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Abstract. The growth of malignant tumors is associated with mechanisms of immune escape and inhibition of apoptosis. Livin is a novel member of the inhibitors of apoptosis (IAP) protein family that inhibits cell apoptosis. Livin is specifically expressed by the majority of tumor cells, but it is not expressed in normal adult tissues. In this study, we used umbilical cord blood (UCB)-derived dendritic cells (DCs) infected with a recombinant adenovirus encoding the livin gene as a vaccine to activate effector cells such as cytotoxic T lymphocytes (CTLs) to recognize and kill livin-expressing cancer cells in vitro as an improved strategy for overcoming the ability of these cancer cells to escape apoptosis and antitumor immune responses. We employed interferon-γ (IFN-γ) enzyme-linked immunospot assays to confirm that our immunization strategy induced an antigen-specific reaction to livin and flow cyto-metric analysis of staining with Annexin V and PI to measure the cytotoxic activity of the effector cells against the livin-expressing lung cancer cell lines A549 and H460. Our results show that the recombinant adenovirus was able to promote the maturation of the UCB-derived DCs. This DC vaccine could activate antigen-specific T cells to produce IFN-γ upon recog-nition of livin peptide in the context of the appropriate HLA molecule. The antigen-specific T cells mediate significant cytotoxicity against the cancer cells, but are unlikely to cause an autoimmune reaction against the human bronchial epithelia cells (HBE), which do not express livin. Introduction
The acquisition of resistance to apoptosis and the ability to escape from immunological surveillance are the hallmarks of tumor cells (1,2). The inhibitors of apoptosis proteins (IAPs) inhibit apoptosis by interacting with the pro-apoptotic caspases via their baculoviral IAP repeat (BIR) (3). Previous studies indicated that downregulating the expression of IAPs is beneficial to the treatment of cancer and may be a neces-sary component of the antitumor activities of conventional treatments such as radiation and chemotherapy (4-6). Livin is a novel member of the IAP family; it has two isoforms that are termed livin α (298-amino acid residues) and livin β (280- amino acid residues). Each isoform contains a single BIR domain and a RING finger domain (7). In contrast to other members of the IAP family, such as cIAP-1, cIAP-2, XIAP and NAIP, which are expressed at low levels in normal adult tissues, livin is not expressed in normal adult tissues but is overexpressed in primary lung cancer and other malignant tumor cells (8,9). Livin expression levels correlate to some degree with the stage and pathological type of the cancer, and livin expression can induce tumor cells to become resistant to antineoplastic drugs and are characteristic of a cancer with a poor prognosis (9-12). Evidence suggests that interfering with either the expression of the livin gene or the function of the livin protein could provide a potential therapeutic avenue to induce apoptosis in tumor cells and to significantly improve antitumor responses (13-15).
Since anti-livin autoantibodies have been identified in the serum of the majority of cancer patients, immunologic tolerance to livin is thought to be very weak (16,17), making livin a promising candidate target for immunotherapy. Dendritic cells (DCs) are classic antigen-presenting cells (APCs) and can efficiently take up and present tumor antigens as well as provide the co-stimulatory molecules required to activate naive T lymphocytes (18,19). In addi-tion to antigen modulation and immune evasion (20), cancer cells may cause DCs to downregulate their expres-sion of MHC class I molecules when the two cell types are co-cultured (21). This reduces the ability of the DCs to activate CD8+ T cells and leads to immune escape. In the present study, we showed that DCs that have been modified to express the livin α gene could provide stable presentation of livin epitopes and could induce T-cell activation. We developed a cell-transfer therapy based on these observations in which pre-sensitized tumor-specific effector cells were expanded in vitro and transplanted into
The antitumor effect of human cord blood-derived dendritic cells modified by the livin α gene in lung cancer cell lines
HAO CHEN1,2, YANG JIN1,2, TING CHEN1,2, MINGQIANG ZHANG1,2,
WANLI MA1,2, XIANZHI XIONG1,2 and XIAONAN TAO1,2
1Department of Respiratory Diseases, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan; 2Key Laboratory of Pulmonary
Diseases of the Ministry of Health of China, Wuhan, Hubei 430022, P.R. China
Received August 1, 2012; Accepted August 16, 2012
DOI: 10.3892/or.2012.2133
Correspondence to: Professor Xiaonan Tao, Department of Respiratory
Diseases, Union Hospital, Tongji Medical College, Huazhong University
of Science and Technology, Wuhan, Hubei 430022, P.R. China
E-mail: taoxn2004@https://www.doczj.com/doc/d6185685.html,
Key words: livin α gene, DC vaccine, recombinant adenovirus, lung
cancer
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autologous hosts (22,23). Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells (24) and is resis-tant to replicative senescence and immunosenescence (25). HLA-mismatched cord blood-derived transplants are associated with a lower incidence and severity of acute or chronic graft-versus-host disease (GVHD) (26). Thus, UCB cells are an ideal source of cells for allogeneic antitumor transplants (27). Due to the biological activity of livin, we used a replication-defective recombinant adenovirus (rAd) vector encoding the livin α gene to infect UCB-derived DCs to generate an antigen-specific vaccine that can be applied to cancer immunotherapy (28-30).
