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Division of Neurosurgery and
Department of Anatomy, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA 23198
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) but not the Th2 cytokine, IL-4, when
cocultured with T9.F stimulator cells. Rats that had rejected s.c.
T9.F/IL6/hi tumors displayed a delayed-type hypersensitivity response
when injected with viable T9.F cells in the contralateral flank.
Passive transfer of spleen cells from these animals transferred glioma
immunity to naive recipients and depletion of CD3+ T cells,
before transfer, completely abolished immunity, whereas depletion of
CD8+ T cells had moderate inhibitory effects on the
transfer of immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Novel approaches using immunotherapy are currently being explored for the treatment of GBM as well as other brain and peripheral solid tumors (see Refs. 6, 7, 8, 9 for review). In these studies, the tumor cells are genetically engineered to produce immune-enhancing cytokines. The presence of these cytokines, in most cases, is believed to counteract the immunosuppressive microenvironment of the tumor, resulting in the activation local immune cells and the initiation of an antitumor response when the cytokine-secreting tumor cells are implanted into a host animal. In many studies, the genetically modified tumor cells are destroyed, and in some cases, the animal develops tumor-specific immunity against the nonmodified, parental tumor.
Pioneering studies demonstrating that tumor cells genetically altered
to secrete cytokines can induce an antitumor response were reported in
1989 by Tepper et al. with IL-4-secreting plasmacytoma cells and by
Fearon et al. in 1990 with IL-2-producing colon carcinoma cells
(10, 11). Since these initial reports, numerous additional
studies have shown similar results using different murine tumors
engineered to secrete a variety of cytokines, for example 1)
TNF-
-secreting sarcoma cells in C57BL/6 mice (12) and
plasmacytoma cells in BALB/c mice (13); 2) IL-4-secreting
renal cell carcinoma cells and colon carcinoma cells in BALB/c mice
(14); 3) IL-2-secreting mammary adenocarcinoma
(15), renal cell carcinoma (16), and melanoma
(16, 17), all performed in BALB/c mice, and fibrosarcoma
in C57BL/6 mice (18); 4) IL-7-secreting fibrosarcoma in
C3Hf/Sed mice (19); 5) IL-1-secreting fibrosarcoma in
BALB/c mice (20); and 6) GM-CSF-secreting melanoma cells
in C57BL/6 mice (21) and colon adenocarcinoma in BALB/c
mice (22).
Reports of cytokine-secreting glioma cells are fewer, and the results
are not as extensive as murine studies with other types of tumors. Ram
et al. (23) demonstrated that IL-2 secreted from
transduced 9L rat glioma cells resulted in reduced s.c. tumor
formation, but was completely lethal and conferred no survival
advantage when implanted in the brain. Tjuvajev et al.
(24) reported similar results with RG-2 rat glioma cells
also genetically altered to secrete IL-2 or IFN-. In this regard,
both the IL-2- and the IFN--secreting RG-2 cells demonstrated
attenuated s.c. tumor growth, although no s.c. tumor regression was
observed, and no increase in survival was noted when the IL-2- and
IFN--secreting RG-2 cells were implanted in the central nervous
system. Another group reported that intratumoral transfection and
expression of the TNF- gene resulted in s.c. growth
inhibition of a human glioma in nude mice (25). Thus,
cytokine gene therapy of intracranial (i.c.) gliomas presents a
formidable endeavor that may be complicated by the immunologically
privileged location.
These studies have begun to reveal the complexity of the mechanisms of the induced antitumor responses and how the primary antitumor response may ultimately result in tumor-specific immunity. However, investigations into the mechanisms are often obscured by such variables as the etiology of the tumor transfected with the cytokine gene, the specific cytokine used, the level of cytokine produced by the tumor cells, and the phenotypic makeup of the cellular infiltrate at the implantation site of the cytokine-secreting tumor cells. The early phase of an antitumor response induced by cytokine-secreting tumor cells may involve an influx of nonspecific immune effector cells, such as macrophages or granulocytes, into the implantation site (13, 14, 19, 21, 26) followed by lymphocytic infiltrate at later stages (14, 21, 26) or can be directly dependent upon T lymphocytes (11). For example, it was shown that IL-4-secreting plasmacytoma tumor cells induce massive eosinophilic infiltration into the growing tumor nodules; this results in rapid and apparently nonspecific destruction of the tumor, and lymphocytes are not involved (10, 27). Golumbeck et al. reported that the early infiltrate of murine renal cell carcinoma cells also genetically engineered to secrete IL-4 was composed of macrophages and eosinophils. T cells appeared to migrate into the rejecting tumor site during the second week, which subsequently induced tumor-specific immunity dependent upon CD8+ T cells (14). The primary antitumor responses in these two reports conform to the danger model of immunity proposed by Matzinger et al. in which an inflammatory environment is needed for the induction of antitumor immunity (28, 29). In contrast, Fearon et al. demonstrated that the antitumor response induced by IL-2-secreting colon carcinoma cells was largely dependent upon CD8+ T cells, indicating that a proinflammatory environment may not always be required (11). Dranoff et al. demonstrated, in studies involving murine melanoma cells that have been transduced with the GM-CSF gene, that the recruitment and differentiation of APCs at the tumor site is a crucial step in the elicited antitumor response, and Huang et al. showed that the generation of tumor-specific immunity is dependent on both CD4+ and CD8+ T cells in the GM-CSF-secreting tumor model (21, 30).
