Synergistic antitumor effect by coexpression of chemokine CCL21/SLC and costimulatory molecule LIGHT

Abstract

To establish a more efficient treatment for immunotherapy against solid tumors, we have evaluated the antitumor effect by coexpression of a chemokine CCL21/secondary lymphoid tissue chemokine and a costimulatory molecule LIGHT in colon carcinoma C26. C26 cells expressing either CCL21 or LIGHT exhibited a significantly reduced tumor growth in vivo, and mice inoculated with these cells showed a prolonged survival, but eventually all these mice died. In contrast, C26 cells expressing both CCL21 and LIGHT exhibited a minimal tumor growth in vivo, and all these mice survived healthily with a tumor remission and consequently acquired a strong protective immunity. A markedly increased infiltration of mature dendritic cells (DCs), and CD8+ T cells was observed in the tumor mass, and their spleen cells showed a greatly enhanced cytotoxic T lymphocyte (CTL) activity against C26 tumor and interferon (IFN)-γ production. Neutralization of IFN-γ or depletion of CD8+ or CD4+ T cells significantly reduced the antitumor activity. These results suggest that the combined treatment with CCL21 and LIGHT is able to induce a synergistic antitumor effect to eradicate tumor completely by greatly enhancing tumor-infiltration of lymphocytes including mature DCs and CD8+ T cells, resulting in markedly augmented CTL activity and IFN-γ production.

Main

Generation of an antitumor immune response is a complex process depending on coordinate interaction of different subsets of effector cells, and dendritic cells (DCs) play a central role in this event.1 One of the most ultimate treatment regimens designed to elicit a strong immune response against malignant cells is considered to induce both efficient recruitment and strong activation of effector cells including DCs and different subsets of T cells in tumor mass. To achieve this strategy, a combined treatment with chemokines and costimulatory molecules in tumor should be the most effective approach for the immunotherapy against solid tumors.

CCL21/secondary lymphoid organ chemokine (SLC) is a CC chemokine expressed on high endothelial venules and within T-cell zones of spleen and lymph nodes, and strongly recruits naive T cells and mature DCs.2,3,4,5 DCs are professional antigen-presenting cells (APCs) that play a crucial role in initiation of the immune response of both helper and cytotoxic T lymphocytes.1 DCs reside in many tissues in an immature state with the ability to capture and process antigens, and acquire a mature phenotype with increased cell surface expression of MHC and costimulatory molecules upon uptake of antigen and response to stimuli such as inflammatory cytokines, necrotic cells, and microbial products. CCL21 recruits and colocalizes naive T cells and antigen-stimulated DCs into T-cell zones of secondary lymphoid organs resulting in cognate T-cell activation6 through two specific chemokine receptors, CCR7 and CXCR3.7,8 Whereas CCR7 is expressed on naive T cells and mature DCs,7,9 CXCR3 is expressed preferentially on T helper cell (Th) 1 type cytokine-producing lymphocytes with a memory phenotype.8

LIGHT, a recently identified member of the TNF superfamily,10 is expressed on activated T cells and immature DCs,11,12 and binds three receptors, herpes virus entry mediator (HVEM), lymphotoxin (LT) βR, and decoy receptor 3/TR6.10,13 HVEM is broadly expressed on T and B cells, NK cells, DCs, and endothelial cells.14,15,16 LTβR plays a key role in the development and organization of lymphoid tissue, but it is absent on mature T and B cells, primary monocytes, and peripheral DCs.17 LIGHT serves as a costimulatory molecule for T-cell activation, leading to enhanced proliferation, Th1-type cytokine production, NF-κB translocation, and induction of cytotoxic T lymphocyte (CTL).12,18 Signaling through HVEM in immature DCs drives the maturation of DC in cooperation with CD40 signaling, resulting in an increased expression of costimulatory molecules and cytokine production.19

