| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1
* Department of Microbiology, State University of New York, Buffalo, NY 14214;
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912;
Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263; and
Lymphoma Biology Program, Wilmot Cancer Center, University of Rochester Medical Center, Rochester, NY 14642
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Serum levels of human IFN- in mice
bearing rhIL-12-treated tumor xenografts correlate directly with the
degree of tumor suppression, while neutralizing Abs to human IFN-
abrogate the IL-12-mediated tumor suppression. Gene expression
profiling of tumors responding to intratumoral rhIL-12 demonstrates an
up-regulation of IFN- and IFN--dependent genes not observed in
control-treated tumors. Genes encoding a number of proinflammatory
cytokines, chemokines (and their receptors), adhesion molecules,
activation markers, and the inducible NO synthase are up-regulated
following the introduction of rhIL-12, while genes associated with
tumor growth, angiogenesis, and metastasis are decreased in expression.
NO contributes to the tumor killing because an inhibitor of inducible
NO synthase prevents IL-12-induced tumor suppression. Cell depletion
studies reveal that the IL-12-induced tumor suppression, IFN-
production, and the associated changes in gene expression are all
dependent upon CD4+ T cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Work in animal tumor models, however, has suggested that
CD4+ T cells have a much broader role in
mediating antitumor effector functions that are independent of
CD8+ T cells (4, 5, 6). For example, it
has been established in mice that CD4+ T cells
can reject tumors without coparticipation of CD8+
T cells, and in the absence of direct interaction between effector T
cells and tumor cells. In such studies, adoptive transfer of
CD4+ T cells (in the absence of
CD8+ T cells) led to the complete elimination of
tumor cells that failed to express MHC class II Ags, indicating an
indirect mechanism of tumor killing (7, 8, 9, 10). The indirect
effects of CD4+ T cells appear to be mediated
through cytokines produced by both Th1 and Th2 cells, including IFN-
(11), which leads to the activation of accessory cells
including eosinophils and tumoricidal macrophages (12).
CD4+ T cells may be particularly important in the
immune response to tumors that have lost MHC class I, because direct
CD8+ T cell tumor recognition is precluded in the
absence of MHC class I expression. This has been demonstrated in mice
in which vaccination with tumor cells lacking MHC class I resulted in
tumor rejection that was dependent upon CD4+ T
cells and NK cells, but not CD8+ T cells
(13). Although these in vivo animal models have been used
effectively to establish and define a significant role for
CD4+ T cells in the antitumor response in mice
and rats, it has heretofore not been possible to design and conduct
similar in vivo studies on human cancer patients for obvious ethical
reasons.
We have recently evaluated the antitumor activity of cancer patients’
lymphocytes in vivo by coengrafting PBL with the patient’s autologous
tumor cells into SCID (3) mice. Using this human-mouse
chimeric (SCID-Winn) model, it was established that
CD4+ T cells mediated an IL-12- and
IFN--dependent suppression of tumor growth in vivo that was
independent of CD8+ CTL (14).
Although the SCID-Winn model does provide valuable insights with
respect to the role of human CD4+ T cells in
tumor immunity that cannot be studied using strictly in vitro systems,
there are several limitations of this approach. Because the tumor cells
and PBL are injected together as single cell suspensions, the antitumor
activity of the human leukocytes is initiated in the absence of a true
tumor microenvironment. As a result, the SCID-Winn model fails to
account for the many complex interactions among the tumor-associated
inflammatory leukocytes, stromal fibroblasts, and endothelial cells, as
well as the potential tumor-induced immunosuppression that may evolve
from the production and release of angiogenic factors, chemokines,
cytokines, or other bioactive molecules such as matrix
metalloproteinases
(MMPs)3 and PGs. To
overcome these limitations, a more physiologically relevant model has
been developed in which nondisrupted pieces of human tumor biopsy
tissues (obtained from patients with primary lung tumors) are
surgically implanted into SCID mice. The resulting xenografts maintain
an intact human tumor microenvironment consisting of tumor cells,
inflammatory cells, fibroblasts, an extracellular matrix, and blood
vessels of human origin (15). Previous work in this
primary tumor xenograft SCID model has established that the patients’
tumor-associated inflammatory cells remain viable and responsive to
cytokine stimulation for prolonged periods (16).
In this study, we demonstrate that CD4+ T cells
present within the tumor microenvironment of human tumor xenografts are
activated by the local and sustained release of human rIL-12 (rhIL-12)
to secrete human IFN-, which results in the suppression of tumor
growth in vivo. Changes in gene expression within the microenvironment
of rhIL-12-treated tumor xenografts have been characterized, which
suggest several indirect IFN--dependent mechanisms contribute to the
observed tumor suppression, including the activation of inducible NO
synthase (iNOS).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C.B-17 scid/scid mice were obtained from the breeding
colony at Roswell Park Cancer Institute. The original breeding stock
was provided by B. Phillips (Hospital for Sick Children, Toronto,
Ontario, Canada) with permission from M. Bosma (Fox Chase Cancer
Center, Philadelphia, PA). SCID mice were maintained in microisolation
cages (Lab Products, Maywood, NJ) under pathogen-free conditions.
Animals of both sexes were used for tumor engraftment at 8–12 wk of
age; however, all mice within a single experiment were age and sex
matched. SCID mice were depleted of NK cells with a mAb (TM-
1) to
the murine IL-2R -chain (17). A single i.p. injection
of TM-1 ascites fluid (100 µl, diluted 1/2 in PBS) was given 
30
min before surgical implantation of tumor tissue. The TM-1-producing
hybridoma was kindly provided by T. Tanaka (Tokyo Metropolitan
Institute of Medical Science, Tokyo, Japan).
Patient tumor specimens
Fresh surgical specimens of human lung tumors (non-small cell lung carcinomas) were obtained from the Tissue Procurement Service at Roswell Park Cancer Institute under sterile conditions. All tumor specimens were from patients who gave their informed consent before surgery, and definitive diagnoses for each specimen were obtained from the postsurgery pathology report. Each experiment was repeated with at least three different patient tumors with similar results; however, in some of the figures, representative data from a single patient’s tumor are presented.
Preparation of microspheres
rhIL-12 (1.7 x 107 U/mg) was a generous gift from S. Wolf (Genetics Institute, Cambridge, MA). A phase-inversion nanoencapsulation technique was used for encapsulation of cytokines into biodegradable microspheres, as previously described (18). Briefly, BSA (RIA grade; Sigma-Aldrich, St. Louis, MO), polylactic acid (m.w. 24,000; Birmingham Polymer, Birmingham, AL), with or without rhIL-12 in methylene chloride (Fisher, Pittsburgh, PA), was rapidly poured into petroleum ether (Fisher) for formation of microspheres (1–5 µm). Microspheres were filtered and lyophilized overnight, for complete removal of solvent. Two formulations containing 10% BSA (w/w) were produced: 1) control (no cytokines), and 2) IL-12 (1.0 mg (1.7 x 107 U)/mg polylactic acid). For injection into mice, microspheres were weighed out into sterile microfuge tubes, resuspended in PBS + 0.5% BSA, and sonicated twice for 15 s to achieve hydration. Suspended, rehydrated microspheres were then injected directly into tumor xenografts (see below).
