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FRACTALKINE TRANSGENE INDUCES T-CELL-DEPENDENT...
2006-03-29 guojun上一篇 | 下一篇

    FRACTALKINE TRANSGENE INDUCES T-CELL-DEPENDENT ANTITUMOR IMMUNITY THROUGH CHEMOATTRACTION AND ACTIVATION OF DENDRITIC CELLS


    Jun GUO1, Minghui ZHANG1, Baocheng WANG2, Zhenglong YUAN1, Zhenhong GUO1, Taoyong CHEN1, Yizhi YU1,Zhihai QIN1 and Xuetao CAO1* 1Institute of Immunology, Second Military Medical University, Shanghai, P.R. China 2Jinan General Hospital of P.L.A, Jinan, P.R. China

    Fractalkine (FK, also called neurotactin or CX3CL1) is a CX3C chemokine that can chemoattract T lymphocytes,monocytes and NK cells. In our study, we investigated the induction of antitumor response by FK gene transfer. FK gene-modified 3LL lung carcinoma cells (3LL-FK) could both secrete soluble form and express membrane-bound form of FK. The tumor growth of 3LL-FK was decreased. Vaccination with 3LL-FK was effective in the induction of protective immunity and CTL. In vivo depletion analysis demonstrated that CD8_ T cells are the main participating cells of the antitumor response. Obvious infiltrations of CD8_ T cells, CD4_ Tcells and dendritic cells (DC) were observed in the tumor sites, suggesting that 3LL-FK might induce antitumor immunity through chemoattraction and activation of T cells and DC. Then we investigated the chemoattraction and activation of DC by 3LL-FK. Chemotaxis assay showed that the supernatants of 3LL-FK could chemoattract immature DC,which were found to express FK receptor CX3CR1, and the immature DC could obviously adhere to 3LL-FK. Adherence of DC to 3LL-FK resulted in phenotypic maturation and upregulated IL-12 secretion of DC, and more strong stimulation of allogeneic T-cell proliferation by DC. The increased production of IL-2 and IFN_ in 3LL-FK tumor tissue was also observed. Our data suggested that FK gene transfer to tumor cells could induce T-cell-dependent antitumor immunity through chemoattraction and activation of DC. 

    Key words: fractalkine; antitumor immunity; dendritic cells; T cells; lung carcinoma

    Chemokines including 4 subgroups (CC, CXC, C and CX3C) are extensively involved in the inflammatory/immunologic responses due to their unique ability to selectively recruit leukocyte subsets.1,2 Chemokines have been implicated in regulation of nomal leukocyte recirculation and homing, in certain physiologic and pathogenic processes and also in tumor cell growth,3 angiogenesis, 4 the host immune response against malignant cells5 and may promote metastasis by acting directly on tumor cell migration  and invasion.6 However, chemokine gene modification has been demonstrated to play important roles in antitumor immunity. It has been shown that monocyte chemotactic protein-1 (MCP-1) could increase infiltration of macrophages/monocytes to the tumor site and reduce tumor growth.7 Adenovirus-mediated gene transfer of macrophage-inflammatory protein-3_ (MIP-3_) to tumors induced local accumulation of dendritic cells (DC) and inhibited growth of preexisting tumors.8 Macrophage-inflammatory protein-1_ (MIP- 1_), macrophage-inflammatory protein 3_ (MIP-3_), RANTES, secondary lymphoid tissue chemokine (SLC) and lymphotactin have all been shown to be capable of inhibiting tumor growth or inducing tumor rejection.9-12 All these data suggested that chemokines might mediate antitumor immunity by recruiting monocytes/ macrophages, DC and T cells to the tumor site and induce specific and nonspecific antitumor responses. Fractalkine (FK, CX3CL1) is a CX3C chemokine bearing a-C-X-X-X-C- motif at its N-terminus.13 FK can uniquely exist in 2 forms: either as a 95 Kda membrane-anchored protein or as a secreted chemokine upon protease cleavage from the mucin stalk. After its release from cell surface, FK can chemoattract monocytes, NK cells and T lymphocytes potently.14,15 It can also adhere to the chemoattracted cells tightly through FK-CX3CR1 