutic antitumor reactivity.
An important in vitro correlate of graft or tumor rejection, the MLR is able to discriminate lymphocyte activation that occurs as a function of disparate MHC expression. IL-10 has previously been reported to potently decrease the capacity of alloreactive cells to proliferate in MLR (27). Our analysis of MLR, with IL-10 transgenic mice-derived APC serving as stimulators, revealed a significant decrease in the proliferative response of normal BALB/c lymphocyte responder cells. In contrast, normal levels of MHC alloreactive proliferation were seen when IL-10 transgenic T cells were used as responders in MLR with normal BALB/c APC. This indicates APC obtained from the IL-10 transgenic mice have an impaired capacity to generate alloreactivity, but that the transgenic T lymphocyte responses remain relatively intact. Thus, although injection of IL-10 transgenic lymphocytes transferred the immune deficit to normal mice, these results suggest that the deficit mediating immune suppression may be predominantly related to lymphocyte-induced modification of APC function and phenotype. In accordance with these findings, APC surface phenotype in IL-10 transgenic mice differed significantly from the phenotype in control mice. We found a significant decrement in surface expression of CD11c, CD40, B7-1, B7-2, and MHC class I and II.
A decreased capacity for Ag presentation by DC has been suggested to be a critical problem responsible for limitations in tumor immunity (46). To overcome this problem, investigators are using the administration of gene-modified or Ag-pulsed, in vitro activated DC to treat tumors in vivo (47, 48, 49). Although specific peptide Ag-pulsed DC from normal mice were highly effective in treating established tumors in control mice, bone marrow-derived DC from IL-10 transgenic mice were ineffective in mediating antitumor responses. This suggests that the exposure to T cell-derived IL-10 in vivo limited the capacity of precursors to undergo functional maturation despite culture in optimal concentrations of IL-4 and GM-CSF in vitro even in the presence of blocking IL-10 mAb. In accordance with these findings, the in vitro generated DC from the IL-10 transgenic mice revealed diminished surface expression of the costimulatory and MHC molecules known to be requisite for fully functional DC Ag presentation. In contrast to our studies evaluating prolonged in vivo exposure of bone marrow-derived DC, DeSmedt et al. (50) found that short-term IL-10 exposure in vitro did not alter surface phenotype. In parallel experiments, we also assessed the capacity for Ag-pulsed DC derived from normal control mice to restore antitumor reactivity in IL-10 transgenic mice. Despite this intervention, the majority of mice succumbed to progressive tumor growth, indicating a profound suppression in the capacity to react to optimal Ag presentation in vivo.
Previous studies have suggested pathways whereby IL-10 may limit antitumor responses. Pretreatment with IL-10 protects target cells from lysis by tumor-specific cytotoxic T cells (51, 52). IL-10 has been found to be released by lymphocytes infiltrating several different tumors, including ovarian (53), lung (11, 20), and cutaneous carcinomas (21). Elevated concentrations of IL-10 within the tumor environment have been suggested to limit access of DC to the tumor site (30) and to promote DC apoptosis (36). We have found IL-10 production in cutaneous basal and squamous carcinomas and documented that this cytokine provides a mechanism for evasion of the local T cell-mediated immune response (4). In a mouse model, Wang et al. (54) demonstrated that tumor cells transfected with the IL-10 gene produce local immunosuppression and prevent the induction of CTL. These results suggest that the reported effects of IL-10 on in vitro assays of Ag presentation may correlate with its effects in situ in the tumor environment. However, the literature also contains reports from studies in murine models suggesting that high local concentration of IL-10 from IL-10-transfected tumors leads to inhibition of tumor growth (55, 56, 57). These findings appear to be consistent with the fact that IL-10 has also been shown to be a positive factor for T cell differentiation, chemoattraction, and costimulation, and is a survival factor for IL-2-deprived T cells (59, 60, 61, 62, 63) The apparent contradiction may be explained by differences in IL-10-mediated outcomes due to concentration-dependent effects. For example, the report from Giovarelli et al. (55) showed IL-10-mediated tumor reduction in transfected murine tumors producing 2000 ng/ml of IL-10/106 cells/48 h. These pharmacologic cytokine levels are not comparable with physiologic concentrations, and thus may be expected to have different effects. In normal mice-bearing 3LL, we find that tumors produce 0.6 ± 0.024 ng/ml of IL-10 per gram of tumor tissue. In contrast to studies such as Giovarelli et al. (55), in our IL-10 transgenic mouse model, we find that 3LL tumor nodules produce 14.8 ± 0.075 ng/ml of IL-10 per gram of tumor tissue. Thus, while recent studies indicate that pharmacological levels of rIL-10 or high level production of IL-10 from transduced tumors promote tumor regression, our results indicate that physiologic levels of lymphocyte-derived IL-10 inhibit tumor immunity. Thus, as suggested by Suzuki et al. (56), when IL-10 is expressed at very high local concentrations in transfected tumors, the stimulatory effects on T cells may obscure the immunosuppressive effects on Ag-presenting and accessory cell functions of DC and macrophages. As recently reported by Groux et al. (58), a similar T cell stimulatory effect may be evident in a transgenic model in which APC overproduce IL-10. In their study, IL-10 caused a biphasic effect on antitumor reactivity, initially causing tumors to grow rapidly, but eventually leading to their complete rejection.
Thus, although this cytokine has complex effects, the induction of lymphocyte IL-10 in the tumor-bearing host is likely to be a critical determinant preventing effective tumor immunity. Furthermore, our data indicate that exposure of DC to IL-10 in vivo may have long-term effects on the capacity of these cells to present Ag. This may limit the effectiveness of autologous DC in clinical trials. Understanding the pathways that regulate tumor-mediated induction of IL-10 will be of central importance in the therapeutic modulation of this immunosuppressive network.
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2 Address correspondence and reprint requests to Dr. Steven M. Dubinett, Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, 37-131 Center for Health Sciences, 10833 LeConte Avenue, Los Angeles, CA 90095-1690. E-mail address:
3 Abbreviations used in this paper: 3LL, Lewis lung carcinoma cells; TD50, tumor dose at which 50% of mice develop tumors; DC, dendritic cell; MCF, mean channel fluorescence; SEB, staphylococcal enterotoxin B.
Received for publication March 17, 1999. Accepted for publication August 25, 1999.
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S. Sharma, M. Stolina, J. Luo, R. M. Strieter, M. Burdick, L. X. Zhu, R. K. Batra, and S. M. Dubinett Secondary Lymphoid Tissue Chemokine Mediates T Cell-Dependent Antitumor Responses In Vivo J. Immunol., May 1, 2000; 164(9): 4558 - 4563. [Abstract] [Full Text] [PDF]
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