Surgical implantation of lung tumor tissue into SCID mice
Human lung tumor tissue (from a fresh surgical specimen for
primary engraftment, or harvested from SCID mice for secondary passage)
was carefully examined, and all macroscopically normal or necrotic
tissue was removed. Tissue was cut into pieces (1–2
mm3 using a surgical scalpel), and kept
moist with sterile PBS. Mice were injected with Avertin (Aldrich
Chemical, Milwaukee, WI; 0.012 g/ml, i.p.) to induce anesthesia. A
small incision was made on the abdominal skin (ventral midline) and
undermined to produce a s.c. pocket (5–7 mm). Tumor pieces (4–5
pieces for a total of 50 mg/mouse) were implanted into the pocket,
and the incision was closed with a surgical staple. Each mouse received
pieces of tumor tissue chosen randomly from different areas of the
original tumor specimen to ensure that the distribution of tumor and
tumor-infiltrating lymphocytes among mice was equivalent. Samples of
the original patient tumor specimen (i.e., pre-engraftment) were
processed for analysis of gene expression and/or immunohistochemical
staining (see below). Secondary (i.e., tumor-infiltrating
lymphocyte-depleted) xenografts were established by serial passage of
tumor tissue from one SCID mouse to another (19). Tumor
tissue was passaged three to four times before use in an experiment,
and the absence of human CD45+ cells was
confirmed by immunohistochemistry (see below).
Treatment of xenografts, tumor measurements, and statistics
Seven days after surgical implantation of tumor tissue, mice were randomized into control and treatment groups (4–5 mice/group) and given a single intratumoral injection of microspheres (2.0 mg/100 µl/mouse) using a 281/2-gauge needle attached to a 0.5-ml insulin syringe. Following treatment on day 0, s.c. tumors were measured every 5–7 days (using engineer’s calipers) for 90 days or until tumor diameter reached >10 mm in two dimensions, at which point all mice in the experiment were sacrificed. Tumor volumes were calculated using the formula: (A2 x B)/2, in which A and B are the length (in mm) of the shortest and longest dimensions, respectively. Statistical significance among the different treatment groups was determined using the unpaired Student’s t test to compare mean final tumor volumes.
Processing of tumor tissue and mouse sera
Mice were sacrificed at different time points postmicrosphere treatment (e.g., 6 h, 24 h, 5 days, etc.), and xenografts were removed for subsequent analyses. Tissue to be used for gene expression studies (i.e., for RNA isolation) was transferred to a sterile cryopreservation vial containing 1 ml RNAlater (Ambion, Austin, TX) and stored at -20°C. Tissue was saved for histologic analysis (immunohistochemistry) by fixation in 10% neutral buffered Formalin. Tumor tissue to be analyzed by flow cytometry was harvested from SCID mice and immediately subjected to enzymatic digestion for generation of single cell suspensions (see below). In some experiments, xenografted mice were bled at different times (before and after microsphere treatment) using capillary pipettes to obtain 100–150 µl blood from the orbital sinus under anesthesia. Mouse sera were stored frozen at -80°C until analysis by ELISA (see below).
Gene array studies
Global patterns of gene expression within the tumor microenvironment were analyzed using the human cytokine gene expression (macro) array (R&D Systems, Minneapolis, MN; GA001), which represents a collection of 375 different human genes encoding cytokines, chemokines, immunostimulatory molecules, activation markers, angiogenic factors, growth factors, proteases, etc., and their corresponding cell surface receptors. The protocols used for array experiments were based upon the manufacturer’s recommendations, and unless otherwise stated, all reagents used were from R&D Systems. A brief description of the experimental protocols, including cDNA synthesis, hybridization, imaging, and data analysis, is given below.
Total RNA was isolated from the original patient tumor specimen or the
surgically excised xenografts using the TRIzol reagent (Life
Technologies, Grand Island, NY), according to the manufacturer’s
protocol. Following isolation, the RNA pellet was redissolved in the
RNA storage solution (Ambion), quantified by measuring the absorbance
at 260 nm, diluted to a final concentration of 0.25 µg/µl, and was
stored at -80°C. For cDNA synthesis, 8.0 µl human
cytokine-specific primers (R&D Systems; GAC11; an equimolar mixture
containing an antisense primer corresponding to each cDNA on the human
cytokine gene expression array) were first added to total RNA (4.0
µg/22.0 µl) and denatured for 3 min at 90°C, cooled at room
temperature for 5 min, and then equilibrated to 42°C in a water bath
for 5 min. A master mix for the reverse transcription (RT mix) was
prepared with the final concentration of each reagent as follows: 1x
reverse-transcriptase buffer, 333 µM dGTP, 333 µM dTTP, 1.67 µM
dCTP (unlabeled), 1.67 µM dATP (unlabeled), 100 U/75 µl
anti-RNase (Ambion), 25 µCi
[
-33P]dCTP, 25 µCi
[-33P]dATP, and 250 U/75 µl avian
myeloblastosis virus reverse transcriptase. A total of 30 µl RT mix
was added to each 30 µl RNA/primer sample. Samples were then
incubated in a 42°C water bath for 3 h to generate
33P-labeled cDNA. Unincorporated radioactive
nucleotides were removed from the cDNA by centrifugation (1100 x
g for 4 min) through Sephadex G-25 spin columns (R&D
Systems). A 2.0-µl aliquot of column-purified cDNA was removed to
assess efficiency of 33P labeling using a
scintillation counter (Beckman LS58001; Beckman Instruments, Fullerton,
CA). The activity of a 2.0 µl cDNA sample was typically
1.5–2.0 x 106 cpm.
Macroarrays were rinsed for 5 min in 2x standard saline citrate
phosphate/EDTA buffer, sealed individually in plastic bags
(Seal-A-Meal; Dazey, Industrial Airport, KS) with 1-cm
margins, and prehybridized (10 ml hybridization solution per bag) for
1–2 h at 65°C. Column-purified cDNA (75–80 µl) was denatured
in a boiling water bath for 5 min and then added to each array in a
total volume of 6 ml hybridization solution. Hybridization was
performed for 12–14 h in a rotating water bath set to 65°C with
continuous rotation (35 rpm). All posthybridization washes were
performed in a plastic wash container according to the manufacturer’s
instructions. Washed arrays were placed face up on blotting paper and
air dried for 5 min, after which they were wrapped individually in
plastic saran wrap and exposed to a low energy phosphor screen
(Molecular Dynamics, Sunnyvale, CA) for a period of 4 days.