interaction in its membrane-immobilized form without the involvement of integrin or other adhesion molecules.16 And the receptor CX3CR1 of FK has been detected to be expressed predominantly on NK cells, CD8_ T cells and CD14_ monocytes,which is consistent with its potent chemotactic activity on these cells.17–19 It has been reported that FK was expressed on the IL-1_ or TNF_- activated endothelium cells,13,15 as well as on DC and mast cells20–22and can be an amplification circuit of polarized Th1 response.14 It is well documented that DC play an important role in the induction of antitumor immunity, and more potent antitumor effects might be obtained by recruiting DC to the tumor site.23–25 DC are the most potent antigen-presenting cells (APC) and possess the unique capacity to induce primary T-cell response in vivo, which depends on their antigen uptake and processing capacity as well as their maturation and migration characteristics. Local administration of DC inhibits the growth of established tumor, indicating that DC accumulated within tumor could exhibit a potent antitumor effect.26,27 Intratumoral injection of DC genetically modified to express some cytokines or chemokines could induce regression of established murine tumor, which demonstrated that efficient DC maturation, migration and activation might be of great importance for the induction of antitumor immunity. Because FK is such a unique chemokine that can act both as chemoattractant and adhesion molecule, we hypothesized that FK may play a certain role in the antitumor response. So we then investigated the antitumor effects of FK gene transfer. Up to now, there is no report on the induction of antitumor immunity by FK gene transfer and the role of DC in this effect. In our study, our data suggested that FK gene transfer into tumor cells could induce T-cell-dependent antitumor immunity through chemoattraction and activation of DC.
    Grant sponsors: TRAPOYT; Grant sponsor: National Natural Science Foundation of China; Grant numbers: 30028022, 30121002; Grant sponsor: National Key Basic Research Program of China; Grant number: 2001CB510002. The first two authors contributed equally to this work. *Correspondence to: Institute of Immunology, Second Military Medical University, 800 Xiang Yin Road, Shanghai, 200433, P.R. China. Fax: _86-21-6538-2502. E-mail:caoxt@public3.sta.net.cn
    Received 19 March 2002; Revised 29 April, 26 July 2002; Accepted 26 September 2002 DOI 10.1002/ijc.10816Int. J. Cancer: 103, 212–220 (2003) . 2002 Wiley-Liss, Inc. Publication of the International Union Against Cancer MATERIAL AND METHODS Animals and cell line
    Female C57BL/6 (H-2Kb) or BALB/c (H-2d), 6–8 weeks of age, were purchased from Joint Ventures Sipper BK Experimental Animal Co., Shanghai, China. Mice were housed under specific pathogen-free conditions for at least 1 week before being used in the following experiments. 3LL, a murine Lewis lung carcinoma cell line (ATCC, Manassas, VA) derived from C57BL/6 mice (H-2Kb) were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 50 mmol/l2-mercaptoethanol, 100 _g/mL streptomycin, 100 U/mL penicillin. All culture media were purchased from Gibco-BRL (Gaithersburg, MD).
    Generation of DC from bone marrow
    DC were obtained from mouse bone marrow precursors as described previously.28,29 In brief, erythrocyte-depleted murine bone marrow cells harvested from femurs of C57BL/6 mice were plated in RPMI 1640 complete media supplemented with 20 ng/ml recombinant murine GM-CSF (Sigma, St. Louis, MO) and 1 ng/ml recombinant murine IL-4 (PeproTech, Rocky Hill, NJ). On day 3, floating cells were gently removed and fresh media were added. On day 6 of the culture, nonadherent and loosely adherent cells with the typical morphologic features of DC were collected and used as immature DC. To prepare mature DC, immature DC were further incubated in the presence of LPS (1 _g/ml) (Sigma) for 24 hr.