Hariu et al demonstrated that stimulating the periph-eral blood lymphocytes (PBMCs) of HLA-A24+ lung cancer patients in vitro with livin peptides in the context of matched HLA molecules induced peptide-specific cytotoxic T lymphocytes (CTLs) that showed cytotox-icity against livin-expressing lung cancer cell lines in an HLA-A24-restricted manner (11). However, the previous study could not be universally applied in oncotherapy in individuals with various HLA subtypes as the livin-expressing DC vaccine generated responses that were restricted to the HLA-A24 MHC subtype. We previously confirmed that human livin α gene-modified DC vaccines can induce tumor-specific CTLs and mount significant anti-Lewis lung cancer responses in mice (28). In addi-tion to CTLs, there are multiple cell types in the human immune system that can mediate non-specific antitumor effects, such as macrophages, NK and LAK cells (31,32). In this study, we expanded on previous work to show that livin α gene-modified DC vaccines derived from UCB can activate multiple innate effector cell types in the human immune system in addition to CTLs to exert antitumor effects.
Materials and methods
Cell lines and culture media. The lung cancer cell lines A549 and H460 (HLA-A2+/A24+) were established in the Labora-tory Center of the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. HBE cells (HLA-A2+/A24-) were kindly provided by Dr Shanshan Song (Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology). HEK-293 cells were purchased from AGTC Gene Technology Company Ltd., Beijing, China. A549 and H460 cells were maintained in RPMI-1640 while HBE and HEK cells were maintained in DMEM. Both culture media were supplemented with 10% heat-inactivated fetal bovine serum (HyC lone; Thermo Scientific, Waltham, MA, USA), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml peni-cillin and 100 μg/ml streptomycin (all from Sigma, St. Louis, MO, USA) in a humidified incubator at 37?C with 5% CO2.
Construction of recombinant adenoviral vector encoding the livin α gene. Replication-defective type-5 adenoviruses (rAd5) in which the E1 and E3 region were deleted and that encoded enhanced green fluorescent protein (EGFP) were used as vectors in this study. Our lab used the shuttle plasmid pDC316-EGFP-cmv-livin α, which was synthesized by Dr Junping Xie. The helper plasmid pBHGlox_E1,3Cre was purchased from AGTC Gene Technology Company Ltd. HEK-293 cells were co-transfected with the shuttle plasmid and the helper plasmid to produce the recombinant adeno-virus.
After three rounds of amplification, the crude lysates were purified by density gradient centrifugation and dialysis. The titer of the rAd-livin α was calculated using the 50% tissue culture infective dose (TCID50) assay.
PCR. Polymerase chain reaction (PCR) was used to evaluate whether the rAd-livin α vector had been generated success-fully. We utilized an empty rAd vector as a negative control and pDC316-EGFP-cmv-livin α as a positive control. Briefly, amplification was performed in 25 μl of PCR mixture containing 1 μl of the template, 0.5 μl of primers (50 μM) and 0.25 μl of Taq DNA polymerase. The PCR mixture was initially incubated at 94?C for 4 min, followed by 30 cycles of denaturation at 94?C for 30 sec, annealing at 56?C for 40 sec and extension at 70?C for 50 sec, followed by a final extension at 72?C for 10 min. PCR products were analyzed by electro-phoresis on a 1% agarose gel.
The forward primer 5'-GCCATGGGGCCTAAAGACA GTG-3' and the reverse primer 5'-CTGCTAGGACAGGAAG GTGCG-3' were used according to the manufacturer's protocol for the specific detection of livin α. The expected size of PCR product for livin α is 897 bp.
Isolation and induction of UCB-derived DCs in vitro. Heparin anti-coagulated (20 U/ml) HLA-A2+/A24- and HLA-A2+/A24+ UCB samples were isolated following cesarean sections with the approval of the ethics committee of the Tongji Medical School, Huazhong University of Science and Technology. All patients provided informed consent prior to the surgery, which was performed at the Obstetrics Department, Wuhan Union Hospital, China. The HLA class was determined using polymerase chain reaction with sequence-specific primers (PCR-SSP).
