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[Cancer Research 61, 6406-6412, September 1, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Secondary Lymphoid Organ Chemokine Reduces Pulmonary Tumor Burden in Spontaneous Murine Bronchoalveolar Cell Carcinoma1

Sherven Sharma, Marina Stolina, Li Zhu, Ying Lin, Raj Batra, Min Huang, Robert Strieter and Steven M. Dubinett2

University of California Los Angeles School of Medicine, Wadsworth Pulmonary Immunology Laboratory, Veterans Administration Greater Los Angeles Healthcare System [S. S., M. S., L. Z., Y. L., R. B., M. H., S. M. D.], and Jonsson Comprehension Cancer Center [S. M. D.] and Division of Pulmonary and Critical Care Medicine, University of California Los Angeles School of Medicine [R. S., S. M. D.], Los Angeles, California 90073


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The antitumor efficiency of secondary lymphoid organ chemokine (SLC), a CC chemokine that chemoattracts both dendritic cells (DCs) and T lymphocytes,was evaluated in SV40 large T-antigen transgenic mice that develop bilateral multifocal pulmonary adenocarcinomas. Injection of recombinant SLC in the axillary lymph node region led to a marked reduction in tumor burden with extensive lymphocytic and DC infiltration of the tumors and enhanced survival. SLC injection led to significant increases in CD4 and CD8 lymphocytes as well as DC at the tumor sites, lymph nodes, and spleen. The cellular infiltrates were accompanied by the enhanced elaboration of Type 1 cytokines and the antiangiogenic chemokines IFN-{gamma} inducible protein 10, and monokine induced by IFN-{gamma} (MIG). In contrast, lymph node and tumor site production of the immunosuppressive cytokine transforming growth factor ß was decreased in response to SLC treatment. In vitro, after stimulation with irradiated autologous tumor, splenocytes from SLC-treated mice secreted significantly more IFN-{gamma} and granulocyte macrophage colony-stimulating factor, but reduced levels of interleukin 10. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for additional evaluation of SLC in regulation of tumor immunity and its use in lung cancer immunotherapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effective antitumor responses require both APCs3 and lymphocyte effectors (1) . Because tumor cells often have limited expression of MHC antigens and lack costimulatory molecules, they are ineffective APCs (2) . In addition, tumor cells secrete immunosuppressive mediators that contribute to evasion of host immune surveillance (3, 4, 5) . To circumvent this problem, investigators are using ex vivo generated DCs to stimulate antitumor immune responses in vivo. In experimental murine models, DCs pulsed with tumor-associated antigenic peptides (6) or transfected with tumor RNA have been shown to induce antigen-specific antitumor responses in vivo (7) . Similarly, fusion of DCs with tumor cells or intratumoral injection of cytokine-modified DCs has also been shown to enhance antitumor immunity (8, 9, 10) . Consequently, it has been suggested that effective anticancer immunity may be achieved by recruiting professional host APCs for tumor antigen presentation to promote specific T-cell activation (11) . Thus, chemokines that attract both DCs and lymphocyte effectors to lymph nodes and tumor sites could serve as potent agents in cancer immunotherapy.

Chemokines, a group of homologous, yet functionally divergent proteins, directly mediate leukocyte migration and activation and play a role in regulating angiogenesis (12) . Chemokines also function in maintaining immune homeostasis and secondary lymphoid organ architecture (13) . Several chemokines are known to have antitumor activity. Tumor rejection has been noted in various murine tumor models in which tumor cells have been modified with chemokines including MIP1{alpha}, RANTES, lymphotactin, TCA3, JE/MCP-1/MCAF, MIP3{alpha}, MIP3ß, and IP-10 (14, 15, 16, 17, 18, 19, 20, 21, 22) . In this study, we evaluated the antitumor properties of a CC chemokine, SLC, in a spontaneous murine model of lung cancer. In the SV40 TAg transgenic mice, adenocarcinomas develop in an organ-specific manner and, compared with transplantable tumors, the pulmonary tumors in these mice more closely resemble human lung cancer. SLC, normally expressed in high endothelial venules and in T-cell zones of spleen and lymph nodes, strongly attracts naive T cells and DCs (23, 24, 25, 26, 27, 28, 29, 30) . The capacity of SLC to chemoattract DCs (16) is a property shared with other chemokines (17, 18, 19) . However, SLC may be distinctly advantageous because of its capacity to elicit a Type 1 cytokine response in vivo (31) . DCs are uniquely potent APCs involved in the initiation of immune responses (32) . Serving as immune system sentinels, DCs are responsible for Ag acquisition in the periphery and subsequent transport to T-cell areas in lymphoid organs where they prime specific immune responses. SLC recruits both naïve lymphocytes and antigen-stimulated DCs into T-cell zones of secondary lymphoid organs, colocalizing these early immune response constituents and culminating in cognate T-cell activation (23) . In this study, using transgenic mice that develop lung cancer spontaneously, we demonstrate that SLC mediates potent antitumor responses in vivo leading to a significant reduction in tumor burden.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Clara cell lung tumor cells (CC-10 Tag and H-2q) were derived from freshly excised lung tumors that were propagated in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS (Gemini Bioproducts, Calabasas, CA), penicillin (100 units/ml), streptomycin (0.1 mg/ml), and 2 mM of glutamine (JRH Biosciences, Lenexa, KS) and maintained at 37°C in humidified atmosphere containing 5% CO2 in air. After two in vivo passages, CC-10 TAg tumor clones were isolated. The cell lines were Mycoplasma free, and cells were used up to the tenth passage before thawing frozen stock cells from liquid N2.