Images of each array were generated by scanning the exposed phosphor screen with the STORM 860 PhosphorImager (Molecular Dynamics) at 50 µm pixel size resolution and creating a .gel file using ImageQuant software version 5.1 (Molecular Dynamics). To quantify hybridization signal intensity, a template grid consisting of rectangles drawn around each pair of duplicate spots was constructed using ImageQuant. (Note that the template grid covered all areas of the array and included blank areas in which no DNA was spotted on the membrane.) For each rectangle in the template, the average pixel number per unit area (area) was calculated, and raw data were exported to Microsoft Excel for analysis.
Data were analyzed by first dividing the hybridization-signal intensity of each gene (averaged from duplicate spots) by the average background value for that particular array to generate values representing fold expression over background. Next, 1x background was subtracted to calculate the relative hybridization-signal intensity (RSI) of each gene. A ratio of total signal intensity (of all spots on the array) divided by average background was used to normalize RSIs between individual arrays generated from control and treated specimens. Changes in gene expression between arrays were considered significant if the magnitude of increase or decrease was >2-fold (i.e., ratio of normalized RSIs >2.0 or <0.5). The minimum threshold intensity used to differentiate expressed genes from nonexpressed genes was determined empirically to be 2 SDs above average background. In some experiments, a particular gene was not expressed above threshold in control-treated tissue, but was highly expressed (i.e., >3-fold over background) after IL-12 treatment. To calculate the ratio of gene expression in these cases, the minimum threshold value (i.e., background + 2 SD) was used in place of 0 for the value of the nonexpressed gene. Because human tissue was maintained in a SCID mouse host, macroarrays were tested for cross-reactivity with murine cDNA generated from both SCID mouse spleen tissue and murine lung tumor tissue (line 1 alveolar carcinoma) treated with murine rIL-12. A total of nine cross-reactive genes (of 375 total genes) were identified in these experiments, which were excluded from all analyses of human tumor xenografts.
Human IFN- ELISA
The concentration of human IFN- in serum of tumor-bearing
mice was determined using a sandwich ELISA, as described (19, 20). Briefly, microtiter plates were coated with an
anti-IFN- mAb (M700A against human IFN-; Endogen, Cambridge,
MA). Mouse sera and human IFN- standards (Pierce Endogen, Rockford,
IL) were added to the plates, followed immediately with
biotinylated secondary anti-IFN- Ab (M700B; Endogen). The bound
Ab was detected with 1 µg/ml streptavidin-conjugated HRP
(Sigma-Aldrich; A3151) and 3, 3', 5, 5'-tetramethylbenzidine
(Sigma-Aldrich). This ELISA was determined to detect human IFN- and
not murine IFN- (data not shown).
In vivo neutralization/depletion studies
For in vivo neutralization of human IFN-, SCID mice bearing
established primary lung tumor xenografts were given i.p. injections of
the anti-human IFN- mAb, B133.3.1 (21), starting
the day of microsphere treatment. Control mice received the same dose
of the mAb, 2C3 (mouse IgG, 1, 
, anti-hapten phthalate), as
an isotype control (22). The first dose of Ab was given
6 h before intratumoral microsphere injections. Subsequent i.p.
injections of Ab were given daily thereafter for a total of 10
consecutive injections. Abs were prepared as ascites fluid diluted 1/4
in a total of 200 µl sterile PBS for injections. The monoclonal
anti-human IFN--secreting hybridoma, B133.3.1, was a gift from
G. Trinchieri (Schering-Plough Laboratory of Immunological Research,
Dardilly, France).
To deplete human CD4+ cells from established
primary xenografts, tumor-bearing SCID mice were injected i.p. with Abs
to human CD4 (-globulin purified from OKT4 ascites; 200 µg/200
µl PBS/mouse) 1 day before microsphere treatment (i.e., 6 days after
tumor engraftment). A second group of mice bearing established primary
xenografts received 2C3 as an isotype control.
For inhibition of iNOS activity in vivo, SCID mice bearing established
primary lung tumor xenografts were injected i.p. with
N-nitro-L-arginine methyl ester
(L-NAME), or the inactive isoform
N-nitro-D-arginine methyl ester (both
from Sigma-Aldrich) as a control. Mice received 10 daily injections
(0.2 mg/200 µl PBS), with the first injection 6 h before
microsphere treatment (23).
Histology/immunohistochemistry
Tissues (from the pre-engraftment specimen or tumor xenografts removed from mice) were fixed in 10% buffered Formalin, processed in an automated tissue processor, and embedded in paraffin. Serial sections were cut at 5 µm, placed on charged slides, and dried at 60°C for 1 h. Slides were deparaffinized in xylene and rehydrated through graded alcohol. Quenching of endogenous peroxidase was done using 3% aqueous H2O2 for 30 min. Slides were then rinsed in PBS and loaded onto a DAKO (Carpenteria, CA) automated Immunostainer. Slides were incubated for 1 h with a mAb to human CD45 (Zymed Laboratories, South San Francisco, CA; 18-0166) or mouse IgG (Sigma-Aldrich; M-5284) as a negative control (2.5 µg/ml). Biotinylated secondary Ab (Vector Laboratories, Burlingame, CA; PK-6200) was added for 30 min, followed by a PBS wash, and then Elite ABC Reagent (Vector; 6200) for 30 min. After a final PBS wash, diaminobenzidine chromagen (DAKO K3466) was added for 5 min. Slides were counterstained with Harris hematoxylin (Poly Scientific, Bay Shore, NY; s212) for 2 min, dehydrated, cleared, and mounted. Digital images were obtained using an Axioskop 2 microscope (Carl Zeiss, Thornwood, NY) with a SPOT camera (Diagnostic Instruments, Sterling Heights, MI). All images were taken under the x10 objective lens (x100 magnification). The anti-human CD45 Ab was tested for cross-reactivity with murine CD45 (using a SCID mouse spleen) and was found not to stain murine leukocytes (data not shown).
Flow cytometry
Four-color flow cytometry was used to quantify human leukocyte subsets in single cell suspensions generated from primary lung tumor xenografts. Microsphere-treated xenografts were surgically excised from mice and digested using an enzyme mixture (10 ml/0.25 g tissue) consisting of 0.5 mg/ml collagenase A (Boehringer Mannheim, Indianapolis, IN; 1088 785), 0.2 mg/ml hyaluronidase type V (Sigma-Aldrich; H-6254), and 0.1 mg/ml DNase I (Sigma-Aldrich; D-5025), in PBS + 0.5% BSA (filter sterilized and stored at -20°C as 10x stock). After incubation at 37°C for 30 min on rotating platform, undigested pieces of tumor were allowed to settle by gravity, and the remaining supernatant (containing cells in suspension) was carefully transferred to a new 15-ml tube. Cells were washed twice in cold PBS + 0.5% BSA + 2 mM EDTA, resuspended in 14 ml cold PBS, placed on ice, and taken to the Laboratory of Flow Cytometry, Roswell Park Cancer Institute, for Ab staining and FACS analysis.