Previous works demonstrated that CCL21 induces colocalization of DCs and T cells within tumor nodules and T-cell-dependent tumor rejection.20,21,22 CCL21 was also reported to inhibit angiogenesis as other CXCR3 ligands, resulting in a strong antitumor effect.21,23 In addition, LIGHT was demonstrated to costimulate T-cell activation and DC maturation and elicit a tumor-specific CTL activity.18 To establish a more efficient treatment aiming at a complete tumor remission, we here evaluated the antitumor effect by coexpression of CCL21 and LIGHT in tumor. We have found that coexpression of these costimulatory molecule and chemokine is able to induce a synergistic antitumor activity leading to a complete tumor remission by greatly enhancing tumor infiltration of mature DCs and CD8+ T cells, resulting in markedly augmented CTL activity against tumor and IFN-γ production.

Materials and methods

Cell culture and mice

Colon carcinoma C26 cell line was cultured in RPMI 1640 medium supplemented with 10% FBS. Spleen cells and primary T cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 50 μM 2-mercaptoethanol. Female BALB/c mice, 6–10 weeks old, were purchased from Japan SLC (Hamamatsu, Japan).

Preparation of C26 cells expressing CCL21, LIGHT, or both CCL21 and LIGHT

Mouse CCL21 cDNA was isolated by reverse transcriptase (RT)–PCR using total RNA prepared from concanavalin A-activated spleen cells and cloned into a cytomegalovirus (CMV) promoter-driven expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). Mouse LIGHT cDNA cloned in pcDNA3.1 was prepared as described previously.18 Bicistronic expression vector of mouse CCL21 and LIGHT cDNAs cloned in pcDNA3.1 was prepared using the internal ribosome entry site (IRES) sequence in a bicistronic retroviral vector pMX-IRES/EGFP (kindly provided by Dr T Kitamura, University of Tokyo, Tokyo, Japan),24 by standard PCR methods. C26 cells were then transfected with these expression vectors or the empty pcDNA3.1 vector by using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) and selected with Geneticin (G418).

RT–PCR analysis

Total RNA was extracted from each transfectant, cDNA was prepared using oligo(dT) primer, and RT–PCR was performed using Taq DNA polymerase as described.25 Cycle conditions were 94°C for 30 s, 60°C for 30 s, and 72°C for 1 minute for 30 cycles. Following primers were used; CCL21 sense primer, 5′-IndexTermGGGAATTCATGGCTCAGATGATGACTC-3′; CCL21 antisense primer, 5′-IndexTermCCGAATTCCTATCCTCTTGAGGGCTG-3′ (PCR product size, 418 bp); LIGHT sense primer, 5′-IndexTermgcatcaacgtcttggagaca-3′; LIGHT antisense primer, 5′-IndexTermatacgtcaagcccctcaaga-3′ (PCR product size, 202 bp); HPRT sense primer, 5′-IndexTermGTTGGATACAGGCCAGACTTTGTTG-3′; HPRT antisense primer. 5′-IndexTermGAGGGTAGGCTGGCCTATAGGCT-3′ (PCR product size, 352 bp).

T-cell costimulation and IFN-γ production assays

Primary T cells were purified by passing BALB/c mouse spleen cells depleted of erythrocytes through nylon wool and Sephadex G-10 columns. Purified T cells (2 × 106 cells/ml) were stimulated with 0.3 μg/ml plate-coated anti-CD3 (145-2C11, American Type Culture Collection (ATCC), Manassas, VA) in the presence of C26 transfectants (2 × 105 cells/ml) irradiated with 150 Gy for 48 hours and pulsed with 3H-thymidine for last 12 hours. For IFN-γ production assay, purified T cells (5 × 106 cells/ml) were stimulated with plate-coated anti-CD3 (0.3 μg/ml) in the presence of C26 transfectants (5 × 105 cells/ml) irradiated with 150 Gy. After 48 hours, culture supernatants were harvested and IFN-γ concentrations were measured by ELISA as described.25

Chemotaxis assay

C26 transfectants (1 × 106 cells/ml) were cultured in RPMI containing 5% FBS for 48 hours, and culture supernatants were collected to test their chemotactic activities in a two-chamber transmigration assay using 5-μm-pore Transwells (Costar, Cambridge, MA). Collected supernatants (600 μl) were placed in lower chamber, and 1 × 106 purified T cells (100 μl) prepared as described above were placed in the upper chamber. After incubation for 12 hours at 37°C, migrated cells were collected and analyzed by FACS Calibur flow cytometry (Becton Dickinson, Mountain View, CA). The number of cells acquired during 50 s was used to determine the relative number of migrated cells.