    Construction of FK expression vector
    The full-length cDNA encoding murine FK was generated by RT-PCR from total mRNA of bone marrow-derived mature DC. Total RNA was extracted and cDNA was synthesized by using reagents from Gibco according to manufacturer’s instructions. For construction of expression vector, the cDNA were used as templates for PCR (95°C for 20 sec, 56°C for 30 sec, 72°C for 1 min, 30 cycles) using primers 5_-GGAATTCATGGCTCCCTCGCCG-3_ (upstream) and 5_-GGAATTCTCACACTGGCACCAGGAC-3_ (downstream) to which EcoR_ site was added. Then the products were ligated into pGEX-T Easy vector (Invitrogen, Carlsbad, CA) and sequenced with Sp6/T7. The right clone was digested with EcoRI and further cloned into pcDNA 3.1/Myc-HisA (Invitrogen) to construct FK-expressing vector pcDNA-FK. pcDNA3.1/Myc-HisA with reverse insertion of FK utilized as mock control. Fk gene transfer and g418 selection of FK-expressing tumor cells Gene transfer of 3LL cells with pcDNA-FK and pcDNA-mock was performed by using LipofectAMINE (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after gene transfer, fresh media containing 10% FCS and 700 _g/ml G418 (Sigma) was added. The G418 selection continued for a total of 4 weeks and the positive clones were obtained and named 3LL-FK, 3LL-mock, respectively. Expression of FK was confirmed by RT-PCR.
    Detection of FK expression by 3LL-FK cells
    To confirm the expression of soluble FK protein in the culture supernatants of 3LL-FK cells, different dilution of supernatants with carbonate buffer (Na2CO3, 1.59 g/L; NaHCO3, 2.93g/L 50% NaHCO3) were added into the 96-well assay plate (Falcon, Becton Dickinson, Mountain View, CA). Recombinant mouse FK (R&D Systems, Minneapolis, MN) was used as standard FK. After coated at 4°C for 12 hr, 100 _l (0.5 _g/ml) of goat against mouse FK polyclonal antibody (M-18, Santa Cruz Biotech, Santa Cruz, CA) was added and incubated for 1 hr at room temperature, then 100 _l HRP-conjugated anti-goat IgG (0.5 _g/ml, Santa Cruz) were added and incubated for 30 min, read at 450 nm after TMB and stop solution was added. To examine the expression of the membrane-bound form of FK on 3LL-FK cells, 3LL-FK cells were washed twice with PBS, fixed in 4%paraformaldehyde for 10 min at room temperature and incubated with goat anti-mouse FK polyclonal antibody (M-18, Santa Cruz). Then the FITC-labeled anti-goat IgG (PharMingen, San Diego, CA) were used as a second antibody sequentially and were examined by fluorescent microscopy and by FACSCalibur flow cytometry (Becton Dickinson) and data were analyzed using CellQuest software (Becton Dickinson).
    Tumorigenicity of 3LL-FK cells
    3LL-FK cells, 3LL-mock or parental 3LL cells were taken from continuous culture and resuspended in PBS without FCS for inoculation into mice. Female C57BL/6 mice (8 mice/group) were inoculated s.c. with 3LL-FK or control cells into the anterior chest wall at the dose of 1 _ 105 or 5 _ 105 cells. The other 2 groups
    of mice were vaccinated with 5 _ 104 3LL-FK cells or 3LL-mock cells and were rechallenged s.c. with 5 _ 104 parental 3LL cells 4 weeks later. Tumor growth was monitored and the length and width of the tumor were measured with calipers 3 times a week after tumor inoculation and the tumor size was calculated 20 days later and was expressed as diameter (length _ width) / 2. Tumor metastases were evaluated by calculating lung nodules on dissected lung lobes contrasted with black India ink under a stereoscopicmicroscope.30
    Cytotoxicity assay
    Splenic lymphocytes were isolated from tumor-bearing mice 12 days after inoculation with 5 _ 105 3LL-FK cells, 3LL-mock cells or parental 3LL cells and were cocultured at 1 _ 107 cells/ml with 1 _ 106 cells/ml inactivated 3LL tumor cells for 7 days in the presence of mIL-2 20 IU/ml (Genzyme, Cambridge, MA) and then collected as CTL effector cells. The CTL activity was determined by a standard 4 hr 51Cr-release assay utilizing parental 3LL cells as targets. Two million 3LL cells in 0.5 ml RPMI-1640 with 20% FCS were labeled with 200 uCi Na51CrO4 (Amersham, Arlington Height, IL) for 2 hr. The labeled cells were washed 3 times in serum-free medium. Ten thousand target cells were then mixed with effector cells for 4 hr at 37°C at the ratio indicated. For the maximal 51Cr-release control, 0.1 ml of 0.1 N HCl was added to the target cells, and for the spontaneous 51Cr control, 0.1 ml of medium were added to the labeled cells. The syngeneic EL4 lymphoma cells were used as control target cells. The amount of 51Cr release was determined by _ counting on a 1275 Minigamma Counter(Lkb-Wallac, Turku, Finland), and the percentage of specific lysis was calculated as follows: CTL activity (%) (experimental c.p.m. spontaneous c.p.m.) / (maximal c.p.m.  spontaneous c.p.m.) X100.
