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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

Polo-like kinase 1 (Plk1), a serine–threonine kinase, plays a key role in the regulation of the cell cycle. Elevated Plk1 expression in various cancers is correlated with poor prognosis and poor patient survival rates. Several Plk1 inhibitors are currently being developed as potential treatments for cancer. In the present study, we investigated whether dendritic cells (DC) electroporated with mouse Plk1RNA (mPlk1RNA/DC) can induce Plk1-specific immune responses and exert antitumor effects in various murine tumor models. Overexpression of Plk1 protein was confirmed in several mouse and human tumor cell lines and various cancer tissues. Furthermore, Plk1-specific CD4+ and CD8+ T cells were induced by vaccination with mPlk1RNA/DC and the cytotoxic activity of the T cells was demonstrated against several Plk1-expressing tumor cell lines. Vaccination with mPlk1RNA/DC inhibited the growth of MC-38 and B16F10 tumors in C57BL/6 mice and the growth of CT26 tumors in BALB/c mice. Depletion of CD8+ T cells reversed the inhibition of tumor growth by mPlk1RNA/DC vaccination. Homologous human Plk1RNA-electroporated DC also inhibited tumor growth in MC-38 tumor-bearing mice. In addition, Plk1-specific cytotoxic T lymphocytes from PBMC of healthy donors could be induced using autologous monocyte-derived DC electroporated with RNA encoding the whole gene of human Plk1. Taken together, the results of the present study suggest that Plk1 could be a universal tumor antigen recognized by cytotoxic T lymphocytes for cancer immunotherapy. (Cancer Sci 2011; 102: 1448–1454)

Polo-like kinase 1 (Plk1), a serine–threonine kinase, has a key role in the regulation of cell division, including mitotic entry, spindle formation, chromosome segregation, and cytokinesis.(1–3) The expression and activity of Plk1 display a cell cycle-dependent pattern, peaking from the late S phase to mitosis.(1–3) During fetal development, Plk1 is expressed in all tissues, whereas in adult tissues Plk1 expression is detected in highly proliferating tissues, including the placenta, testis, ovary and spleen.(4,5) Forced overexpression of Plk1 in NIH3T3 cells promotes transformation and tumor formation in nude mice,(6) suggesting that Plk1 is implicated in the origin or progression of tumors. Overexpression of Plk1 has been reported in approximately 80% of human cancers(7–14) and elevated Plk1 expression is correlated with a poor prognosis and poor patient survival in a variety of cancers.(9,13) Moreover, downregulation of Plk1 by antisense oligonucleotides and siRNA results in a marked reduction in proliferation and increase in apoptosis in tumor cells, but not in normal cells in vitro, and has been demonstrated to be a powerful suppression of tumor growth in a xenogenic model.(15–17) Several Plk1 inhibitors, including scytonemin, B-2536, HMN-214, ON-01910, and poloxin, are under development as potential treatments for cancer, with some of them in clinical trials.(18–21)

Because many tumor antigens are tolerogenic as self-antigens, it is often difficult to induce a specific immune responses against them. Dendritic cells (DC), as the most potent antigen-presenting cells, can break tolerance against self-antigens by effectively priming naïve T cells in vitro and in vivo. We have reported previously that DC electroporated with the RNA of tumor antigens, such as mouse survivin, telomerase reverse transcriptase (TERT), and topoisomerase IIα (TopIIα), successfully induce antitumor immunity and antigen-specific CD4+ and CD8+ T cell responses in murine tumor models.(22–24) The induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T cells in vitro using human DC transfected with RNA has also been reported.(25)

Of the molecules that regulate the cell cycle, Aurora A, TopIIα, Foxm1, and Ran have been reported to be tumor antigens. Aurora A was defined as a novel target of cellular immunotherapy for leukemia;(26) the immunogenicity of TopIIα was demonstrated in a mouse system by vaccination with mRNA-electroporated DC;(24) and Foxm1 and Ran peptide can induce cytotoxic T lymphocytes (CTL) activity in human PBMC and in human leukocyte antigen (HLA)-A2 transgenic mice.(27,28) Because Plk1 is tightly associated with these proteins as a key cell cycle protein,(29–32) we investigated whether DC electroporated with mouse Plk1RNA (mPlk1RNA/DC) can induce Plk1-specific immune responses and exert antitumor effects in various murine tumor models. These results suggest that Plk1 could be a new target of T cells that could function as a universal tumor antigen for cancer immunotherapy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