We have previously reported, in a modified rat T9 glioblastoma model, that T9 glioblastoma cells genetically altered to express IL-6 are less tumorigenic when implanted in the brain of syngeneic animals (31). When the IL-6-secreting glioma cells are injected s.c., they are rejected and the animals are protected against a subsequent i.c. challenge with the parental, noncytokine-secreting T9 glioma but not against a mammary adenocarcinoma. In this study, we investigate the cellular mechanisms of the IL-6-induced antiglioma response, subsequent activation of tumor-specific effector cells, and their involvement in glioma immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred female Fischer 344 rats weighing 140–160 g and ranging in age from 4–6 mo were obtained from Harlan Breeders (Indianapolis, IN). Animals were housed in a climate-controlled, American Association of Laboratory Animal Care-approved vivarium and were provided free access to rat chow and water. All experimental animal procedures have been approved by members of the Institutional Animal Care and Use Committee.
Cell lines and cell culture
All tumor cell lines were cultured in complete medium consisting of DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Life Technologies) and nonessential amino acids. Tumor cell lines were maintained as adherent monolayers in culture flasks, incubated at 37°C in an atmosphere of 5% CO2 and 95% air, and passed biweekly using 0.5% trypsin. The T9 glioblastoma and the MadB106 adenocarcinoma cell lines were provided by Dr. Gale A. Granger (Department of Molecular Biology and Biochemistry, University of California, Irvine, CA) and Dr. John C. Hiserodt (Meyers Pharmaceuticals, Irvine, CA), respectively. The T9 glioblastoma tumor was originally induced by the repeated i.v. injection of N-nitrosomethylurea in a Fischer 344 rat (32). It has been reported that T9 glioma cells are derived from the 9L glioma cell line (33). The MadB106 mammary adenocarcinoma is a selected pulmonary metastases produced by the i.v. injection of the MadB100 parental adenocarcinoma (34), which was induced in a Fischer rat given an oral dose of 9,10 dimethyl-1,2-benzanthracene by Dr. Saburo Sone (35). The Fischer rat-derived RT-2 glioma was induced by the avian sarcoma virus, and the glioma cell line was provided by Dr. Yancy Gillespi (University of Alabama, Birmingham, AL) (36). Clone T9.F was isolated from the T9 glioblastoma cell line in a clonogenic assay. T9.F glioma cells were transduced with the LXSN retroviral expression vector containing the human IL-6 cDNA and a T9.F subclone, which secreted a high level of IL-6, and IL-6 was identified and termed T9.F/IL6/hi. In culture, T9.F/IL6/hi constitutively produced IL-6 at a level of 35 ng/106 cells/48 h (31). Tumor cells were routinely monitored for contamination by mycoplasma, bacteria, and fungus.
Subcutaneous tumor implantation
Tumor cells for s.c. implantation were trypsinized, counted on a hemacytometer, and checked for viability by trypan blue exclusion. Cells were washed twice in PBS, and a final suspension of viable cells was made in PBS. The injection site of recipient animals was shaved and wiped with 70% ethanol. Tumors were induced by injecting a suspension of 1 x 106 viable cells in 100 µl of PBS, and tumor growth was monitored by measuring tumor diameter using calipers.
