T-Cell-Dependent Antitumor Effects Produced by CD40 Ligand

T-cell-dependent antitumor effects produced by CD40 ligand expressed on mouse lung carcinoma cells are linked with the maturation of dendritic cells and secretion of a variety of cytokines Yuji Tada,1,2 Jiyang O-Wang,1 Ling Yu,1 Osamu Shimozato,1 Yan-Qing Wang,1 Yuichi Takiguchi,2 Koichiro Tatsumi,2 Takayuki Kuriyama,2 Keizo Takenaga,3 Shigeru Sakiyama,1 and Masatoshi Tagawa1 1Division of Pathology, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan; 2Department of Respirology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan; and 3Division of Chemotherapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan.

CD40/CD40 ligand (CD40L) interaction plays an essential role in cell-mediated immune responses. We examined whether expression of CD40L in murine lung carcinoma (A11) cells could produce antitumor effects. The proliferation rate in vitro of A11 cells transfected with the murine CD40L gene (A11/CD40L) was not different from that of parent cells; however, half of the immunocompetent mice inoculated with A11/CD40L cells did not form tumors and the growth of A11/CD40L tumors developed in the rest of mice was significantly retarded compared with that of parent tumors. Protective immunity was also induced in the mice that had rejected A11/CD40L cells. In T-cell-defective nude mice, these antitumor effects were not observed. Bone-marrow-derived dendritic cells (DCs), when cultured with A11/CD40L cells, formed clusters with the tumors and showed upregulated CD86 expression. Expression of the interleukin-23 (IL-23) p19, IL-12p35, IL-18, interferon-g (IFN-g) and Mig (monokine induced by IFN-g) genes was induced in the DCs that were cultured with A11/CD40L but not with A11 cells, and P40, the subunit of both IL-12 and IL- 23, was secreted from the cocultured DCs. These data directly showed that the expression of CD40L in tumors facilitated the interaction between DCs and the tumors, enhanced the maturation of DCs, induced secretion of cytokines, and consequently produced T-cell-dependent systemic immunity. Cancer Gene Therapy (2003) 10, 451–456. doi:10.1038/sj.cgt.7700584 Keywords: CD40 ligand; dendritic cells; IL-12; IL-23; lung cancer

D40 is a receptor of CD40 ligand (CD40L) and is highly regulated thereby plays an essential role(s) in Cexpressed on antigen-presenting cells such as B cells initiating immune responses.4 and dendritic cells (DCs). CD40L is transiently expressed Forced expression of CD40L on nonlymphoid could on activated CD4+ T cells and the CD40/CD40L activate systemic immune responses and previous studies interaction triggers a number of events that are pro- demonstrated that transfer of CD40L gene into solid foundly related with cardinal processes of antigen tumors produced antitumor effects against the tumors.5–7 presentation; secretion of IL-12,1 which can polarize Expressed CD40L on B-cell leukemia also induced immune responses to a T helper type 1 (Th1)-dominant antitumor activity and the strategy has been investigated state; production of proinflammatory cytokines;2 and for its clinical feasibility and effectiveness to treat chronic upregulation of costimulatory molecules such as CD86, lymphocytic leukemia.8 The mechanism of CD40L- which enhances cognate interactions between T and mediated antitumor effects was suggested to be because antigen-presenting cells.3 CD40L whose expression is of the activation of DCs and subsequent generation of cytotoxic T cells.5–8 Recent studies also showed that adenovirus-mediated transfer of the CD40L gene into 9,10 Received December 6, 2002. DCs produced antitumor effects. However, the Address correspondence and reprint requests to: Dr Masatoshi direct interaction between CD40L-expressed tumors Tagawa, Division of Pathology, Chiba Cancer Center Research and DCs has not been precisely analyzed. Inoculation of Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan. E-mail: tumors alone may produce inflammatory cyotokines [email protected] that activate DCs and transduction with empty Antitumor effects by expressed CD40L Y Tada et al 452 adenovirus alone can activate DCs and enhance the Production of IFN-g antigen acquisition.11 Spleen cells from naive or A11/CD40L-rejected mice In this study, we examined the antitumor effects of (4 Â 107) were cultured for 11 days with A11 cells murine lung carcinoma A11 cells transduced with the irradiated with 100 Gy (4 Â 106). Living cells were then CD40L gene and investigated the interaction between the collected with lympholyte-M (Cedarlane, Hornby, Ont., CD40L-expressed tumors and bone marrow-derived DCs. Canada) and they (2 Â 106/well) were cultured with 100- We found that purified DCs were directly activated by the Gy irradiated A11 cells (2 Â 105/well) in 24-well plates for CD40L-expressed tumors and secreted a variety of 24 hours. The amounts of IFN-g in the culture super- cytokines including a novel cytokine interleukin-23 (IL- natants were measured with an enzyme-linked immuno- 23), which enhances proliferation of memory T cells and sorbent assay (BioSource, Camarillo, CA). production of IFN-g from activated T cells.12