    In vivo depletion of T-cell subsets
    To understand which are the cellular mediators of antitumor activity, mice were injected i.p. with rat IgG2b mAb (1 mg/mouse/ injection) against murine CD4(GK1.5, TIB-207, ATCC) or CD8 (2.43, TIB210, ATCC) on day 2, 0, _2 ,_4 of 3LL-FK cell inoculation. Normal rat IgG (Sigma) was given as negative control. This procedure was shown to be effective for specific depletion of T cells in blood and spleen, and long-term T-cell subset depletion was confirmed by flow cytometry analysis (data not shown). Tumor volume was measured as described above. Immunohistochemistry analysis of 3LL-FK tumor Twelve days after inoculation of 3LL-FK cells, 3LL-mock cells or parental 3LL cells, the tumor-bearing mice were sacrificed and the tumor nodules were harvested and embedded in OCTcompound (IEC, Needham HTS, MA). The frozen sections (6 _m) were fixed in acetone and incubated with 1% H2O2 for 10 min at room temperature. Slides were then preincubated for 30 min with a 1/10 dilution of serum from the same species of the secondary antibody (Dako, Glostrup, Denmark). Slides were then incubated with the optimal dilution of the primary mAbs, rat anti-mouse mAb against CD4 (clone GK1.5, PharMingen, San Diego, CA), FK GENE TRANSFER INDUCES ANTITUMOR IMMUNITY 213 CD8 (clone 53.6.72, PharMingen), CD11c (clone HL3, PharMingen), DEC205 (clone NLDC-45, ATCC). Mouse IgG2a and rat IgG1 (all from PharMingen) were used as isotype control. Sections were preincubated with rabbit serum and sequentially incubated with optimal dilution of biotinylated rabbit anti-rat IgGs or goat anti-mouse IgG2a and streptavidin-HRP (all from Santa Cruz).
    Each incubation lasted 30 min and was followed by a 10 min wash in Tris-buffered saline. Sections were then stained with CN/DAB substrate kit (Pierce, Rockford, IL) according to the manufacturer’s instructions and finally lightly counterstained with hematoxylin.The number of immunostained cells was examined by light microscopy at 200_ magnification and expressed as mean cell numbers detected under 5 high-resolving power from different sections derived from different tumors. Chemotaxis and adhesion assay of DC Approximately 1 _ 106 DC were resuspended in 0.1 ml of RPMI1640 medium containing 0.5% BSA and loaded into the upper well of a transwell chamber (3 _ pore size; Falcon, Franklin Lakes, NJ). Different dilutions with the same buffer of the supernatant of 3LL-FK, 3LL-mock or parental 3LL cells were added to the lower well in a volume of 0.6 ml. The chamber was incubated for 4 hr at 37°C. Directed migration was expressed as the number of cells that had migrated to the lower chamber, which were seen in 5 high-power fields. For blocking experiment, FK polyclonal antibody (M-18, Santa Cruz) was added to the supernatant of 3LL-FK cells at the final concentration of 2 _g/ml for 1 hr at room temperature,then were used for the chemotaxis assay. Each experiment was performed in triplicate at least 3 times. The data are presented as mean _ SE. Adhesion assay was carried out as previously described31with some modifications. In brief, 3LL-FK and control cells were eeded at 3 _ 104 cells/well in 24-well culture dishes and cultured overnight to form confluent monolayers. DC were fluorescently labeled with the red fluorescence marker PKH-26 (Sigma Chemical, St. Louis, MO) according to the manufacturer’s protocol.32 The labeling procedure using PKH26 had little influence on cell viability as determined by trypan blue exclusion (96% viability) and on the phenotype of DC determined by flow cytometry (data not shown). The labeled cells were added to each well (3 _ 104 cells/well) in a final volume of 100 _l and incubated for 60 min at 37°C. After removal of nonadherent cells, the numbers of fluorescently labeled cells were calculated by fluorescent microscopy, seen in 5 high-power fields. For the blocking experiment,FK polyclonal antibody (M-18, Santa Cruz ) was added to the cultured 3LL-FK cells at the final concentration of 2 _g/ml for 1 hr at 37°C, then were used for the adhesion assay. Each experiment was performed in triplicate at least 3 times. The data are presented as mean _ SE.