Cell suspensions were stained with different Ab panels consisting of
four different Abs (each with a different fluorochrome label) to human
leukocyte surface markers, including CD45, CD3, CD4, CD8, CD19, and
CD33. Anti-human CD45 (present on all human leukocytes except plasma
cells) (24) was included in every panel so that data could
be gated on human leukocytes. For detection of intracellular IFN-,
single cell suspensions of tumor tissue were incubated for 2 h in
brefeldin A (20 µg/ml final concentration), and then surface stained
with Abs to human CD45, CD3, and CD4 or CD8. After fixation with 2%
formaldehyde, cells were permeabilized with 100 µl permeabilization
reagent (Caltag, Burlingame, CA), stained with an anti-human
IFN- Ab (clone MCH-IFG01; Caltag), and then analyzed by flow
cytometry. All data were acquired on a FACSCalibur flow cytometer (BD
Biosciences, San Jose, CA), and analyzed by C. Stewart (Director,
Laboratory of Flow Cytometry, Roswell Park Cancer Institute) using the
WinList software package (Verity Software House, Topsham, ME). Between
10,000 and 20,000 events were collected for each sample analyzed. In
preliminary experiments, the Ab panels used were found to reliably
stain human PBL subsets after short (20-min) treatment with enzyme
mixture, and did not demonstrate any cross-reactivity with SCID mouse
spleen cells (data not shown).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The implantation of small nondisrupted pieces of human lung tumor
biopsy tissues into the subcutis of SCID mice results in the
engraftment of a tumor microenvironment that includes both tumor cells
and human CD45+ inflammatory cells (Fig. 1
A, inset). To
determine whether the inflammatory cells within the xenograft retain
functional capacity, their response to the proinflammatory cytokine
IL-12 was assessed. One week after engraftment, biodegradable polymer
microspheres loaded with rhIL-12 were injected directly into the tumor
xenografts. Cytokine-free microspheres were used as a control. As shown
in Fig. 1A, the local and sustained release of rhIL-12
completely suppressed tumor growth over a period of 45 days
postengraftment. In contrast, tumor xenografts inoculated with control
microspheres grew progressively (Fig. 1A). Similar results
were observed in 18 of 20 different patient tumors, including three
different histological types of non-small cell lung cancer (i.e.,
squamous cell carcinomas, adenocarcinomas, and adenosquamous cell
carcinomas).
|
B, rhIL-12 treatment had no effect on
the growth of leukocyte-depleted xenografts (established from the same
patient’s tumor as in Fig. 1A). The absence of human
CD45+ cells was confirmed by immunohistochemistry
in Fig. 1B inset. These results are
representative of experiments conducted with five different patients’
tumors. Thus, we conclude that the human CD45+
inflammatory cells present within the lung tumor xenografts are
responsive to cytokine stimulation, and are necessary for tumor
suppression following local rhIL-12 therapy. rhIL-12 treatment promotes the maintenance, infiltration, and expansion of human inflammatory cells within the microenvironment of lung tumor xenografts
IL-12 is known to stimulate the proliferation of activated
lymphocytes (27, 28), and may also serve to protect cells
from apoptosis (29, 30). To evaluate the effects of local
IL-12 treatment on the cellular events occurring within the tumor
microenvironment, xenografted tissue was removed from control and
experimental mice for histological and immunohistochemical analyses.
The presence of human inflammatory cells in each tissue sample was
assessed by staining with a mAb to human CD45, and a serial section of
each specimen was stained with mouse IgG as a control (data not shown).
As illustrated in Fig. 2, a greater
number of CD45+ cells was observed in the
rhIL-12-treated xenograft, compared with control-treated
tumor, as early as 5 days after treatment (Fig. 2, A and
B). By day 24, human CD45+ cells were
essentially absent from the control-treated xenograft (Fig. 2C), which was now densely packed with tumor cells. In
contrast, the rhIL-12-treated xenograft (Fig. 2D) was
heavily infiltrated by CD45+ cells and showed
little evidence of viable tumor. Data from additional patient tumors
have yielded similar results, and human CD45+
cells have been detected up to 90 days postengraftment in IL-12-treated
xenografts (data not shown).
|
We conclude that IL-12 has a significant effect upon the proliferation
and longevity of the tumor-associated inflammatory cells and may
contribute to their infiltration into the tumor parenchyma. Although
each of these IL-12-associated effects may facilitate the human
leukocyte-mediated killing, the data presented to date provide little
insight with respect to the effector mechanisms responsible for the
observed antitumor activity. It is now well established that IL-12
stimulates both activated CD4+ and
CD8+ T cells (and NK cells) to secrete the
proinflammatory cytokine IFN-, which in turn mediates tumor killing
via several different effector mechanisms (28). Therefore,
experiments were conducted to determine whether rhIL-12 was inducing
the production of IFN- in one or more subsets of
CD45+ human leukocytes within the lung tumor
xenografts.
rhIL-12 stimulates tumor-associated T cells to produce human
IFN- in vivo
Single cell suspensions were obtained from tumor xenografts
16 h after treatment with either rhIL-12-loaded or control
microspheres. The 16-h time point was selected for comparison to allow
sufficient time for IFN- induction, while minimizing the effects of
rhIL-12 on lymphocyte proliferation. Following surface staining with
Abs to human CD45, CD3, and CD4 or CD8, the cells were then
permeabilized and stained intracytoplasmically with a mAb to human
IFN-. As shown in Table I,
IFN--producing CD3+ T cells were identified in
both control- and rhIL-12-treated tumors, with
CD4+ T cells accounting for the majority of
IFN- production (86 and 87% of all IFN-+ T
cells in control- and IL-12-treated tumors, respectively). IL-12
treatment increased the frequency of IFN-+
CD4+ T cells 3.6-fold (4.2–15.0%), while the
frequency of IFN-+ CD8+
T cells increased 2.3-fold (2.5–5.7%) in response to IL-12 (Table I).
Similar results were obtained using two additional patient tumors (data
not shown). These results indicate that both CD8+
and CD4+ T cells within the microenvironment of
human lung tumors produce IFN-, and that these IFN--producing
cells are increased in response to local IL-12 stimulation. The data
also suggest that CD4+ T cells are the more
important source of IFN- under basal conditions, and following IL-12
treatment.
|
was detectable in the sera of SCID
mice bearing primary lung tumor xenografts, mice were bled at different
time points after intratumoral microsphere treatment. As shown in Fig. 3, human IFN- was detected in the sera
of mice treated with rhIL-12-loaded microspheres, but not in
the sera of mice treated with control microspheres. This
representative kinetic analysis revealed that human IFN- was present
in the SCID serum as early as 1 day after injection of rhIL-12
microspheres, continued to increase until day 5, and was no longer
detectable by day 10 posttreatment (Fig. 3). Human IFN- was not
detected in the sera of mice bearing tumor xenografts depleted of
inflammatory leukocytes even after treatment with IL-12-loaded
microspheres (data not shown). Thus, rhIL-12 treatment of primary
xenografts stimulates human inflammatory cells (i.e.,
CD4+ and CD8+ T cells)
within the tumor microenvironment to produce human IFN-
protein, which can be detected in the sera of SCID mice bearing the
primary xenografts. Additional experiments revealed that the serum
level of human IFN- 5 days after rhIL-12 microsphere treatment of
the xenograft correlated directly with the degree of tumor growth
suppression in individual mice that was observed 90 days after
engraftment (Fig. 4). These data
establish a link between IFN- production and tumor suppression.
