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Originally published online as doi:10.1189/jlb.0306212 on September 15, 2006

Published online before print September 15, 2006
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(Journal of Leukocyte Biology. 2006;80:1434-1444.)
© 2006 by Society for Leukocyte Biology

Targeted in vivo expression of IFN-{gamma}-inducible protein 10 induces specific antitumor activity

Xiuli Yang*,1, Yiwei Chu*,1, Ying Wang*, Ruihua Zhang* and Sidong Xiong*,{dagger},2

* Department of Immunology of Shanghai Medical College and Institute for Immunobiology, Fudan University, Shanghai, People’s Republic of China; and
{dagger} Immunology Division, E-Institutes of Shanghai Universities, Shanghai, People’s Republic of China

2 Correspondence: Department of Immunology, Shanghai Medical College of Fudan University, 138 Yixueyuan Rd., Shanghai 200032, P.R. China. E-mail: sdxiongfd{at}126.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although it is known that the chemoattractant effect of IFN-{gamma} inducible protein 10 (IP-10), a CXC chemokine (CXCL10), plays an important role in T cell-mediated antitumor immunity in vivo, whether IP-10 is involved in modulating the proliferation, survival and functional activation of tumor-specific T cells remains poorly investigated. Using an experimental mouse tumor model, we demonstrated that the in vivo growth of 4T1 tumor cells harboring IP-10 gene (4T1-IP-10) was inhibited. Mice inoculated with 4T1-IP-10 tumor cells expressing functional IP-10 survived over 90 days, whereas mice injected with control parental 4T1 cells and mice of control 4T1 cells transduced with control plasmid all succumbed to the tumor by day 38 after tumor inoculation. Mechanical analysis showed that targeted expression of IP-10 in 4T1 tumor cells markedly enhanced the infiltration of tumor-specific T cells into the 4T1-IP-10 tumor. These tumor infiltrating T lymphocytes (TILs) recruited by IP-10 were potent cytolytic killers against 4T1 tumor cells and were able to proliferate and produce high levels of IFN-{gamma} in response to 4T1 cells. In vivo administration of IP-10-recruited TILs induced vigorous proliferation of these TILs in situ in the 4T1-IP-10 tumor but not in the 4T1-pcDNA3 and parental 4T1 tumors. Furthermore, culture of TILs together with recombinant IP-10 significantly enhanced the proliferation and expansion of IP-10-recruited TILs in response to 4T1 tumor antigens. These results suggest that IP-10 is not only able to chemoattract tumor-specific T cells into the local tissue, but also enhance the proliferation, survival, and functional activation of these TILs, leading to the tumor regression. Thus, targeted expression of IP-10 in vivo will allow for the development of a novel approach for immunotherapy of tumor.

Key Words: CXCL10 • anti-tumor immunity • 4T1 • lymphoproliferation • recruitment


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines comprise a superfamily of small (8-10 kDa) cytokines that control the migration of leukocytes and regulate the expression of adhesion molecules [1 2 3 ]. During an inflammatory response, leukocytes exit from circulation and infiltrate into inflamed local tissues in response to chemokines [4 , 5 ]. Blockade of leukocyte migration into local tissues inhibits inflammation [6 ]. Interestingly, injection of the chemokine responsible for the migration of T lymphocytes into established tumor markedly enhances the antitumor effect and results in prolonged survival of tumor-bearing animals [7 , 8 ]. Mechanical analyses in these experimental models demonstrate that this chemokine-mediated antitumor effect was associated with enhanced recruitment of antitumor T lymphocytes into the local tissues [1 2 3 ] and with inhibited angiogenesis in the tumor site [9 ]. These data are in agreement with recent observations that a successful in vivo antitumor effect relies on not only the traffic of T lymphocytes into the local tumor tissues but also the persistence and survival of these antitumor T lymphocytes [10 11 12 13 ]. Thus, targeted delivery of chemokine to tumor sites will prove to be valuable for developing novel approaches for eradicating tumor.

IFN-{gamma} inducible protein 10 (IP-10) is a CXC chemokine produced by certain types of cells, including activated monocytes, fibroblasts, endothelial cells, and keratinocytes. The primary effect of IP-10 is regulating the migration of CXCR3+ T lymphocytes [1 , 7 , 13 , 14 ]. It has been shown that IP-10 involves in multiple Th1-type inflammatory diseases such as rheumatoid arthritis [15 ], multiple sclerosis [16 ], psoriasis [17 ], and allograft rejection [18 ], indicating that IP-10 plays an important role in regulating T cell-mediated immune responses and inflammatory reactions. During the progression of these diseases, IP-10 induces the migration of T cells via its receptor CXCR3 that is mainly expressed on activated T cells and NK cells, resulting in the localization and accumulation of inflammatory cells in local tissues and the subsequent tissue injury [19 , 20 ]. Previous studies show that IP-10 is able to significantly enhance antitumor immunity. Intratumoral transgene expression of IP-10 mediated by adenoviral vector augments the infiltration of T cells into the tumor in mice and the subsequent tumor eradication [7 ]. However, it remains to be investigated whether in addition to its chemoattractant effect, IP-10 plays an important role in expanding and sustaining T lymphocytes that are recruited into local tissues.