Immunohistochemistry

Tumor was removed from each mouse, embedded in OCT compound (Sakura Finetechnical Co., Tokyo, Japan) and snap-frozen in liquid nitrogen. Cryostat sections were mounted onto glass slides and fixed, and endogenous peroxidase activity was blocked by treatment with 0.6% H2O2 and 0.2% sodium azide. Slides were incubated with 2% normal rat serum and 1% BSA and then sequentially with biotinylated hamster anti-mouse CD11c monoclonal antibody (mAb, N418, ATCC) and streptavidin-peroxidase (Zymed Laboratories Inc., San Francisco, CA), followed by development with diaminobenzidine substrate. The slides were counterstained with hematoxylin to detect cell nuclei.

Isolation of tumor-infiltrating cells

Tumor was removed from each mouse 2 weeks after inoculation, and 0.1 g section of each tumor was minced into small pieces and digested with 500 U/ml collagenase (type IV, Roche Molecular Biochemicals) for 1 hour at 37°C under agitation. The resultant cells were passed through nylon mesh to remove debris, and viable cells were separated by using Lympholite-M (Cedarlane Laboratories, Ontario, Canada). Tumor-infiltrating DCs were further purified using CD11c+ microbeads and autoMACS following the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany).

Flow cytometric analysis

Single-cell suspensions were incubated with anti-Fc receptor (24G2, ATCC) to block Fc receptors and stained with various mAbs conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC) in PBS containing 2% FBS and 0.1% sodium azide. Labeled cells were analyzed by FACS Calibur flow cytometry with CellQuest software (Becton Dickinson). The mAbs to CD4 (GK1.5), CD8 (53-5.8), CD11c (HL3), CD80 (1G10), CD86 (GL1), and MHC class II (M5/114) were used and purchased from PharMingen.

Cytotoxicity assay

Spleen was removed from each mouse 2 weeks after tumor inoculation, and spleen cells (5 × 106 cells/ml) were restimulated in vitro by coculture with C26 parental tumor cells (5 × 105 cells/ml) irradiated with 150 Gy for 5 days and used as effector cells in a standard 51Cr release assay. These culture supernatants were also collected and assayed for INF-γ production by ELISA as described above. C26 parental tumor cells were labeled by incubating in 100 μCi 51Cr for 1 hour at 37°C and washing three times. Labeled target cells (1 × 104 cells/200 μl/well) and serial dilutions of effector cells were incubated in RPMI 1640 containing 10% FBS in 96-well U-bottom plate at 37°C for 4 hours. The supernatants were then analyzed in a scintillation counter. The percentage of lysis was determined for each triplicate experiment as (experimental count−spontaneous count) × 100/(maximal count−spontaneous count). Results were expressed as the percentage of specific lysis.

Neutralization of IFN-γ and depletion of CD8+ and CD4+ T cells with mAbs

For depletion or neutralization with mAbs, each mouse was injected i.p. with 0.5 mg rat anti-mouse CD8 (53-6.7), anti-CD4 (GK1.5), anti-IFN-γ (XMG1.2, all from ATCC) mAbs, or normal rat IgG (Sigma Chemical Co., St Louis, MO) as control Ab in 200 μl PBS 1 day before tumor inoculation, once daily for following 3 consecutive days and then twice a week.

Statistical analysis

Statistical analysis was performed by Student's t-test, except for survival. Survival was evaluated by generation of Kaplan–Meier plots and log-rank analysis. A P-value of less than 0.05 was considered to indicate statistical significance.