    Analysis of DC phenotype
    For cultivation with 3LL-FK, 3LL-mock or wild-type 3LL cells, DC cultured on day 6 were incubated with these cells at a ratio of 3:1 in the culture medium containing GM-CSF (20 ng/ml) and IL-4 (1 ng/ml). After a 24 hr incubation, DC were harvested by gently washing with PBS containing 10 mmol/L EDTA, purified with Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, Sweden) and washed twice with PBS. For analysis of relatively DC-specific phenotypes, DC were incubated with FITC- or PE-conjugated mAbs (5 _g/ml) specific for mouse CD86, CD80, CD40, Ia-Kb or isotype control antibodies for 30 min at 4°C in PBS. Labeled cells were analyzed by FACSCaliber flow cytometry and Cell Quest software (Becton Dickinson).
    Mixed lymphocyte reaction (MLR)
    The function to activate naive T cells by DC cultivated with 3LL-FK or control cells for 24 hr was examined in allogeneic MLR. Splenic T lymphocytes of BALB/c mice were enriched with a nylon wool column. Triplicates of 3 _ 105 of these T cells were incubated with DC of C57BL/6 mice irradiated at 3,000 rad in 200 _l 10% FCS culture medium in round-bottom 96-well plates for 5 days at 37°C. 3[H] thymidine (0.5 _Ci/well, Amersham, Les Ulis, France) was added at the final 16 hr of the culture period. Cells were harvested using glass fiber filters, and thymidine incorporation was assessed using a beta-scintillation counter (Wallac1409,Turku, Finland).
    Assay for cytokine production by DC and tumor nodules
    DC cultured on day 6 were incubated with 3LL-FK, 3LL-mock or parental 3LL cells at a ratio of 3:1 in the culture medium containing GM-CSF (20 ng/ml) and IL-4 (1 ng/ml). After 24 hr incubation,DC were harvested and washed as described above, then cultured for another 12 hr in the culture medium without GM-CSF and IL-4. Next, the DC (1 _ 106 cells) were evaluated for the production of IL-12 P70 by ELISA (R&D Systems) in the supernatants.
    The cytokine production in tumors was determined 12 days after inoculation of 3LL-FK cells, 3LL-mock cells or parental 3LL cells as described previously.12 In brief, nonnecrotic tumors were harvested, cut into small pieces and passed through a sieve. Singlecell suspension (5 _ 106cells/ml) was evaluated for the production of IL-2 and IFN_ by ELISA in the supernatants after an overnight culture. Mouse IL-2 and IFN_ ELISA kits were obtained from R&D Systems.
    Detection of CX3CR1 expression on DC
    To test the expression of CX3CR1 on mature and immature DC, RT-PCR analysis was performed. Total cellular RNA of DC was isolated using Trizol reagent (Life Technologies, Rockville, MD) and then was reverse transcribed into cDNA. Primers 5_-TTCGGTCTGGTGGGAAATCTG- 3_ and 5_-CGTCTGGATGATGCGGA AGTAG-3_ were used to specifically amplify the corresponding sequence of CX3CR1 in a thermocycler PCR9600 (Perkin- Elmer, Foster City, CA), with the expected product of 1188 bp. The reactions were incubated for 10 min at 98°C, followed by 25 cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at 56°C and extension at 72°C for 30 sec. Statistical analysis
    The data are presented as mean _ SE. Statistical analysis was performed using Student’s t-test. Statistical significance was determined at p _ 0.05.