Intracranial tumor implantation
Tumor cells for i.c. implantation were prepared as previously mentioned for s.c. inoculation, and a final suspension of 2 x 106 viable cells per 100 µl of PBS was made. Animals were anesthetized by an i.p. injection of ketamine-HCl (87 mg/kg) and xylazine (6.5 mg/kg). The scalp hair was shaved and wiped with betadine, and an incision was made over the cranial midline. Animals were placed in a stereotactic apparatus, and bregma was located and used as a reference point for injections. A hand-held Dremel drill was used to create a shallow depression 4 mm to the right of the sagittal suture and 1 mm posterior to the coronal suture. Five microliters of the tumor cell suspension (1 x 105 cells) was injected into the posterior parietal lobe of the brain at a depth of 3.5 mm using a Hamilton syringe and a 26-gauge needle secured to the arm of the stereotactic apparatus. The needle track was sealed with bone wax to prevent tumor cell extravasation, and the incision was closed with surgical staples.
FACS analysis of tumor-infiltrating leukocytes
Animals were implanted s.c. with 1 x 106 tumor cells or i.c. with 1 x 105 cells. Ten or 17 days post inoculation, parental T9.F or T9.F/IL6/hi tumor nodules were surgically excised and forced through a 70-µm nylon cell strainer to generate a single cell suspension. The resulting cell suspension was washed twice with cold 5% FBS-PBS, and the viable leukocytes were enumerated using a hemacytometer and trypan blue exclusion. The concentration of leukocytes was adjusted to 1 x 106/100 µl and stored on ice.
Tumor-infiltrating leukocytes were analyzed by FACS using Ab markers for T cells (CD3, CD4, CD8), B cells (CD45RA), granulocytes (HIS48) (all obtained from PharMingen, San Diego, CA), and monocytes (ED1; obtained from SeroTec, Raleigh, NC). mAbs were either directly conjugated to fluorescent markers (FITC or PE) or were biotinylated and were used at dilutions recommended by the supplier.
Cell surface staining was performed using standard methodology. Briefly, 1 x 106 leukocytes were stained in a V-bottom 96-well microplate in a volume of 50 µl of 5% FBS-PBS containing a mixture of three different mAbs (0.5 µg of each purified mAb) for 30 min on ice. Cells were then washed twice with 200 µl of 5% FBS-PBS and, in the case of mAbs directly conjugated to fluorescent markers, were resuspended in 200 µl of 5% FBS-PBS containing 1% paraformaldehyde and stored in the dark at 4°C. In the case of biotinylated mAbs, cells were then incubated on ice for 30 min in a 50-µl volume of 5% FBS-PBS with a streptavidin-peridinin chlorophyll protein conjugate (Becton Dickinson Immunocytometry Systems, San Jose, CA) used at a 1:10 dilution. Cells were then washed twice with 250 µl of 5% FBS-PBS and were resuspended in 200 µl of 5% FBS-PBS containing 1% paraformaldehyde and stored in the dark at 4°C. Three-color FACS analysis was performed using a Coulter Epics XL-MCL Flow Cytometer (Miami, FL).
Histology
Subcutaneous tumors were carefully excised from sacrificed animals and were preserved in a 10% neutral buffered formaldehyde solution. Tissue samples were next embedded in paraffin, sectioned into 5-µm slices, mounted on glass slides, and stained with hematoxylin and eosin for microscopic analysis and photography.
Induction of neutropenia
Rats were made neutropenic by i.p. injection of methotrexate (2 mg/ml PBS, pH 7.5, Sigma) for 5 consecutive days (37, 38). The next day, animals were injected s.c. with 1 x 106 T9.F/IL6/hi cells. Rats received additional injections of methotrexate on days 5, 6, 12, 13, 19, and 20 post-T9.F/IL6/hi inoculation. The spleens of sentinel animals were analyzed by FACS using a granulocyte marker (HIS48) on the day of T9.F/IL6/hi injection and after the first and second weeks to confirm the duration of neutropenia.
In vitro tumor-specific effector cell responses
Spleens were aseptically removed from animals with 17-day-regressing T9.F/IL6/hi s.c. tumors or age-matched control rats and forced through a 70-µm nylon cell strainer to generate single cell suspensions. Erythrocytes were lysed with Tris-buffered ammonium chloride (5 ml/spleen); spleen cells were washed twice with PBS and counted on a hemacytometer, then viability was assessed by trypan blue exclusion. Spleen cells were cultured at a concentration of 1 x 106 cells/ml in RPMI 1640 (Life Technologies) supplemented with 10% FBS, 0.05 mM 2-ME, and 50 U/ml of recombinant human IL-2 (Chiron Therapeutics, Emoryville, CA) in the presence of irradiated T9.F, RT-2, or MadB106 stimulator cells adjusted to 50:1 (splenocyte/stimulator cell) ratio and were used in several immune assays to detect the presence and functions of tumor-specific effector cells.