Results

Characterization of C26 transfectants in vitro

Colon carcinoma C26 cells were stably transfected with pcDNA3.1-CCL21 cDNA (C26-CCL21), pcDNA3.1-LIGHT cDNA (C26-LIGHT), pcDNA3.1-LIGHT cDNA/IRES/CCL21 cDNA (C26-C+L), or the empty pcDNA3.1 vector (C26-Vector). Expression of CCL21 and LIGHT at mRNA levels in each transfectant was confirmed by RT–PCR (Fig 1a). Then, their costimulatory activities for T-cell proliferation and IFN-γ production were determined. Consistent with the mRNA expression, C26-LIGHT and C26-C+L cells enhanced T-cell proliferation, but C26-CCL21 cells did not (Fig 1b). Similar enhancement of IFN-γ production was observed when C26-LIGHT and C26-C+L cells were used (Fig 1c). The culture supernatant of each transfectant was analyzed for chemotactic activity to mouse primary T cells using a two-chamber transmigration assay. The supernatants of C26-CCL21 and C26-C+L cells increased the chemotactic activity (Fig 1d). But, unexpectedly, the supernatant of C26-LIGHT cells also showed a slightly, but significantly and constantly increased chemotactic activity (Fig 1d). Thus, C26-LIGHT and C26-C+L cells express bioactive LIGHT, and the expression of CCL21 does not affect the costimulatory activities for T-cell proliferation and IFN-γ production. On the other hand, C26-CCL21 and C26-C+L cells secrete bioactive CCL21, and C26-LIGHT cells appear to secrete molecule(s) which has a chemotactic activity to T cells as well. Furthermore, there was no difference in cell growth in vitro and surface expression of MHC class I among these C26 transfectants (data not shown).

Figure 1
figure1

Characterization of C26 transfectants in vitro. (a) Expression of LIGHT and CCL21 at mRNA level in nontreated C26 tumor cell line, C26-Vector, C26-LIGHT, C26-CCL21, and C26-C+L was analyzed by RT–PCR. (b) Enhanced T-cell proliferation by costimulation with C26-LIGHT and -C+L. Purified T cells were stimulated with plate-coated anti-CD3 in the presence of irradiated C26 transfectants, and 3H-thymidine incorporation was measured in triplicate. Data are shown as the mean±SD. * indicates P<1 × 10−3, compared with C26-Vector. Similar results were obtained in five independent experiments. (c) Augmented IFN-γ production by costimulation with C26-LIGHT and -C+L. Purified T cells were stimulated with plate-coated anti-CD3 in the presence of irradiated C26 transfectants, and culture supernatants were assayed for IFN-γ by ELISA in triplicate. Data are shown as the mean±SD. * indicates P<1 × 10−4, compared with C26-Vector. Similar results were obtained in five independent experiments. (d) Enhanced chemotactic activity by culture supernatants of C26-CCL21, -LIGHT and -C+L. Culture supernatant from each C26 transfectant was tested for chemotactic activity to T cells in a two-chamber transmigration assay. The number of cells acquired during 50 s by FACS was used to determine the relative number of migrated cells. Data are shown as the mean±SD. * and ** indicate <1 × 10−4 and P<1 × 10−5, respectively, compared with C26-Vector. Similar results were obtained in five independent experiments.

Synergistic antitumor effect on C26 tumor by coexpression of CCL21 and LIGHT in vivo

C26 transfectants were injected s.c. into syngenic BALB/c mice, and in vivo cell growth and survival rate were compared. As reported previously,18,20,21 both C26-CCL21 and C26-LIGHT cells showed a significantly reduced cell growth in vivo (Fig 2a), and mice inoculated with these cells survived longer than those with C26-Vector cells (Fig 2b). But, these tumors were never eradicated, and all these mice died eventually within 6 weeks after tumor inoculation. In contrast, C26-C+L cells never became more than 50 mm3 in tumor volume and disappeared soon (Fig 2a). All mice inoculated with C26-C+L cells survived healthily with a complete tumor remission (Fig 2b). These results suggest that coexpression of CCL21 and LIGHT in C26 tumor induces a much more strong antitumor effect in vivo than single expression of either CCL21 or LIGHT in the tumor.