Using experimental 4T1 breast cancer murine model [21 , 22 ], we studied the mechanisms by which IP-10 enhanced antitumor effect of T lymphocytes in vivo. We found that targeted IP-10 not only enhanced the trafficking of T lymphocytes into the tumor but also significantly augmented the expansion of antitumor T lymphocytes in local tissues. Thus, targeted in vivo expression of IP-10 will prove to be an invaluable approach of antitumor therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and reagents
4T1 cells derived from a Balb/c spontaneous mammary carcinoma [21 , 22 ], Renca cells derived from a Balb/c spontaneous renal carcinoma cell line and L929 fibroblast cells were all kindly donated by Dr. Hong-Ming Hu from EACRI, OR). They were cultured at 37°C under 5% CO2 in complete RPMI 1640 (GIBCO, Grand Island, NY) medium containing 10% heat-inactivated FBS, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin sulfate. Recombinant mouse IFN-{gamma} and anti-mouse IP-10 antibody were purchased from R&D systems (Minneapolis, MN). Recombinant IP-10 and ITAC protein were purchased from PeproTech (Rocky Hill, NJ). Anti-CD3 and anti-CD28 antibodies were purchased from eBioscience (eBioscience, San Diego, CA). Goat antibody (Ab) to mouse CXCR3 was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). FITC-conjugated goat Ab to mouse Thy1, FITC-conjugated goat Ab to mouse IgG, FITC-conjugated donkey Ab to goat IgG, PerCP-conjugated hamster Ab to mouse CD4 and PE-conjugated hamster Ab to mouse CD8 were purchased from PharMingen (San Diego, CA). Fluorescence dye 5- and CFSE was purchased from Fluka (Buchs, Switzerland). Other reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) into tumor-inoculated mice
TILs were separated as described previously [23 ]. In brief, mice were inoculated s.c. with 1 x 105 4T1-IP-10 cells on day 0. Fourteen days after injection, tumors were sectioned, cut into pieces, and suspended in RPMI1640 containing 1 mg/ml collagenase IV. The resultant single-cell suspension was loaded onto Ficoll for gradient centrifugation and separation of tumor-infiltrating lymphocytes. The TILs were then labeled with 5 µM CFSE in vitro And after that, 1x107 cells/mouse CFSE-labeled lymphocytes in a 0.2-ml volume were injected intravenously into the tumor-bearing mice, which had been inoculated with 1 x 105 4T1-IP-10, parental 4T1, or 4T1-pcDNA3 cells, respectively. Subsequent to the adoptive transfer in vivo, the bulk lymphocytes, including CFSE-labeled and unlabeled were isolated from tumors at the indicated time points.

Plasmid construction and in vitro transfection
Total RNA was extracted from IFN-{gamma} (1000 U/ml)-stimulated L929 cell line using Trizol (Invitrogen, Carlsbad, CA). The oligonucleotides used for RT-PCR were as follows: IP-10 forward (5'-CGGAATTCATCAGCACCATGAACCCAAGT-3'), and backward (5'-GCGTGGCTTCTCTCCAGTT-3'), and GAPDH forward (5'-CTGCACCACCAACT GCTTAG-3') and backward (5'-GTCTGGGARGGAAAR RGRGA-3'). IP-10 cDNA was amplified with RT-PCR and subsequently subcloned into the EcoRI and XhoI sites of the ampicillin-selectable mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA). 4T1 cells were transfected with pcDNA3-IP-10 plasmid by electroporation and selected in complete RPMI1640 medium containing 0.8 mg/ml G418 for two weeks. Limiting dilution was performed to select single G418-resistant 4T1-IP-10 cell clone that produces high levels of IP-10 protein. 4T1 cells transfected with pcDNA3 plasmid were selected as controls.

Detection of IP-10 expression via ELISA
The detection of IP-10 expression from the cultured supernatant of 4T1-IP-10, 4T1-pcDNA3, and 4T1 was performed by ELISA. All regents for ELISA were from R&D Systems (Minneapolis, MN) and were performed according to the manufacturer’s protocol. Briefly, 2 µg/ml anti-mIP-10 antibody was coated overnight at 4°C. After coating, wells were blocked with 1% BSA in PBS for 2 h. Wells were incubated with supernatants from various cultured cells at 1:10 dilution for 2 h at 37°C in PBS containing 1% BSA and 0.05% Tween-20. Add 100 µl of mice IP-10 conjugate to each well for 2 h at room temperature. Add 100 µl substrate solution for 30 min and stopped by stop solution. The results were determined by using a microplate reader set to 450 nm to measure the concentration of IP-10 from the supernatant, according to the mIP-10 standard curve.

Inoculation of tumor cells
Female Balb/c mice at 5–6 weeks age were purchased from the Center of Experimental Animal, Fudan University (Shanghai, China). Mice were housed in a pathogen-free animal care facility in our institution. All animal experiments were performed according to the guidelines for the care and use of laboratory animals (Ministry of Health, People’s Republic of China, 1998) and the guidelines of the Shanghai Medical Laboratory Animal Care and Use Committee. Mice were injected subcutaneously (s.c.) with 1 x 105 4T1-IP-10 cells, 4T1-pcDNA3, or parental 4T1 cells to induce tumor. Mice were monitored for evidence of tumor growth by palpation and inspection twice a week until they were killed at the indicated time after tumor cell inoculation.