CC10TAg Mice.
The transgenic CC-10 TAg mice, in which the SV40 large TAg is expressed under control of the murine Clara cell-specific promoter, were used in these studies (33) . All of the mice expressing the transgene developed diffuse bilateral bronchoalveolar carcinoma. Tumor was evident bilaterally by microscopic examination as early as 4 weeks of age. After 3 months of age, the bronchoalveolar pattern of tumor growth coalesced to form multiple bilateral tumor nodules. The CC-10 TAg transgenic mice had an average life span of 4 months. Extrathoracic metastases were not noted. Breeding pairs for these mice were generously provided by Francesco J. DeMayo (Baylor College of Medicine, Houston, TX). Transgenic mice were bred at the West Los Angeles Veteran Affairs vivarium and maintained in the animal research facility. Before each experiment using the CC-10 TAg transgenic mice, presence of the transgene was confirmed by PCR of mouse tail biopsies. The 5'primer sequence was SM19-TAG: 5'-TGGACCTTCTAGGTCTTGAAAGG-3', and the 3' primer sequence was SM36-TAG: 5'-AGGCATTCCACCACTGCTCCCATT-3'. The size of the resulting PCR fragment is 650 bp. DNA (1 µg) was amplified in a total volume of 50 µl, which contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 µM each deoxynucleotide triphosphates, 0.1 µM primers, 2.5 mM MgCl2, and 2.5 units of Taq polymerase. PCR was performed in a Perkin-Elmer DNA thermal cycler (Norwalk, CT). The amplification profile for the SV40 transgene consisted of 40 cycles, with the first cycle denaturation at 94°C for 3 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min, followed by 39 cycles with denaturation at 94°C for 1 min, and the same annealing and extension conditions. The extension step for the last cycle was 10 min. After amplification, the products were visualized against molecular weight standards on a 1.5% agarose gel stained with ethidium bromide. All of the experiments used pathogen-free CC-10 TAg transgenic mice beginning at 4–5 week of age.

The SLC Therapeutic Model in CC-10 TAg Mice.
CC-10 TAg transgenic mice were injected in the axillary node region with murine recombinant SLC (0.5 µg/injection; Pepro Tech, Rocky Hill, NJ) or normal saline diluent, which contained equivalent amounts of murine serum albumin (Sigma Chemical Co., St. Louis, MO) as an irrelevant protein for control injections. Beginning at 4–5 weeks of age, SLC or control injections were administered three times per week for 8 weeks. The endotoxin level reported by the manufacturer was <0.1 ng/µg (1 endotoxin unit/µg) of SLC. The dose of SLC (0.5 µg/injection) was chosen based on our previous studies (31) and the in vitro biological activity data provided by the manufacturer. Maximal chemotactic activity of SLC for total murine T cells was found to be 100 ng/ml. For in vivo evaluation of SLC-mediated antitumor properties we used 5-fold more than this amount for each injection. At 4 months, mice were sacrificed, and lungs were isolated for quantification of tumor surface area. Tumor burden was assessed by microscopic examination of H&E-stained sections with a calibrated graticule (a 1-cm2 grid subdivided into 100 1-mm2 squares). A grid square with tumor occupying >50% of its area was scored as positive, and the total number of positive squares was determined as described previously (4) . Ten separate fields from four histological sections of the lungs were examined under high-power (x20 objective). Ten mice from each group were not sacrificed so that survival could be assessed.