Animals and tumor cell culture.  Female C57BL/6 mice (H-2b; 6–8 weeks old) and BALB/c mice (H-2d; 6–8 weeks old) were purchased from Orient Bio (Kapung, Korea). The 11 different tumor cell lines (B16F10, Renca, CT26, EL4, YAC-1, LoVo, MCF7, T98G, SK-MEL5, HeLa and K562) were purchased from American Type Culture Collection (ATCC, Manassa, VA, USA). GL26 and MC38 were kindly provided by Dr. John S Yu (Cedars Sinai Medical Center, Los Angeles, CA, USA) and Dr. J Schlom (Division of Tumor Immunology and Biology, National Institutes of Health, Bethesda, MD, USA), respectively. The tumor cell lines were cultured under conditions recommended.

Western blotting and immunohistochemical staining.  Western blotting using an anti-Plk1 antibody (Cell Signaling Technology, Danvers, MA, USA) was performed as described previously.(24) In addition, tissues from patients with colon cancer (18 cases), stomach cancer (eight cases), ovarian cancer (14 cases), and malignant lymphoma (17 cases) were stained immunohistochemically using an anti-Plk1 antibody (Millipore, Temecula, CA, USA), as described previously.(33)

Electroporation of mRNA into DC.  For the in vitro transcription (IVT) of mRNA, the mouse Plk1/pcDNA3.1 and human Plk1/pcDNA3.1 plasmids were linearized with ScaI and NdeI, respectively. The linearized DNA was used as a template and transcripted in vitro using the mMessage mMachine T7 Ultra Kit (Ambion, Austin, TX, USA).

After IVT, the mRNA (20 μg) was electroporated into DC and the DC were placed immediately in complete medium containing granulocyte–macrophage colony stimulating factor (20 ng/mL), interleukin (IL)-4 (20 ng/mL), and lipopolysaccharide (1 μg/mL) to allow the DC to fully mature for 24 h. Both the generation of DC from the bone marrow of C57BL/6 mice or BALB/c mice and electroporation into the DC were performed as described previously.(24)

Isolation of CD4+ and CD8+ T cells.  To isolate CD4+ and CD8+ T cells, splenocytes were incubated with magnetic beads conjugated to CD4- or CD8-specific mAbs for 15 min at 4°C and processed through a magnetic antibody cell sorter (MACS) magnetic separation column. The purity of each T cell population after sorting was >90%, as determined by FACS analysis.

Enzyme-linked immunospot assay.  An interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay was performed as described previously.(24) The targets were mPlk1RNA/DC, DC electroporated with RNA encoding mouse survivin (mSuvRNA/DC), DC electroporated with RNA encoding CEA (CEARNA/DC), and DC.

Cytotoxicity assay.  A 51Cr-release assay was performed as described previously.(24) In these experiments, MC-38, GL26, mPlk1RNA/DC, CEARNA/DC, DC, YAC-1, CT26, and normal splenocytes labeled with 3.7 GBq 51Cr-sodium chromate per 1 × 106 cells were used as targets.

Production of tumor lysate.  To produce tumor lysate, MC-38 or B16F10 cells (1 × 107 cells/mL PBS) were repeatedly freeze–thawed four times. After centrifugation (70g, 10 min, 4°C), the supernatant was collected and filtered (0.2 μm) and protein concentration was determined using a bicinchoninic acid assay. The DC were incubated with the MC-38 or B16F10 tumor lysate (100 μg/mL) for 16–18 h to pulse with the MC-38 tumor lysate.