We generated UCB-derived DCs in vitro using a combi-nation of previously described protocols (33-36). Briefly, mononuclear cells (MNC) were obtained using a Ficoll-Hypaque (TBD Biotechnology developing center, relative density 1.077 at 20?C) density gradient. The precursor DCs were isolated using anti-CD34 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). These cells were cultured in RPMI-1640 medium supplemented with 20% FBS. The culture media were also supplemented with 50 ng/ml of GM-CSF (PeproTech Inc., Rocky Hill, NJ, USA), 50 ng/ml of SCF (PeproTech) and 20 ng/ml of IL-4 (PeproTech) to induce the development of UCB-derived DCs. Half of the culture medium was replaced with fresh medium containing the appropriate concentration of cytokines every other day. The cultured cells were then separated into adherent cells and non-adherent fractions and the non-adherent cells were supplemented with 100 U/ml of IL-2 (Peprotech) to induce the development of effector cells, which were predominantly CTLs.
Preparation of the stimuli and DC vaccines. On Day 5, DCs were harvested and washed with RPMI-1640 medium, then
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pulsed with a variety of tumor-associated antigens (TAAs), either rAd-livin α or rAd-c at a multiplicity of infection (MOI) of 200, and 30 μg/106 cells of A549 and H460 lysates that had undergone 3 cycles of freezing and thawing between -80 and 37?C and had been passed through a 0.22-μm filter. The cultures were incubated for 2 h in 0.3 ml of serum-free medium supplemented with GM-CSF and IL-4 but not SCF, then supplemented with complete medium. On Day 7, 50 ng/ml GM-CSF, 20 ng/ml IL-4, 10 ng/ml TNF-α (all PeproTech) and 1 μg/ml PGE (Sigma) were added to the cultures to induce DC maturation and the cells were cultured for an additional 2 days.
On Day 9, IL-2-induced cells were co-cultured with mature DCs at a ratio of 10:1 in culture medium supplemented with IL-2 and IL-12 (PeproTech). The cells were cultured together for an additional 48 h before the cytotoxic activity of the effector cells was measured.
Evaluation of the infection efficiency of restructured adenovi-ruses in DCs and cell viability. DCs infected rAd at MOI 50, 100, 200 and 400. As the viral vector was modified to encode EGFP, the efficiency of infection can be correlated to the proportion of EGFP-positive cells detected by flow cytometry. Viable cells cannot be stained with trypan blue and trypan blue staining was used to evaluate cell viability.
Differentiation and maturation of DC cells measured by flow cytometry. The expression of DC cell surface molecules (CD1a, CD80, CD86 and HLA-DR) was analyzed by flow cytometry. The DC cells were divided into precursor DC cells, mature DC cells, and DC cells infected with either rAd-control or rAd-livin α. Cells were harvested and incu-bated with monoclonal antibodies (anti-human CD86 Alexa Fluor 488, anti-human CD80 PE-Cy5, anti-human CD1a PE and anti-human HLA-DR APC) (eBioscience Inc., San Diego, CA, USA) at 4?C for 30 min. The lysates were centrifuged at 1,000 rpm for 6 min, the supernatant was then discarded and the cells were washed with PBS. The cells were centrifuged again at 1,000 rpm for 6 min, then fixed in 4% paraformalde-hyde and analyzed by flow cytometry.
Western blot analysis. We used western blots to analyze livin expression in H460, HBE and A549 cells, in rAd-c-infected and rAd-livin α-infected DCs and in MNCs. Amino acids 264-280 of the short isoform or amino acids 281-298 of the long isoform of human livin (Abcam, Cambridge, MA, USA) were used as positive controls. Cells were lysed in lysis buffer (12.5 mM HEPES, 200 mM KCl, 5 mM EDTA, 50 mM NaF, 0.5% NP-40, 0.5 mM PMSF, 5 μg/ml leupeptin, 5 μg/ml aprotinin and 2 mM Na3VO4). The protein content of the supernatant was determined using the Bio-Rad Protein Assay kit. Whole cell extract (50 μg) was denatured in SDS sample buffer and loaded onto a 12% SDS-PAGE gel. Following electrophoresis, the proteins were transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in Tween-20 in TBS (TBST, 15 mM Tris-HCl, pH 7.5, 200 mM NaCl and 0.1% Tween-20) for 2 h at room temperature. Subsequently, the membranes were incubated overnight with a rabbit anti-livin polyclonal antibody (Abcam) at 4?C. The primary antibodies were detected using a horse-radish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The signal was detected by treating the membrane with ECL reagent and exposing it to X-ray film. Protein loading was assessed by GAPDH.