Cytokine Determination from Tumor Nodules, Lymph Nodes, and Spleens.
The cytokine profiles in tumors, lymph nodes, and spleens were determined in both SLC and diluent-treated mice as described previously (4) . Non-necrotic tumors were harvested and cut into small pieces and passed through a sieve (Bellco, Vineland, NJ). Axillary lymph nodes and spleens were harvested from SLC-treated tumor-bearing, control tumor-bearing, and normal control mice. Lymph nodes and spleens were teased apart, RBC depleted with ddH2O, and brought to tonicity with 1 x PBS. After a 24-h culture period, tumor nodule supernatants were evaluated for the production of IL-10, IL-12, GM-CSF, IFN-{gamma}, TGF-ß, VEGF, MIG, and IP-10 by ELISA and PGE-2 by EIA. Tumor-derived cytokine and PGE-2 concentrations were corrected for total protein by Bradford assay (Sigma Chemical Co.). For cytokine determinations after secondary stimulation with irradiated tumor cells, splenocytes (5 x 10 6 cells/ml), were cocultured with irradiated (100 Gy, Cs137 {gamma}-rays) CC-10 TAg tumor cells (105 cells/ml) at a ratio of 50:1 in a total volume of 5 ml. After a 24-h culture, supernatants were harvested and GM-CSF, IFN-{gamma}, and IL-10 determined by ELISA.

Cytokine ELISA.
Cytokine protein concentrations from tumor nodules, lymph nodes, and spleens were determined by ELISA as described previously (34) . Briefly, 96-well Costar (Cambridge, MA) plates were coated overnight with 4 µg/ml of the appropriate antimouse mAb to the cytokine being measured. The wells of the plate were blocked with 10% FBS (Gemini Bioproducts) in PBS for 30 min. The plate was then incubated with the antigen for 1 h, and excess antigen was washed off with PBS/Tween 20. The plate was incubated with 2 µg/ml of biotinylated mAb to the appropriate cytokine (PharMingen) for 30 min, and excess antibody was washed off with PBS/Tween 20. The plates were incubated with avidin peroxidase, and after incubation in O-phenylenediamine substrate to the desired extinction, the subsequent change in color was read at 490 nm with a Molecular Devices Microplate Reader (Sunnyvale, CA). The recombinant cytokines used as standards in the assay were obtained from PharMingen. IL-12 (Biosource) and VEGF (Oncogene Research Products, Cambridge, MA) were determined using kits according to the manufacturer’s instructions. MIG and IP-10 were quantified using a modification of a double ligand method as described previously (35) . The MIG and IP-10 antibodies and protein were obtained from R&D (Minneapolis, MN). The sensitivities of the IL-10, GM-CSF, IFN-{gamma}, TGF-ß, MIG, and IP-10 ELISA were 15 pg/ml. For IL-12 and VEGF the ELISA sensitivities were 5 pg/ml.

PGE2 EIA.
PGE2 concentrations were determined using a kit from Cayman Chemical Co. (Ann Arbor, MI) according to the manufacturer’s instructions as described previously (3) . The EIA plates were read by a Molecular Devices Microplate reader (Sunnyvale, CA).

Flow Cytometry.
For flow cytometric experiments, two or three fluorochromes (PE, FITC, and Tri-color; PharMingen) were used to gate on the CD3 T-lymphocyte population of tumor nodule, lymph node, and splenic single cell suspensions. DCs were defined as the CD11c and DEC 205 bright populations within tumor nodules, lymph nodes, and spleens. Cells were identified as lymphocytes or DCs by gating based on forward and side scatter profiles. Flow cytometric analyses were performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) in the University of California, Los Angeles, Jonsson Cancer Center Flow Cytometry Core Facility. Between 5,000 and 15,000 gated events were collected and analyzed using Cell Quest software (Becton Dickinson).