Mouse tumor models and DC vaccination.  To create the tumor models, mice were injected subcutaneously in the right flank with 2 × 105 MC-38, B16F10 or CT26 cells. After 1 or 2 days after tumor inoculation, mice were vaccinated subcutaneously in the opposite flank once a week for 3 weeks with 1 × 106 mPlk1RNA/DC, DC pulsed with the tumor lysate from MC-38 cells or B16F10 cells, mSuvRNA/DC, or unpulsed DC. To test the effects of xenogenic human Plk1, 2 days after tumor inoculation MC-38-tumor bearing mice were vaccinated three times, at intervals of 7 days, with 1 × 106 human Plk1RNA/DC or DC. In these experiments, survivin was used as a positive control. Changes in tumor volume were measured every 5 days using calipers and calculated using the following equation:

  • image

where a and b are the major and minor tumor axes, respectively.

In vivo depletion of the T cell subset.  Depletion of CD4+ or CD8+ T cells was achieved as described previously(24) using an anti-CD4 mAb (GK1.5; eBioscience, San Diego, CA, USA) or an anti-CD8 mAb (2.43; a gift from Professor Byungsuk Kwon, Ulsan University, Ulsan, Korea).

In vitro generation of human Plk1-specific CTL from PBMC.  Antigen-specific CTL were generated using with autologous DC as described previously.(34) Purified CD8+ and CD4+ T cells were stimulated with human DC electroporated with human Plk1RNA (40 μg) at a ratio of 1:10. After restimulation twice with 10 U/mL IL-2 (Genzyme, Cambridge, MA, USA), cells were assayed with an IFN-γ ELISPOT.

This study was approved by the Institutional Review Board of The Catholic University of Korea, College of Medicine and all donors provided written informed consent.

Statistical analysis.  Results are shown as the mean ± SD. Data were analyzed by Student’s t-test. P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

Expression of Plk1.  The expression of Plk1 was analyzed in mouse and human tumor cells lines and in normal mouse tissues by western blotting. Mouse Plk1 strongly expressed in most tumor cell lines including B16F10 (H-2b; melanoma), EL4 (H-2b; thymoma), MC-38 (H-2b; colon cancer), GL26 (H-2b; gliomas), Renca (H-2d; renal cancer), and CT26 (H-2d; colon cancer), but weakly expressed in the YAC-I (H-2a; lymphoma) (Fig. 1a). Human Plk1 was also expressed in human tumor cell lines that originated from a range of different organs, including SKOV3 (ovarian cancer), MCF7 (breast cancer), T98G (glioblastoma), LoVo (colon cancer), SK-MEL5 (melanoma), K562 (leukemia), and HeLa (cervical cancer) cells (Fig. 1a). We also investigated Plk1 expression in tissue from patients with colon cancer, stomach cancer, ovarian cancer, and malignant lymphoma using immunohistochemical staining. In these experiments, Plk1 protein was detected in all colon and stomach tumors (n = 18 and 8, respectively), in five of 14 cases of ovarian cancer, and in 14 of 17 cases of malignant lymphoma. Figure 1(b) showed strong Plk1 expression of in both the nucleus and cytoplasm of colon cancer cells.

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Figure 1.  Expression of Plk1 in mouse and human tumor cell lines, human colon cancer tissue, and normal mouse tissues. (a) Expression of Plk1 protein, as determined by western blotting, in mouse and human tumor cell lines. (b) Immunohistochemical staining against Plk1 in human colon cancer tissues. Representative results are shown. T, tumor cells; N, normal cells. (c) Plk1 expression in normal mouse tissues. Western blotting was analyzed in the brain, lymph node (LN), spleen, bone marrow (BM), lung, liver, kidney, and small and large intestine (intestine) of a normal C57BL/6 mouse (H-2b). The results shown are representative of three independent experiments.

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Plk1 was not detected in the brain, lymph node, bone marrow, lung, liver, kidney, or small and large intestines of normal C57BL/6 mice, but was weakly expressed in the spleen (Fig. 1c). These results confirm that Plk1 protein is strongly expressed in most tumor cells.