Spleen cells from naive or T9.F/IL6/hi-primed animals that were cultured for 4 days with T9.F stimulator cells were used in a 5-h cytotoxicity assay with 51Cr-labeled T9.F, RT-2, or MadB106 viable target cells at various E:T ratios. Briefly, 1 x 106 viable target cells were incubated with 100 µCi of Na2CrO4 (Amersham Pharmacia Biotech, Piscataway, NJ) in 200 µl of PBS supplemented with 20% FBS for 3 h at 37°C, washed twice with PBS, and resuspended to a final concentration of 1 x 106 cells/ml in RPMI 1640 supplemented with 10% FBS, 0.05 mM 2-ME, and 10 mM HEPES (Sigma, St. Louis, MO). Five thousand target cells were added to each well of a 96-well V-bottom microplate. Spleen cells were suspended in the same medium and were added to the wells to achieve E:T ratios of 100:1, 50:1, and 25:1 in a final volume of 200 µl/well. The microplates were briefly centrifuged to initiate cell contacts and were incubated at 37°C. At the end of 5 h, the microplates were centrifuged, 100 µl of the cell-free supernatants were harvested, and the amount of 51Cr (experimental release, ER) was determined in a beta counter. Spontaneous 51Cr release (SR) was assessed from wells containing target cells only and total release (TR) was determined from similar wells receiving 1% SDS. Percent cytotoxicity was calculated from the following formula: % cytotoxicity = [(ER) - (SR)]/[(TR) - (SR)] x 100. Data are expressed as % cytotoxicity of the mean of triplicate wells ± SD.
The production of cytokines by the spleen cells in response to the
various tumor stimulator cells was assessed. Briefly, naive or
T9.F/IL6/hi-primed spleen cells (1 x
106/ml) were cultured for 3 days in the presence
of T9.F, RT-2, or MadB106 stimulator cells. The cell-free supernatants
were analyzed by ELISA for the presence of IL-2, IL-4 (both obtained
from PharMingen), and IFN- (BioSource International, Camarillo, CA).
Cultures were set up in triplicate, and the concentration of cytokines
is expressed as pg/ml ± SD. In addition, the spleen cells
cultured for 4 days with T9.F stimulator cells were stained with mAbs
for the detection of intracellular IFN- by FACS analysis. Briefly,
spleen cells were stained for phenotypic identification as described
above using anti-CD4 (PE conjugated) and anti-CD8 (peridinin
chlorophyll protein conjugated). Cells were then fixed, permeabilized,
and stained using a Cytofix/Cytoperm Plus Kit (PharMingen) and a mAb
for IFN- (FITC conjugated; SeroTec).
The proliferation of naive or T9.F/IL6/hi-primed spleen cells in
response to T9.F, RT-2, or MadB106 stimulator cells was assessed by the
incorporation of [3H]TdR. Immediately after
spleen cells were harvested from naive or T9.F/IL6/hi-primed rats,
cells were placed into culture in a 96-well flat-bottom tissue culture
plate at a concentration of 2 x 105 cells
per well. Stimulator cells were added to the wells in a 50:1
splenocyte-stimulator cell ratio in a final volume of 200 µl/well and
were cultured for 4 days. During the last 15 h of culture, spleen
cells were pulsed with 1 µCi of [3H]TdR
(Amersham Pharmacia Biotech). The cultures were then stored at
-20°C. Incorporation of [3H]TdR was used as
a measure of proliferation and was analyzed using a 96-well plate
harvester and a 
-plate reader (Packard, Meriden, CT). Data are
expressed as cpm of the mean of triplicate wells ± SD.
Detection of delayed-type hypersensitivity (DTH) responses
For the assessment of a DTH response, 5 wk after animals had rejected an initial s.c. injection of 1 x 106 T9.F/IL6/hi cells, rats were injected in the contralateral flank or hind foot pad with 1 x 107 viable T9.F parental cells. In the case of foot pad injections, the DTH response was monitored by daily measurements of foot pad swelling with a thickness gauge micrometer. The swelling response was calculated by subtracting the thickness of the noninjected hind foot pad from that of the experimental foot pad to yield a corrected foot pad swelling value.
Depletion of CD3+ or CD8+ T cells and passive transfer of tumor immunity
Spleens were harvested from T9.F/IL6/hi-primed animals 10 days
after injection of parental T9.F cells in the contralateral flank and
the appearance of a DTH reaction. The generation of single cell
suspensions and the lysis of erythrocytes were performed as described
above. The concentration of spleen cells was adjusted to 1 x
108 cells/ml PBS. For the depletion of
CD8+ T cells, 1 x 108
spleen cells were added to individual tubes in a volume of 1 ml PBS,
then 50 µg of purified anti-rat CD8 IgG2 (clone G28;
PharMingen) was added to each tube and incubated for 45 min on ice.
