Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8+ cytotoxic T-cell activation and antitumor immunity

Abstract

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in their activation and is essential for induction of antigen-specific T-cell responses. In the present study, we investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express CD40L and pulsed with Mut1 tumor peptide. Our data show that transfection of DCs with recombinant adenovirus AdV-CD40L resulted in activation of DCs with up-regulated expression of proinflammatory cytokines (IL-1β and IL-12), chemokines (RANTES, IP-10, and MIP-1α), and immunologically important cell surface molecules (CD54, CD80, and CD86). Our data also demonstrate that DCs transfected with AdV-CD40L (DCCD40L) are able to stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8+ cytotoxic T-cell responses in vitro. Vaccination of mice with Mut1 peptide-pulsed control virus–transfected DC (DCpLpA) could only protect mice from challenge of a low dose (0.5×105 cells per mouse, 8/8 mice), but not a high dose (3×105 cells per mouse, 0/8 mice) of 3LL tumor cells. However, vaccination of Mut1 peptide-pulsed AdV-CD40L–transfected DCCD40L induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of 3LL tumor cells. Thus, DCs engineered to express CD40L by adenovirus-mediated CD40 ligand gene transfer may offer a new strategy in production of DC cancer vaccines.

Main

Dendritic cells (DCs) are the most potent stimulators of primary immune responses. They migrate as precursors from the bone marrow (BM) into various organs, where they usually reside in an inactive state. Within the internal organs, they can efficiently endocytose and process antigens.1 Upon activation, they undergo a differentiation process that results in decreased antigen processing capacity and enhanced expression of major histocompatibility complex (MHC) and costimulatory molecules, following which they migrate to lymphoid organs to interact with, or activate, naive T cells.2 Because of the critical roles DCs have in the generation of primary immune responses, an important avenue of investigation is their potential for modulating immune functions such as the induction of tumor immunity. Recently, it has been shown that DCs pulsed with tumor-derived MHC class I–restricted peptides or tumor lysates are able to induce significant cytotoxic T-lymphocyte (CTL)–dependent antitumor immune responses in vitro as well as in vivo.3,4

CD40 ligand (CD40L) is a 33-kDa type II membrane protein, a member of the tumor necrosis factor (TNF) gene family that is preferentially expressed on activated CD4+ T cells.5,6 The receptor for CD40L is CD40, a member of the TNF receptor family.7 CD40 is expressed on antigen-presenting cells (APCs) including DCs. The CD40–CD40L interaction was initially identified in relation to T- and B-cell interactions relevant to humoral immune responses.8 It has recently been reported that stimuli from CD4+ T helper (Th) cells, via the CD40–CD40L interaction, are also essential in activation of DCs with up-regulation of costimulatory CD80 molecule and intercellular adhesion molecule (ICAM-1), which can then autonomously trigger CD8+ CTL (Tc) responses.9,10,11 In addition, the CD40–CD40L interaction also induces the production of cytokines that favor the development of Th1 type immune response.10,11,12

In this study, we used adenovirus-mediated gene transfer to engineer DCs to express CD40L, pulsed the engineered DCs with an important MHC class I–restricted tumor peptide (Mut1), and then investigated the antitumor immunity derived by vaccination of mice with these cells.

Materials and methods

Cell lines, antibodies, chemokines, peptides, and animals

The 3LL cell line is a poorly immunogenic Lewis lung carcinoma line; EL4 is a T-cell lymphoma line of C57BL/6 mouse (H-2Kb) origin. Both were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS). Mouse monoclonal antibodies against MHC class I and II molecules (H-2Kb and Iab) as well as monoclonal rat antimouse CD11b, CD11c, CD40, CD80, CD86, and ICAM-I (CD54) antibodies were obtained from Pharmingen (San Diego, CA). Fluorescein isothiocyanate (FITC)–conjugated goat antimouse and antirat IgG antibodies were purchased from Bio/Can Scientific (Missassauga, Ontario, Canada). Recombinant mouse interleukin 4 (IL-4) and granulocyte–macrophage colony-stimulating factor (GM-CSF) were purchased from Endogen (Woburn, MA) or R&D Systems (Minneapolis, MN). The MHC class I–restricted Mut1 peptide (FEQNTAQP), comprising amino acids 52–59 of the mutated connexin 37 protein expressed by 3LL tumor cells,13 was synthesized by Multiple Peptide Systems (San Diego, CA). Female C57BL/6 (H-2Kb) and BALB/c (H-2Kd) mice were obtained from Charles River Laboratories (St. Laurent, Quebec, Canada). All mice were housed in the animal facility of the Cancer Research Unit, Saskatchewan Cancer Agency. Following receipt of approval by the Animal Care Ethics Review Committee, experiments were conducted according to the guidelines of the Canadian Council for Animal Care.