Induction of Plk1-specific immune responses.  The cytotoxic activity of splenocytes vaccinated with mPlk1RNA/DC was evaluated in Plk1-expressing and non-expressing targets. Cytotoxic activity was evident only against H-2-matched Plk1-expressing targets (H-2b; MC-38, B16F10, GL26, and mPlk1RNA/DC; Fig. 2a–d) and not against targets not expressing Plk1 (DC and CEARNA/DC) or H-2-mismatched targets (H-2d; CT26; Fig. 2e–g). The YAC-I cells targeted by natural killer (NK) cells and normal splenocytes did not show significant lysis (Fig. 2h,i).

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Figure 2.  Induction of Plk1-specific immune responses following vaccination with dendritic cells (DC) electroporated with mouse Plk1 RNA (mPlk1RNA/DC). (a–i) Plk1-specific cytotoxic activity, as determined by the 51Cr release assay at different effector:target ratios after vaccination with either DC (♦) or mPlk1RNA/DC (▪). Both Plk1-expressing targets (MC38, B16F10, GL26, mPlk1RNA/DC, and normal splenocytes: H-2b, CT26:H-2d) and non-expressing targets (DC and DC electroporated with RNA encoding carcinoembryonic antigen [CEARNA/DC]: H-2b) were used. The YAC-I cell line (H-2a) was a target for natural killer cell activity, and the CT26 cell line was a Plk1-expressing, but H-2b-mismatched, target. Specific lysis, as a percentage, was calculated from four wells. The data are representative findings from three independent experiments with similar results. *P < 0.05 (t-test). (j–l) Induction of Plk1-specific interferon (IFN)-γ-producing T cells. Splenocytes from mice with 1 × 106 mPlk1RNA/DC, DC electroporated with RNA encoding mouse survivin (mSuvRNA/DC), or unpulsed DC were stimulated with mPlk1RNA/DC, mSuvRNA/DC, or CEARNA/DC as targets. The frequency of IFN-γ-producing T cells was measured by ELISPOT assay. Results show the mean ± SD number of IFN-γ spots per 5 × 104 cells from individually tested mice. *P < 0.05 (t-test).

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To further define Plk1-specific T cell responses, IFN-γ-producing T cells were measured by ELISPOT assay. Mice were vaccinated with mPlk1RNA/DC, mSuvRNA/DC, or unpulsed DC. Splenocytes from vaccinated mice were stimulated with mPlk1RNA/DC, mSuvRNA/DC, and CEARNA/DC in vitro for the ELISPOT assay. We found that mPlk1RNA/DC and mSuvRNA/DC vaccination induced Plk1- and Suv-specific IFN-γ-producing T cells, respectively (Fig. 2j,k). However, mPlk1-specific T cells did not respond significantly to mSuvRNA/DC and CEARNA/DC as irrelevant targets (Fig. 2k,l). These results indicate that Plk1-specific immune responses can be induced by mPlk1RNA/DC vaccination.

Antitumor effects of mPlk1RNA/DC vaccination in various tumor models.  To demonstrate the potential of Plk1 as a target tumor antigen for cancer immunotherapy, the antitumor effects of mPlk1RNA/DC vaccination were evaluated and compared with those of mSuvRNA/DC vaccination in various tumor models. As shown in Figure 3, vaccination with mPlk1RNA/DC provided a significant therapeutic effect in MC-38 and B16F10 tumor models in C57BL/6 mice 20 days after tumor inoculation. In addition, mPlk1RNA/DC vaccination inhibited tumor growth in the C26 tumor model in BALB/c mice. The inhibition of tumor following mPlk1RNA/DC vaccination was similar to that seen following mSuvRNA/DC vaccination. These results suggest that Plk1 may be used as a tumor antigen, such as survivin, which is already recognized as a universal tumor antigen.

