Induction of Antitumor Immunity by Transduction of CD40 Ligand Gene

D2001 Nature Publishing Group 0929-1903/01/$17.00/+0 www.nature.com/cgt Induction of antitumor immunity by transduction of CD40 ligand gene and interferon- gene into lung cancer Masahiro Noguchi,1 Kazuyoshi Imaizumi,1 Tsutomu Kawabe,1 Hisashi Wakayama,1 Yoshitsugu Horio,1 Yoshitaka Sekido,2 Toru Hara,1 Naozumi Hashimoto,1 Masahide Takahashi,3 Kaoru Shimokata,2 and Yoshinori Hasegawa1 1First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan; Departments of 2Clinical Preventive Medicine and 3Pathology, Nagoya University School of Medicine, Nagoya, Japan. CD40±CD40 ligand (CD40L) interaction is an important costimulatory signaling pathway in the crosstalk between T cells and antigen-presenting cells. This receptor±ligand system is known to be essential in eliciting strong cellular immunity. Here we demonstrate that murine lung cancer cells (3LLSA) transduced with the CD40L gene (3LLSA-CD40L) were rejected in syngeneic C57BL/6 mice, but grew in CD40-deficient mice to the same extent as control tumor cells. Immunohistochemical study showed that inflammatory cells, including CD4+, CD8+ T cells and NK cells, infiltrated into the inoculated 3LLSA-CD40L tumor tissue. Inoculation of 3LLSA-CD40L cells into mice resulted in the induction of 3LLSA-specific cytotoxic T-cell immunity, and the growth of parental 3LLSA tumors was inhibited when 3LLSA cells were inoculated into C57BL/6 mice mixed with 3LLSA-CD40L cells or when they were rechallenged 4 weeks after 3LLSA-CD40L cells were rejected. Furthermore, co-inoculation of interferon (IFN)- ± transduced cells (3LLSA-IFN ) with 3LLSA-CD40L cells enhanced the antitumor immunity efficiently in vivo. These results indicate that the in vivo priming with CD40L- and IFN- gene±transduced lung cancer cells is a promising strategy for inducing antitumor immunity in the treatment of lung cancer. Cancer Gene Therapy (2001) 8, 421±429 Key words: Antitumor immunity; CD40 ligand; interferon- ; cytotoxic T lymphocyte; lung cancer. espite recent increases in therapeutic options, lung surface proteins expressed on B cells, macrophages, and Dcancer remains one of the leading causes of cancer dendritic cells. It binds a CD40 ligand (CD40L/CD154), death.1 This malignancy is often resistant to chemotherapy which is a member of the TNF superfamily expressed on and frequently presents metastasis to distant organs in the activated T cells, basophils, and mast cells.7 Although the early phase of the disease. Even if the primary tumor is CD40±CD40L interaction is primarily implicated in the removed surgically, relapse at the primary or distant sites establishment of humoral immunity,8 recent reports dem- frequently occurs. To overcome these problems, new onstrated that the CD40±CD40L interaction also plays a therapeutic approaches including cancer immunogene critical role in the induction of antitumor immunity,9 therapy have recently been investigated.2 Several reports especially for the priming of tumor-specific T cells.10 We have identified tumor-specific antigens for some tumors, previously reported that alveolar macrophages were and tumor-specific cytotoxic T cells (CTLs) have been activated by stimulation through CD40±CD40L interaction successfully isolated from patients with cancer.3 However, and developed tumoricidal activity against lung cancer most malignant tumors, including lung cancer, evade host cells, and that interferon (IFN)- induced a high immune surveillance.4 It has also been suggested that expression of CD40 molecule on the surface of alveolar antigen-specific T cells may be rendered tolerant early in macrophages.11 the course of tumor progression.5 Recent studies have Considering these findings, we hypothesized that CD40L revealed that the state of activation and/or maturation of gene transduction into lung cancer cells could activate APCs antigen-presenting cells (APCs) may determine whether T surrounding the tumor tissue, and induce antitumor cells are primed or rendered tolerant.6 CD40 is a member immunity against parental lung cancer cells in vivo, although of the tumor necrosis factor (TNF) receptor family of cell lung cancer cells generally reveal low antigenicity, and it seems to be difficult to induce lung cancer±specific cellular immunity, unlike in melanoma cells. In the present study, we Received March 22, 2001. studied the effect of CD40L gene transduction combined Address correspondence and reprint requests to Yoshinori Hasegawa, with IFN- gene transduction into lung cancer cells for the MD, PhD, First Department of Internal Medicine, Nagoya University efficient induction of antitumor immunity against lung School of Medicine, Nagoya 466-8550, Japan. cancer. Cancer Gene Therapy, Vol 8, No 6, 2001: pp 421±429 421 422 NOGUCHI, IMAIZUMI, KAWABE, ET AL: INDUCTION OF ANTITUMOR IMMUNITY BY CD40L MATERIALS AND METHODS designated as pcDNA-IFN . 