Chemotaxis assay
Chemotactic assay was performed as described previously [24 ]. In brief, 27 µl supernatants harvested from 1 x 106 cultured 4T1-IP-10 cells, 4T1-pcDNA3, or parental 4T1 cells for 48 h were added in triplicate into the lower chamber of 48-well chemotaxis plate with 5-µm pores (Corning Costar, Corning, NY) in a medium of RPMI-1640 containing 0.1% BSA used as a negative control. Recombinant IP-10(1 ng/ml) and ITAC (1 ng/ml) were set for the positive control. In addition, a neutralizing anti-IP-10 antibody (10 µg /ml), which was added in the lower chamber to block the function of IP-10, was also performed as the control. The upper chambers of the chemotaxis plate were filled with 50 µl of IL-2-stimulated activated splenocytes suspension (1x107cells/ml) that had previously incubated without or with 10 µg/ml mCXCR3-specific Ab (Y-16) for 30 min at 4°C. IL-2-stimulated splenocytes preincubated with 10 µg/ml control IgG for 30 min were identically added as controls. The chemotaxis plate was incubated at 37°C. Four hours later, the numbers of cells that migrated into the lower chambers were numerated. The chemotaxis index (CI) was calculated by dividing the number of cells migrated in response to testing supernatants by the number of cells migrated in response to negative control medium.

Real-time RT-PCR
The quantitative analysis of IP-10 expressing in tumor tissues was done using a LightCyclerTM (Roche Diagnostic Company, Mannheim, Germany) [25 ]. For relative quantitative analysis, housekeeper gene GAPDH was used as internal control, and relative quantity of IP-10 mRNA was represented by the ratio of the expression quantity of IP-10 to that of GAPDH. The reaction mixture was consisted of 2 µl of LightCyclerTM DNA Master SYBR Green I (Roche Diagnostic Company). The cycling program had a 10-min initial denaturation at 95°C and then entered a cycle of an instant 95°C denaturation, 10 s of annealing at 60°C, and 12 s of extension at 72°C with transition rate of 20°C/s between temperature plateaus for a total of 40 cycles. Quantification of data was analyzed using the LightCycler analysis software version 4.5.

Flow cytometry analysis
Lymphocytes (1x106) were sequentially stained with goat Ab to mouse CXCR3 antibody, FITC conjugated donkey Ab to goat IgG, PE-CD8 (53-6.7), and PerCP-CD4 (RM4-5) at the dosage of 1 µl/0.5 ml volume each, followed by flow cytometry analysis using CellQuest software (Becton Dickinson, San Jose, CA).

Immunofluorescence microscopy
Tumor tissues were collected and fixed in 4% paraformaldehyde and embedded for cryosection. Tumor sections (6 µm) were fixed for 20 min in cold acetone, hydrated in PBS, and treated with 3% BSA in PBS for 30 min at 37°C to block nonspecific bindings. Then these slides were incubated with FITC-conjugated goat anti-mouse Thy1 Ab (1:50 dilution) at 37°C for 30 min followed by overnight incubation at 4°C. Slides were checked for fluorescence using Olympus microscope (Tokyo, Japan). In indicated experiments, tissue sections were H&E stained for histologic examination.

In vivo and in vitro IP-10 depletion assay
To block IP-10 in vivo, mice inoculated with 4T1-IP-10, 4T1-pcDNA3, and 4T1 tumor were injected intratumor with 100 µg/ml anti-mouse IP-10 (R&D Systems) for 5 days to neutralize the IP-10 secretion. For in vitro neutralization, 10 µg /ml anti-mouse IP-10 was added in TILs stimulated with the supernatant from 4T1-IP-10 TILs, and the lymphocytes proliferation assay was then performed.

Tumorigenesis experiments
Female Balb/c immunocompetent mice (n=6) and nude mice (n=6) were s.c. injected with 1 x 105 4T1-IP-10, 4T1-pcDNA3, and 4T1 tumor cells, respectively. Mice were monitored for evidence of tumor growth by palpation and inspection twice a week until they were killed on day 30. The survival of the tumor-bearing mice was observed by daily assessment for over 90 days.

Cell proliferation assay
Splenocytes and TILs that were isolated from 4T1-IP-10-, 4T1-pcDNA3-, and 4T1-inoculated tumors were added into the wells of 96-well plate at a number of 5 x 105 cells per well with the existence of 20 U/ml IL-2.Mitomycin C (MMC)-treated 4T1 cells were added into these tumor-infiltrating cells at a dose ranging from 0 to 50,000 cells per well. The plate was incubated for 3 days at 37°C prior to measuring the proliferation of lymphocytes in response to 4T1 target antigen. The proliferation of the lymphocytes stimulated with MMC-treated Renca cells was identically examined as control. 3H-thymidine (Shanghai Atomic Energy Institute, Chinese Academy of Science) was added at 1 µCi per well for the last 18 h of the 3-day culture. 3H-thymidine incorporation was measured in a liquid scintillation counter (Shanghai Atomic Energy Institute, Chinese Academy of Science). Results are expressed as a cpm. In addition, to determine the effect of IP-10 on TIL proliferation, TILs were ex vivo cultured with the addition of different concentrations (0, 10, 100, 1,000 ng/ml) of recombinant IP-10 protein or 10 ng/ml supernatant from cultured TILs isolated from 4T1-IP-10 inoculated tumors. The proliferation of TILs under the existence of IP-10 was determined by [3H]-thymidine incorporation at the final 18 h of culture. Also, naïve T cells activated in vitro with 2 µg/ml anti-CD3 combined with anti-CD28 or rIP-10 were cultured for 72 h. The proliferation of these activated T cells under the existence of IP-10 was determined by [3H]-thymidine incorporation at the final 18 h of culture.