Spleen cells used for complement controls did not receive the addition
of mAbs. Cells were then washed once and resuspended in 5 ml of PBS.
Cedarlane Low-Tox-M rabbit complement (Accurate Chemical & Scientific,
Westbury, NY) was added to the spleen cells at a 1:10 dilution and
incubated at 37°C for 1 h. Cells were then washed once and
resuspended in 1 ml of PBS. For the depletion of
CD3+ T cells, the same procedure was followed
using 67 µg of purified anti-rat CD3 IgM (clone 1F4; SeroTec).
Depletion of CD8+ T cells was verified by FACS,
and depletion of CD3+ T cells was verified by
hemacytometer and trypan blue exclusion counts. Naive animals were
injected with 1 x 108 complement control
spleen cells, spleen cells depleted of CD8+ or
CD3+ T cells. The next day, animals were
implanted i.c. with 1 x 104 parental T9.F
cells.
Statistics
Statistical analysis was performed using the Student paired t test, and differences with a p value of <0.05 were considered significant. In the case of survival plots, results were analyzed using the LIFETEST procedure (SAS Institute, Cary, NC) and significance was determined using the Log-Rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To understand the potential cellular mechanisms of the antitumor
response elicited by IL-6-secreting T9.F glioma cells, we studied the
cellular infiltrate of s.c. and i.c. tumor nodules at an early (day 10)
and a late (day 17) time point. The leukocytic tumor infiltrate was
analyzed by FACS using mAb markers for T cells (CD3, CD4, and CD8),
B cells (CD45RA), granulocytes (HIS48), and monocytes (ED1).
For the study of the s.c. antitumor response, animals were implanted
with 1 x 106 parental T9.F or T9.F/IL6/hi
cells (three rats per group), and the tumor nodules were excised 10
days postinjection. Representative dot plots of the leukocytic
infiltrate of T9.F and T9.F/IL6/hi tumors stained with mAbs for
granulocytes and monocytes are shown in Fig. 1
and illustrate a significant increase
of the number of granulocytes present in the T9.F/IL6/hi tumor as
compared with the non-IL6-secreting T9.F parental tumor
(p = 0.013). At this time point, there were no
significant differences in the infiltration of
CD4+ or CD8+ T cells, B
cells, and monocytes/macrophages between the IL-6-secreting T9.F
gliomas and the parental T9.F gliomas (data not shown). Histological
examination of the parental T9.F and T9.F/IL6/hi day 10 tumors, also
shown in Fig. 1, revealed that neutrophils were the predominant subset
of granulocytes infiltrating the T9.F/IL6/hi tumors. Neutrophils were
noticeably absent in the parental T9.F tumors. FACS analysis was also
performed on the leukocytic infiltrate of 17-day s.c. tumors. At this
time point, parental T9.F tumors are progressing (tumor diameter 
10
mm), whereas T9.F/IL6/hi tumors are rapidly regressing (tumor diameter
5 mm) (32). The granulocytic infiltration of
T9.F/IL6/hi tumors that had been observed at day 10 was absent and
appeared to be replaced by a T cell infiltrate (Fig. 2). Day 17 parental T9.F s.c. gliomas
were also void of granulocytes and only modestly infiltrated with T
cells (data not shown).
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. At day 10, there was a modest degree
of infiltration by granulocytes in the IL-6-secreting tumor, which
increased considerably by day 17. This is in contrast to s.c.
T9.F/IL6/hi tumors, where the granulocytic infiltrate had subsided by
day 17. The difference in the time period of the granulocytic
infiltrate may be explained by the observation that immune reactions
tend to be delayed in the brain. Interestingly, we have shown
that the survival of animals implanted in the brain with
T9.F/IL6/hi cells was significantly extended, although the animals
ultimately succumbed to their i.c. tumor whereas 70% of the animals
implanted s.c. with T9.F/IL6/hi cells completely rejected their tumors
(32).
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To provide support for a functional role for the presence of
neutrophils in the IL-6-induced antitumor response, rats were depleted
of neutrophils with methotrexate treatment for 5 consecutive days
before the s.c. implantation of T9.F/IL6/hi cells. Animals received two
additional treatments of methotrexate per week for the duration of the
study. Control animals did not receive methotrexate injections. Tumor
development and growth were monitored by daily measurements of tumor
diameter. The results of a 3-wk study are shown in Fig. 4. At the conclusion of the experiment,
100% (5/5) of the neutropenic had progressively growing T9.F/IL6/hi
tumors at the injection site (mean tumor diameter = 10.9 ±
2.2 mm SD). Over the same period, all but one of the control animals
(5/6) had completely rejected their T9.F/IL6/hi tumors.
