In Vitro Immune Response Against Lung Cancer Induced By Dendritic Cells Transfected With DNp73α Recombinant Adenovirus

Background:Lung cancer is one of the most aggressive carcinoma. With the incidence of lung caner rasing rapidly in China, it becomes the leading cause of cancer-related deaths. Despite of therapeutic advances in surgery, chemotherapy or radiation, the survival rate of patients with lung cancer has not markedly improved over the past decades. The overall 5-year-survival rate is still less than 15%. Besides surgery, chemotherapy and radiation, biotherapy was thought the 4th way to create an opportunity to develop new strategies against this disease. Its main mechanism was to transfer exogenous antisense gene or micromolecule into tumor cell, so as to interfere or even inhibit its growth; or activate denfence system and secrete some factors to induce growth arrest or apoptosis.Among varied biotherapy patterns, immunotherapy is the most attractive one. It was confirmed that specific anti-tumor immune response was done by activated T lymphocyte. So it is the key point to activate the specific cytotoxic T lymphocyte (CTL). Dendritic cells (DC) are the most potent professional antigen-presenting cells and only ones initiating primary T lymphocyte. It is capable of uptaking (endocytosis and phagocytosis), presenting tumor associated antigen in the context of both MHC classâ… and classâ…¡in association with costimulatory molecules. Much attention has been directed to the problem of how and what antigens should be pulsed to DC in their clinical application as immunotherapy.Many methods for priming antigen to DC have been attempted to induce an anti-tumor immune response. The most common approaches included the pulsing of DC with whole tumor cell lysate or tumor-derived MHC classâ… -restricted peptides. However, whole tumor cell preparations are difficult to standardize and are of limited availability for some tumor cells, and pulsing DC with synthetic peptides is limited by identification of specific HLA-restricted epitopes. Recently, DCs transfected with total TAA cDNA were demonstrated to induce strong TAA-specific immune responses. Gene modification of DC with the TAA gene has the potential to present various, known and unknown, TAA epitopes. The endogenous processing and presentation of TAA peptides may be more efficient for cell surface presentation than exogenous loading of synthetic TAA peptides. At present, the most efficient method for genetic modification of DC is viral vectors. In particular, recombined adenoviral (rAd) vectors have been shown to be most effective in transducing DC.The DNp73αprotein, a protein of the p53 family, was recently identified to be generated from the p73 gene under an alternative promoter in intron 3, and thus DNp73αprotein lacks a transactivation domain presenting in p73. DNp73αis not expressed in normal tissues but overexpressed in lung cancer, ovarian cancer, vulval cancer, neuroblastoma and breast cancer cell lines; it acts as a potent transdominant inhibitor of the wild-type p53 and the transactivation-competent TAp73. Several studies found that positive expression of DNp73αwas a significant independent factor for predicting a poor prognosis, and considered it an important molecular target for effective therapy. However, few studies have reported experimental manipulations targeting DNp73α.This study was aimed to investigate the effects of dendritic cells transfected with recombined adenovirus vector encoding DNp73αon induction of immunity against DNp73α-overexpressing tumor cells.Methods:1. A recombinant replication-deficient adenovirus vector carrying a HA tagged full-length human DNp73αcDNA driven by a CMV promoter, was prepared using the AdEasy system and referred to as Adv-DNp73α. In brief, human DNp73αcDNA was cloned into adenovirus transfer vector pAdTrack-CMV. The resulting plasmid, pAdTrack-CMV-DNp73α, was then linearized with PmeI and transformed into E. coli strain B J5183 containing pAdEasy-1, the viral DNA plasmid, for homologous recombination. The recombinant adenoviral construct was then cleaved with PacI and transfected into 293T cells to produce viral particles. The same adenoviral vector backbone carrying the marker gene EGFP, Adv-EGFP, was used as the control vector. Titers of the viral stocks were determined with a plaque-forming assay.2. Immature dendritic cells generated in the presence of interleukin-4 and granulocyte/macrophage colony-stimulating factor from human umbilical cord blood were transfected with the adenovirus vector with centrifugal force method. Following up expression of GFP, we detected the infective efficiency of varied M.O.I. recombined adenovirus. Combined with viability of DC evaluated through FCM, we then ascertained the optimal infection programmer and M.O.I..3. RT-PCR and Western Blot were use to detect the level of