|
|
is causally linked to IL-12-induced tumor suppression
To determine whether human IFN- was required for the
IL-12-induced tumor suppression observed in this model, SCID mice
bearing primary lung tumor xenografts were injected i.p. with either a
neutralizing mAb to human IFN- or an isotype-matched control Ab
before the intratumoral injection of rhIL-12-loaded or control
microspheres. Representative data presented in Fig. 5 demonstrate that anti-IFN- Abs
reversed the rhIL-12-induced tumor suppression, but had no effect upon
the growth of tumors receiving control microspheres. In mice receiving
rhIL-12, anti-IFN- treatment also reduced serum levels of human
IFN- to undetectable levels, but did not inhibit the induction of
IFN- mRNA as detected by RT-PCR (data not shown). These data
establish that human IFN- is a necessary component in the
suppression of human lung tumor xenografts, rather than simply a marker
of leukocyte activation, following local rhIL-12 therapy.
|
upon tumor growth in
vivo. Nevertheless, IFN- is known to alter the expression of a
number of genes that have the potential to indirectly alter tumor
growth. To gain further insight with respect to possible antitumor
effector mechanisms that contribute to IL-12-induced tumor suppression,
gene expression patterns within xenografts were monitored following
treatment with either control or rhIL-12-loaded microspheres. Changes in gene expression patterns in tumor xenografts associated with rhIL-12 treatment and tumor suppression
A human gene expression array that monitors the expression of 375
genes encoding cytokines, chemokines, adhesion molecules, other
immunomodulatory factors, and their receptors was used to determine
which genes, if any, demonstrated altered expression within the tumor
xenografts after treatment with rhIL-12.
33P-labeled cDNA was generated from RNA isolated
from tumor xenografts 5 days after treatment with rhIL-12- or
BSA-loaded microspheres, and hybridized to the array (see
Materials and Methods for details). The RSIs for each gene
were determined from phosphor images of the arrays, and values from the
rhIL-12 microsphere-treated xenografts were plotted against values
obtained from xenografts treated with control microspheres.
Representative data presented in Fig. 6A show that rhIL-12 treatment
of a primary xenograft induced significant changes (i.e., 
2-fold
increase or decrease) in 56 of 243 (23%) of expressed genes. In
contrast, no significant change in gene expression with rhIL-12 was
observed in leukocyte-depleted xenografts established from the same
patient’s tumor. Similar results were observed with xenografts
established from additional patient tumors and at different time points
posttreatment (e.g., 6 h, 24 h, 3 days, 10 days; data not
shown); however, the most significant changes were seen 5 days after
rhIL-12 treatment.
|
. Each of the six symbols represents a
different patient’s lung tumor whose gene expression was profiled 5
days after treatment of xenografts with either rhIL-12-loaded
microspheres or control microspheres. The data establish that rhIL-12
treatment significantly increased the expression of proinflammatory
chemokines and cytokines (e.g., monokine induced by IFN- (MIG),
IFN--inducible protein 10 (IP-10), monocyte chemoattractant
protein-1 (MCP-1), macrophage-inflammatory protein-1, RANTES,
IFN-, TNF-, IL-16, etc.) in the majority of tumors studied
(Fig. 7, A and B). In addition, immunostimulatory
factors, leukocyte activation markers, and adhesion molecules (e.g.,
iNOS, CD40, Fas, CD27, VCAM, integrin 4, etc.)
were found to be increased in response to rhIL-12 (Fig. 7C).
By contrast, the expression of immunosuppressive factors such as
TGF-1 and macrophage migration inhibitory factor (Fig. 7B), as well as proangiogenic and vascular-associated
factors (e.g., vascular endothelial growth factor (VEGF),
platelet endothelial cell adhesion molecule (PECAM), CD34,
epithelial-derived neutrophile-activating peptide-78,
platelet-derived growth factor (PDGF)-Ra/b, etc.; Fig. 7D)
decreased after IL-12 treatment. Decreases in the expression of a
number of growth factors and tumor-associated molecules (e.g.,
insulin-like growth factor-I/II, decorin, osteopontin, epithelial cell
adhesion molecule, etc.; Fig. 7E) were observed in
IL-12-treated tumors compared with control-treated tumors. Several MMPs
(MMP-7, MMP-9, MMP-10, MMP-12, MMP-13) were down-regulated, while the
MMP inhibitor, tissue inhibitor of metalloproteinase-1, and the
proapoptotic factor, caspase-1, were up-regulated following IL-12
treatment (Fig. 7F).
|
-responsive genes) in five of the six
tumors evaluated (Fig. 7, A–F, open symbols). None of the
patients from whom these five IL-12-responsive xenografts were
established had been treated with radiation or chemotherapy before
surgical resection of their tumor. However, primary xenografts
established from a single patient who was treated with high dose
chemotherapy before surgical resection of his tumor (i.e., neoadjuvant
chemotherapy) demonstrated a markedly attenuated response to IL-12
therapy (Fig. 7, •). Moreover, the poor response of this patient’s
xenografts to IL-12 in terms of changes in gene expression was
paralleled by a lack of IL-12-induced tumor growth suppression in vivo
(data not shown). The cytotoxic effects of chemotherapy may account for
the relative unresponsiveness of the inflammatory cells within these
xenografts to IL-12 immunotherapy. Interestingly, primary xenografts
established from this IL-12-unresponsive tumor appeared to have a
similar degree of leukocytic infiltration as that of the tumors
obtained from patients who were chemotherapy naive, suggesting that the
effect of chemotherapy was not in reducing the number of
tumor-associated inflammatory cells, but rather in decreasing their
functional capacity.