Figure 2
figure2

Synergistic antitumor effect on C26 tumor by coexpression of CCL21 and LIGHT in vivo. (a) Minimal tumor growth of C26 tumor by coexpression of CCL21 and LIGHT in vivo. BALB/c mice (n=5) were injected s.c. with C26 transfectants (2 × 105 cells) and tumor growth was monitored. Tumor volume was calculated using the volume equation 0.5(ab2), where a is the long diameter and b is the short diameter. Data are shown as the mean±SD. * and ** indicate P<1 × 10−3 and <1 × 10−4, respectively, compared with C26-Vector. Similar results were obtained in five independent experiments. (b) Prolonged survival time of mice inoculated with C26-C+L cells. BALB/c mice (n=23) were injected s.c. with C26 transfectants (2 × 105 cells) and survival time was monitored. * indicates P<1 × 10−4, compared with C26-Vector. (c) Induction of a strong protective immunity by coexpression of CCL21 and LIGHT. Recovered mice (n=5) from inoculation with C26-C+L cells and nontreated mice (n=5) were injected s.c. with C26 parental tumor cells (2 × 105 cells), and tumor growth was monitored. * indicates P<1 × 10−5, compared with nontreated mice. Similar results were obtained in two independent experiments.

Then, mice recovered from inoculation with C26-C+L cells and nontreated mice were challenged by the parental C26 tumor and tumor volume was monitored. Tumor inoculated into these recovered mice never became palpable (Fig 2c) and all these mice survived very healthily, while tumor inoculated into nontreated mice grew vigorously and all these mice died soon (data not shown). These results suggest that coexpression of CCL21 and LIGHT in C26 tumor induces a strong protective immunity to the following challenge with its parental tumor.

Greatly enhanced infiltration of mature DCs, CD8+ T cells, and B cells in tumor by coexpression of CCL21 and LIGHT

To examine the mechanism underlying the synergistic antitumor effect, we first of all analyzed the phenotype of infiltrating cells in tumors 2 weeks after inoculation by immunohistochemistry. Compared with C26-Vector and also C26-CCL21 or -LIGHT tumors, C26-C+L tumor was characterized by a much more rich infiltrate of lymphocytes and DCs (Fig 3a). To further analyze the percentage of infiltrating cells quantitatively, tumor masses were removed 2 weeks after inoculation, digested with collagenase, and immunostained for a two-color analysis using FACS. Consistent with the previous reports,20,21 percentage of infiltrating cells such as DCs and CD8+ T cells into C26-CCL21 tumor was significantly increased (Fig 3b). Similar increase in the percentage of DCs and CD8+ T cells was also observed in C26-LIGHT tumor, that would agree with the in vitro chemotactic activity of the culture supernatant of C26-LIGHT cells (Fig 1d). In striking contrast, percentage of infiltrating cells including DCs and CD8+ T cells, and also B cells into C26-C+L tumor was markedly increased compared with those into C26-CCL21 or C26-LIGHT tumors (Fig 3b). No significant increase in the percentage of tumor-infiltrating CD4+ T cells and NK1.1+ cells was observed.

Figure 3
figure3

Greatly enhanced tumor infiltration of mature DCs and CD8+ T cells by coexpression of CCL21 and LIGHT. BALB/c mice (n=3) were injected s.c. with C26 transfectants (5 × 105 cells). After weeks, tumor was removed from each mouse, and immunohistochemical analysis of tumor sections using anti-CD11c was performed (a). Each section was counterstained with hematoxylin to detect cell nuclei. Cells stained dark brown represent DCs. The arrowheads indicate infiltrated lymphocytes. Scale bars represent 50 μm. Then, 0.1 g section of each tumor was minced into small pieces and digested with collagenase, and the resultant tumor-infiltrating cells were analyzed by using FACS (b). Tumor-infiltrating DCs were further purified using CD11c+ microbeads and autoMACS, and expression of surface makers for DC maturation (CD80, CD86, and MHC Class II) was analyzed by using FACS (c). The number of cells acquired during 20 s by FACS was used to determine the relative number of positive cells for expression of these markers. Data are shown as the mean±SD. * and ** in (b) indicate P<0.05 and <0.01, respectively, compared with C26-Vector. * and ** in (c) indicate P<1 × 10−3 and P<1 × 10−4, respectively, compared with C26-Vector. Similar results were obtained in three independent experiments.