Intracellular Cytokine Analysis.
T lymphocytes from single cell suspensions of tumor nodules, lymph nodes, and spleens of SLC-treated and diluent-treated CC-10 TAg transgenic mice were depleted of RBC with distilled, deionized H2O and were evaluated for the presence of intracytoplasmic GM-CSF and IFN-{gamma}. Cell suspensions were treated with the protein transport inhibitor kit GolgiPlug (PharMingen) according to the manufacturer’s instructions. Cells were harvested and washed twice in 2% FBS/PBS. Cells (5 x 105) were resuspended in 200 µl of 2% FBS/PBS with 0.5 µg of FITC-conjugated mAb specific for cell surface antigens CD3, CD4, and CD8 for 30 min at 4°C. After two washes in 2% FBS/PBS, cells were fixed, permeabilized, and washed using the Cytofix/Cytoperm kit (PharMingen) following the manufacturer’s protocol. The cell pellet was resuspended in 100 µl of Perm/Wash solution and stained with 0.25 µg of PE-conjugated anti-GM-CSF and anti-IFN-{gamma} mAb for intracellular staining. Cells were incubated at room temperature in the dark for 30 min and washed twice, resuspended in 300 µl of PBS/2% paraformaldehyde solution, and analyzed by flow cytometry.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SLC Mediates Potent Antitumor Responses in a Murine Model of Spontaneous Bronchoalveolar Carcinoma.
We evaluated the antitumor efficacy of SLC in a spontaneous bronchoalveolar cell carcinoma model in transgenic mice in which the SV40 large TAg is expressed under control of the murine Clara cell-specific promoter, CC-10 (33) . Mice expressing the transgene develop diffuse bilateral bronchoalveolar carcinoma and have an average life span of 4 months. SLC (0.5 µg/injection) or the same concentration of murine serum albumin was injected in the axillary lymph node region beginning at 4 weeks of age, three times per week and continuing for 8 weeks. At 4 months when the control mice started to succumb because of progressive lung tumor growth, mice were sacrificed in all of the treatment groups, and lungs were isolated and paraffin embedded. H&E staining of paraffin-embedded lung tumor sections from control-treated mice revealed large tumor masses throughout both lungs with minimal lymphocytic infiltration (Fig. 1A and C)Citation . In contrast, SLC-treated mice had significantly smaller tumor nodules with extensive lymphocytic infiltration (Fig. 1, B and D)Citation . Mice treated with SLC had a marked reduction in pulmonary tumor burden as compared with diluent-treated control mice (Fig. 1E)Citation . SLC-treated mice had prolonged survival compared with mice receiving control injections. Median survival was 18 ± 2 weeks for control-treated mice, whereas mice treated with SLC had a median survival of 34 ± 3 weeks (P < 0.001).



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  		Fig. 1. SLC mediates potent antitumor responses in a murine model of spontaneous lung cancer. The antitumor efficacy of SLC was evaluated in the spontaneous bronchogenic carcinoma model in transgenic mice in which the SV40 large T Ag is expressed under control of the murine Clara cell-specific promoter, CC-10 (41) . Mice expressing the transgene develop diffuse bilateral bronchoalveolar carcinoma and have an average life span of 4 months. SLC (0.5 µg/injection) or the same concentration of murine serum albumin was injected in the axillary lymph node region of 4-week-old transgenic mice three times a week for 8 weeks. At 4 months when the control mice started to succumb because of progressive lung tumor growth, mice in all of the treatment groups were sacrificed, and their lungs were isolated and embedded in paraffin. H&E staining of paraffin-embedded lung tumor sections from control-treated mice evidenced large tumor masses throughout both lungs without detectable lymphocytic infiltration (A and C). In contrast, the SLC therapy group evidenced extensive lymphocytic infiltration with marked reduction in tumor burden (B and D). Arrows in D depict tumor (*1) and infiltrate (*2). (A and B, x32; C and D, x320) E, reduced tumor burden in SLC-treated mice. Tumor burden was quantified within the lung by microscopy of H&E-stained paraffin-embedded sections with a calibrated graticule (a 1-cm2 grid subdivided into 100 1-mm2 squares). A grid square with tumor occupying >50% of its area was scored as positive, and the total number of positive squares was determined. Ten separate fields from four histological sections of the lungs were examined under high-power (x20 objective). There was reduced tumor burden in SLC-treated CC-10 mice compared with the diluent-treated control group. Median survival was 18 ± 2 weeks for control-treated mice. In contrast, mice treated with SLC had a median survival of 34 ± 3 weeks. (P < 0.001; n = 10 mice/group).

 
SLC Treatment of CC-10 TAg Mice Promotes Type 1 Cytokine and Antiangiogenic Chemokine Release and a Decline in the Immunosuppressive Cytokines TGF-ß and VEGF.
On the basis of previous reports indicating that tumor progression can be modified by host cytokine profiles (36 , 37) , we evaluated the cytokine production from tumor sites, lymph nodes, and spleen after SLC therapy. Cytokine profiles in the lungs, spleens, and lymph nodes of CC-10 TAg mice treated with recombinant SLC were compared with those in diluent-treated control mice bearing tumors as well as nontumor bearing controls. SLC treatment of CC-10 TAg mice led to systemic induction of Type 1 cytokines but decreased production of immunosuppressive mediators. Lungs, lymph node, and spleens were harvested, and after a 24-h culture period, supernatants were evaluated for the presence of VEGF, IL-10, IFN-{gamma}, GM-CSF, IL-12, MIG, IP-10, and TGF-ß by ELISA and for PGE-2 by EIA. Compared with lungs from the diluent-treated group, CC-10 TAg mice treated with SLC had significant reductions in VEGF (3.5-fold) and TGF-ß (1.83-fold) but an increase in IFN-{gamma} (160.5-fold), IP-10 (1.7-fold), IL-12 (2.1-fold), MIG (2.1-fold), and GM-CSF (8.3-fold; Table 1Citation ). Compared with the diluent-treated group, splenocytes from SLC-treated CC-10 TAg mice revealed reduced levels of PGE-2 (14.6-fold) and VEGF (20.5-fold) but an increase in GM-CSF (2.4-fold), IL-12 (2-fold), MIG (3.4-fold), and IP-10 (4.1-fold; Table 1Citation ). Compared with diluent-treated CC-10 TAg mice, lymph node-derived cells from SLC-treated mice secreted significantly enhanced levels of IFN-{gamma} (2.2-fold), IP-10 (2.3-fold), MIG (2.3-fold), and IL-12 (2.5-fold) but decreased levels of TGF-ß (1.8-fold; Table 1Citation ). The immunosuppressive mediators PGE-2 and IL-10 were not altered at the tumor sites of SLC-treated mice; however, there was a significant reduction in the level of PGE-2 in the spleen of SLC-treated mice.