Among the genes whose expression was most significantly increased by
rhIL-12 treatment in the five IL-12-responsive tumors analyzed, several
proinflammatory chemokines were identified. Quantitative data presented
in Fig. 8 indicate the level of
expression of the C-X-C chemokines MIG and IP-10, as well as the C-C
chemokines MCP-1 and RANTES under different conditions. Although none
of these genes was highly expressed in the pre-engraftment tumor
specimen or the control-treated xenografts, all four genes were induced
by treatment with rhIL-12-loaded microspheres (Fig. 8). In mice treated
with neutralizing Abs to human IFN-, the IL-12-mediated induction of
MIG, IP-10, RANTES, and (to a lesser extent) MCP-1 was inhibited (Fig. 8). Moreover, in secondary xenografts (i.e., depleted of inflammatory
leukocytes) established from the same tumor specimen, rhIL-12 treatment
failed to up-regulate the expression of these proinflammatory factors
(Fig. 8). Similar results were obtained with xenografts established
from additional patient tumor specimens (data not shown). Thus, we
conclude that local and sustained delivery of rhIL-12 to the
microenvironment of human lung tumor xenografts stimulates
leukocyte-dependent and IFN--dependent changes in gene expression,
including the induction of the proinflammatory chemokines MIG, IP-10,
RANTES, and MCP-1.
|
has been observed as
early as 6 h after treatment with rhIL-12, reaching a peak of
110-fold increase in IFN- message 2 days after the cytokine
treatment. Similar increases in the expression of MIG and its receptor
CXCR-3 were also detected by RT-PCR analysis. No increase in the
expression of these genes was observed in xenografts treated with
control microspheres (compared with the untreated pre-engraftment
specimen), and the IL-12-induced increases in MIG and CXCR-3 expression
were abrogated by treatment with Abs to human IFN-.
Although the changes in gene expression patterns induced by IL-12
correlate with tumor suppression, they do not define the effector
mechanisms responsible for the observed inhibition of tumor growth.
Previous studies in mice have demonstrated that the IL-12-induced
suppression of established tumors is associated with activated
macrophages in the tumor microenvironment that are expressing high
levels of iNOS, and the importance of NO as an effector molecule has
been confirmed in mouse tumor models (33). Because
the gene encoding iNOS was expressed in six of six tumor xenografts
tested, and its expression increased in four of six tumors after
treatment with rhIL-12 (Fig. 7C), the possible role of NO in
mediating rhIL-12-induced tumor regression was now investigated.
IL-12-induced tumor suppression is mediated in part by NO
To address the potential role of NO in the suppression of tumor in
lung tumor xenografts, mice were treated systemically with
L-NAME, an inhibitor of iNOS activity, during rhIL-12
therapy. Data presented in Table II show
that treatment of mice with L-NAME resulted in a
significant loss of rhIL-12-induced tumor suppression, compared with
mice treated with N-nitro-D-arginine
methyl ester (an inactive isoform of
L-NAME). Despite the loss of rhIL-12-induced
tumor suppression, serum levels of human IFN- were not altered by
L-NAME treatment (Table II). Moreover,
L-NAME had no effect on the growth of xenografts
treated with control microspheres (Table II). Similar results were
observed with one additional patient tumor. These data suggest that
following local rhIL-12 treatment, human IFN- mediates the
suppression of human lung tumor xenografts at least in part through an
indirect mechanism requiring the induction of iNOS.
|
production, and
changes in gene expression are all dependent upon CD4+ T
cells
The role of CD4+ T cells in tumor growth
suppression, IFN- production, and changes in gene expression was
assessed by the depletion of these cells from the xenograft before
treatment of the tumor-bearing mice with rhIL-12-loaded microspheres.
To deplete the CD4+ T cells from established
primary tumor xenografts, mice were injected i.p. with 200 µg of
anti-human CD4 (OKT4) Ab 6 days after tumor engraftment. A second
control group of tumor-bearing mice received 200 µg of an isotype
control Ab. One day after receiving the Ab, both groups of mice were
treated intratumorally with rhIL-12-loaded microspheres. Five days
later, all mice were bled, and their sera were assayed for levels of
human IFN-. At this time, two mice from each group were sacrificed,
and RNA was isolated for gene expression analysis. The remaining mice
were monitored weekly for 11 wk for tumor growth. The results presented
in Table III indicate that CD4 depletion
reversed the rhIL-12-induced tumor suppression in two of three animals
and significantly decreased the serum levels of human IFN- in five
of five mice tested. The altered gene expression patterns observed with
rhIL-12 were partially or completely reversed in xenografts
depleted of human CD4+ T cells. Following
rhIL-12 treatment, message levels for IFN-, TNF-, MIG, IP-10,
MCP-1, and RANTES were 3–5-fold lower in
CD4+-depleted xenografts compared with
nondepleted xenografts. The expression levels of genes that were
suppressed as a result of treatment of rhIL-12 in the control
Ab-treated group (i.e., MMPs, VEGF, PECAM, and CD34) were found to be
higher in the anti-CD4-treated group compared with the control
group. Genes encoding the human IL-12R (i.e., IL-R1 and IL-12R2)
that were expressed in the control (i.e., nondepleted) group were below
the level of detection in xenografts depleted of human
CD4+ T cells. The CD4+ T
cell depletion experiment was repeated with a second tumor with similar
results. In this repeat experiment, there were four mice in the control
group and four mice in the experimental group. Tumor growth was
suppressed in three of four mice in the control group treated with
rhIL-12-loaded microspheres and an isotype control Ab. In the
IL-12-treated CD4+ T cell-depleted experimental
group, tumor suppression was observed in only one of four animals. No
IFN- was detected in the sera of mice from the
CD4+ T cell-depleted group, while three of
four mice were positive for IFN- in the control group. These data
establish a significant role for human CD4+ T
cells in the rhIL-12-induced tumor growth suppression, IFN-
production, and associated changes in gene expression pattern.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in response to a local and sustained
release of IL-12, resulting in significant suppression or complete
eradication of the tumor. Based upon the rapid response to IL-12
(changes in gene expression patterns are observed as early as 6 h
after rhIL-12 treatment) and the dependence upon
CD4+ T cells, the responding T cells are assumed
to have been previously activated (most likely by the tumor itself) and
may represent an effector memory subset of CD4+ T
cell. This assumption is based upon the fact that the functional IL-12R
(IL-12R1 and 2) is only expressed on T cells after activation
(26, 28). The role of these IL-12-responsive
CD4+ T cells in mediating the tumor growth
suppression was established by cell depletion studies. Adoptive cell
transfer experiments in mice have previously established that
tumor-sensitized, but not naive T cells are essential for tumor
rejection that is induced by IL-12 (34). The findings
presented in this work are the first to show that a similar activated T
cell exists within human lung tumors and that this cell can be
reactivated by rhIL-12.
In addition to mobilizing CD4+ T cells to kill
tumor cells in situ by the release of IFN-, rhIL-12 was observed to
enhance the longevity of the CD45+
tumor-associated inflammatory cells, which would be expected to have a
significant long-term effect upon the ability of these inflammatory
cells to control tumor progression. We previously documented
proliferation of human leukocytes within established primary xenografts
by 5-bromo-2'-deoxyuridine uptake (31) and now have
confirmed by FACS analysis the expansion of the
CD45+ cells, including CD4+
T cells, CD8+ T cells, and
CD19+ B cells. IL-12 is known to stimulate the
proliferation of activated murine lymphocytes (28) and has
been shown to protect these cells from apoptosis (35, 36).
Our finding of dense accumulations of human CD45+
inflammatory cells in rhIL-12-treated (but not in control) tumor
xenografts as long as 90 days after engraftment is consistent with
these earlier studies in mice.