Quantitation of specific lysis by the CFSE/7-AAD cytotoxicity assay
Cytotoxicity assay was performed as described previously with minor modification [26 ]. Briefly, TILs were stimulated with MMC-treated 4T1 target cells for four days as effector cells. After that, these TILs were labeled with 5 µM CFSE [27 ] and seeded with specific target 4T1 cells (1x104 /well) in a 96-well plate, respectively. The ratios of effector vs. target (E:T) were at 10:1, 20:1, and 40:1. In parallel, target cells were incubated alone to measure basal apoptosis. Six hours after culture, cells were collected and incubated in PBS/1% BSA containing 20 µg/ml 7-AAD (Sigma) for 20 min at 4°C in the dark. These cells were then fixed in 4% paraformaldehyde (Sigma) followed by flow cytometry analysis. CFSE fluorescence and 7-AAD emission were detected in the FL-1 and FL-3 channels, respectively. For each E:T ratio, 20,000 target cells were acquired. Analysis was performed with the Cell Quest software (BDIS). The formula to calculate the percentage of specific target cell lysis is: % specific lysis=100 x (% sample lysis-% basal lysis)/(100-% basal lysis).

ELISPOT assay
ELISPOT was performed according to the instruction of the manufacturer (BD Biosciences). In brief, TILs (1x105/well) were plated on 96-well plates that had been previously coated with 2 µg/ml anti-mouse IFN-{gamma} Ab. MMC-treated 4T1 cells (1x103 /well) were added as stimulators into tumor-infiltrating lymphocyte cultures. The plates were incubated 37°C for 18 h. To measure the cells producing IFN-{gamma}, 1 µg/ml secondary biotin-conjugated Ab to mouse IFN-{gamma} was added into the plate at 37°C for 1 h after washing away the cells, followed by 50 µl addition of alkaline phosphatase-conjugated streptavidin at 37°C for 1 h. Spots were visualized by adding 30 µl 5-bromo-4-chloro-3-indolyl phosphate, and counted using the KS ELISPOT Imagine System (Carl Zeiss, Thornwood, NY).

Statistical analysis
Statistical analyses of the data were performed with the aid of analysis programs in SPSS10.0 software. Statistical evaluation was performed using two-way ANOVA (ANOVA; P<0.05) using the program PRISM 4.0 (GraphPad Software, San Diego, CA). All results with a P value less than 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transfection of 4T1 cells with IP-10 gene
To determine the in vivo antitumor effect of IP-10, 4T1 cells were transfected with plasmid-encoding IP-10. G418-resistant, IP-10-transfected 4T1 cell clones (termed 4T1-IP-10) were selected based on the expression of IP-10. Higher levels of IP-10 mRNA (Fig. 1A ) and IP-10 protein from cultured supernatant (Fig. 1B) were detected in 1x106 48 h-cultured 4T1-IP-10 cells compared with nontransfected parental 4T1 cells and the 4T1 cells transfected with mock plasmid (termed 4T1-pcDNA3) (P<0.01). To further determine the function of IP-10 protein produced by 4T1-IP-10 cells, supernatants from cell cultures were collected for measuring their chemotactic activity against IL-2-stimulated T lymphocytes that expressed high level of CXCR3 (Fig. 1C) . 4T1-IP-10 cell-derived supernatants chemoattracted 2 times more IL-2-stimulated CXCR3+ T lymphocytes than did the supernatants derived from parental 4T1 and 4T1-pcDNA3 cells (Fig. 1D , P<0.01). To confirm that the phenomenon was mediated by the expression of IP-10, anti-IP-10 neutralizing antibody was used. Results showed a significant decrease in chemotaxis index after using anti-IP-10 neutralizing antibody. However, it is worth noting that the chemotaxis index was not blocked when anti-IP-10-neutralizing antibody had an effect on another chemokine, ITAC, which shared the CXCR3 receptor of IP-10 and functioned to chemoattract CXCR3+ T cells (Fig. 1D , P<0.01), indicating that the chemoattractant to CXCR3+ T was caused by IP-10. On the other hand, addition of neutralizing antibody to CXCR3 inhibited the chemoattracting effect of 4T1-IP-10 cells-derived supernatants on IL-2-activated T lymphocytes (Fig. 1E , P<0.01). These results suggest that functional expression of IP-10 is established in 4T1-IP-10 tumor cells.