Recombinant adenoviral vectors

A 1-kb cDNA fragment coding for the full open reading frame of mouse CD40L gene was cloned by reverse transcription polymerase chain reaction (RT-PCR) from a cDNA library of Con A–stimulated mouse spleen T cells using the TaqI polymerase. Two primers specific for the mouse CD40L gene were used, namely, the sense primer (5′ ctcca ttggc tctag attcc 3′) and the antisense primer (5′ cctca tgagc cacat aatac 3′). The cloned cDNA fragment was ligated into the pCR2.1 vector (Invitrogen, Carlsbed, CA) to form pCR2.1-CD40L. The CD40L sequence was verified by the dideoxy nucleotide sequencing method. The cDNA fragment of CD40L (XbaI/HindIII) from the pCR2.1-CD40L vector was further ligated into the pLpA to form the adenoviral vector pLpA-CD40L. The construction of recombinant adenovirus AdV-CD40L from pLpA-CD40L and pJM17 vectors was performed as previously described.14 The adenoviruses AdV-LacZ expressing the Escherichia coli β-galactosidase and AdV-pLpA (i.e., with no gene insert) used as marker and control adenoviral vector, respectively, were constructed in our laboratory as previously reported.14 These E1-deleted replication-deficient recombinant adenoviruses were amplified in 293 cell line (adenoviral E1 transformed human embryonic kidney cells), under the control of cytomegalovirus (CMV) early/immediate promoter/enhancer purified by cesium chloride ultracentrifugation and stored at −80°C.

Generation of DCs

A slightly modified procedure from that described previously15 was used to generate DCs from BM cultures. Briefly, BM cells prepared from femurae and tibiae of normal C57BL/6 mice were depleted of red blood cells with 0.84% ammonium chloride and plated in DC culture medium [DMEM plus 10% FCS, GM-CSF (10 ng/mL) and IL-4 (10 ng/mL)]. On day 3, nonadherent granulocytes and T and B cells were gently removed and fresh media added. Two days later, the loosely adherent proliferating DC aggregates were dislodged and replated. On day 7, the immature, nonadherent cells were harvested and used for in vitro AdV-CD40L transfection. The DCs generated in this manner displayed: (a) typical morphologic features of DCs (i.e., numerous dendritic processes) and (b) significant expression of MHC class I (H-2Kb) and II (Iab) antigens, costimulatory molecules (CD80 and CD86), and adhesion molecules (CD11b, CD11c, CD40, and CD54) (data not shown).

Adenoviral transfection

In order to test the susceptibility of DCs to adenoviral infection, serial dilutions of AdV-LacZ stock (2×1010 pfu/mL) were added to DCs seeded in triplicate in 96-well plates (1×105 cells/well) to form different “multiplicities of infection” (MOI). The cells were incubated with the adenovirus in 293 serum-free medium (Gibco) for 2 hours at 37°C; the medium was then replaced with DMEM/10% FCS and the cells incubated for an additional 24 hours at 37°C. To assess β-galactosidase expression,13 the cells were fixed in formaldehyde/glutaraldehyde, then stained and counterstained with X-gal and nuclear fast red, respectively. The proportions of positive (i.e., blue staining) cells were determined from triplicate wells and taken as the percentage of transduction. Control DCs transfected with AdV-pLpA (termed DCpLpA) did not exhibit any intrinsic β-galactosidase activity or false-positive staining. We observed a dose-dependent response to the infecting dose of adenovirus, with maximal staining (82%) at an MOI of ≥100. Therefore, an MOI of 100 was selected for transfection of DCs with AdV-CD40L in this study. Transfection of DCs with AdV-CD40L at 100 MOI was performed as follows: after viral adsorption for 1 hour at 37°C in 24-well culture plates, the DC culture medium was replaced with DMEM/20% FCS and the cells (3×106 cells per well) incubated for another 24 hours at 37°C. The DCs transfected with AdV-CD40L (DCCD40L) were then harvested for examination of CD40L expression in RT-PCR, phenotypic analysis by flow cytometry, and Mut1 peptide pulsing in vitro, respectively.