Studies in mice have shown that IFN- is required for in vivo immune
responses against tumors (37), virus-infected cells
(38, 39), self Ags in autoimmune diseases (40, 41), and parasite-infected cells (42). A common
characteristic of all of these murine studies is that IFN- is
necessary, but not sufficient for the immune response. By linking the
IFN- dependency of these T cell-mediated responses to the production
of NO by macrophages as an effector mechanism, others have attempted to
explain the necessary, but not sufficient, role of IFN- because this
is only one of several cytokines and factors that are required for the
macrophage-mediated NO production (43, 44, 45, 46). Our findings
that the gene encoding human iNOS is expressed in human lung tumor
xenografts and is elevated after treatment with rhIL-12 suggest that NO
may be an antitumor effector molecule in humans also. The data showing
that inhibition of iNOS partially reverses rhIL-12-induced tumor
suppression provide the first direct evidence of NO-mediated tumor
killing in vivo in humans. However, while NO is one of the effector
mechanisms, we have not ruled out other factors within the tumor
microenvironment that may be contributing to tumor arrest in response
to local rhIL-12 therapy.
Changes in gene expression patterns observed in the primary tumor
xenografts following rhIL-12 therapy are consistent with what one would
expect from inflammatory cells in response to this cytokine, and
suggest a number of other mechanisms that may be contributing to the
tumor arrest (28). The consistent and significant
enhancement of two IFN--inducible chemokines IP-10 and MIG could be
contributing to tumor arrest in a number of different ways. In addition
to their ability to attract activated T cells, these C-X-C chemokines
have been shown to have antiangiogenic properties (47).
Both MIG and IP-10 can inhibit tumor growth by preventing the
generation of new vessels needed for tumor expansion
(48, 49, 50). Although we do not yet have evidence to sustain
this possibility directly, our data showing rhIL-12-induced decreases
in the expression of genes associated with angiogenesis (i.e., VEGF,
PECAM, CD34, epithelial-derived neutrophile-activating
peptide-78, growth-related oncogene-, Ephrin A4,
pleiotrophin, PDGFRa, and PDGFRb) suggest that inhibition of tumor
neovascularization may be an important effector mechanism in the
suppression of human lung tumor growth.
The suppression observed in several other genes that was associated with IL-12 treatment and tumor arrest needs further study and may be of considerable potential importance clinically. Ten different growth factors or lung tumor-associated genes were suppressed, and the expression of five MMP genes was significantly decreased in tumor xenografts treated with rhIL-12. A decrease in tumor-promoting growth factors within the tumor microenvironment could obviously contribute to a decrease in tumor growth, while the reduction in MMPs would be expected to have a number of different inhibitory effects upon tumor progression (51). MMPs have been shown to contribute to virtually all stages of cancer evolution and progression including metastasis (51). It therefore seems likely that the rhIL-12-induced decrease in MMPs and the increase in tissue inhibitor of metalloproteinase-1 (an inhibitor of MMP function) would have a significant impact upon tumor progression.
Our studies using this human/SCID chimeric model sustain the hypothesis
that inflammatory cells associated with human lung tumors are
functional, and able to respond to a proinflammatory cytokine when
studied in situ. Previous studies addressing the potential functional
capacity of human leukocytes in tumors have been primarily conducted on
cells isolated from disrupted tumor tissues (52, 53, 54, 55, 56, 57, 58). The
isolation of these cells requires the disruption of the tumor
microenvironment, resulting in the loss of cell-cell and
cell-extracellular matrix interactions that may be critical to
leukocyte function and response to cytokines. It is becoming
increasingly apparent that the extracellular matrix and stromal cells
within microenvironments play a key role in programming immunocompetent
cells and coordinating T cell activation and migration of both
leukocytes and tumor cells (59, 60, 61, 62). Using nondisrupted
fresh human tumor tissue explants, it was recently established by
others that rhIL-12 induced the production of IFN-
(63). However, because these studies were done in vitro,
the long-term effect of rhIL-12 treatment and the release of human
IFN- on tumor growth could not be assessed. The one obvious
advantage of the human SCID chimeric model used in this study is that
one is able to monitor within an intact microenvironment the cellular
and molecular events associated with IL-12 treatment and correlate
these events with in vivo tumor growth. The advantages, limitations,
and pitfalls of using this and other human SCID chimeric models to
study the tumor microenvironment and to evaluate therapeutic approaches
to human cancer have been reviewed elsewhere (15).
The data presented in this work suggest that a local and sustained release of IL-12 into the tumor microenvironment of a single tumor nodule may be exploited clinically in the treatment of human cancer. The cytokine delivery may be accomplished either by the intratumoral injection of IL-12-loaded biodegradable microspheres, as reported in this work, or by the injection of irradiated cells transfected to produce IL-12, as reported previously (20). An assumption is being made that the IL-12-dependent CD4 T cell-mediated antitumor response locally, results in tumor cell lysis and the release of tumor Ags that induce a systemic tumor-specific immune response. The systemic antitumor response would be expected to eradicate uninjected tumors at other sites throughout the body. These predictions and assumptions have been tested and sustained using a completely murine lung tumor model that is syngeneic to BALB/c mice, i.e., the line 1 tumor. In this experimental model, it was established that the local and sustained release of IL-12 (or a combination of IL-12 and GM-CSF) into a single primary tumor nodule led to the eradication of multiple established metastatic tumors (32, 64). These studies also revealed that the local and sustained release of cytokines into a single tumor microenvironment provoked a tumor-specific systemic and protective antitumor immunity (32). A potential limitation of the in situ tumor therapy as a general therapeutic approach to be used clinically is that at least one accessible tumor nodule is required. In the case of lung cancer, most patients do not have any readily accessible tumor nodules. However, it would be possible to access a primary tumor nodule within the lung with the aid of a stereotactically directed needle. Such an approach is already used routinely for performing lung tumor biopsies and could be easily modified to inject IL-12-secreting cells or IL-12-loaded microspheres into the primary tumor.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Richard B. Bankert, Department of Microbiology, 138 Farber Hall, State University of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214. E-mail address: rbankert{at}buffalo.edu
3 Abbreviations used in this paper: MMP, matrix metalloproteinase; iNOS, inducible NO synthase; IP-10, IFN--inducible protein 10; L-NAME, N-nitro-L-arginine methyl ester; MCP, monocyte chemoattractant protein; MIG, monokine induced by IFN-; PDGF, platelet-derived growth factor; PECAM, platelet endothelial cell adhesion molecule; rh, recombinant human; RSI, relative hybridization intensity; VEGF, vascular endothelial growth factor.