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  		Table 1 SLC treatment of CC-10 TAg mice promotes Type 1 cytokine and antiangiogenic chemokine release and a decline in the immunosuppressive and angiogenic cytokines TGF-ß and VEGF

Following axillary lymph node region injection of SLC, pulmonary, lymph node, and spleen cytokine profiles in CC-10 TAg mice were determined and compared with those in diluent-treated tumor bearing control mice and nontumor bearing syngeneic controls. Lungs were harvested, cut into small pieces, passed through a sieve, and cultured for 24 h. Splenocytes and lymph node-derived lymphocytes (5 x 106 cells/ml) were cultured for 24 h. After culture, supernatants were harvested, cytokines quantified by ELISA, and PGE-2 determined by EIA. All determinations from lung were corrected for total protein by Bradford assay, and results are expressed in pg/milligram total protein/24 h. Cytokine and PGE-2 determinations from the spleen and lymph nodes are expressed in pg/ml. Compared with lungs from diluent-treated CC-10 tumor-bearing mice, CC-10 mice treated with SLC had significant reductions in VEGF and TGF-ß but a significant increase in IFN-{gamma}, IP-10, IL-12, MIG, and GM-CSF. Compared with diluent-treated CC-10 TAg mice, splenocytes from SLC-treated CC-10 mice had reduced levels of PGE-2 and VEGF but significant increases in GM-CSF, IL-12, MIG, and IP-10. Lymph node-derived cells from SLC-treated mice secreted significantly enhanced levels of IFN-{gamma}, IP-10, MIG, and IL-12 but decreased TGF-ß levels as compared with diluent-treated CC-10 mice. Values given reflect mean ± SE for six mice/group.

 
To determine whether SLC administration induced significant specific systemic immune responses, splenocytes from SLC and diluent-treated CC-10 TAg mice were cocultured in vitro with irradiated CC-10 TAg tumor cells for 24 h, and GM-CSF, IFN-{gamma}, and IL-10 were determined by ELISA. After secondary stimulation with irradiated tumor cells, splenocytes from SLC-treated tumor-bearing mice secreted significantly increased levels of IFN-{gamma} (5.9-fold) and GM-CSF (2.2-fold). In contrast, IL-10 secretion was reduced 5-fold (Table 3)Citation .


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  		Table 3 Systemic induction of type 1 cytokines and downregulation of IL-10 after SLC treatment

Splenic lymphocytes (5 x 106 cells/ml) were cultured with irradiated CC-10 (105 cells/ml) tumors at a ratio of 50:1 in a total volume of 5 ml. After overnight culture, supernatants were harvested and GM-CSF, IFN-{gamma}, and IL-10 were determined by ELISA. After stimulation with irradiated tumor cells, splenocytes secreted significantly more IFN-{gamma} and GM-CSF but reduced levels of IL-10 from SLC-treated mice compared to diluent-treated tumor-bearing mice. Results are expressed in pg/ml (a P < 0.01 compared with diluent-treated mice as well as SLC-treated constitutive levels). Values given reflect mean ± SE for five mice/group.

 
SLC Treatment of CC-10 TAg Mice Leads to Enhanced DC and T-Cell Infiltrations of Tumor Sites, Lymph Nodes, and Spleen.
To determine the cellular source of GM-CSF and IFN-{gamma}, single cell suspensions of tumors, lymph nodes, and spleens were isolated from SLC and diluent control-treated CC-10 TAg mice. T-lymphocyte infiltration and intracellular cytokine production were assessed by flow cytometry. The cells were also stained to quantify DC infiltration at each site. Compared with the diluent-treated control group, the SLC-treated CC-10 TAg mice showed significant increases in the frequency of cells expressing the DC surface markers CD11c and DEC 205 at the tumor site, lymph nodes, and spleen (Table 2)Citation . Similarly, as compared with the diluent-treated control group, there were significant increases in the frequency of CD4 and CD8 cells expressing IFN-{gamma} and GM-CSF at the tumor sites, lymph nodes, and spleen of SLC-treated CC-10 TAg mice (Table 2)Citation .