    RESULTS
    Fk expression by FK gene-modified tumor cells The full-length FK cDNA was obtained by RT-PCR from total mRNA of murine bone marrow-derived DC, then cloned into pcDNA 3.1/Myc-HisA to construct pcDNA-FK. Murine Lewis lung carcinoma cell line 3LL, which did not express FK itself (as detected by RT-PCR; data not shown), was used as a tumor model to stably express FK. Positive clones were selected via G418 resistance and limiting dilutions. Clone expressing high level of FK was named as 3LL-FK, as evidenced by ELISA measurement for the soluble form (Fig. 1a), by FACS and fluorescent microscopy for membrane-bound form (Fig. 1b–d). These data indicated that the FK gene-modified 3LL cells could efficiently express both soluble form and membrane form of FK in vitro. The effect of the FK gene modification on the in vitro growth of 3LL cells was evaluated by [H3]TdR incorporation for 48 hr and the result showed that the proliferation of 3LL-FK cells remained unchanged compared to that of parental 3LL or 3LL-mock cells (data not shown), indicating that FK gene transfer itself does not affect the in vitro growth of 3LL cells.
    Induction of T-cell-dependent antitumor immunity
    First, we investigated the tumorigenicity of FK gene-modified 3LL cells. The results showed that 12.5% (1/8) of mice inoculated with 3LL-FK cells and 100% of mice inoculated with mock-vector transferred 3LL cells (3LL-mock) developed tumors at the dose of 1 _ 105 cells/mouse. In order to determine whether antitumor 214 GUO ET AL. immunity could be induced in the mice inoculated with 3LL-FK, the tumor-free mice (7/8) after inoculation with 3LL-FK were rechallenged with 5 _ 105 parental 3LL cells in the contralateral flank subcutaneously. After the rechallenge with parental 3LL cells, 3/7 mice developed tumor while 4/7 mice were still free of tumor, indicating that FK-transduced 3LL cells can induce antitumor response to some extent. When 5 _ 105 3LL-FK or 3LL-mock cells were inoculated for each mouse, 100% of the mice developed progressively growing tumors in both groups. Tumor growth of the 3LL-FK mice was significantly inhibited and the survival time of the mice was significantly prolonged compared to that of the 3LL-mock mice (Fig. 2). As 3LL cells were capable of metastasizing to lung, we examined the lung metastasis 28 days later after 3LL-FK inoculation. The lung metastasis of 3LL-FK was reduced markedly compared to that of 3LL-mock or parental 3LL cells (Table I).

    Then we wanted to know the induction of protective immunity by vaccination of FK gene-modified 3LL cells. Four weeks after the injection with irradiated (50 Gy) 5 _ 104 3LL-FK cells, 3LL-mock cells or parental 3LL cells, the mice were challenged s.c. with 1 _ 105 parental 3LL cells. No mice vaccinated with 3LL-FK developed tumors, but 7/8 mice vaccinated with 3LLmock or parental 3LL cells developed tumor, indicating that the protecting effect was effectively induced by vaccination with 3LLFK. To further characterize the involvement of T-cell subsets in the in vivo induction of antitumor response by FK gene-modified 3LL cells, anti-CD4 or anti-CD8 monoclonal antibodies were used to specifically deplete CD4_ or CD8_ T cells. After subcutaneous inoculation with 3LL-FK cells or 3LL-mock cells, we evaluated the incidence of tumor development and the tumor volume of the mice. As shown in Figure 3, after immunodepletion of CD8_ T cells, the antitumor effect induced by FK gene-modified 3LL cells was significantly impaired (p _ 0.01 as compared to 3LL-FK vaccination). Antitumor activity was also impaired in the mice immunodepleted of CD4_ T cells (p _ 0.05 as compared to 3LL-FK vaccination). Therefore, CD8_ T cells play the most important roles in the FK-induced antitumor immunity. Also, CD4_ T cells are involved in the antitumor activity induced by FK gene-modified tumor cells.


    Since the data have shown that reduced tumorigenicity or delayed progression of tumor cells after FK gene transfer and vaccination with FK gene-modified tumor cells could protect mice from the challenge of parental tumor cells, we further investigated whether specific immunity was induced by vaccination with FK gene-modified 3LL cells. After vaccination with 3LL-FK cells as well as the control cells, the splenic lymphocytes were isolated from the tumor-bearing mice and then used in cytotoxic assays. The results showed that the CTL activities in mice vaccinated with 3LL-FK cells increased markedly when compared to those in mice vaccinated with 3LL-mock or parental 3LL cells (Fig. 4). In determining CTL activity, no cytotoxicity on syngeneic EL4lymphoma cells were found with these induced lymphocytes (data not shown). These data suggested that the specific CTL was induced potently and might be involved in the antitumor response of the FK gene transfer.