3LLSA cells were transfected with pcDNA-IFN using the lipofection method described Mice above. Stable transfectants were obtained by selection in 600 C57BL/6 mice (H-2b) were purchased from SLC (Shi- g/mL Zeocin (Invitrogen). These selected clones were zuoka, Japan). CD40-deficient mice were generated by a seeded into a 24-well culture dish (105 per well) and gene-targeting technique previously reported.8 A CD40+ / cultured for 24 hours, then supernatants from each well were À mouse was produced by backcrossing the originally collected to measure IFN- concentration using a murine described CD40À / À mouse to a C57BL/6 mouse. The IFN- enzyme-linked immunosorbent assay kit (R&D heterozygous littermates were intercrossed to generate the Systems, Minneapolis, MN) following the manufacturer's CD40À / À mice used in this study. These mice were instructions (data not shown). The clone that produced the genotyped by a polymerase chain reaction (PCR) of highest amount of IFN- was selected (designated as genomic DNA obtained from a tail biopsy using primers to 3LLSA-IFN ) and used in the following experiments. identify the rearranged CD40 locus, as described previ- 3LLSA-MOCK, which was established by transfecting the ously.8 All these mice were maintained at the Institute for BCMGS-neo plasmid without a cDNA insert, was used in Laboratory Animal Research of the Nagoya University this study as a control. All these transfectants were cultured School of Medicine. in RPMI 1640/10% FCS. Tumor cells Tumor growth A murine lung carcinoma cell line (Lewis lung cancer cells; Parental 3LLSA or transfectants (3LLSA-CD40L, 3LLSA- 3LLSA), which was originally established from the lung of a IFN or 3LLSA-MOCK) (106 cells/mouse) were inocu- C57BL mouse bearing a tumor, was obtained from the lated subcutaneously (s.c.) into the right shoulder of Japanese Cancer Research Resources Bank. A murine C57BL/6 mice or CD40À / À mice (8±10 weeks old). melanoma cell line, B164A5, which also originated from a In some experiments, 106 cells of 3LLSA were inoculated C57BL/6 mouse,12,13 was obtained from Riken Cell Bank with 106 cells of one of the transfectants (3LLSA-CD40L, (Tsukuba, Japan). 3LLSA was maintained in RPMI 1640 3LLSA-IFN or 30 Gy±irradiated 3LLSA-MOCK) simul- supplemented with 1% L -glutamine, 1% penicillin±strepto- taneously into C57BL/6 mice at the same or distant sites. In mycin, and 10% fetal calf serum (RPMI/10% FCS). other experiments, 3LLSA (106 cells) were injected s.c. B164A5 was maintained in high glucose±Dulbecco's simultaneously with a mixture of transfectants [3LLSA- modified Eagle's medium (DMEM) (Gibco BRL, Grand CD40L (106 cells)+3LLSA-IFN (106 cells) or 3LLSA- Island, NY) containing 10% fetal calf serum (DMEM/10% CD40L (106 cells)+30 Gy±irradiated 3LLSA-MOCK FCS). (106 cells)] at the same or distant sites. Similarly, B164A5 (106 cells) were inoculated into C57BL/6 mice 6 cDNA and transfection with 3LLSA transfectant [3LLSA-CD40L (10 cells), 3LLSA-IFN (106 cells), or 30 Gy±irradiated 3LLSA- The expression vector containing murine CD40L cDNA MOCK (106 cells)] or a mixture of the transfectants encoding the entire coding region (BCMGS-neo-CD40L) [3LLSA-CD40L (106 cells)+3LLSA-IFN (106 cells) or was a kind gift from Dr. Hideo Yagita (Department of 3LLSA-CD40L (106 cells)+30 Gy±irradiated 3LLSA- Immunology, Juntendo University School of Medicine, MOCK (106 cells)] as described above. The mice were Tokyo, Japan). BCMGS-neo-CD40L was transfected into monitored for tumor growth weekly, and tumor size was 11 3LLSA cells as previously reported. In brief, transfections determined by measuring two perpendicular diameters with were performed on 100-mm plates using 10 g of plasmid a caliper. DNA per plate by the lipofection method with Lipofectace reagent (Gibco BRL, Gaithersburg, MD), according to the Tumor rechallenges manufacturer's instructions. After 24 hours of exposure, cells were washed three times with medium and cultured in 10 mL Either 3LLSA transfectant (3LLSA-CD40L, 3LLSA- of normal medium. Twenty-four hours after medium IFN , or 30 Gy±irradiated 3LLSA-MOCK) or a mixture exchange, cells were selected in medium containing 800 of the transfectants (3LLSA-CD40L+3LLSA-IFN or g/mL G418 (Gibco, Grand Island, NY). After 2 weeks of 3LLSA-CD40L+30 Gy±irradiated 3LLSA-MOCK) was injected s.c. into the left shoulder of C57BL/6 mice. Four selection, G418-resistant clones were selected randomly 6 from the surviving colonies. Expression of the cell surface weeks later, these mice were rechallenged with 10 cells of CD40L of these selected clones was analyzed by flow the parental 3LLSA cells at the right shoulder. Tumor size cytometry with an FITC-conjugated anti-murine CD40L at the right shoulder was measured weekly as described mAb (PharMingen, San Diego, CA). The clone that showed above. the highest expression (designated as 3LLSA-CD40L) was selected and used in the following experiments. Murine Histological evaluation and immunohistochemistry IFN- cDNA subcloned into pBluescript KS+ (pBluescript 3LLSA-CD40L or 3LLSA-MOCK was inoculated into KS+mIFN- RDB No. 1482) was obtained from Riken C57BL/6 mice. Seven days after inoculation, tumors were Gene Bank (Tsukuba, Japan).

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