Preparation of DCs and culture with tumors Materials and methods Bone marrow cells from tibias and femurs were depleted of erythrocytes with hypotonic buffer containing 0.15 M Cells and mice NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA (pH 7.4). High-metastatic A11 cells were established from Lewis Macrophages, T and B cells were removed with biotiny- lung carcinoma.13 They were cultured in Dulbecco’s lated antibodies for CD4, CD8, B220, TER119, and modified Eagle’s medium supplemented with 10% heat- CD11b, and streptavidin-conjugated magnetic beads. inactivated fetal calf serum. C57BL/6 and BALB/c nu/nu These cells were further incubated at 371C for 2 hours to nude mice (6- to 8-week-old female) were purchased from remove adherent cells and then cultured in RPMI1640 Japan SLC (Hamamatsu, Japan). medium supplemented with recombinant mouse GM-CSF and IL-4 (20 ng/ml each, PeproTech, London, UK), and Establishment of CD40L-expressed cells 10% fetal calf serum for 10 days. Nonadherent cells were harvested and used as DCs. PKH67-labeled tumor cells A11 cells were transfected with the full-length mouse (2 Â 105/well) were cultured with DCs (6 Â 105/well) in 24- CD40L cDNA and then G418 (Life Technologies, well plates for 24 hours and the number of cluster was Gaithersburg, MD) resistant cells were selected. They examined with a fluorescence microscope. were examined for their expression of CD40L with flow cytometry. G418-resistant A11 cells transfected with vector DNA pMKITneo were used as a control. Detection of secreted P40 subunit and polymerase chain reaction (PCR) Flow cytometry DCs (6 Â 105/well) were cultured with tumor cells 5 Tumor cells were incubated with fluorescein-isothiocya- (2 Â 10 /well) in 24-well plates for 24 hours and the nate (FITC)-conjugated anti-CD40L antibody (PharMin- concentrations of P40 in the culture supernatants were gen, San Diego, CA) at room temperature for 20 minutes. determined with an enzyme-linked immunosorbent assay Isotype-matched control antibody labeled with FITC was (BioSource, Camarillo, CA). First strand cDNA was used for the background staining. Bone marrow-derived synthesized with RNA that was extracted from cultured DCs were cultured with tumor cells that were labeled with cells. Amplification of an equal amount of respective PKH67 (Sigma, St Louis, MO) for 12 hours and then cDNA was performed for 30 cycles with the following stained with phycoerythrin-conjugated anti-CD86 anti- primers and conditions: for p19 gene expression, forward (50-CACAGAGCCAGCCAGATCTGAGAAGC-30) and body (Caltag, Burlingame, CA). As a control, DCs were 0 also incubated with 10 mg/ml lipopolysaccharide (LPS) for reverse (5 -CCATGGGAACCTGGGCATCCTTAAGC- 0 1 12 hours. The stained cells were analyzed with FACScan 3 ) primers, and 15 seconds at 94 C for denaturation/ 1 1 (Becton Dickinson, Mountain View, CA) with CellQuest 30 seconds at 60 C for primer annealing/1 minute at 72 C software (Becton Dickinson). for primer extension: for p35 gene expression, forward (50-ACCTGCTGAAGACCACAGATG-30) and reverse (50-TTTCACTCTGTAAGGGTCTGC-30) primers, and Animal experiments 15 seconds at 941C/30 seconds at 531C/1 minute at Parent or transfected cells (2 Â 105) were subcutaneously 721C; for IL-18 gene expression, forward (50-ATTACTC- inoculated into syngeneic or nude mice. Tumor volumes GAGGAACAATGGCTGCCATGTC-30) and reverse were calculated according to the formula, tumor (50-ATTACTCGAGAAGGCGCATGTGTGCTAATC- volume ¼ 0.5 Â a Â b2, where a and b are the larger and 30) primers, and 30 seconds at 941C/30 seconds at 601C/ the smaller diameter, respectively. For spontaneous lung 1 minute at 721C; for IFN-g gene expression forward metastasis, parent and transfected cells (2 Â 105) were (50-TGCGGCCTAGCTCTGAGACAATG-30)andreverse subcutaneously injected into the abdominal flank of mice. (50-TGAATGCTTGGCGCTGGACCTG-30) primers, The lungs were fixed with Bouin’s solution and the and 30 seconds at 941C/30 seconds at 601C/1 minute at number of metastatic nodules was counted on day 28.13 721C; for monokine induced by IFN-g (Mig) gene Statistical analysis was performed by one-way analysis of expression, forward (50-GACATTCTCGGACTT- variance (ANOVA). CACTC-30) and reverse (50-GATTCAGGGTGCTTGT-