RT-PCR analysis of CD40L expression

Total RNA was obtained from DCs. First-strand cDNA synthesis of RT-PCR was performed with 5 μg of RNA using a commercial kit (Stratagene, La Jolla, CA), in accordance with the manufacturer's instructions. The PCR primers specific for the mouse CD40L were as described above. PCR primers specific for glyceraldehyde phosphate dehydrogenase (GAPDH) include the sense (5′ caggt tgtct cctgc gactt 3′) and antisense primers (5′ cttgc tcagt gtcct tgctg 3′). The PCR conditions comprised one cycle at 94°C (5 minutes), 54°C (1 minute), and 72°C (1 minute) as well as 40 cycles at 94°C (1 minute), 54°C (1 minute), and 72°C (1 minute). All PCR reaction products were resolved on 1% agarose gels with ethidium bromide staining.

Immunophenotypic analysis

For phenotypic analyses, DCs were stained for 30 minutes on ice with antibodies (5 μg/mL each) specific for H-2Kb, Iab, CD11b, CD11c, CD40, CD54, CD80, and CD86, respectively. The cells were then washed twice with phosphate-buffered saline (PBS) prior to incubation for an additional 30 minutes on ice with FITC-conjugated goat antimouse and antirat IgG antibodies (1:60), respectively. After three more washes with PBS, the cells were then analyzed by flow cytometry. Isotype-matched monoclonal antibodies were used as controls.

RNase protection assay

DCs were subjected to RNase protection assays using a commercial kit (RiboQuant Multi-Probe kit; Pharmingen) in accordance with manufacturer's instructions. RNA was extracted from the cells using a commercial kit (Pharmingen). 32P-UTP labeled probes (Amersham Canada, Oakville, Ontario, Canada) were generated by in vitro transcription of cytokine/chemokine-related multiprobe template sets (Pharmingen®) using T7 RNA polymerase. The labeled probes were first purified by phenol–chloroform extraction and ethanol precipitation, their counts adjusted to 3×105 cpm/μL, then hybridized to the RNA samples (5 μg each). The reaction samples were subsequently digested with RNase, followed by Proteinase K treatment and phenol–chloroform extraction. Following ethanol precipitation with 4 M ammonium acetate, the protected samples were resuspended in ×1 loading buffer and realized on 5.7% acrylamide–bisacrylamide urea gels. The gels were absorbed to filter paper, dried under vacuum, and exposed to Kodak X-AR film with intensifying screens at −80°C. The relative expressions of cytokine and chemokine encoding mRNA were measured by scanning densitometry (Molecular Dynamics, Sunnyvale, CA) on subexposed autoradiograms, and further normalized using the housekeeping gene value (GAPDH).

Allogeneic mixed lymphocyte reactions (MLRs)

T cells were purified from BALB/c mouse spleen lymphocytes as nylon wool nonadherent cells.16 Primary MLRs were performed as previously described.17 Briefly, graded doses of irradiated DCs (3000 cGy) were cocultured in 96-well plates with constant numbers (2×105) of allogeneic T cells from BALB/c mice. After 2 days, T-cell proliferation was measured using an overnight 3H-thymidine (1 mCi/mL; Amersham Canada) uptake assay (1 μCi/well). The levels of 3H-thymidine incorporation into the cellular DNA were determined by liquid scintillation counting.

Peptide pulsing of transfected DCs

For peptide pulsing, 1–2×106 DCs were resuspended in 1 mL of DMEM containing 20 μM Mut1 peptide. After 3 hours of incubation at 37°C with gentle shaking every 30 minutes, the peptide-pulsed DCs were washed twice with PBS and resuspended in PBS for vaccination of mice.