Figure 1
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  		Figure 1. IP-10 expression and its chemotactic activity from the supernatant of 4T1-IP-10 cells. (A) Total RNA was extracted and reverse transcribed with random primers. The cDNA was subjected to RT-PCR. The level of IP-10 mRNA expression in parental 4T1 cells (open bars), 4T1-pcDNA3 cells (hatched bars), and 4T1-IP-10 cells (closed bars) was normalized to the level of GAPDH; P < 0.01. (B) IP-10 secretion from the supernatant of 4T1 transfected with various plasmids. Supernatants from 1 x 106 4T1-IP-10 (solid bar), 4T1-pcDNA3 (hatched bar), and 4T1 (open bar) tumor cells, which were cultured for 48 h, were collected, and IP-10 production was detected via ELISA with IP-10-specific antibody; P < 0.01. (C) The expression of CXCR3 was analyzed by flow cytometry. Lymphocytes stimulated without (left) or with (right) IL-2 were collected and stained without (thin trace) or with CXCR3 antibody followed with FITC-IgG, (thick trace). Staining with the secondary antibody alone was also shown (dotted line). (D) Chemotaxis assay of naïve (open bars) and IL-2-stimulated lymphocytes (closed bars) as well as IL-2 stimulated T cells treated with anti-IP-10 antibody (hatched bar) in response to cultured supernatants of 4T1-IP-10, 4T1-pcDNA3 and parental 4T1 cells was performed. 1 ng/ml recombinant IP-10 (rIP-10) or recombinant ITAC served as positive controls. Chemotaxis index (CI) was calculated by dividing the number of cells migrated in response to testing supernatants or positive controls by the number of cells migrated in response to negative control medium. A significant chemoattractant activity was exhibited in 4T1-IP-10 and rIP-10 or ITAC groups (P<0.01). (E) Chemotaxis assay of IL-2-stimulated lymphocytes preincubated with anti-CXCR3 antibody (open bars), isotype IgG (solid bars), or without any treatment (hatched bar) in response to the cultured supernatants of 4T1-IP-10, 4T1-pcDNA3, or parental 4T1 cells was performed. The experiments were repeated three times. Data represent means ± SD. There was a significant difference between 4T1-IP-10 group and 4T1-pcDNA3 or parental 4T1 group (P<0.01).

 
Inhibited growth of IP-10 gene-transfected 4T1 tumor cells in vivo
To investigate whether the expression of IP-10 in 4T1 tumor cells enhanced in vivo antitumor activity, 1 x 105 4T1, 4T1-pcDNA3, and 4T1-IP-10 tumor cells were injected s.c. into Balb/c mice, respectively. Both Balb/c mice receiving 4T1 cells and Balb/c mice receiving 4T1-pcDNA3 cells developed tumors, in which the tumor progressively grew (Fig. 2A ). In contrast, the in vivo growth of 4T1-IP-10 tumor cells was markedly inhibited (P<0.01). By day 30 after tumor inoculation, the tumor weight in mice receiving 4T1-IP-10 cells was 3 times less than that in mice inoculated with parental 4T1 or 4T1-pcDNA3 cells (Fig. 2B , P<0.01). Mice injected with 4T1-IP-10 tumor cells survived much longer than mice receiving 4T1 cells and mice of 4T1-pcDNA3 cells (median survival time, 53 days vs. 38 days and 38 days, respectively). As many as 33% of mice inoculated with 4T1-IP-10 tumor cells survived more than 90 days, whereas all control mice succumbed to the tumor by day 38 after tumor inoculation (Fig. 2C , P<0.05). To further determine that the forced expression of IP-10 played an important role in the tumor regression, specific anti-mouse IP-10 antibody was injected intratumor to neutralize the IP-10 secretion. Results showed that the significant progress of tumor growth could be observed in 4T1-IP-10 tumor inoculation, whereas no changes of tumor growth in 4T1-pcDNA3 and 4T1 tumor (Fig. 2D , P<0.05), indicating that blocking the function of IP-10 greatly hampered the antitumor immunity.


Figure 2
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  		Figure 2. Effects of IP-10 expression on inhibiting 4T1 tumor growth in vivo. (A) Balb/c mice were injected s.c. with 1 x 105 4T1-IP-10 cells, 4T1-pcDNA3 cells, and parental 4T1 cells. Tumor diameters were expressed as an average of the longest and perpendicular diameter ± SD (n=6). (B) Tumors were sectioned at day 30. The tumor weight was measured among the three groups; P < 0.01. (C) Long-term survival obtained in 4T1-IP-10 inoculated tumor. Mice (n=6) were inoculated with 4T1, 4T1-pcDNA3, and 4T1-IP-10 on day 0. Survival was observed by daily assessment for more than 90 days. The percentage of survival in each group was calculated. (D) Inhibition of antitumor efficacy after using neutralizing IP-10 Ab in vivo. Mice (n=6 per group) inoculated with 4T1-IP-10 and 4T1 tumor cells were injected intratumor with anti-mouse IP-10 monoclonal antibody at 5-day intervals. Tumor size was monitored with a caliper every other day. Each of the curves shown represents the mean tumor size per group (P<0.05). The experiments were repeated twice.

 
To rule out the possibility that IP-10 gene transduction per se affects the survival and growth of 4T1 cells, parental 4T1, 4T1-pcDNA3, and 4T1-IP-10 cells were cultured in vitro under the same conditions. As shown in Fig. 3 , each of these cell lines had the same growth rate in the cultures over a period of 6 days (Fig. 3 , P>0.05), indicating that IP-10 gene transduction per se did not change the tumorigenicity of 4T1 cells. Taken together, these results indicate that prolonged survival of mice inoculated with 4T1-IP-10 cells was associated with the functional expression of IP-10.


Figure 3
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  		Figure 3. Effect of IP-10 gene introduction on the growth of 4T1 cells in vitro. The tumor growth was observed by plating 1 x 104 tumor cells in individual plates in triplicate. The 4T1-IP-10 cells, 4T1-pcDNA3, and parental 4T1 cells were harvested and counted at the designated time points (P>0.05). Values shown are the means ± SD of triplicate wells from one representative experiment of at least three.