IFN-γ ELISPOT assays. An IFN-γ ELISPOT assay was performed to detect livin-specific effector T cells (37). According to the manufacturer's instructions, UCB-derived effector cells were resuspended in Lympho-Spot? serum free medium at a final concentration of 1.0x106 cells/ml. Lympho-Spot serum free medium (200 μl/well) was added to 48-well PVDF plates (DKW, China) that had been pre-coated with anti-IFN-γ antibody. After 10 min, the serum free medium was replaced and the effector cells were moved to suspension cultures in plates (1.5x105 cells/well). The cells were then stimulated with a final concentration of 10 μg/ml of either a livin polypeptide (KWFPSCQFLL) or a livin-unrelated polypeptide (RYLRDQQLLGI), both of which have previously been demonstrated to be restricted to HLA-A24 (11). Lympho-Spot serum free medium alone was used as a negative control and PHA (plant hemagglutinin, 2 μg/ml) was used as a positive control. The PVDF plates were placed in a humidified incubator at 37?C for 20 h. The media were then removed from the plates and the cells were lysed with cold ddH2O. After washing the plates, each well was incubated first with biotinylated anti-IFN-γ antibody (100 μl/well) and then with streptavidin-HRP (100 μl/well) at 37?C for 1 h. Then, the PVDF plates were treated with AEC (100 μl/well) at room temperature in the dark for 25 min. After washing with ddH2O, the number of spot-forming cells (SFC) was analyzed using a Biosys-Bioreader. The results are expressed as SFC/105 effector cells = (SFC number/well) x 105/(number of cells/well).
Cytotoxicity assay. The cytotoxic activity of the effector cells was measured by flow cytometry as previously described (38-41). Briefly, the target cells were resuspended in PBS supplemented with 0.1% BSA at a final concentra-tion of 2x106 cells/ml. The target cells were incubated in 1 μM CFSE at 37?C for 10 min. Then, 5 volumes of ice-cold culture media were added to quench the staining. The CFSE-stained target cells were centrifuged, resuspended and mixed with the effector cells at effector/target ratios of 5:1, 10:1 and 20:1. The cells were co-cultured in a humidified incubator at 37?C with 5% CO2.
After 4 h, the cells were resuspended, incubated with 5 μl of Annexin V-APC and 5 μl of PI and examined by flow cytometry. Annexin V staining was assessed on CFSE-positive cells to measure target cell death. Annexin V-/PI+ cells were defined as necrotic and were excluded from the analysis. The percentage of specific lysis was calculated as follows: (% experimental lysis - % basal lysis)/(% maximum lysis - % basal lysis) x 100.
Statistical analysis. Values are expressed as the means ± SE. Statistical analysis was performed using the Student's t-test or one way analysis of variance (ANOVA). Values of P≤0.05 were considered to indicate statistically significant differences.
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Figure 1. Construction and identification of rAd-livin α. (A) HEK-293 cells before co-transfection (magnification, x200). (B) HEK-293 cells show typical CPE after co-transfection, (magnification, x200). (C) GFP expression was detected after the infection of HEK-293 cells with rAd-livin α (magnification, x200).
(D) Identification of rAd-livin α by PCR. Lane M, DNA ladder marker; lane 1, empty adenovirus as a negative control; lane 2, pDC316-EGFP-cmv-livin α as a positive control; lane 3, rAd-livin α. (E) Western blot analysis of livin protein expression in NMCs, rAd-c-infected DCs, rAd-livin α-infected DCs, A549, H460 and HBE cells.
Figure 2. Morphology of and surface marker expression on DCs. (A) DC precursors, (B) mature DCs, (C) rAd-c-infected DCs, (D) rAd-livin α-infected DCs, (magnification, x400). (E) Expression levels of cell surface molecules on DCs (CD1a, CD80, CD86, HLA-DR). Data are presented as the means ± SD of 3 separate tests by ANOVA. *P<0.05.
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Results
Verification of adenovirus encoding the livin α gene. Seven days after co-transfecting HEK-293 cells (Fig. 1A) with the shuttle plasmid pDC316-EGFP-cmv-livin α and the backbone plasmid pBHGlox_E1,3Cre, the cells acquired an appearance typical of cytopathic effects (CPE) and viral plaques were visible under microscopic examination (Fig. 1B). Moreover, the cells appeared beaded and to have undergone botryoid changes, and the majority of the cells had detached from the surface of the plate. The expression of green fluorescent protein was visualized using a fluorescent microscope (Fig. 1C), and the results indicated that the recombination adenovirus had successfully infected the target cells and that the adenovirus encoded genes were being expressed.