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  		Table 2 SLC treatment of CC-10 TAg mice leads to enhanced dendritic and T cell infiltrations of tumor sites, lymph nodes, and spleen

Single-cell suspensions of tumor nodules, lymph nodes, and spleens from SLC and diluent-treated tumor-bearing mice were prepared. Intracytoplasmic staining for GM-CSF and IFN-{gamma} and cell surface staining for CD4 and CD8 T lymphocytes were evaluated by flow cytometry. DCs that stained positive for cell surface markers CD11c and DEC205 in lymph node, tumor nodule, and spleen single-cell suspensions were also evaluated. Cells were identified as lymphocytes or DCs by gating based on the forward and side scatter profiles; 15,000 gated events were collected and analyzed using Cell Quest software. Within the gated T-lymphocyte population from mice treated with SLC, there was an increase in the frequency of CD4+ and CD8+ cells secreting GM-CSF and IFN-{gamma} in the tumor sites, lymph nodes, and spleens compared with those of diluent-treated tumor-bearing control mice. Within the gated DC population, there was a significant increase in the frequency of DCs in the SLC-treated tumor-bearing mice compared with the diluent-treated control tumor-bearing mice.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Host APC are critical for the cross-presentation of tumor antigens (1) . However, tumors have the capacity to limit APC maturation, function, and infiltration of the tumor site (38, 39, 40, 41) . Thus, molecules that attract host APC and T cells could serve as potent agents for cancer immunotherapy. A potentially effective pathway to restore Ag presentation is the establishment of a chemotactic gradient that favors localization of both activated DC and Type 1 cytokine-producing lymphocytes. SLC, a CC chemokine expressed in high endothelial venules and in T-cell zones of spleen and lymph nodes, strongly attracts naive T cells and DCs (23, 24, 25, 26, 27, 28, 29, 30) . Because DCs are potent APCs that function as principle activators of T cells, the capacity of SLC to facilitate the colocalization of both DC and T cells may reverse tumor-mediated immune suppression and orchestrate effective cell-mediated immune responses. In addition to its immunotherapeutic potential, SLC has been found to have potent angiostatic effects (11) , thus adding additional support for its use in cancer therapy. On the basis of these dual capacities we speculated that SLC would be an important protein for evaluation in cancer immunotherapy. Using two transplantable murine lung cancer models, we have shown previously that the antitumor efficacy of SLC is T cell-dependent. In both models, recombinant SLC administered intratumorally led to complete tumor eradication in 40% of the treated mice. The SLC-mediated antitumor response was dependent on both CD4 and CD8 lymphocyte subsets and was accompanied by DC infiltration of the tumor. The results of our earlier findings were recently substantiated by Vicari et al. (42) in the C26 colon cancer model. Using C26 colon carcinoma cells transduced with the SLC cDNA, Vicari et al. (42) demonstrated that the SLC-transduced tumor cells had reduced tumorigenicity that was attributed to both immunological and angiostatic mechanisms (42) . In recent studies that directly support the antiangiogenic capacity of this chemokine, Arenberg et al. (43) have reported that SLC inhibits human lung cancer growth and angiogenesis in a SCID mouse model.

In the models reported previously, the antitumor efficacy of SLC was determined using transplantable murine or human tumors propagated at s.c. sites. We embarked on the current studies to determine the antitumor properties of SLC in a clinically relevant model of lung cancer in which adenocarcinomas develop in an organ-specific manner. Transgenic mice expressing SV40 large TAg transgene under the control of the murine Clara cell-specific promoter, CC-10, develop diffuse bilateral bronchoalveolar carcinoma and have an average life span of 4 months (33) . The antitumor activity of SLC was determined in the spontaneous model for lung cancer by injecting recombinant SLC into the axillary lymph node region of the transgenic mice. The efficacy of injecting immune stimulators in the vicinity of the lymph nodes for the treatment of cancer has been demonstrated in recent studies; vaccination with tumor cell-DC hybrids in the lymph node region led to regression of human metastatic renal cell carcinoma (44) . Our rationale for injecting SLC in the lymph node region was to colocalize DC to T-cell areas in the lymph nodes where they can prime specific antitumor immune responses. In many clinical situations access to lymph node sites for injection may also be more readily achievable than intratumoral administration. Our results show that this approach is effective in generating systemic antitumor responses. SLC injected in the axillary lymph node regions of the CC-10 TAg mice evidenced potent antitumor responses with reduced tumor burden and a survival benefit as compared with CC-10 TAg mice receiving diluent control injections. The reduced tumor burden in SLC-treated mice was accompanied by extensive lymphocytic as well as DC infiltrates of the tumor sites, lymph nodes, and spleens.