 
Targeted in vivo expression of IP-10 enhances the recruitment of lymphocytes into tumor site
We next determined the mechanisms by which the in vivo growth of 4T1-IP-10 tumor cells was inhibited. 4T1-IP-10, 4T1-pcDNA3 and 4T1 tumor cells were injected s.c. into Balb/c mice, and tumors were removed after 14 days. RT-PCR assay showed that the expression of IP-10 was substantially detected in tumor tissues from mice injected with 4T1-IP-10 but not in those injected with 4T1-pcDNA3 or 4T1 cells (Fig. 4A , P<0.001). Histologic examination showed the number of lymphocytes in the tumor from mice injected with 4T1-IP-10 cells increased 2.5-fold more than those from mice received the parental 4T1 and 4T1-pcDNA3 cells (Fig. 4 B and C , P<0.05). Immunofluorescence assay showed that among the infiltrated lymphocytes, the number of T cells in the tumors of the mice received 4T1-IP-10 increased significantly compared with that from the mice that received the parental 4T1 cells and mice of control 4T1-pcDNA3 tumor cells (25±3 vs. 4±2, and 6±1 cells per field) (Fig. 4 D and E , P<0.05). Flow cytometry analyses revealed that the increased infiltrating T lymphocytes consisted of both CD4+ and CD8+ T cells (Fig. 4F and 4G) . Thus, in vivo expression of IP-10 in tumor tissues enhances the recruitment of both CD4+ and CD8+ T lymphocytes into the tumor tissues and the augmentation of antitumor activity.


Figure 4
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  		Figure 4. Increased lymphocytes infiltrating mediated by IP-10 expression in tumor site. (A) Expression of IP-10 mRNA in tumor tissues was analyzed by real-time PCR. The level of IP-10 was normalized to the level of GAPDH; *, P < 0.01 (B) Tumor sections were stained with H&E for detection of lymphocytes infiltration in (a) parental 4T1, (b) 4T1-pcDNA3 and (c) 4T1-IP-10 tumor tissues. (C) Tumor tissues were digested and the infiltrated lymphocytes were separated. The amounts of the lymphocytes infiltrated in the tumors was counted and normalized to tumor mass; *, P < 0.05. (D) Tumor sections were stained with FITC-conjugated anti-mouse Thy1 antibody for the detection of T lymphocytes infiltration in the (d) parental 4T1, (e) 4T1-pcDNA3, and (f) 4T1-IP-10 tumors. (E) The number of T lymphocytes per field under fluorescent microscopy was counted; *, P<0.05 (F) The lymphocytes isolated from various tumors were stained with PE-CD8 and PerCP-CD4 antibodies. Subsets of the lymphocytes infiltrated in tumors were analyzed by flow cytometry. (G) The percentage of the CD4+ (open bar) and CD8+ (solid bar) T cells in TILs was assessed. Staining is representative of three independent experiments. Significant differences are indicated between 4T1-IP-10 group and 4T1-pcDNA3 or parental 4T1 group.

 
TILs recruited by IP-10 are cytolytic killers against 4T1 cells
To test the antitumor ability of TILs recruited by IP-10, lymphocytes were isolated from the tumor and cultured in the presence of MMC-treated 4T1 target tumor cells for measuring their proliferation, cytokine secretion, and cytotoxic activity in vitro, with TIL cultures stimulated with irrelevant Renca cells as controls. As shown in Fig. 5A , TILs from 4T1-IP-10 tumors proliferated greater than those TILs from mice of parental 4T1 cells and mice injected with control 4T1-pcDNA3 cells in response to MMC-treated 4T1 tumor cells (P<0.05) but not to irrelevant control Renca cells (Fig. 5B) . A specific cytotoxicity assay showed that TILs from 4T1-IP-10 tumors displayed more potent cytolytic killing activity against 4T1 target cells, as compared with TILs derived from mice receiving parental 4T1 tumors and mice receiving 4T1-pcDNA3 tumors (Fig. 5C) . Furthermore, a higher level of IFN-{gamma} was detected in the culture supernatant of TILs that were isolated from mice injected with 4T1-IP-10 cells than those of TILs derived from mice injected with 4T1 and mice of control 4T1-pcDNA3 tumor cells (61±7 vs. 34±7, and 36±4, respectively, Fig. 5D , P<0.05). All of these results suggest that IP-10 derived from 4T1-IP-10 tumor cells not only enhances the trafficking of tumor-specific TILs into the site of the tumor but also augments the functional expansion of TILs.


Figure 5
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  		Figure 5. Specific immune response of TILs against 4T1 tumor cells. (A) 5 x 105 TILs derived from 4T1, 4T1-pcDNA3, or 4T1-IP-10 tumors were cocultured with different amounts of MMC-treated 4T1 cells in vitro. The proliferation of the TILs to 4T1 cells was determined by [3H]-thymidine incorporation. (B) 5 x 105 TILs in response to different amounts of MMC-treated irrelevant Renca cells in vitro. The proliferative responses were determined by [3H]-thymidine incorporation. (C) Cytolytic activity of TILs derived from IP-10 against 4T1 parent tumor. TILs derived from 4T1-IP-10, 4T1-pcDNA3, and 4T1 inoculated tumor were labeled with 5 µM CFSE and coincubated with 4T1 target cells at 1 x 104/well. The E:T ratio were 10:1, 20:1, and 40:1. The percentage of cytotoxicity was detected by counting the apoptotic target cells labeled with 7-ADD and analyzed by FACS. *, P < 0.05 denotes significant difference between 4T1-IP-10 experiment group and 4T1-pcDNA3 or 4T1 parent tumor experiment group. (D) The number of IFN-{gamma}-producing lymphocytes in the 1 x 105 TILs was measured by ELISPOT after being stimulated without or with MMC-treated 4T1 cells in vitro. The representative results are from three independent experiments. *Significant differences are indicated between between 4T1-IP-10 group and 4T1-pcDNA3 or parental 4T1 group (P<0.05).