The target gene detected by PCR was consistent with the expected gene livin α (897 bp) (Fig. 1D). The titer of the rAd-livin α was 2x109 TCID 50/ml, and the titer of the rAd-control was 2x1011 TCID 50/ml, as determined by the TCID 50 method.Livin is expressed in tumor cell lines and in DC cells infected by rAd-livin α, but not in HBE cell lines or mononuclear cells. We determined the expression of endogenous protein livin in A549, H460, HBE, rAd-c DC, rAd-livin α DC and MNC by western blot analysis. We confirmed that the livin protein is expressed in A549 and H460 cells and rAd-livin α-infected DCs, but not in HBE cells, rAd-c-infected DCs or MNCs (Fig. 1E).
Optimal MOI for recombinant adenovirus infection of DC cells. DC cells were infected with rAd-livin α after 7 days of culture. The expression levels of EGFP were determined by flow cytometry after 48 h. We determined that the optimal MOI value was 200, at which 88% of the cells expressed EGFP and 69% of the cells were viable.
Expression of DC surface molecules is increased when DCs mature or are infected with adenovirus. DC precursors were isolated using anti-CD34 microbeads. Freshly isolated precursor cells were small, round, transparent and non-adherent (Fig. 2A). After 3 days of culture with cytokines, the majority of the cells developed multiple surface protrusions of
various lengths. The cultured cells appeared to proliferate and to increase in size and granularity and became either adherent or semi-adherent. Over time, the cultured DC cells developed longer protrusions and a more irregular morphology. After being cultured with TNF-α for 3 days, the majority of DC cells became non-adherent, scattered uniformly throughout the medium, and acquired a morphology typical of mature DCs (Fig. 2B). DCs infected with either rAd-c or rAd-livin α fluoresced green when viewed using a fluorescent microscope (Fig. 2C and D).
The expression of CD1a, CD80, CD86 and HLA-DR are important markers of immunocompetence and maturity in DCs. As shown in Fig. 2E, DC precursors expressed low levels of CD80, CD86 and HLA-DR. The expression levels of these surface markers increased significantly when these cells were cultured in the presence of cytokines and were further increased by infection with adenovirus although this difference did not reach significance for CD1a and CD86. The mature DCs exhibited the same phenotype regardless of whether they were infected with rAd-livin α or rAd-c. These results suggest that infection with rAd can promote the matu-ration of UCB-derived DCs.
Percentage of CD4 and CD8 T-cell populations in the effector cells. After culturing the cells in the presence of IL-2, non-adherent NMCs were used as effector cells. Flow cytometric analysis revealed that 72% of the effector cells were CD3+ lymphocytes. Further characterization of the T-cell subsets demonstrated that 78% of the CD3+ population was CD3+CD8- (CD4 T cells) and 22% of the CD3+ population was CD3+CD8+ (CD8 T cells) (Fig. 3).
Effector cells activated by rAd-livin α-infected DCs respond specifically to livin polypeptides in an HLA-restricted manner. We assessed the response of T lymphocytes to livin poly-peptides using ELISPOT assays to detect IFN-γ production. Effector cells that had been stimulated by HLA-24+ rAd-livin α-infected DCs and were then exposed to HLA-24 presenting livin polypeptide generated a statistically greater number of IFN-γ spots than either cells stimulated by rAd-c-infected DCs or the blank control (Fig. 4B). By contrast, HLA-24- lympho-
cytes incubated with the peptide did not produce a significant
Figure 3. CD3 staining on effector cells. Gating on the CD3 positive T cells (72.3%), 22.2% were CD8 positive and the others (77.8%) were considered to be CD4 positive T cells.
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number of IFN-γ spots. Furthermore, when the effector cells were exposed to irrelevant peptides, there were very few IFN-γ spots regardless of the HLA type, as shown in the bottom row of Fig. 4A.