The cytokine production from tumor sites, lymph nodes, and spleens of the CC-10 TAg mice was altered as a result of SLC therapy. The following cytokines were measured: VEGF, IL-10, PGE-2, TGF-ß, IFN-{gamma}, GMCSF, IL-12, MIG, and IP-10 (Table 1)Citation . The production of these cytokines was evaluated for the following reasons: the tumor site has been documented to be an abundant source of PGE-2, VEGF, IL-10, and TGF-ß, and the presence of these molecules at the tumor site has been shown to suppress immune responses (3 , 38 , 45) . VEGF, PGE-2, and TGF-ß have also been documented previously to promote angiogenesis (46, 47, 48) . Antibodies to VEGF, TGF-ß, PGE-2, and IL-10 have the capacity to suppress tumor growth in in vivo model systems. VEGF has also been shown to interfere with DC maturation (38) . Both IL-10 and TGF-ß are immune inhibitory cytokines that may potently suppress Ag presentation and antagonize CTL generation and macrophage activation (4 , 45) . Although at higher pharmacological concentrations IL-10 may cause tumor reduction, physiological concentrations of this cytokine suppress antitumor responses (4 , 49, 50, 51) . Before SLC treatment in the transgenic tumor-bearing mice, the levels of the immunosuppressive proteins VEGF, PGE-2, and TGF-ß were elevated when compared with the levels in normal control mice. There was no such increase with IL-10. Similarly there were not significant alterations in IL-4 and IL-5 after SLC therapy (data not shown). SLC-treated CC-10 TAg mice showed significant reductions in VEGF and TGF-ß. The decrease in immunosuppressive cytokines was not limited to the lung but was evident systemically. SLC treatment of CC-10 TAg transgenic mice led to a decrease in TGF-ß in lymph node-derived cells and reduced levels of PGE-2 and VEGF from splenocytes. Thus, possible benefits of a SLC-mediated decrease in these cytokines include promotion of antigen presentation and CTL generation (4 , 45) , as well as a limitation of angiogenesis (46, 47, 48) .

It is well documented that successful immunotherapy shifts tumor-specific T-cell responses from a type 2 to a type 1 cytokine profile (52) . Responses depend on IL-12 and IFN-{gamma} to mediate a range of biological effects, which facilitate anticancer immunity. IL-12, a cytokine produced by macrophages (53) and DC (54) , plays a key role in the induction of cellular immune responses (55) . IL-12 has been found to mediate potent antitumor effects that are the result of several actions involving the induction of CTL, Type 1-mediated immune responses, and natural killer activation (53) , as well as the impairment of tumor vascularization (56) . IP-10 and MIG are CXC chemokines that chemoattract activated T cells expressing the CXCR3 chemokine receptor (57) . Both IP-10 and MIG are known to have potent antitumor and antiangiogenic properties (14 , 58, 59, 60) . The lungs of SLC-treated CC-10 TAg mice revealed significant increases in IFN-{gamma}, IL-12, IP-10, MIG, and GM-CSF. MIG and IP-10 are potent angiostatic factors that are induced by IFN-{gamma} (59 , 61 , 62) and may be responsible in part for the tumor reduction in CC-10 TAg mice after SLC administration. Because SLC is documented to have direct antiangiogenic effects (11 , 63) , the tumor reductions observed in this model maybe attributable to T cell-dependent immunity as well as participation by T cells secreting IFN-{gamma} in inhibiting angiogenesis (62) . Hence, an increase in IFN-{gamma} at the tumor site of SLC-treated mice could explain the relative increases in IP-10 and MIG. Both MIG and IP-10 are chemotactic for stimulated CXCR3-expressing T lymphocytes that could additionally amplify IFN-{gamma} at the tumor site (64) . Flow cytometric determinations revealed that both CD4 and CD8 cells were responsible for the increased secretion of GM-CSF and IFN-{gamma} in SLC-treated mice. An increase in GM-CSF in SLC-treated mice could enhance DC maturation and antigen presentation (32) . Additional studies are necessary to precisely define the host cytokines that are critical to the SLC-mediated antitumor response.