    Increased T-cell and DC infiltration at tumor sites after FK gene transfer To investigate which cells other than T cells are also involved in the antitumor effects of FK gene transfer, the immune cell infiltration in 3LL-FK and 3LL-mock tumors growing progressively in vivo were analyzed. The results demonstrated that more DC were accumulated at the tumor site in 3LL-FK tumors compared to that of 3LL-mock or parental 3LL tumors, as shown in Fig. 5a (p _ 0.01). The results also demonstrated that CD8_ T cells significantly accumulated within the tumors of 3LL-FK tumors, as shown in Fig. 5b (p _ 0.05).These data suggested that the induction of antitumor immunity of FK gene-modified tumor cells might be due to the chemoattraction and activation of T cells and DC in vivo. Increased chemotaxis and adherence of DC induced by FK gene-modified 3LL cells in vitro Since we had detected DC infiltration at the 3LL-FK tumor sites, we further investigated whether the FK gene-modified 3LL tumor cells had the effect on the chemotaxis and adhesion of DC in vitro. The supernatants from parental 3LL, 3LL-mock or 3LL-FK were tested for the chemotaxis of immature DC. As shown in Figure 6a, the supernatant of 3LL-FK cells could chemoattract DC more significantly than that of the 3LL-mock cells and the parental 3LL cells(p _0.01). Specific anti-FK polyclonal antibody could efficiently block the increased chemotactic activity of the supernatant of 3LL-FK cells. Then we tested the adherence of DC to 3LL-FK cells. As shown in Figure 6b, more significant adherence of DC to 3LL-FK was observed (p _ 0.01), and the preincubated of 3LLFK cells with anti-FK polyclonal antibody could efficiently block this adherence. These data suggested that DC could be efficiently chemoattracted and adhered by FK gene-modified tumor cells. As shown in Figure 6c, RT-PCR analysis demonstrated that CX3CR1 could be detected in immature DC, and the result is consistent with the chemotaxis and adhesion analysis of immature DC to 3LL-FK cells.

    Activation of DC by FK-expressing tumor cells
    As we have demonstrated DC could be chemoattracted and adhered to FK gene-modified tumor cells, we further studied whether the DC could be activated by the FK gene-modified tumor cells after chemoattracted and adhered. After a 24 hr incubation of immature DC with 3LL-FK, 3LL-mock or parental 3LL cells, the phenotype of DC was analyzed by flow cytometry. As shown in Figure 7, MHC-II molecule and costimulatory molecules including CD86, CD80 and CD40 were markedly upregulated on DC incubated with 3LL-FK cells.When DC was cocultured with supernatants from 3LL, 3LL-mock and 3LL-FK or when DC was added to the upper well of the transwell while 3LL, 3LL-mock and 3LL-FK cells were cultured in the lower well, phenotype of DC was unaffected (data not shown). This suggested that the contact and adhesion of DC with 3LL-FK was necessary for the induction of DC maturation. Then we investigated the capacity of DC to stimulate the proliferation of T cells. As shown in Figure 8, DC cocultured with 3LL-FK cells were found to be more effective in stimulating the proliferation of T cells compared to that cocultured with 3LLmock cells or parental 3LL cells (p _ 0.01). Given that cytokines are essential mediators in the induction of antitumor immunity, we intended to know the expression of these immunoregulatory cytokines secreted by DC or produced in 3LLFK tumor sites. IL-12 P70 secretion of DC after coculturing with 3LL-FK cells was upregulated to 216.37 _ 34.21 pg/ml compared to that of DC cocultured with 3LL-mock cells (42.68 _ 5.32 pg/ml) and DC cocultured with parental 3LL cells (36.75 _ 4.62 pg/ml) (p _ 0.01). As shown in Figure 9, the increase of IL-2 and IFN_ production was observed in the overnight cultured supernatants of 3LL-FK (p _ 0.01) homogenated tumor tissues 12 days after tumor cell inoculation compared to that of 3LL-mock tumor tissues or parental 3LL tumor tissues. Increased Th1 cytokine production indicated that the antitumor cellular immunity in the tumor-bearing host might be induced efficiently in 3LL-FK inoculated mice. These experiments strongly implied that DC were not only chemoattracted but also activated by FK-expressing tumor cells, which may contribute to the induction of specific antitumor immunity by FK gene transfer.