rAd-livin α-infection of DCs enhances tumor-specific CTL cytotoxicity and decreases autoimmunity. HLA-A2+ UCB-derived DCs were pulsed with different forms of antigen from either A549 or H460 cells that had undergone multiple freeze-thaw cycles or rAd-c or rAd-livin α to study whether rAd-livin α-infected DCs could enhance tumor-specific cyto -toxicity against livin protein-expressing tumor cells (A549, H460) without inducing autoimmune responses against livin protein negative cells (HBE). Cytotoxicity against A549, H460 and HBE cells was examined by flow cytometry by staining for Annexin V/PI. The data are presented in Fig. 5. Effector cells stimulated with rAd-livin α-infected DCs mediated more cytotoxicity than effector cells stimulated with rAd-c DC or the blank control at effector/target ratios of 5:1, 10:1 and 20:1. Effector cells stimulated with rAd-livin α-infected DCs or with proteins that had undergone multiple freeze-thaw cycles mediated similar levels of cytotoxicity but effector cells stimulated with rAd-livin α-infected DCs mediated less non-specific cytotoxicity than those stimulated with proteins from A549 or H460 cells that had undergone multiple freeze-thaw cycles. Discussion
A variety of immune cells and immune molecules participate in antitumor immune responses. CD8+ CTLs and CD4+ Th1 cells play key roles in antitumor activity (42). We propose
using DCs loaded with tumor antigen as a DC vaccine to induce or enhance antitumor immune responses. CTLs require not only appropriately presented antigen and co-stimulatory signals from APCs for their activation, but also co-stimulatory signals from activated CD4+ T cells. In addition, a few CD4+ T cells show MHC II-restricted cytotoxicity towards cancer cells (43-46). Due to these immune mechanisms and the in vivo immune environment, we chose non-adherent UCB-derived MNCs cultured with IL-2 as effector cells instead of CD8+ T lymphocytes sorted from MNCs. The effector cell population was composed predominantly of CD4+ and CD8+ T lympho-cytes, with a minor contribution from other immune cells, which mounted weak specific cytotoxic responses against the targets. When DC vaccines are administered either simultaneously with or sequentially in combination with other anticancer agents, they can induce synergistic antitumor activity while minimizing the toxicity to normal hematopoietic or other organs (47-49). There are several methods to load DCs with tumor antigens. DC cells can either be pulsed with tumor-asso-
ciated antigens (TAAs) or tumor cell lysates or be modified to
Figure 4. T lymphocytes stimulated by rAd-livin α-infected DCs are specific for livin and livin peptide (KWFPSCQFLL) is HLA-A24 restricted. (A) IFN-γ ELISPOT of UCB-lymphocytes primed by rAd-livin α-infected DCs or rAd-c-infected DCs and restimulated with an HLA-A24 restricted livin peptide or irrelevant peptide. (B) ELISPOT wells are shown for comparison. y-axis, the number of IFN-γ spots per 10,000 cells. Data are presented as the means ± SD of 3 separate tests by ANOVA. P<0.05.
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express TAA genes (30,50-52). Tumor proteins are known to be involved in the degradation and recycling of MHC molecules and tumor-associated polypeptides are only weakly immuno-genic in the context of HLA restriction. Tumor cell extracts also contain typical host antigens and may induce autoimmune response when used as vaccines (53). In the present study we showed that although DC cells pulsed with lysates from A549 or H460 cells can activate lymphocytes and induce a strong cytotoxic response against A549 or H460 cells, they also induce the significant non-specific killing of HBE cells. This indicates that unmodified proteins, polypeptides or tumor cell extracts may not be optimal for making DC vaccines. A key goal in cancer immunotherapy is to present TAA in an appropriate form that can be widely applied to induce anti-tumor immunity.It is not sufficient that the peptide epitope be recognized by an allo-reactive CTL; it is important to target tumor antigens that play a key role in the growth and metabolism of malignant tumors. As the livin protein contributes directly to the phenotype of malignant tumors, it is a rational target for novel therapeutic strategies. As livin α contains virtually all the antigenic epitopes of livin β, we infected DCs with rAd-livin α. Our strategy allows the DCs to express the livin protein endogenously, process it and present it in the context of MHC I to activate CTLs. Furthermore, we observed that infection with rAd can promote DC maturation and enhance their expression of co-stimulatory molecules (28,46,54).
T lymphocytes specifically recognize antigen peptides
presented by APCs in the context of MHC I molecules and
Figure 5. Cytotoxicity assays of various forms of the DC vaccines. (A) Plots are gated on the CFSE-labeled target cells. For simplicity, an E:T ratio of 10:1 is shown in the figures. (a and b) The cytotoxicities of the primitive T cells against A549 and HBE cells. (c and d) The cytotoxicity of the effector cells stimulated by rAd-c-infected DCs and rAd-livin-infected DCs against the A549 cells. (e and f) The cytotoxicity of the effector cells stimulated by rAd-livin-infected DCs and A549 lysate-pulsed DCs against H460 and HBE cells, respectively. (g) Background levels of apoptosis of A549 cells. (B) The cytotoxicity assay at E:T ratios of 5:1, 10:1 and 20:1. Data are presented as the means ± SD of 3 separate tests by ANOVA.