Cancer Gene Therapy Antitumor effects by expressed CD40L Y Tada et al 453 TGGT-30) primers, and 5 seconds at 951C/10 seconds at Table 1 Production IFN-g from spleen cells that were stimulated 551C/1 minute at 721C; for glyceraldehyde 3-phosphate with irradiated A11 cells dehydrogenase (GAPDH) 0 gene expression, forward (5 - 7 ACCACAGTCCATGCCATCAC-30) and reverse (50- Spleen cells from IFN-g SE (pg/ml) TCCACCACCCTGTTGCTGTA-30) primers, and 15 sec- 7 a onds at 941C/15 seconds at 601C/40 seconds at 721C. For Naive 100.27 19.53 A11/CD40L-rejected 1749.747197.66a quantitative analysis of p19 and p35 mRNA, diluted b None 9.2675.96 cDNA was amplified for 35 cycles under the condition described above. a Po.01. bSecretion from irradiated A11 cells.

Results Table 2 Number of lung metastatic foci in the mice that were Expression of CD40L on A11 cells subcutaneously inoculated with parent or CD40L-expressed A11 cells Lewis lung carcinoma-derived A11 cells were transfected with the mouse CD40L gene and a clone expressing a Number of lung foci (average7SE) large amount of CD40L on cell surface (A11/CD40L) was Cells injected (2 Â 105) on day 28 selected (Fig 1). Expression of the class I antigens of the major histocompatibility complexes on A11 cells was A11/parent 5, 6, 13, 18, 19, 64 (20.878.96)a undetectable with flow cytometry and was not upregu- A11/CD40L 0, 0, 0, 0, 0, 1 (0.16770.167)a lated after the forced expression of CD40L. The a proliferation rate in vitro of A11/CD40L cells was not Po.05. different from that of parent cells (data not shown).

Antitumor effects produced by A11/CD40L cells We subcutaneously inoculated A11, A11/CD40L, or vector DNA-transfected A11 cells into immunocompetent syngeneic mice (Fig 2a). All of the mice inoculated with parent and vector DNA-transfected A11 cells developed tumors and the growth in vivo was not different between the two groups. In contrast, seven out of 14 mice injected with A11/CD40L cells did not form tumors and the growth of A11/CD40L tumors developed in the rest of mice was significantly retarded compared with that of parent or vector DNA-transfected A11 tumors (Po.01, days 18, 21 and 24). All of the mice that had rejected A11/ CD40L cells were resistant to parent but not irrelevant tumors subsequently challenged (data not shown). When spleen cells of the mice that had rejected A11/CD40L cells were cultured with irradiated A11 cells, significant amounts of IFN-g were produced compared with naive mice (Table 1). The CD40L-mediated antitumor effects were also examined with the colony number of spontaneous lung metastasis (Table 2). Subcutaneous inocula-

Figure 2 In vivo tumor growth of parent and transfected cells. (a) Syngeneic C57/BL6 mice were subcutaneously inoculated with A11 (n ¼ 7), A11/CD40L (n ¼ 14), or vector DNA-transfected A11 (n ¼ 7). The average volume of A11/CD40L tumors was calculated based on the developed tumors (n ¼ 7). (b) Nude mice were inoculated with either A11 (n ¼ 6) or A11/CD40L cells (n ¼ 6). Standard error bars are also shown.