Since mature DCs but not immature DCs play an important role in the activation, recruitment, and expansion of effector cells at the vicinity of their targets,1 we next analyzed the phenotype of DCs infiltrating in tumor by FACS. CD11c+ DCs were isolated using CD11c microbeads from 0.1 g tumor section digested with collagenase, and stained for various surface markers of DC maturation such as CD80, CD86, and MHC class II (Fig 3c). More than 60% of DCs were positive for these markers in C26-C+L tumor, suggesting these DCs had a mature phenotype. Although DCs infiltrating in the other tumors, C26-Vector, -CCL21, and -LIGHT, also had a mature phenotype with similar level of expression of these markers, the relative cell number of mature DCs infiltrating in C26-C+L tumor was more than five times larger than that in C26-Vector tumor, and also much larger than those in C26-CCL21 or C26-LIGHT tumors. These results suggest that coexpression of CCL21 and LIGHT greatly enhances infiltration of mature DCs, CD8+ T cells, and B cells in tumor.

Markedly augmented CTL activity and increased IFN-γ production by coexpression of CCL21 and LIGHT

Since LIGHT was demonstrated to serve as a costimulatory molecule for T-cell activation and DC maturation resulting in induction of specific CTL,12,18 we then analyzed CTL activity induced by C26-Vector, -CCL21, -LIGHT, and -C+L. Spleen was removed from each mouse 2 weeks after tumor inoculation, and spleen cells were restimulated in vitro by coculture with irradiated C26 parental tumor cells for 5 days. These cells were then used as effector cells, and their cytolytic capacities were assessed against 51Cr-labeled C26 tumor targets in a standard 51Cr release assay (Fig 4a). Spleen cells from mice inoculated with C26-CCL21 or -LIGHT exerted a slightly increased CTL activity against parental tumor C26 compared with those with C26-Vector. In contrast, spleen cells from mice inoculated with C26-C+L strikingly enhanced the CTL activity. The culture supernatants obtained form these cocultures were also collected and assayed for INF-γ production by ELISA (Fig 4b). Correlating with the induction level of CTL activity, much higher production of IFN-γ and less but significant production of IFN-γ were observed in spleen cells from mice inoculated with C26-C+L, and C26-CCL21 or -LIGHT, respectively. These results suggest that coexpression of CCL21 and LIGHT in tumor markedly augments CTL activity against tumor and IFN-γ production as well.

Figure 4
figure4

Markedly augmented CTL activity and increased IFN-γ production by coexpression of CCL21 and LIGHT. Spleen was removed from each mouse (n=3) 2 weeks after tumor inoculation, and spleen cells (5 × 106 cells/ml) were restimulated in vitro by coculture with C26 parental tumor cells (5 × 105 cells/ml) irradiated with 150 Gy for 5 days. These cells were then used as effector cells, and their cytolytic capacities were assessed against 51Cr-labeled C26 tumor targets in a standard 51Cr release assay (a). The culture supernatants obtained form the coculture were also collected and assayed for INF-γ production by ELISA (b). Data are shown as the mean ± SD. * and ** in (A) indicate P<0.05 and <0.01, respectively, compared with C26-Vector. * and ** in (B) indicate P<1 × 10−4 and <1 × 10−5, respectively, compared with C26-Vector. Similar results were obtained in five independent experiments.