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become activated and secrete cytokines (55). The IFN-γELISPOT assay shows that homologous effector cells sensi-tized by rAd-livin α-infected DCs respond specifically to livin polypeptide presented in the context of the appropriate HLA molecules. HLA-A24-/HLA-A2+ T cells that had been sensitized by rAd-livin α-infected DCs were able to recognize and kill A549 and H460 cells. We hypothesize that rAd-livin α-infected DCs expressing the complete livin gene present a wide variety of molecular epitopes in the context of MHC I, so the rAd-livin α-infected DCs may also present other HLA-restricted livin antigens in either HLA-A2 or other HLA subtypes. Thus, rAd-livin α-infected DCs could be widely applied to the treatment of cancer, regardless of HLA subtype. The cytotoxicity test demonstrated that all target cells were susceptible to a low level of non-specific killing by effector cells. In addition to activated CTLs, there are other types of effector cells that can exert nontumor antigen-specific or MHC-restricted cytolytic activity against the tumors, which include macrophages, NK cells and LAK cells. The relatively small number of apoptotic cells observed in the control wells indicates that the majority of cytolytic activity is mediated by the CTLs and Th1 cells, rather than by the NK cells or the macrophages. As shown in Fig. 4, homologous effector cells sensitized by rAd-livin α-infected DCs show significant cytotoxicity against A549 and H460 cells, but only moderate cytotoxicity against HBE cells. Effector cells stimulated with rAd-livin α-infected DC demonstrated the same level of cytotoxicity against HBE cells as the control, indicating that effector cells alone are mildly non-specifically cytotoxic to HBE cells.
The present study demonstrated for the first time that UCB-derived DCs infected with rAd-livin α can sensitize autologous T lymphocytes to effectively kill livin-expressing tumor cells without inducing autoimmunity against normal tissue. This antitumor strategy shows great potential, but needs to be studied further in vivo. In particular, the antitumor effect of UCB-derived rAd-livin α-infected DC vaccines in allogenic cancer patients warrants further study. Acknowledgements
This study was supported by the Hubei Key Laboratory of Biological Targeted Therapy.
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Unit 1How can I get there? 第一课时 一、教学内容 Part A Let's try & Let's talk 二、教学目标 1．能够听、说、读、写句子：“Where is the museum shop？”“It's near the door.”。 2．能够听、说、认读单词ask、sir和句型“Is there a…？”“I want to…”“What a great museum！”。 三、教学重难点 1．学习句子“Where is the museum shop？”“It's near the door.”。 2．正确使用方位介词。 四、教学准备 单词卡、录音机、磁带。 五、教学过程 Step 1 热身(Warming－up) Let's do Go to the bookstore.Buy some books. Go to the post office.Send a letter. Go to the hospital.See the doctor. Go to the cinema.See a film.
Go to the museum.See some robots. Step 2 新课呈现(Presentation) 1．学习Let's try (1)打开课本读一读Let's try中呈现的问题和选项。 (2)播放录音，让学生听完后勾出正确的选项。 (3)全班核对答案。 2．学习Let's talk (1)播放Let's talk的录音，学生带着问题听录音：Where is the museum shop？Where is the post office？听完录音后让学生回答这两个问题，教师板书：It's near the door.It's next to the museum.教师讲解：near表示“在附近”，next to表示“与……相邻”，它的范围比near小。最后让学生用near和next to来讲述学校周围的建筑物。 (2)讲解“A talking robot！What a great museum！”，让学生说说这两个感叹句的意思。 (3)再次播放录音，学生一边听一边跟读。 (4)分角色朗读课文。 Step 3 巩固与拓展(Consolidation and extension) 1．三人一组分角色练习Let's talk的对话，然后请一些同学到台前表演。 2．教学Part A：Talk about the places in your city/town/village.