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Figure 3.  Antitumor effects of vaccination with dendritic cells (DC) electroporated with mouse Plk1 RNA (mPlk1RNA/DC) in various murine tumor models. (a) The MC-38 tumor model. At 2, 9, and 16 days after inoculation of 2 × 105 MC-38 tumor cells, C57Bl/6 mice were vaccinated with mPlk1RNA/DC, DC electroporated with RNA encoding mouse survivin (mSuvRNA/DC), DC pulsed with MC-38 tumor lysate (MC38TL/DC), or unpulsed DC (n = 14 in each group). (b) The B16F10 tumor model. At 1, 8, and 15 days after inoculation of 2 × 105 B16F10 tumor cells, C57Bl/6 mice were vaccinated with mPlk1RNA/DC, mSuvRNA/DC, DC pulsed with B16F10 tumor lysate (B16F10TL/DC), or unpulsed DC (n = 10 per group). (c) The CT26 tumor model. At 1, 8, and 15 days day after inoculation of 2 × 105 CT26 tumor cells, BALB/c mice were vaccinated with mPlk1RNA/DC, mSuvRNA/DC, or unpulsed DC (n = 9 in each group). Tumor volume was determined every 5 days. Results are given as the mean ± SD. *P < 0.05 (t-test).

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Role of CD4+ and CD8+ T cells in the antitumor effects.  Given that mRNA expressing the whole molecule of Plk1 was used as an antigen, we examined the immune responses of CD4+ and CD8+ T cells. The CD4+ and CD8+ T cells were sorted from splenocytes of mice vaccinated with either mPlk1RNA/DC or unpulsed DC and were then stimulated with mPlk1RNA/DC or CEARNA/DC. Both the CD4+ and CD8+ T cell populations exhibited a high frequency of IFN-γ-producing T cells against mPlk1RNA/DC, but not against CEARNA/DC in mice vaccinated with mPlk1RNA/DC (Fig. 4a).

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Figure 4.  CD4+ and CD8+ T cell responses induced by vaccination with dendritic cells (DC) electroporated with mouse Plk1 RNA (mPlk1RNA/DC). (a) Detection of CD4+ and CD8+ interferon (IFN)-γ-producing T cells specific for Plk1. The CD4+ and CD8+ T cells were isolated from the splenocytes of mice vaccinated with either mPlk1RNA/DC (▪) or unpulsed DC (□) using magnetic beads conjugated with anti-CD4- and anti-CD8-specific mAbs. Each T cell population was stimulated with mPlk1RNA/DC or DC electroporated with RNA encoding carcinoembryonic antigen (CEARNA/DC) as the target and IFN-γ-producing T cells were then measured by the ELISPOT assay. Results are given as the mean ± SD. *P < 0.05 (t-test). (b) Depletion of CD4+ and CD8+ T cells in MC-38 tumor-bearing mice. Mice were treated with anti-CD4 or anti-CD8 Ab 2 days before each mPlk1RNA/DC vaccination. Vaccination with unpulsed DC was used as a control for normal tumor growth. Results are given as the mean ± SD (n = 7 mice in each group). *P < 0.05 (t-test).

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To determine which T cell subset was involved in the antitumor effects induced by vaccination with mPlk1RNA/DC in the MC-38 tumor model, CD4+ or CD8+ T cells were depleted by treatment with anti-CD4 or anti-CD8 mAbs, respectively. Depletion of CD8+ T cells significantly reversed the inhibition of tumor growth in mice vaccinated with mPlk1RNA/DC, whereas depletion of CD4+ T cells did not (Fig. 4b). These data suggest that CD8+ T cells are the main effector cells involved in antitumor immunity in vivo.