 
IP-10 enhances the in vivo expansion of TILs
The finding that TILs derived from mice of 4T1-IP-10 cells displayed much more potent ability to proliferate and produce IFN-{gamma} than those TILs derived from mice of control 4T1 cells is surprising to us. To determine whether targeted expression IP-10 stimulates the expansion of TILs in vivo in the tumor, TILs were isolated at day 14 from the primary recipient mice of 4T1-IP-10 tumor cells, labeled with CFSE, adoptively transferred into the secondary recipient mice that had previous subcutaneous inoculation with 4T1-IP-10, control 4T1-pcDNA3, or parental 4T1 cells for 14 days. As shown in Fig. 6A , the % of CFSE-labeled TILs among all TILs per milligram of tumor tissue was remarkably increased in 4T1-IP-10-inoculated tumors on days 2, 6, and 14 after adoptive transfer, as compared with mice receiving 4T1 and mice injected with 4T1-pcDNA3 (P<0.05). Interestingly, CFSE-labeled TILs proliferated much more vigorously, as evidenced by cell divisions in the 4T1-IP-10 tumor, than did TILs in the parental 4T1 and 4T1-pcDNA3 tumors (Fig. 6B , P<0.05).


Figure 6
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  		Figure 6. IP-10-conditioned microenvironment on the infiltration and proliferation of the TILs. Balb/c mice bearing subcutaneous 4T1-IP-10, parental 4T1, or 4T1-pcDNA3 tumors were infused with CFSE-labeled TILs isolated from 4T1-IP-10 tumors. (A) After 2, 6, and 14 days, tumors were excised and weighed for 4T1-IP-10 (solid bars), 4T1-pcDNA3 (hatched bars), and 4T1 (open bars), respectively; then, the TILs were isolated from the tumor, including CFSE-labeled, adoptively transferred 4T1-specific splenocytes and CFSE-unlabeled TILs. The % of CFSE labeled cells in total TILs per microgram tumor tissue was calculated. *Significant differences are indicated between 4T1-IP-10 group and 4T1-pcDNA3 or parental 4T1 group (P<0.05). (B) CFSE-labeled lymphocytes proliferation in vivo. On days 2 (top) and 6 (bottom), after adoptive transfer of CFSE-labeled lymphocytes isolated from 4T1-IP-10 tumors, TILs were isolated, and their proliferation of the TILs as measured by CFSE dilution was shown. The experiments were repeated three times with similar results.

 
To further confirm whether IP-10 acted as a stimulator to directly enhance the proliferation of TILs, TILs isolated from the 4T1-IP-10, 4T1-pcDNA3, and 4T1 tumors, as well as splenocytes isolated from 4T1-IP-10 tumor-inoculated mice were cultured ex vivo in the presence of recombinant IP-10 at a concentration ranging from 0 to 1,000 ng/ml. The addition of recombinant IP-10 significantly enhanced the proliferation of TILs in a dose dependent manner (Fig. 7A ). On the other hand, other than T cells derived from antigen inoculation in vivo, activated lymphocytes treated with {alpha}CD3 followed by recombinant IP-10 in vitro exhibited proliferation similar to lymphocytes treated with {alpha}CD3 followed by {alpha}CD28 (Fig. 7B .), indicating IP-10 functioned as a stimulating-factor to elicit T cell proliferation. Therefore, combining the in vivo and in vitro data, our results strongly demonstrated that besides the chemoattractant function of chemokine, more importantly, as a general property, IP-10 could act as a stimulator to induce lymphocytes proliferation.


Figure 7
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  		Figure 7. The effects of IP-10 protein on the proliferation of the lymphocytes. (A) IP-10 promotes the proliferation of antigen-primed lymphocytes. Tumor-infiltrated lymphocytes derived from 4T1, 4T1-pcDNA3, and 4T1-IP-10 were stimulated with MMC-treated 4T1 parental tumor cells and various concentrations of rIP-10 in vitro for 72 h. Splenocytes harvested from 4T1-IP-10 were treated with the same procedure as above. Proliferative activity was determined by [3H] thymidine incorporation (cpm). The experiment was repeated three times. (B) IP-10 promotes in vitro activated lymphocytes proliferation. Naïve splenocytes at 2 x 105 per well were stimulated with {alpha}CD3 (open bar), {alpha}CD3 plus rIP-10 (hatched bar), and {alpha}CD3 plus {alpha}CD28 (solid bar) for 72 h. 1 µci/well [3H] thymidine was added at the final 18 h of culture. The proliferation of splenocytes was determined by [3H] thymidine incorporation (cpm). The experiment was repeated three times.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The infiltration, expansion, and persistence of T cells in tumor are critical for a successful T cell immunotherapy [28 , 29 ]. We demonstrate here that targeted delivery of IP-10 into tumor significantly enhances T cell-mediated antitumor effect. We found that adoptively transferred tumor-specific T cells were quickly recruited into the tumor expressing IP-10 but not into the control 4T1-pcDNA3 and parental 4T1 tumors in vivo. Although this recruitment of tumor-specific T cells into the tumor was obviously attributable to the chemoattracting effect of IP-10, we found that IP-10 also enhanced the survival, proliferation, and antitumor activity of TILs in vivo. Moreover, TILs derived from tumors expressing transduced IP-10 gene were potent cytolytic cells against tumor cells and produced high levels of IFN-{gamma} upon reencounter of tumor cells. Ex vivo stimulation of TILs with IP-10 markedly enhanced their proliferation in response to tumor antigens. Thus, in vivo targeted expression of IP-10 not only can potently recruit tumor-specific T lymphocytes into the tumor but also enhance the expansion, as well as the IFN-{gamma} production, of these TILs, leading to tumor regression.