    have antitumor activity either by chemoattracting NK cells and monocytes/macrophages or by accumulating DC.2,33 FK is a unique membrane-anchored chemokine that can act also as an adhesion molecule. In vitro, FK has been shown to have multiple activities including chemotaxis and adhesion of monocytes, NK cells and T lymphocytes.13–17 So, an underlying possibility exists that FK can mediate antitumor immunity through chemoattracting and immobilizing DC and T lymphocytes. In this article, we demonstrated for the first time that FK could induce potent antitumor immunity through chemoattracting and activating DC. After subcutaneous injection of FK gene-modified 3LL cells at the doses of 1 _ 105 or 5 _ 105 into immunocompetent mice, tumorigenicity or growth of 3LL-FK cells was significantly reduced. Tumor growth inhibition was comparable to that reported on MIP-3_ gene-modified C3L5 tumor cells and MCP-3 gene-modified P815 tumor cells.34,35 Although the used chemokine and tumor cell model were different, the remarkable reduction of tumor formation was observed. Giovarelli et al.30 reported that LEC-modified TSA cells could elicit tumor rejection and immune memory by locally released LEC chemokine. Rejection was associated with an impressive infiltration of macrophages, DC, T cells and polymorphonuclear leukocytes. However FK could not only chemoattract DC and T cells but also adhere DC and T cells, thus possibly eliciting a more efficient antigen presentation and potent tumor rejection.

    It has been demonstrated that chemokines can mediate tumor rejection through accumulating host immune cells. In our study, immunohistochemistry analysis of established 3LL-FK tumor showed that CD8_ T cells and a fraction of CD4_ T cells infiltrated within the tumor. More importantly, a number of DC were also detected within the tumor site. The G protein-coupled receptor CX3CR1 has been shown to be expressed in NK cells,36 monocytes, 37 CD8_ (CD45RO_ and CD45RO-) T cells, CD4_ (CD45RO_) T cells,38 microglia of brain,39 mast cells and THP-1 cells.22,40 We demonstrated by RT-PCR that murine immature DC expresses CX3CR1 mRNA, which is consistent with a study by Dichmann et al.41 So it was speculated that the function of FK on immature DC might be mediated through CX3CR1. This interaction between FK and immature DC was confirmed by in vitro chemotactic and adhesion analysis by using 3LL-FK supernatant and 3LL-FK cells, respectively. It has been reported that DC could express FK and FK is upregulated upon DC maturation.20,21 So it is interesting that immature DC express CX3CR1 while mature DC express FK. This is similar to reports on MDC and CCR4, as well as CCR7 and MIP-3_.Since immature DC is efficient for antigen capture and mature DC is efficient for antigen presentation and activation of naive T cells, therefore it is possible that the immature DC adhered to 3LL-FK cells were undergoing an intermediate stage of maturation. This hypothesis was supported by the upregulated expression of Iab, CD40, CD80 and CD86 on DC after DC cocultured and adhered to 3LL-FK cells. More potent stimulation of T-cell proliferation by this kind of DC just reflected this process and showed the improved functional status of DC through interaction with 3LL-FK cells. In conclusion, FK gene-modified tumor cells could chemoattract and activate immature DC and are capable of inducing protective immunity and CTL response. We also demonstrated that the observed antitumor immunity was due to the unique characterization of FK, which, in its soluble form, could chemoattract DC, CD8_ T cells and a fraction of CD4_ T cells, and in its membrane-bound form, could adhere and activate the chemoattracted DC. ACKNOWLEDGEMENTS We thank Dr. D. Ju, Dr. R. Zhang, Dr. S. Liu, Dr. W. Wang and Dr. H. Li for their expert technical assistance.
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