Antitumor effects of human Plk1 in the murine tumor model.  Because human survivin and TERT share considerable amino acid sequence similarity with mouse survivin and TERT, respectively, vaccination with human survivin or TERT has been reported to induce antitumor effects in the mouse tumor model.(35) First, to investigate the cross-reactivity between mouse and human Plk1, splenocytes from mice vaccinated with either mPlk1RNA/DC or unpulsed DC were stimulated with humanPlk1RNA/DC, mPlk1RNA/DC, or CEARNA/DC in vitro for an IFN-γ ELISPOT assay. The mPlk1-specific T cells recognized human Plk1 at levels similar to mPlk1, but did not recognize CEA (Fig. 5a). Conversely, to evaluate the antitumor effects of human Plk1 vaccination, MC-38 tumor-bearing mice were vaccinated with DC electroporated with human Plk1RNA. As shown in Figure 5(b), mice vaccinated with humanPlk1RNA/DC exhibited significant inhibition of tumor growth, similar to that observed in response to mPlk1RNA/DC. These data indicate that because of the high degree of amino acid homology between mouse and human Plk1, human Plk1RNA/DC can trigger effective Plk1-specific immune responses in mice, both in vitro and in vivo.

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Figure 5.  Cross-reactivity between mouse (m) and human (hu) Plk1 in murine tumor models. (a) Recognition of huPlk1 following mPlk1RNA/DC vaccination. Splenocytes from mice vaccinated with 1 × 106 dendritic cells (DC) electroporated with mouse Plk1 RNA (mPlk1RNA/DC; ▪) or unpulsed DC (□) were stimulated with either huPlk1RNA/DC, mPlk1RNA/DC, or DC electroporated with RNA encoding carcinoembryonic antigen (CEARNA/DC), and the frequency of interferon (IFN)-γ-producing T cells was measured by the ELISPOT assay. Results are given as the mean ± SD. *P < 0.05 (t-test). (b) Mice were vaccinated with 1 × 106 huPlk1RNA/DC (▪), mPlk1 RNA/DC (bsl00066) or unpulsed DC (•) at 2, 9, and 16 days after inoculation with 2 × 105 MC-38 tumor cells. Tumor volume was determined every 5 days. Results are given as the mean ± SD (n = 8 mice per group). *P < 0.05 (t-test).

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In vitro generation of human Plk1-specific CTL from the PBMC of healthy donors.  We attempted to generate human Plk1-specific CTL from the PBMC of healthy donors using autologous monocyte-derived DC electroporated with RNA encoding the whole gene of human Plk1 (human Plk1RNA/DC). After three stimulations, the IFN-γ-producing T cells were measured by ELISPOT assay. In four healthy donors, IFN-γ-producing T cells exhibited high frequencies against human Plk1RNA/DC, but not against unpulsed DC and CEARNA/DC (Fig. 6a). To confirm that the induced CTL recognized the target cells in an HLA Class I-restricted manner, we blocked the binding of MHC Class I molecules using the anti-HLA Class I antibody (W6/32). The IFN-γ production induced by human Plk1-specific CTL was significantly inhibited by W6/32 (Fig. 6b). These results suggest that Plk1 has immunogenicity to induce CTL in humans following stimulation with human Plk1RNA/DC in vitro.

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Figure 6. In vitro generation of Plk1-specific CTL with human Plk1RNA/DC from the PBMC of healthy donors. (a) Human Plk1-specific CTL generated from the PBMC of healthy donors using autologous monocyte-derived dendritic cells (DC) electroporated with RNA encoding the whole gene of human Plk1 (huPlk1RNA/DCs). Human Plk1-specific CTL were stimulated with huPlk1RNA/DC (▪), DC electroporated with RNA encoding carcinoembryonic antigen (CEARNA/DC; bsl00023), or unpulsed human DC (□) and interferon (IFN)-γ-producing T cells were measured by the ELISPOT assay. (b) Recognition of targets by the induced CTL in a human leukocyte antigen (HLA) Class I-restricted manner, the binding of MHC Class I molecules was blocked using the anti-HLA Class I antibody (W6/32) and IFN-γ-producing T cells measured by ELISPOT assay. (□), with W6/32; (bsl00001), without W6/32. Data show individual responses obtained with the PBMC from four healthy donors (#1–#4).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