The capability of chemokines to direct the trafficking of lymphocyte into the local tissue has encouraged many efforts to develop novel approaches for enhancing T cell-mediated antitumor activity. Previous studies have demonstrated that in vivo intratumor delivering IP-10 gene encoded by Ad vector combined with adoptive T cell transfer enhanced T cell infiltrating into the tumor site and prolonged the survival of tumor-bearing mice [7 ]. This was further confirmed that targeted expression of IP-10 in the tumor induced the accumulation of T lymphocytes in the local tissues, which was believed to be responsible for the inhibition of tumor growth [30 ]. However, in these mouse tumor models, merely enhancing the trafficking of T cells into the tumor in vivo does not result in the elimination of tumor in these mice, with only a few number of them survived tumor free [7 , 30 ]. In our experiments, we found that TILs recruited by IP-10 expressed in the tumor were activated and potent cytolytic killers that were able to proliferate, expand, and persist in vivo in the tumor, which account for the elimination of tumor in as many as 33% of mice. Thus, targeted delivery of chemokine with the ability to augment tumor-specific T cell expansion and survival in the tumor will allow for the development of novel approaches for immunotherapy.

It is intriguing to investigate the direct effect of IP-10 on regulating T cell survival and proliferation. Our findings indicate that IP-10 may have multiple functions in mediating antitumor immunity. Data from other studies indicate that besides their chemoattractant effect, chemokines have the ability to modulate the proliferation and functional differentiation of T lymphocytes. For instance, MIG/CXCL19 stimulated allogeneic T lymphocyte proliferation and increased the number of IFN-{gamma}-producing T lymphocytes both in vivo and in vitro [31 ], whereas IP-10/CXCL10 is important in enhancing the effector functions of T cells by using IP-10/CXCL10 knockout mice [13 ]. In our studies, we found that despite the chemoattracting effect of IP-10 on CXCR3+ T lymphocytes, IP-10 was also involved in regulating the proliferation and cytolytic activity, as well as the cytokine production of TILs. In response to IP-10, tumor-specific T cells were vigorously proliferated and sustained in the tumor, as many as 14 days after their adoptive transfer. More importantly, TILs isolated from 4T1-IP-10, 4T1-pcDNA3, and 4T1 were proliferated ex vivo with either the recombinant IP-10 or supernatant IP-10 from culture 4T1-IP-10 tumor. And this efficacy could be blocked by adding anti-mouse IP-10-neutralized antibody. More interestingly, naïve T cells, when treated with {alpha}CD3 followed by recombinant IP-10 in vitro, exhibited proliferation similar to lymphocytes treated with {alpha}CD3 followed by {alpha}CD28 (Fig. 7B) , indicating IP-10 functioned as a stimulating-factor to elicit T cell proliferation. Therefore, combining the in vivo and in vitro data, our results strongly demonstrated that besides the chemoattractant function of chemokine, more importantly, as a general property, IP-10 could act as a stimulator to induce lymphocytes proliferation. Further exploring the molecular mechanism by which IP-10 modulates the proliferation and functional differentiation of T cells will be important for further understanding the biology of tumor-specific T cells.

In clinical settings, the major obstacle to successful immunotherapy is the lack of efficient T cell infiltration and activation in the tumor site [32 , 33 ]. The trafficking of lymphocytes from the systemic circulation into the tumor site is mostly dependent on the chemoattractant effect by chemokines. The expanding and proliferating of attracted lymphocytes inside the tumor mainly relies on the interaction between tumor and T cells. So, a successful in vivo antitumor effect needs the combination of recruitment and proliferation lymphocytes in the tumor site. Given that intratumor expression of IP-10 can recruit tumor-specific T lymphocytes into the local tumor tissues and enhance the proliferation and survival of these antitumor T lymphocytes, it will be interesting to further test whether targeted delivery of IP-10 can be translated into a clinical trial for tumor therapy.

In summary, we demonstrated that target delivery of IP-10 gene into tumor cells can significantly augment T cell-mediated antitumor effect in vivo. These findings suggest that, in addition to its recruitment of CXCR3+ T cells into the tumor site, IP-10 also acts as a stimulator to promote the proliferation of recruited T cells and sustain the specific T cells in the local tissues, leading to tumor regression. Identification of the multiple functions of IP-10 in T cell-mediated antitumor immunity should yield further insight into the molecular and cellular mechanisms by which tumor-specific T cells eliminate solid tumor, and novel chemokine-based tumor therapy.


   ACKNOWLEDGEMENTS
 
This work was supported by the grants from the program of the Science and Technology Commission of Shanghai University (04XD14003, 04DZ14902) and the program for Outstanding Medical Academic Leader and the Major State Basic Research Development Program of the People’s Republic of China (2001CB510005) and the National Natural Science Foundation of China (30571713). We thank Dr. Yi Zhang (Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA) for his helpful suggestions and discussion in the paper.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received March 18, 2006; revised July 6, 2006; accepted August 5, 2006.


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