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The results from the previous experiments suggested that neutrophils may play a role in the antitumor response elicited by T9.F/IL6/hi cells; however, animals ultimately develop long-lasting tumor-specific immunity. We used several in vitro immune assays to detect the presence of activated tumor-specific effector cells in the circulation 17 days after s.c. inoculation of T9.F/IL6/hi cells. Spleen cells were obtained from naive or T9.F/IL6/hi-primed rats and were cocultured with several syngeneic, irradiated stimulator cells (T9.F glioma, RT-2 glioma, or MadB106 adenocarcinoma) at a 50:1 (splenocytes/stimulator) ratio, and tumor-specific effector functions were evaluated in terms of cytotoxicity, cytokine secretion, and proliferation.
The supernatants from 3-day cocultures were assayed by ELISAs specific
for the following rat cytokines: IL-2 and IFN- (Th1 associated) and
IL-4 (Th2 associated). Fig. 5 illustrates
that T9.F/IL6/hi-primed spleen cells secrete significant levels of IL-2
and IFN- in response to T9.F stimulator cells. IL-4 was not detected
in the coculture medium. T9.F/IL6/hi-primed spleen cells cocultured
with T9.F stimulator cells for 4 days were stained for FACS analysis
using mAbs (anti-CD4 and CD8) for phenotypic identification,
followed by intracellular staining for IFN- using a mouse
anti-rat IFN- mAb. The results shown in Fig. 5 indicate that
both CD4+ helper T cells and
CD8+ CTLs produce IFN- when stimulated by T9.F
glioma cells. The results of a proliferation assay in which spleen
cells were cocultured with several stimulator cells for 4 days and
pulsed with [3H]TdR for the last 15 h
demonstrated that spleen cells from T9.F/IL6/hi-primed rats
specifically proliferate in response to T9.F stimulator cells (Fig. 6). Naive and T9.F/IL6/hi spleen cells
that were cultured for 4 days with T9.F stimulator cells were used in
5-h cytotoxicity assays with different
51Cr-labeled target cells. These experiments
showed that T9.F/IL6/hi-primed spleen cells specifically killed T9.F
glioma cells and that naive spleen cells were not cytotoxic to T9.F
target cells (Fig. 7).
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Five weeks after the initial implantation of T9.F/IL6/hi cells,
animals were challenged in the contralateral flank or hind foot pad
with 1 x 107 viable parental T9.F cells.
Naive animals developed a tumor at the injection site that grew
progressively. In contrast, T9.F/IL6/hi-primed rats exhibited a DTH
reaction at the site of injection. In animals that were challenged in
the contralateral flank, induration and erythema were detectable within
24 h after the challenge and had reached peak intensity between
days 2 and 3. Fig. 8 shows the evaluation
of the DTH response by measuring the foot pad swelling in response to
the T9.F challenge in the hind foot pad on the opposite side in which
T9.F/IL6/hi cells were initially implanted. Mean foot pad swelling from
the inflammatory reaction was the greatest between the first 24 h
and the 4th day. Swelling began to subside 5 days after the challenge
and by day 10, the DTH response was barely detectable. The appearance
of a DTH response in T9.F/IL6/hi-primed rats indicated the presence of
a tumor-specific, cell-mediated immune memory response.
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Passive transfer of 1 x 108 spleen
cells harvested from T9.F/IL6/hi-primed animals 11 ± 1 days after
injection of parental T9.F cells in the contralateral flank and the
appearance of a DTH reaction into naive recipients resulted in immunity
to i.c. challenge with 1 x 104 T9.F
parental cells (7/7 survivors) as shown in Fig. 9. However, transfer of 1 x
108 spleen cells from the T9.F/IL6/hi-primed rats
depleted of T cells by anti-CD3 Ab and complement completely
abrogated the transfer of immunity (0/6 survivors), and the depletion
of CD8+ T cells only had moderate effects on the
transfer of T9.F immunity in that 29% of the animals succumbed to the
i.c. T9.F challenge (5/7 survivors). These results suggested that the
induced T9.F immune memory was partially dependent upon
CD8+ T cells while being completely
CD3+ T cell dependent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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70% of the animals rejected the IL-6-secreting T9.F glioma, and
tumor rejection was significantly blocked by the addition of
neutralizing anti-IL-6 Abs to the tumor inoculum (31).