Critical involvement of IFN-γ and also CD8+ and CD4+ T cells in induction of synergistic antitumor effect by coexpression of CCL21 and LIGHT

To examine the involvement of particular cytokines or lymphocytes in the inhibition of tumor growth by coexpression of CCL21 and LIGHT, we treated mice with neutralizing anti-IFN-γ mAb or depleting anti-CD8 or anti-CD4 mAbs during tumor challenge, and tumor growth was monitored (Fig 5). Consistent with the increased production of IFN-γ and vigorous infiltration of CD8+ T cells in tumor as shown in Figures 4b and 3b, respectively, treatment of mice with mAb neutralizing IFN-γ and mAb depleting CD8+ T cells significantly recovered tumor growth. Similarly recovered tumor growth was also observed in mice treated with mAb depleting CD4+ T cells. These results suggest that IFN-γ production and also CD8+ and CD4+ T cells are critically involved in the induction of synergistic antitumor effect by coexpression of CCL21 and LIGHT.

Figure 5
figure5

Critical involvement of IFN-γ and also CD8+and CD4+ T cells in induction of synergistic antitumor effect by coexpression of CCL21 and LIGHT. Mice (n=5) were inoculated with C26-C+L or C26-Vector cells and injected i.p. with anti-IFN-γ neutralizing mAb, anti-CD8- or anti-CD4-depleting mAbs, or control Ab as described in Materials and methods, and tumor growth was monitored. Data are shown as the mean±SD. * and ** indicate P<0.05 and <0.01, respectively, compared with C26-C+L + control Ab. Similar results were obtained in two independent experiments.

Discussion

To elicit a strong immune response against malignant cells, a combined treatment with chemokines and costimulatory molecules in tumor is considered to be one of the most effective approaches for immunotherapy against solid tumors. Several previous reports have demonstrated that chemokines or costimulatory molecules induce an effective antitumor response.26,27,28 One of the best candidates for chemokines is CCL21, which has been proposed to play a role in favoring the interactions between DCs and T cells in secondary lymphoid organs through CCR7 receptor.4,7,9,29 In fact, mice deficient in the expression of CCL21 showed defects in lymphocyte homing and DC localization,30 and mice lacking CCR7 receptor also had defects in lymph node architecture.31 Furthermore, it has been demonstrated that CCL21 could participate itself in lymphoid tissue development and organization in a transgenic model where the CCL21 gene was expressed in pancreatic islets.32 In addition, CCL21 has been demonstrated to induce an angiostatic activity through interaction with another receptor CXCR3.21,23 These dual properties are profitable to induce a strong antitumor response. On the other hand, the best candidates for costimulatory molecule are those belonging to the TNF superfamily, which plays a critical role in multiple aspects of the immune system through regulation of lymphoid organ formation, cell apoptosis, B-cell activation, T-cell costimulation, and DC activation.33 Among them, LIGHT is a recently identified TNF superfamily molecule and serves as a costimulatory molecule for T-cell activation and DC maturation leading to the generation of specific CTL.12,18,19 Of note, it has recently been reported that LIGHT-deficient mice show selective impairments of CD8+ T-cell function but not CD4+ T cells.34

According to the strategy described above, we were here able to induce a strong antitumor activity to eradicate tumor completely in all treated mice by a synergistic antitumor effect of CCL21 and LIGHT with a rich infiltrate of mature DCs and CD8+ T cells in tumor and also with a strong CTL activity and increased IFN-γ production. DCs with a mature phenotype play an important role in the activation, recruitment, and expansion of effector cells such as CD8+ T cells.1 The efficient tumor infiltration of mature DCs and CD8+ T cells was presumably attributed to the facts that LIGHT has an ability to induce maturation of DCs19 and CCL21 has a chemotactic activity for mature DCs29,35 and CD8+ T cells.21,36 The enhanced CTL activity and IFN-γ production were mainly due to the rich infiltrate of mature DCs and CD8+ T cells in tumor as well as a costimulatory activity to T cells elicited by LIGHT.12,18