Received for publication September 26, 2002. Accepted for publication November 1, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
elicits antitumor CD4+ MHC class II-restricted T cells that are cytolytic in vitro and tumoricidal in vivo. J. Immunol. 154:4613.[Abstract]
. Proc. Natl. Acad. Sci. USA 96:8633.-dependent suppression of autologous lung tumor xenografts in SCID mice. Cancer Res. 62:2611.-chain: characterization of receptor expression in normal lymphoid cells and EL-4 cells. J. Immunol. 147:2222.[Abstract]
induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect. J. Exp. Med. 188:1603. and tumor necrosis factor have a role in tumor regressions mediated by murine CD8+ tumor-infiltrating lymphocytes. J. Exp. Med. 173:647. interferon-treated mice. J. Virol. 63:2813.. Lymphokine Cytokine Res. 10:353.[Medline]
and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/Wehi mice. J. Clin. Invest. 87:739.
. J. Autoimmun. 4:237.[Medline]
antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major. J. Immunol. 143:266.[Abstract]
-dependent production of reactive nitrogen intermediates mediates IFN- plus IL-2-induced murine macrophage tumoricidal activity. J. Immunol. 149:3290.[Abstract]
producing (Th1) T cell clones: failure of IL-4-producing (Th2) T cell clones to activate effector function in macrophages. J. Immunol. 142:760.[Abstract]
, promotes tumor necrosis in vivo. Blood 89:2635., and tumor necrosis factor gene activation in fresh tumor-infiltrating lymphocytes from lung adenocarcinoma. Cancer Immunol. Immunother. 41:1.[Medline]
and CXC chemokine induction by interleukin 12 in renal cell carcinoma. Clin. Cancer Res. 5:2780.This article has been cited by other articles:
|
|
 |
|
  M. O. Kilinc, T. Gu, J. L. Harden, L. P. Virtuoso, and N. K. Egilmez Central Role of Tumor-Associated CD8+ T Effector/Memory Cells in Restoring Systemic Antitumor Immunity J. Immunol., April 1, 2009; 182(7): 4217 - 4225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Winter, A. H. Schmieder, S. D. Caruthers, J. L. Keene, H. Zhang, S. A. Wickline, and G. M. Lanza Minute dosages of {alpha}{nu}{beta}3-targeted fumagillin nanoparticles impair Vx-2 tumor angiogenesis and development in rabbits FASEB J, August 1, 2008; 22(8): 2758 - 2767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Simpson-Abelson, G. F. Sonnenberg, H. Takita, S. J. Yokota, T. F. Conway Jr., R. J. Kelleher Jr., L. D. Shultz, M. Barcos, and R. B. Bankert Long-Term Engraftment and Expansion of Tumor-Derived Memory T Cells Following the Implantation of Non-Disrupted Pieces of Human Lung Tumor into NOD-scid IL2R{gamma}null Mice J. Immunol., May 15, 2008; 180(10): 7009 - 7018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Nazareth, L. Broderick, M. R. Simpson-Abelson, R. J. Kelleher Jr., S. J. Yokota, and R. B. Bankert Characterization of Human Lung Tumor-Associated Fibroblasts and Their Ability to Modulate the Activation of Tumor-Associated T Cells J. Immunol., May 1, 2007; 178(9): 5552 - 5562. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Watkins, N. K. Egilmez, J. Suttles, and R. D. Stout IL-12 Rapidly Alters the Functional Profile of Tumor-Associated and Tumor-Infiltrating Macrophages In Vitro and In Vivo J. Immunol., February 1, 2007; 178(3): 1357 - 1362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-c. Chen, R. Wang, X. Zhao, Y.-q. Wei, M. Hu, Y.-s. Wang, X.-w. Zhang, R. Zhang, L. Zhang, B. Yao, et al. Prophylaxis against carcinogenesis in three kinds of unestablished tumor models via IL12-gene-engineered MSCs Carcinogenesis, December 1, 2006; 27(12): 2434 - 2441. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Broderick and R. B. Bankert Membrane-Associated TGF-beta1 Inhibits Human Memory T Cell Signaling in Malignant and Nonmalignant Inflammatory Microenvironments. J. Immunol., September 1, 2006; 177(5): 3082 - 3088. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Robertson, H.-C. Chang, D. Pelloso, and M. H. Kaplan Impaired interferon-{gamma} production as a consequence of STAT4 deficiency after autologous hematopoietic stem cell transplantation for lymphoma Blood, August 1, 2005; 106(3): 963 - 970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Stout, C. Jiang, B. Matta, I. Tietzel, S. K. Watkins, and J. Suttles Macrophages Sequentially Change Their Functional Phenotype in Response to Changes in Microenvironmental Influences J. Immunol., July 1, 2005; 175(1): 342 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Broderick, S. J. Yokota, J. Reineke, E. Mathiowitz, C. C. Stewart, M. Barcos, R. J. Kelleher Jr., and R. B. Bankert Human CD4+ Effector Memory T Cells Persisting in the Microenvironment of Lung Cancer Xenografts Are Activated by Local Delivery of IL-12 to Proliferate, Produce IFN-{gamma}, and Eradicate Tumor Cells J. Immunol., January 15, 2005; 174(2): 898 - 906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Radhakrishnan, L. T. Nguyen, B. Ciric, D. Flies, V. P. V. Keulen, K. Tamada, L. Chen, M. Rodriguez, and L. R. Pease Immunotherapeutic Potential of B7-DC (PD-L2) Cross-Linking Antibody In Conferring Antitumor Immunity Cancer Res., July 15, 2004; 64(14): 4965 - 4972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Pelloso, K. Cyran, L. Timmons, B. T. Williams, and M. J. Robertson Immunological Consequences of Interleukin 12 Administration after Autologous Stem Cell Transplantation Clin. Cancer Res., March 15, 2004; 10(6): 1935 - 1942. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, and R. W. Dutton Tc1 and Tc2 Effector Cell Therapy Elicit Long-Term Tumor Immunity by Contrasting Mechanisms That Result in Complementary Endogenous Type 1 Antitumor Responses J. Immunol., February 1, 2004; 172(3): 1380 - 1390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, J. C. Hylind, and R. W. Dutton Effector Cell-Derived Lymphotoxin {alpha} and Fas Ligand, but not Perforin, Promote Tc1 and Tc2 Effector Cell-Mediated Tumor Therapy in Established Pulmonary Metastases Cancer Res., January 1, 2004; 64(1): 406 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Kuppner, A. Scharner, V. Milani, C. von Hesler, K. E. Tschop, O. Heinz, and R. D. Issels Ifosfamide impairs the allostimulatory capacity of human dendritic cells by intracellular glutathione depletion Blood, November 15, 2003; 102(10): 3668 - 3674. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or control (cytokine-free) (•) microspheres. Tumor progression
was assessed in both control- and rhIL-12-treated mice for a period of
45 days. The difference in mean tumor volume at day 45 between rhIL-12-
and control-treated mice was statistically significant
(p = 0.003, unpaired Student’s t
test). In B, the experiment was repeated with tumor
tissue from the same patient that had been depleted of human leukocytes
by serial passage in SCID mice. Tumor tissue was passaged four times
before treatment, and the absence of human CD45+ cells was
confirmed by immunohistochemistry (inset). In both
A and B, n = 5
mice/group, and error bars represent the SD of mean tumor volume.