In this study, we demonstrate that the early antitumor response invoked
by T9.F/IL6/hi cells may be mediated by neutrophils and that
glioma-specific T cells are activated and in circulation 17 days after
the s.c. injection of T9.F/IL6/hi cells. Moreover, animals that reject
T9.F/IL6/hi s.c. tumors develop tumor-specific,
CD3+ T cell-dependent immunity. Other researchers have also reported that IL-6 expression by tumor cells can result in reduced tumorigenicity. Dougherty et al. demonstrated that murine fibrosarcoma cells genetically modified to express human IL-6 produced fewer lung metastases when injected i.v. and attenuated tumor formation when injected s.c. in syngeneic mice (39). They noted a significant infiltration of macrophages in the IL-6-secreting fibrosarcomas growing s.c., suggesting an important role for IL-6 in the recruitment and proliferation of tumor-associated macrophages. In an earlier study, Porgador et al. reported that expression of human IL-6 by Lewis lung carcinoma cells also resulted in decreased s.c. tumor formation and suppressed lung metastasis when injected i.v. in syngeneic mice (40). However, when these IL-6-secreting carcinoma cells were injected in nude mice, the s.c. tumor growth was reduced but there was no suppression of lung metastasis. This observation suggested that T cells, as well as other effector cells, were involved in the IL-6-elicited antitumor response. Interestingly, in the case of murine fibrosarcoma cells transduced with the murine IL-6 gene, Mullen et al. (41) reported a reduction of pulmonary nodule development in an experimental metastasis model in normal, nude, and sublethally irradiated mice, which also suggests a non-T cell-mediated antitumor mechanism. However, when the IL-6-secreting fibrosarcoma cells were injected s.c., reduced tumorigenicity was observed in normal mice but not in nude or sublethally irradiated mice. These results implicate an alternative, T cell-dependent antitumor response, which is supported by the fact that mice that reject the s.c. IL-6-secreting tumors were resistant to a subsequent challenge with wild-type tumor. It is noteworthy that nude mice are void of T cells, and sublethal irradiation of mice primarily affects circulating lymphocytes; therefore, in the above immunocompromised animal models, nonlymphocyte immune cells, such as granulocytes, are functional and may be mediators of the non-T cell antitumor response. Current data suggest that nonspecific immune cells play an integral role in the IL-6-mediated response; however, we believe that we are the first to report a functional role for neutrophils in the IL-6-induced antitumor response.
In the T9.F/IL6/hi glioma model, s.c. tumors progress in size for 12
days postinjection at which point a majority of the tumors begin to
regress, as detected by measurement of the tumor diameter
(31). FACS and cytological analysis of the leukocytic
infiltrate of T9.F/IL6/hi 10-day s.c gliomas revealed that the
IL-6-secreting gliomas were significantly infiltrated by neutrophils.
Rats made neutropenic failed to reject T9.F/IL6/hi s.c. tumors,
suggesting a functional role for neutrophils in the antitumor response.
Although neutrophils are the major leukocytic population targeted by
methotrexate, one must consider that there are marginal side effects on
other cells of the immune system (42); therefore, the lack
of tumor protection in methotrexate-treated animals injected with
T9.F/IL6/hi cells cannot unequivocally be attributed to neutropenia. In
17-day regressing T9.F/IL6/hi s.c. tumors, the granulocytic infiltrate
was not as pronounced as observed in 10-day tumors, whereas the number
of both CD4+ and CD8+ T
cells was increased. Similar FACS analysis was performed on i.c.
T9.F/IL6/hi gliomas. In contrast to s.c. T9.F/IL6/hi gliomas, the
granulocytic infiltrate was moderate in day 10 i.c. gliomas and
was most pronounced at day 17, demonstrating that an antitumor response
can be elicited in the brain by the IL-6-secreting glioma cells, but it
appears to be delayed by 1 wk, suggesting that the immune-privileged
environment of the brain may delay the influx of granulocytes. We have
reported that animals implanted in the brain with the IL-6-secreting
T9.F glioma cells have a significant extension of survival but
ultimately succumb to i.c. glioma burden (31).