CTL assay

Red blood cell–depleted splenic lymphocytes from mice vaccinated with Mut1-pulsed DCs, DCCD40L, and DCpLpA cells were generated using standard methods. These cells were cocultured in each well of 24-well plates with irradiated 3LL cells (20,000 cGy) for 4 days, using 5×106 lymphocytes and 2×105 3LL cells per 2 mL of DMEM/10% FCS. The activated T cells were then harvested and used as effector cells in a chromium release assay against 3LL and EL4 target cells, which were labeled with 51Cr chromate, as previously described.16 These target cells (104/well) were incubated for 8 hours in triplicate cultures with effector cells at various effector/target ratios. Percent specific lysis was calculated using the formula:

The spontaneous count per minute (cpm) release in the absence of effector cells was less than 10% of specific lysis; maximal cpm release was effected by adding 1% Triton X-100 to the cells.

DC peptide vaccination of mice

For evaluation of tumor prevention, mice were vaccinated subcutaneously (s.c.) with 0.5×106 Mut1-pulsed DCCD40L and DCpLpA cells, respectively. On day 11, the mice (8 and 10 mice per group) were challenged by s.c. injection of 0.5×105 or 3×105 3LL tumor cells, respectively. Tumor growth was monitored daily; for humanitarian reasons, mice bearing tumors ≥1.5 cm in diameter were necessarily sacrificed.

Results

CD40L expression of DCCD40L

To examine CD40L expression, RNA extracted from DCCD40L and DCpLpA was subjected to RT-PCR analysis. As shown in Figure 1, significant amounts of CD40 ligand expression were found in the AdV-CD40L–transfected DCCD40L, but not in the control adenovirus–transfected DCpLpA.

Figure 1
figure1

Expression of CD40L. RNA was obtained from DCCD40L and DCpLpA. The first strand cDNA was synthesized from RNA using reverse transcriptase; PCRs were conducted using primers sets for CD40L and the control ‘housekeeping’ gene, GAPDH.

Transfection of DCs with AdV-CD40L induces DC activation

To examine the phenotypic changes induced within DCs by transfection with AdV-CD40L, DCCD40L as well as the control DCpLpA were subjected to phenotypic analysis by flow cytometry and RNase protection assay. We found an evident up-regulated expression of CD54, CD80, and CD86 in DCCD40L, compared to DCpLpA (Fig 2). In addition, DCCD40L also displayed increased and slightly increased expressions of chemokines (RANTES, MIP-1α, and IP-10) and cytokines (IL-1β and IL-12), respectively, compared to control DCpLpA (Fig 3). On the contrary, DCpLpA cells derived from the control adenovirus (AdV-pLpA) infection could induce an evident increase in expression of some cytokine (IL-1β) and chemokines (RANTES, IP-10, and MIP-1α) (Fig 3). However, phenotypic features of these DCpLpA, such as cell surface expression of CD54, CD80, and CD86 molecules (Fig 2), and H-2Kb, Iab, CD11b, CD11c, and CD40 molecules (data not shown) were not significantly altered, compared to uninfected DCs.

Figure 2
figure2

Phenotypic changes of DCCD40L by flow cytometric analysis. DCCD40L and DCpLpA (solid lines) were harvested and analyzed for measurement of surface expression of CD54, CD80, and CD86 molecules using rat antimouse CD54, CD80, and CD86 antibodies, respectively, by flow cytometry. Isotype-matched monoclonal antibodies (dotted lines) were used as controls. One of two representative experiments is shown.

Figure 3
figure3

Enhanced expression of cytokines and chemokines. A: RNase protection assay of DCCD40L and DCpLpA. B: Relative expression of cytokine and chemokine mRNA of DCCD40L (open bars) and DCpLpA (black bars).

DCCD40L significantly enhances tumor-specific T-cell responses in vitro

DCs are potent stimulators of primary MLRs and are able to induce proliferation of allogeneic CD8+ T cells in vitro.18 Therefore, in our assessment of the ability of DCCD40L to stimulate antitumor responses, we first examined the abilities of these cells to stimulate primary MLRs against allogeneic CD8+ T cells. We found that DCCD40L did indeed induce stronger allogeneic T-cell proliferative responses, compared with DCpLpA (Fig 4).