Recently, a combined treatment with CCL21 and another costimulatory molecule CD40 ligand (CD40L) was reported to induce an augmented antitumor activity.37 CD40L plays a central role in activating APCs, including B cells, monocytes, and DCs, and initiation of antigen-specific immune responses.38 CCL21 and CD40L were administered to mice using a herpes simplex virus (HSV) amplicon vector, a gene delivery platform amenable for tumor immunotherapy. Since the experimental conditions are different from ours, it is difficult to compare the efficiency to induce the antitumor immune responses with ours. But a complete remission in all mice was attained by a combined treatment with CCL21 and LIGHT as described in the present study, but not with CCL21 and CD40L.37

Many molecules of the TNF family were reported to affect migration of T cells by regulating expression of chemokines and their receptors.39 Indeed, we observed a chemotactic activity for T cells in the culture supernatant of C26-LIGHT cells (Fig 1d). Consistent with the in vitro result, a more increased infiltration of DCs and CD8+ T cells in tumor in vivo was also observed in mice inoculated with C26-LIGHT cells than those with C26-Vector cells (Fig 3). LIGHT may be secreted into the culture supernatant to exert the chemotactic activity or indirectly exhibit the chemotactic activity by inducing the secretion of chemokines. Moreover, LIGHT may enhance the retention or survival in tumor mass, since LIGHT can promote the activation of lymphocytes and DCs.12,19,34 Further studies are necessary to prove these possibilities.

Consistent with the previous report,21 no significant increased tumor infiltration of CD4+ T cells was observed in C26-C+L, -CCL21, and -LIGHT tumors 2 weeks after tumor inoculation (Fig 3b). However, treatment of mice inoculated with C26-C+L by mAb depleting CD4+ T cells significantly reduced the antitumor activity (Fig 5). It is well established that DCs require CD4+ T-cell help to cross-prime CD8+ T cells properly and this process depends on the stimulation of CD40 receptor on DCs by transiently expressed CD40L on CD4+ T cells.40 CD40L also provides the primary stimulus for induction of IL-12, which is critically important for generation of CTL,41 by mature DCs.42,43,44 In vitro, CCL21 has been reported to be a chemotactic factor for CD4+ T cells.36,45 Therefore, CCL21 and LIGHT may enhance the function of CD4+ T cells without increasing the cell number or the infiltration of CD4+ T cells into tumor may occur in earlier time after tumor inoculation.

In the present study, we have shown that coexpression of chemokine CCL21/SLC and costimulatory molecule LIGHT is able to induce a synergistic antitumor activity by greatly enhancing tumor infiltration of lymphocytes including mature DCs and CD8+ T cells, resulting in markedly augmented CTL activity against tumor and IFN-γ production. Thus, the combined treatment with CCL21 and LIGHT could open up a novel avenue to cancer immunotherapy.

Abbreviations

DC:

dendritic cell

SLC:

secondary lymphoid tissue chemokine

APC:

antigen-presenting cell

Th:

T helper cell

CTL:

cytotoxic T lymphocyte

RT:

reverse transcriptase

IRES:

internal ribosome entry site

ATCC:

American Type Culture Collection

mAb:

monoclonal antibody

C26-CCL21:

C26 cells transfected with pcDNA3.1-CCL21 cDNA

C26-LIGHT:

C26 cells transfected with pcDNA3.1-LIGHT cDNA

C26-C+L:

C26 cells transfected with pcDNA3.1-LIGHT cDNA/IRES/CCL21 cDNA

C26-Vector:

C26 cells transfected with pcDNA3.1 vector alone

CD40L:

CD40 ligand

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Acknowledgements

We thank Drs T Kitamura and E Takada for kindly providing pMX-IRES/EGFP and technical help, respectively.

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Correspondence to Takayuki Yoshimoto.

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Supported by a Grant-in-Aid for Scientific Research on Priority Areas and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

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Hisada, M., Yoshimoto, T., Kamiya, S. et al. Synergistic antitumor effect by coexpression of chemokine CCL21/SLC and costimulatory molecule LIGHT. Cancer Gene Ther 11, 280–288 (2004). https://doi.org/10.1038/sj.cgt.7700676

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Keywords

  • CCL21/SLC
  • LIGHT
  • Antitumor effect
  • DC

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