tion of A11 cells produced metastatic foci in lung, but that Figure 1 Expression of CD40L on parent and A11/CD40L cells. of A11/CD40L cells formed few foci. When T-cell Cells were stained with anti-CD40L antibody or isotype-matched defective nude mice were inoculated with A11/CD40L control antibody. cells, they developed tumors and the growth was not

Cancer Gene Therapy Antitumor effects by expressed CD40L Y Tada et al 454 different from that of parent tumors (P4.05, days 21 and with A11/CD40L cells than on those with parent cells (Fig 27) (Fig 2b). 3b). The upregulated level of CD86 expression on DCs cultured with A11/CD40L cells was almost equal to that on LPS-stimulated DCs (Fig 3b). These data suggested Interaction between DCs and A11/CD40L cells that DCs maturated upon the coculture with A11/CD40L + + We examined the effects of CD40L-expressed tumors on cells. There was no CD86 PKH67 cell population, DCs. Bone marrow-derived DCs used in the present study implying that DCs did not phagocytose tumor cells. showed an immature phenotype such as CD86low. DCs were then cultured with either parent or A11/CD40L cells. Production of cytokines from DCs and tumors We found that DCs formed more clusters with A11/ We examined cytokine production induced by the DCs– CD40L cells than with parent cells (Po.01, Fig 3a) and tumor interaction. Since activated DCs secrete IL-12,1 we that expression level of CD86 was higher on DCs cultured measured the amounts of the P40 subunit in the supernatants of cultured cells (Fig 4a). DCs cultured without tumor cells or those with parent tumors did not secrete P40. When DCs were cultured with A11/CD40L cells, DCs came to secrete a significant amount of P40. We also examined DCs that were cultured with A11 cells expressing Fas ligand (A11/FasL). Since bone marrow-derived DCs were positive for Fas and A11/FasL cells were rejected in the inoculated mice,14 DCs cultured with A11/ FasL cells were comparable to those with A11/CD40L

Figure 3 (a) Cluster formation between DCs and A11 or A11/CD40L Figure 4 (a) Secretion of P40 molecules. DCs were cultured with or cells. The average number of clusters per 30 visual fields that were without tumor cells. (b) Expression of cytokines and chemokine randomly selected is shown. Standard error bars are shown. (b) genes analyzed with PCR. RNA were extracted from DCs and/or Expression of CD86 on DCs. DCs were incubated with PKH67- tumors cells as indicated. GAPDH was used as a control. (c) labeled A11 or A11/CD40L cells, or 10 mg/ml LPS for 12 hours and Quantitative analysis of the IL-23p19 and the IL-12p35 genes then they were analyzed with flow cytometry. Percentages of each expression in DCs that were cultured with A11/CD40L cells. First- fraction are also shown. strand cDNA was diluted up to 16 times ( Â 16).