DNp73αmRNA and protein in the transfected DC to ensure the overexpression of DNp73α.4. At 48 h after Adv-DNp73αtransfection, the expression of DC surface markers, CD83 and HLA-DR, was assessed by FCM analysis. Normal DC and Adv-EGFP transfected DC were used as the control.5. Annexin V-propidium iodide apoptosis detection was utilized to quantificate DC' apoptosis on day 4, 8 and 12 after Adv-DNp73αtransfection, respectively. Normal DC and Adv-EGFP transfected DC were used as the control.6. Autologous T cells were isolated and purified using nylon wool columns. The purified cells were cultured in 96-well U-bottom plates in the presence of cytomycin-C-pretreated transfected or untransfected DC as stimulators for 5 days. During the last 8 h of incubation, 1μCi/well of [³H] thymidine was added. Assays were performed in triplicate. And then the cells were harvested and [³H] thymidine incorporated into T cells was measured by a beta counter.7. T cells (1×10⁶) were co-cultured with Adv-DNp73αmodified DC (5×10⁴) for 72 h to induce cytotoxic T lymphocytes (CTLs). Then the CTLs were collected and used as the effector cells in CTL assays. A549/DNp73αcells, K562 cells, and MCF-7 cells, as the target cells with different DNp73αespression levels determined by real time PCR, were placed in 96-well tissue culture plates at 1×10⁴ cells per well respectively, and co-cultured with effector cells (CTLs) at varied E/T (effect cells: target cells) ratio of 1∶10, 1∶20, 1∶40 and 1∶80. The cytotoxic activities were determined by CytoTox non-radioactive cytotoxicity assay. Normal DC and ??Adv-EGFP transfected DC were used as the control.8. For a single comparison of two groups, Student's t test was used to evaluate the significance of differences. If the data distribution was not normal, the Mann-Whitney rank-sum test was employed for the nonparametric analysis. For all analyses, the level of significance was set at p<0.05. All statistical calculations were performed using the SigmaStat statistical software package SPSS10.0. Data are presented as the mean±SD.Results;1. Via directed cloning and homologous recombination, infection-competent adenovirus was generated in 293T package cell line. Following repeatedly infecting, high titer adenovirus particles yielded ( about 2.5×10¹¹ pfu/L).2. Dendritic cells were confirmed generated in the presence of interleukin-4 and granulocyte/macrophage colony-stimulating factor from human umbilical cord blood, displaying the typical morphology under light microscopy and scan electron microscopy, i.e. great increase in size, irregular shape and multiple dendritic projections; and showing phenotypical characteristics by flow cytometry analysis, CDla⁺, CD83⁺, and HLA-DR⁺.3. The addition of purified Adv-DNp73αvector to the DC culture with centrifugal force method, at an M.O.I. of 200, resulted in a transduction efficiency of nearly 80% without significant cytotoxic effects. Expression of the DNp73αmRNA was demonstrated by a semiquantitative RT-PCR assay at 48 h after infection. Expression of DNp73α(HA tagged) protein was readily detectable in DC 48 h after Adv-DNp73αtansfection by Western blot analysis using an HA antibody. Additionally, the DC transfected with Adv-DNp73αor Adv-EGFP, showed marked up-regulation of CD83 and HLA-DR.4. The percentage of viable cells among the three kinds of DC is no defference at day 4 and 8 after transfection. But at day 12 after transfection, the viable cell percentage of DC transduced with Adv-DNp73αwas 71±2.83% significantly higher than DC transduced with Adv-EGFP or the uninfected mDC (45.50±3.54% and 53.52±0.71% respectively, p＜0.01).5. When Adv-DNp73α-transduced DCs, Adv-EGFP-transdcued DC and DC on day 4 and 8 after transduction were used to induce T cells' proliferation, the two adenovirus-modified DC demonstrated identical strength in stimulation, which was higher than normal mDC (p<0.01) but had no difference between the two adenovius-modified DCs. However, comparing the ability of DC on day 12 after transduction to stimulate T cell proliferation, we found that Adv-DNp73ct-transduced DC exhibited a stronger T cell response than Adv-EGFP-transduced DC (p＜0.01).6. DC/Adv-DNp73αstimulated effector T cells could effectively lyse DNp73α-overexpressing A549/DNp73αand K-562 cells but not the DNp73α-null MCF-7 cells. The untreated T cells had minimal lysis of all the tumor cells. LAK cells showed comparable lytic activity against A549/DNp73αand K-562 cells with that of DC/Adv-DNp73αstimulated T cells, but demonstrated much more effective killing of MCF-7 cells than the DC/Adv-DNp73αstimulated T cells.Conclusions:DNp73α-engineered DC can induce DNp73α-specific CTL against tumor cells over-expressing DNp73α. Expression of DNp73 enhanced DCs' survival and their ability of stimulating T cells at day 12 following transduction, and may make the specific anti-tumor immune responses stronger.