The Plk1 protein was overexpressed in a broad range of mouse and human tumor cell lines and colon cancer tissue (Fig. 1). The CTL induced by mPlk1RNA/DC vaccination specifically killed Plk1-expressing cells and not cells that did not express Plk1 (Fig. 2). Vaccination with mPlk1RNA/DC resulted in antitumor effects in various tumor models (Fig. 3). The inhibitory effects on tumor growth following mPlk1RNA/DC vaccination in tumor models was similar to that induced by mSurRNA/DC targeting survivin, which is already known as a universal tumor antigen. Although the Plk1 protein was expressed at a very low level in normal splenocytes, repeated vaccination with Plk1RNA/DCs may trigger autoimmunity. However, we confirmed no cytotoxicity against normal splenocytes of Plk1-specific CTL (Fig. 2i). In addition, we induced Plk1-specific CTL from PBMC of healthy donors using autologous monocyte-derived DC electroporated with RNA encoding the whole gene of human Plk1. Taken together, our results suggest that Plk1 could be a potent target recognized by T cells and that it could be used as a universal tumor antigen for cancer immunotherapy.

Vaccination with mPlk1RNA/DC effectively induced both CD4+ and CD8+ IFN-γ-producing T cells specific for Plk1, as shown in Figure 4(a). However, we could not demonstrate that in vivo depletion of CD4+ T cells reduced the antitumor effects in mice vaccinated with mPlk1RNA/DC (Fig. 4b). It has been reported that a large number of CD4+CD25+ regulatory T (Treg) cells are present in tumors and draining lymph nodes in MC-38 tumor-bearing mice and that depletion of CD4+CD25+ Treg cells enhances the antitumor immunity.(36) In addition to inhibiting Plk1-specific CD4+ T cells in our tumor models, treatment with the anti-CD4 Ab may also inhibit Treg cells induced by the tumor environment.

For some tumor antigens, amino acid sequences are highly conserved between mouse and human (e.g. 84.3% for survivin, 91.8% for WT-1, and 80% for TRP2), indicating that epitopes capable of inducing tumor antigen-specific CTL in the mouse may be identical in the human. The immunization of rodents with xenogeneic antigens has been reported to overcome tolerance to the corresponding self-antigen and to induce strong immunity against self-tumor antigens.(37–39) Dendritic cells (DC) transduced with adenovirus encoding human survivin provided significant antitumor effects against three different murine tumors.(35) In the present study, tumor growth in MC-38 tumor-bearing mice was decreased significantly in mice vaccinated with human Plk1RNA/DCs (Fig. 5b). Because the Plk1 amino acid sequence is 95% identical in humans and mice, T cells induced by vaccination with mouse or human Plk1 may cross-reactively recognize the naturally processed mouse and human epitopes. It is difficult to directly demonstrate the antitumor effects of candidate tumor antigens in humans. Therefore, elucidating the immunogenicity of human tumor antigens that are highly homologous with that in the mouse in murine models may be an efficient strategy.

Because survivin and TERT are known as an anti-apoptosis protein and an anti-aging ribonucleoprotein, respectively, and Plk1 has a key role in cell division for tumorigenesis, these tumor antigens could be different target molecules for cancer therapy. Therefore, Plk1 could be used for combined immunotherapy targeting multiple tumor antigens, including survivin and TERT, or as a new target antigen for tumors not expressing other universal tumor antigens. Recently, we demonstrated that cotransduction of adenoviral vectors encoding CEA and survivin into DC enhances antitumor immunity in a murine colorectal cancer model.(40) Further studies are needed to demonstrate the effects of vaccination of DC transduced with RNA encoding multiple tumor antigens, including Plk1, in tumor-bearing mice. Although vaccination with mPlk1RNA/DC and DC pulsed with the whole tumor cell lysate resulted in similar antitumor effects, vaccination with whole tumor cell lysate, including undefined self-antigens, could increase the risk of autoimmunity. Therefore, vaccination with DC transduced with defined tumor antigen RNA is suggested to be a better strategy for cancer immunotherapy not only in preclinical animal models, but also in clinical trials.

In conclusion, the present study provides the first evidence that Plk1 is a new target of T cells for cancer immunotherapy. Further studies are needed to demonstrate Plk1-specific CTL in cancer patients and to identify epitopes recognized by T cells.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2010–359-E00010).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References