Figure 4
figure4

Mixed lymphocyte reaction. Irradiated DCCD40L (open circles) and DCpLpA (black circles) starting with (1×104 cells/well) and its reciprocal dilutions were added to 1×105 allogeneic BALB/c T cells. Cells were cocultured for 2 days. [3H]thymidine uptake after overnight incubation is expressed as the mean of three determinations. The SDs of each point were less than 5% of the mean value. Background proliferation of DCs or T cells alone was always below 5000 cpm. One of two representative experiments is shown.

Next, we addressed specific antitumor effector functions induced by vaccination of mice with DCCD40L. We examined CTL activities against 51Cr-labeled 3LL target cells of splenocytes from the vaccinated animals (Fig 5). T cells from mice vaccinated with Mut1-pulsed DCCD40L cells displayed substantially enhanced CTL activity (48% specific killing; E:T ratio=50) relative to analogous cells from animals vaccinated with Mut1-pulsed DCpLpA (34% specific killing; E:T ratio=50). This CTL activity was immunologically specific, in that none of these cells showed cytotoxic activities against the irrelevant EL4 tumor cells; also, similar cells from naive mice had no activity against 3LL cells (data not shown).

Figure 5
figure5

Cytotoxicity assay. Spleen lymphocytes were harvested from mice vaccinated with DCCD40L and DCpLpA pulsed with Mut1 peptide, respectively. T cells were subsequently generated by cocultivation of these spleen lymphocytes with irradiated 3LL cells (20,000 cGy) for 5 days. T cells derived from mice vaccinated with DCCD40L (open circles) and DCpLpA (black circles) were used as effector cells in a chromium release assay, in which 51Cr-labeled 3LL tumor cells were used as target cells. To confirm that T-cell cytotoxicity was 3LL tumor–specific, we also included EL4 cells (open triangles) as a target control and activated T cells from DCCD40L-immunized mice as effector cells. Each point represents the mean of triplicates. One of two representative experiments is shown.

DCCD40L strongly induces antitumor immunity in vivo

To examine whether DCCD40L cells were also capable of inducing enhanced antitumor immunity in vivo, we vaccinated mice with three kinds of DCs (DCCD40L, DCpLpA, and uninfected DCs) pulsed with Mut1 peptide, and then challenged the animals with 3LL tumor cells. As shown in Figure 6, vaccination with Mut1 peptide-pulsed uninfected DCs effectively protected mice from tumor growth after challenge with 0.5×105 (low dose, 8/8 protected), but not with 3×105 (high dose, 0/8 protected) 3LL cells. Adenoviral infection of DCs by itself did not significantly enhance the efficiency of DC vaccination because all mice vaccinated with Mut1 peptide-pulsed DCpLpA died within 5 weeks of inoculation with 3×105 tumor cells. However, vaccination with peptide-pulsed DCCD40L was significantly more effective, completely protecting the mice (8/8) from challenge with either 0.5×105 (low dose) or 3×105 (high dose) 3LL cells.

Figure 6
figure6

Efficient protective tumor immunity derived from vaccination of mice with Mut1-pulsed DCCD40L. A: C57BL/6 mice (eight mice per group) were vaccinated with 0.5×106 DCCD40L, DCpLpA, and uninfected DCs pulsed with Mut1 peptide, respectively. As a control, mice were injected with PBS only. Ten days later, mice were challenged s.c. with 0.5×105 (A) or 3×105 (B) 3LL tumor cells. The survival times of each group of mice were monitored. This animal experiment was repeated once, with similar results.

Discussion

Initiation of immune responses relies on APCs activated by CD40–CD40L interactions. In order to stimulate APCs for induction of tumor-specific cellular immunity, tumor cells were genetically engineered to express CD40L. Grossmann et al19 and Couderc et al20 showed that murine tumor cell lines transduced with retroviral vectors containing murine CD40L cDNA were capable of inducing systemic tumor-specific immune responses. Recently, Nakajima et al21 further demonstrated that CD40L, expressed on engineered tumor cells, was capable of eliciting local and systemic antitumor responses mediated through IL-12 and B7 molecules. These data indicate a potent antitumor effect of CD40L that is mediated via host APC functions.