The increase in the Type 1 cytokines was not limited to the lung but was evident systemically. SLC treatment of CC-10 TAg transgenic mice led to systemic increases in Type I cytokines and antiangiogenic chemokines. Hence, splenocytes from SLC-treated CC-10 TAg mice had an increase in GM-CSF, IL-12, MIG, and IP-10 as compared with diluent-treated CC-10 TAg mice. Similarly, lymph node-derived cells from SLC-treated mice secreted significantly enhanced levels of IFN-{gamma}, IP-10, MIG, and IL-12. Recent studies suggest that the evaluation of type 1 responses at the LN sites may provide insights into antitumor responses in patients receiving immune therapy (65) . The increase in GM-CSF and IFN-{gamma} in the spleen and lymph nodes of SLC-treated mice could in part be explained by an increase in the frequency of CD4 and CD8 cells secreting these cytokines. The increase in Type 1 cytokines was in part attributable to an increase in specificity against the autologous tumor; when cocultured with irradiated CC-10 TAg tumor cells, splenocytes from SLC-treated CC-10 TAg mice secreted significantly increased amounts of GM-CSF and IFN-{gamma} but reduced levels of IL-10. Whereas the T cells secrete cytokines in response to stimulation with CC-10 cells, we have not yet confirmed that this cytokine secretion is tumor-specific. Cell surface staining of CC-10 cells followed by flow cytometry did not show detectable levels of MHC class II molecules. Although the tumor did not show MHC class II expression, CD4+ type 1 cytokine production may have occurred because splenic APC were present in the assay. Although in vitro tumor-stimulated splenic T cells from SLC-treated mice showed reduced expression of IL-10, SLC therapy did not lead to a decrease of IL-10 levels in vivo. The in situ microenvironment may provide other important factors from cellular constituents in addition to T cells that determines the overall levels of IL-10. This may explain the discrepancies in the in vitro and in vivo results.

Taken together, the current study indicates that SLC injected in the axillary lymph node region in the spontaneous lung cancer model leads to the generation of systemic antitumor responses. The antitumor properties of SLC may be attributable to its chemotactic capacity in colocalization of DCs and T cells, as well as the induction of key cytokines such as IFN-{gamma}, IP-10, MIG, and IL-12. Additional studies will be required to delineate the importance of each of these cytokines in SLC-mediated antitumor responses. The potent antitumor properties demonstrated in this model of spontaneous bronchoalveolar carcinoma provide a strong rationale for additional evaluation of SLC regulation of tumor immunity and its use in immunotherapy for lung cancer.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant R01 CA78654, P01 1P50 CA90388 (to S. M. D.), and R01 CA87879 (to R. S.); Medical Research Funds from the Department of Veteran Affairs; the Research Enhancement Award Program in Cancer Gene Medicine; and the Tobacco-Related Disease Research Program of the University of California. Back

2 To whom requests for reprints should be addressed, at Division of Pulmonary and Critical Care Medicine, School of Medicine, 37–131 Center for Health Sciences, 10833 LeConte Avenue, Los Angeles, CA 90095-1690. E-mail address: sdubinett@mednet.ucla.edu. Back

3 The abbreviations used are: APC, antigen-presenting cell; SLC, secondary lymphoid organ chemokine; DC, dendritic cell; IP-10, IFN-{gamma} inducible protein 10; TGF-ß, transforming growth factor ß; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; FBS, fetal bovine serum; mAb, monoclonal antibody; VEGF, vascular endothelial growth factor; EIA, enzyme immunoassay; SV40 TAg, simian virus 40 large T antigen; Ag, antigen; PGE2, prostaglandin E2; PE, phycoerythrin; LN, lymph node. Back

Received 4/23/01. Accepted 7/ 2/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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L. Chun, C.-C. Yin, J.-Z. Song, M.-X. Liu, J.-H. Piao, Q. Lin, X.-B. Wang, and H.-L. Huang
Soluble Expression of Recombinant Human Secondary Lymphoid Chemokine (SLC) in E. coli and Research on Its In Vitro and In Vivo Bioactivity
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I. Treilleux, J.-Y. Blay, N. Bendriss-Vermare, I. Ray-Coquard, T. Bachelot, J.-P. Guastalla, A. Bremond, S. Goddard, J.-J. Pin, C. Barthelemy-Dubois, et al.
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Y.-F. He, G.-M. Zhang, X.-H. Wang, H. Zhang, Y. Yuan, D. Li, and Z.-H. Feng
Blocking Programmed Death-1 Ligand-PD-1 Interactions by Local Gene Therapy Results in Enhancement of Antitumor Effect of Secondary Lymphoid Tissue Chemokine
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L. Zhang, S. Sharma, L. X. Zhu, T. Kogai, J. M. Hershman, G. A. Brent, S. M. Dubinett, and M. Huang
Nonradioactive Iodide Effectively Induces Apoptosis in Genetically Modified Lung Cancer Cells
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C. J. Moran, D. A. Arenberg, C.-C. Huang, T. J. Giordano, D. G. Thomas, D. E. Misek, G. Chen, M. D. Iannettoni, M. B. Orringer, S. Hanash, et al.
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