Cancer Gene Therapy Antitumor effects by expressed CD40L Y Tada et al 455 cells. In contrast to DCs cultured with A11/CD40L cells, should be derived from DCs. Since A11 cells were positive P40 was not detected in the culture supernatants of DCs for both CD40 and Fas13 and A11/CD40L and A11/FasL with A11/FasL cells. cells did not produce the cytokines, stimulation of A11/ A novel cytokine IL-23 has been recently reported: it CD40L and A11/FasL cells by the cocultured DCs could has heterodimeric structure composed of P19 subunit, not induce cytokine production. The undetectable level of which is a new molecule related to P35 of IL-12, and P40 cytokine expression in DCs stimulated with A11/FasL subunit that is shared with IL-12.12 Since DCs can secrete cells was not because of the cell death of DCs, because IL-23 as a P19/P40 heterodimer,12 we examined the DCs were resistant to apoptosis upon the Fas/FasL expression of the p19 and the p35 genes in the cultured ligation despite their Fas expression.15 DCs were in fact cells. Both transcripts were detected from DCs cultured not apoptotic, when they were cultured with A11/FasL with A11/CD40L cells but not from others: DCs alone, cells.14 These data collectively suggested that interaction tumor cells alone, or DCs cultured with parent or A11/ of immature DCs and FasL-expressed tumors could favor FasL cells (Fig 4b). In order to quantitate the amounts of antigen presentation possibly through the cluster forma- both transcripts, we performed PCR using serially diluted tion but did not induce maturation of DCs, whereas cDNA that were prepared from DCs cultured with A11/ interaction of DCs and CD40L-expressed tumors did CD40L cells (Fig 4c). The signal intensity of the p19 facilitate DCs maturation. We presume that maturation product was greater than that of the p35 product in any of of DCs in the experiments of A11/FasL cells-mediated the diluted samples. We also examined the expression of antitumor activity was induced by proinflammatory other types of cytokine and chemokine that were related cytokines, which came to be produced by tumor inocula- with differentiation and function of Th1 cells. Transcripts tion in vivo. of IL-18, IFN-g, and Mig were detected from DCs Detection of P40 molecules in the culture supernatants cultured with A11/CD40L cells but not from the others and expression of both the IL-23p19 and the IL-12p35 (Fig 4b). Neither IL-15 nor IL-21 transcript was detected transcripts suggested that both P35/P40 heterodimer (IL- in any of the cultured cell populations (data not shown). 12) and P19/P40 heterodimer (IL-23) were secreted from the DCs stimulated with A11/CD40L cells. The present data also implied that the amount of IL-23 was greater than that of IL-12; therefore, IL-23 as well as IL-12 could Discussion play a crucial role in DCs–T-cell interactions. DCs can potentially secrete IL-12, IL-18, IFN-g and Mig even in In this study, we showed that forced expression of the the resting state,16–19 however, CD40L-stimulated DCs CD40L gene in tumor cells produced T-cell-dependent have not been shown to increase the production of IFN-g antitumor effects and induced protective immunity. and Mig. We speculate that DCs initially secrete IL-12 Production of a significant amount of IFN-g from All- and IL-18, then IFN-g comes to be expressed by DCs- stimulated spleen cells of the mice that had rejected A11/ derived IL-12 and/or IL-1817 and Mig is induced by IFN- CD40L cells suggested the induction of cytotoxic T cells g.20 These cytokines can shift host immune responses to besides natural killer cells. Previous studies also showed Th1-dominant state and Mig can attract activated T that antitumor effects were achieved by the expression of cells;19 therefore, these soluble factors coordinately CD40L on tumors5–8 and reported that the CD40L enhanced immune responses. IL-15, which augments molecules could stimulate DCs through the CD40/ immunostimuatory ability of DCs,21 was not induced in CD40L interaction. However, these studies did not the present study, although previous studies showed that directly demonstrate that the activation and the matura- IL-15 was secreted from CD40L-stimulated DCs.18 IL-21, tion of DCs were caused by the expressed CD40L on which can promote the activation of T and natural killer tumors. The present study revealed that bone marrow- cells,22,23 was not induced. Both IL-15 and IL-21 may not derived DCs tended to form clusters with A11/CD40L be directly involved in the DCs–tumor cells interactions. cells and that upregulated CD86 expression and cytokine Expression of CD40L did not induce any biological production were induced by the interaction between DCs changes in CD40-positive A11 cells such as cell adhesion, and CD40L-expressed tumors. proliferation, and phosphorylation of intracellular pro- Since DCs were positive for Fas as well as CD40, we tein. A recent study showed that CD40-expressed tumor examined DCs cultured with A11/FasL cells. Our cells could increase their tumor growth by impairing previous study showed that expression of FasL on All immune responses through the downregulation of CD40L cells facilitated cluster formation between DCs and the on activated T cells.24 In addition, A11 cells were less tumors, and induced T-cell-mediated antitumor effects;14 immunogenic because the class I expression on A11 cells however, incubation with A11/FasL cells did not upre- was undetectable. However, forced expression of CD40L gulate CD86 expression on DCs.14 The present study also could induce systemic immunity against A11 by stimulat- showed that the interaction between DCs and A11/FasL ing DCs. In conclusion, we examined the direct interac- cells did not induce the expression of cytokine genes tion between CD40L-expressed tumors and DCs, and tested. The cluster formation thereby was not directly observed the cluster formation between DCs and tumors, correlated with cytokine production or maturation of maturation of DCs and secretion cytokines from DCs. DCs. Although expression of these cytokines was detected The interaction of CD40/CD40L facilitates antigen from the mixture of DCs and the tumors, the transcripts acquisition of DCs and these DCs are further stimulated

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