DCs are one of the most potent APCs for induction of antitumor immune responses currently known and, as such, have been recognized as potentially important tools for cancer vaccine strategies.2,3,4 However, it has also been reported that tumor cells can interfere with host DC differentiation, maturation, and function in vivo.22,23 As a circumvention strategy, investigators have begun to successfully employ BM-derived DCs stimulated with cytokines in vitro in both animal models and in clinical trials.24,25 Indeed, tumor-specific CTL responses have been induced in vivo following vaccination with DCs pulsed in vitro with tumor antigens.2,3,4 As a further strategy to enhance their abilities to induce antitumor responses, the effects of DCs genetically engineered to express cytokines such as IL-7, IL-12, and GM-CSF have been tested. It has been shown that intratumoral administration of adenoviral IL-7 gene-modified DCs can augment specific antitumor immunity and induce tumor eradication.26 In both mouse and human studies, DCs engineered to express IL-12 have been shown to augment priming of antigens delivered in a variety of ways to T cells. These engineered DCs became powerful catalysts for induction of tumor-specific CD4+ Th cells and CD8+ CTLs.27,28 In addition, engineering DCs to express GM-CSF has also been reported to increases antitumor immunity in vivo.17

It has been recently reported that ligation of CD40 onto DCs by incubation with tumor cells, engineered to express CD40L, triggered DC activation resulting in IL-12 secretion.11 As well, triggering of T-helper–mediated CD40 molecules endowed activated DCs with the ability to autonomously stimulate CTL responses.29,30 These findings prompted us to investigate whether enforced expression of CD40L by engineered DCs is capable of activating DCs and augmenting antitumor immunity. We constructed a recombinant adenoviral vector AdV-CD40L and transfected BM-derived DCs with AdV-CD40L. Our data show that (a) activated DCCD40L is able to stimulate stronger allogeneic T-cell proliferative responses in vitro, and (b) vaccination of mice with Mut1 peptide-pulsed DCCD40L is able to induce more efficient Mut1-specific CD8+ CTL responses and antitumor immunity in vivo than the control DCpLpA.

In this study, we further demonstrated that triggering of CD40 on DCs by AdV-CD40L–mediated CD40L expression leads to increased production of several inflammatory cytokines and chemokines (IL-1β, IL-12, RANTES, IP-10, and MIP-1α) and up-regulated expression of immune molecules (CD54, CD80, and CD86), many of which have been reported to affect antitumor responses in other systems. For example, enhanced expression of cell adhesion (CD5431) and T-cell costimulatory (CD80, CD8632,33) molecules by DCCD40L could possibly have played a significant role in enhanced antigen presentation to, and activation of, T cells observed in our experiments. IL-12 is a cytokine that promotes the development of CD4+ Th1 cells.34 In contrast, the chemokines expressed by DCCD40L (IP-10, RANTES, and MIP-1α) are all chemotactic for T cells and macrophages.35 In addition to the chemotactic effect on T-cell migration, MIP-1α is also known to preferentially help DCs to encounter and stimulate rare tumor-specific CD8+ CTLs.36 Therefore, up-regulation of the above cytokines and chemokines may also play a role in the enhanced antitumor immunity in this study.

In conclusion, our study demonstrates that triggering of the CD40 molecule by AdV-CD40L–mediated CD40 ligand expression is capable of inducing DC activation with up-regulated expression of cytokines (IL-1β and IL-12), chemokines (RANTES, IP-10, and MIP-1α) and immune regulatory molecules (CD54, CD80, and CD86). As well, activated CDCD40L stimulated enhanced allogeneic T-cell proliferation and Mut1-specific CD8+ CTL responses in vitro. Vaccination of mice with Mut1-pulsed DCCD40L also augmented antitumor immunity in vivo. Therefore, DCs engineered to express CD40L by adenovirus-mediated gene transfer may offer a new strategy in the development of cancer vaccines.

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Acknowledgements

This work was supported by a research grant (ROP-15151) of the Canadian Institute of Health Research.

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Correspondence to Jim Xiang.

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Liu, Y., Zhang, X., Zhang, W. et al. Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8+ cytotoxic T-cell activation and antitumor immunity. Cancer Gene Ther 9, 202–208 (2002). https://doi.org/10.1038/sj.cgt.7700429

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Keywords

  • engineered dendritic cell vaccine
  • adenoviral vector
  • CD40 ligand
  • antitumor immunity

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