Although it appears that neutrophils may be responsible for the initial
antitumor response elicited by T9.F/IL6/hi cells, the animals
ultimately develop long-lasting, tumor-specific immunity that is T cell
dependent. Therefore, we used several in vitro immune assays to detect
the presence and assay the effector functions of activated,
circulating, tumor-specific lymphocytes 17 days after s.c. inoculation
of T9.F/IL6/hi cells. In a proliferation assay, when spleen cells from
T9.F/IL6/hi-primed animals were cocultured with irradiated T9.F glioma,
RT-2 glioma, or MadB106 adenocarcinoma cells, the splenocytes
specifically proliferated only in the presence of T9.F cells. ELISA
results demonstrated that T9.F/IL6/hi-primed spleen cells secreted IL-2
and IFN- (Th1 cytokines), but not the Th2 cytokine IL-4, when
cocultured with T9.F cells, whereas intracellular FACS of the same
spleen cells showed that CD4+ helper T cells and
CD8+ T cells were positive for IFN- (4.9 and
6.9% positive, respectively). When one considers that in a rat,
35% of total spleen cells are CD3+ and that
1 in 1 x 106 circulating T cells in a
naive animal or 1 in 1 x 104 T cells in a
fully immunized animal is specific for a particular Ag, the low
percentages of CD4+ and
CD8+ T cells staining positive for intracellular
IFN- can be justified (43). Lastly, primed spleen cells
selectively lysed T9.F glioma cells in chromium release assays, which
indicates the generation of glioma-specific CTLs. In a previous study,
we reported that spleen cells obtained from rats primed with T9 glioma
cells expressing the membrane-associated isoform of macrophage-CSF
proliferated in culture when restimulated with T9 glioma cells but
failed to lyse T9 target cells in killing assays (44).
Recently, Sepulveda et al. reported a novel, CD28- and IL-2-independent
pathway in which naive CD8+ T cells could be
activated by TCR ligation and costimulatory signals from IL-6 and
TNF- and that the activated T cells were strongly cytolytic toward
Ag-specific targets (45). It is enticing to consider that
the secreted IL-6 from the T9.F/IL6/hi tumors may play a costimulatory
role in the activation of glioma-specific CTLs via this alternative
pathway.
Subcutaneous injection of viable, parental T9.F glioma cells in the contralateral flank from which T9.F/IL6/hi tumors were rejected induced a DTH reaction, and therefore points to the generation of T9.F glioma-specific, cellular immunity. To identify lymphocyte populations responsible for cellular memory, we conducted a series of passive transfer experiments in which spleen cells obtained from T9.F/IL6/hi-immunized rats were depleted of specific T cell subsets and were injected to naive recipients. The recipients were then implanted i.c. with a lethal dose of parental T9.F glioma cells. The results of these experiments indicated that the induced T9.F cellular memory is partially dependent upon CD8+ T cells and is completely CD3+ T cell dependent.
It is tempting to hypothesize that at the early stage of the
IL-6-induced antitumor response, neutrophils infiltrating the s.c.
T9.F/IL6/hi tumors become activated and initiate tumor destruction by
their effector functions, i.e., respiratory burst, degradative enzyme
release, and TNF- secretion. We are currently investigating the
potential cytolytic activity of neutrophils on T9.F/IL6/hi cells. APCs
may then phagocytize tumor cell debris, migrate to the tumor-draining
lymph node, and present tumor-specific Ags to naive T cells. Activated
T cells then traffic to the tumor site and lyse the remaining tumor
cells. Activation of glioma-specific T cells and their subsequent
effector functions may also be enhanced due to the presence of IL-6. A
fraction of the glioma-specific T cells become memory cells and are
responsible for the maintenance of glioma immunity.
We believe that successful treatment of the GBM will encompass the formation of a cellular immune response consisting primarily of glioma-specific T cells. We have demonstrated in a modified rat T9 glioblastoma model that glioma cells genetically engineered to secrete IL-6 invoke an effective, antitumor response in which the early stages may be mediated by neutrophils. During the later phase of the antitumor response, in which the T9.F/IL6/hi tumor is beginning to regress, activated glioma-specific T cells are present in the circulation and proceed to generate glioma-specific T cell-dependent immunity. These findings may be important in the clinical application of IL-6-transduced glioma cells for the treatment of malignant brain tumors, where such vaccines could be used to prevent tumor recurrence after initial surgical resection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martin R. Graf, Department of Anatomy, Virginia Commonwealth University, Medical College of Virginia Station, P.O. Box 980709, Richmond, VA 23298-0709.
3 Abbreviations used in this paper: GBM, glioblastoma multiforme; i.c., intracranial; 51Cr, 51-chromium; TdR, tritiated thymidine; DTH, delayed-type hypersensitivity.
Received for publication February 22, 2000. Accepted for publication September 29, 2000.
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