小学六年级英语上册期末试卷（含答案) 听力部分 一、L isten and choose. (根据你所听到的内容, 选择相符合的一项，并将其字母编号填在题号 前的括号内。)（10分） ( ) 1. A. always B．often C. aunt ( ) 2. A. actress B. actor C. active ( ) 3. A. buy B. bike C. bus ( )4. A. sell B. same C. say ( ) 5. A. bike B. kite C. side ( ) 6. A. difference B. different C. dictionary ( ) 7. A. writes a letter? B. write a letter C. writer ( ) 8. A. Amy’s uncle B. Lily’s uncle C. Billy’s uncle ( ) 9. A. What is your mother doing? B. What does your mother do? C. What is your mother going to do? ( ) 10. A. Mike works in a DVD company. B. Mike works in a VCD company. C. Mike works in a DVD factory. 二、Listen and judge. (根据所听到的内容, 判断图片或句子是否相符, 相符的在相应的题号前的括号内打“√”, 不相符的打“×”。)（10分） 1. 2. 3. ( ) ( ) ( ) 4. 5. ( ) ( ) 6. Chen Jie is going to be an artist. ( ) 7. Mike often does homework at 7:00. ( ) 8. Mr. Li likes playing the violin. ( ) 9. Feng Gang is a policeman. ( )
人教版六年级英语上册各单元知识点汇总 Unit 1 How do you go to school？一、重点短语： by plane 坐飞机 by ship 坐轮船 on foot 步行 by bike 骑自行车 by bus 坐公共汽车 by train 坐火车 traffic lights 交通灯 traffic rules 交通规则 go to school 去上学 get to 到达 get on 上车 get off 下车Stop at a red light. 红灯停Wait at a yellow light. 黄灯等Go at a green light. 绿灯行 二、重点句型： 1.How do you go to school？你怎么去上学？ https://www.doczj.com/doc/d6185685.html,ually I go to school on foot. Sometimes I go by bus. 通常我步行去上学。有时候骑自行车去。 3.How can I get to Zhongshan Park ？我怎么到达中山公园？ 4.You can go by the No. 15 bus. 你可以坐 15 路公共汽车去。三、重点语法： 1、There are many ways to go somewhere.到一个地方去有许多方法。这里的 ways 一定要用复数。因为 there are 是There be 句型的复数形式。 2、on foot 步行乘坐其他交通工具大都可以用介词by…，但是步行只能用介词 on 。 4、go to school 的前面绝对不能加 the，这里是固定搭配。 5、USA 和 US 都是美国的意思。另外America 也是美国的意思。 6、go to the park 前面一定要加the. 如果要去的地方有具体的名字，就不能再加 the ，如果要去的地方没有具体名字，都要在前面加 the. （ go to school 除外。） 7、How do you go to …？你怎样到达某个地方？如果要问的是第三人称单数，则要用： How does he/she…go to …？ 8、反义词： get on（上车）---get off（下车） near（近的）—far（远的） fast（快的）—slow（慢的） because（因为）—why（为什么） same（相同的）—different（不同的） 9、近义词： see you---goodbye sure---certainly---of course 10、频度副词： always 总是，一直 usually 通常 often 经常 sometimes 有时候 never 从来不 Unit 2 Where is the science museum？一、重点短语： library 图书馆 post office 邮局 hospital 医院 cinema 电影院
分类专项复习卷（一） 听力 建议时间：40分钟满分：100分 一、听录音，选择你所听到的内容，将其序号填入题前括号里。（5分）( ) 1. A. afraid B. amazing C. attention ( ) 2. A. can’t go to school B. count to ten C. don’t be late ( ) 3. A. a TV reporter B. a sports reporter C. a PE teacher ( ) 4. A. I want to buy a postcard. B. I want to send the postcard. C. I want to go to the post office. ( ) 5. A. It’s next to the restaurant B. It’s near the museum shop. C. It’s behind the supermarket. 二、听录音，选择与你所听到的内容相符的图片，将其序号填入题前括号里。（5分） ( ) 1. A. B. C. ( ) 2. A. B. C. ( ) 3. A. B. C. ( ) 4. A. B. C. ( ) 5. A. B. C. 三、听录音，用数字给下列图片排序。（6分）
（）（）（） ( ) ( ) ( ) 四、听录音，判断下列句子与你所听到的内容是否相符，相符的写“√”，不相符的写“×”。（5分） ( ) l. Mike should see a doctor in the morning. ( ) 2. Amy’s father works at sea. ( ) 3. Mike’s brother is a postman. ( ) 4. Peter likes doing kung fu and swimming. ( ) 5. We are going to Beijing next Tuesday. 五、听录音，判断下列图片与你所听到的内容是否相符，相符的打“√”，不相符的打“×”。（6分） 1. ( ) 2. ( ) 3. ( ) 4. ( ) 5. ( ) 6. ( ) 六、听录音，选择相应的答语，将其序号填入题前括号里。（7分） ( ) 1. A. He’s over there. B. Thank you. C. Great! ( ) 2. A. Here you are. B. Sure. C. Thanks. ( ) 3. A. Please turn left. B. It’s near the door.
Unit 1 How can I get there? 一：重点单词和短语 Science科学, museum博物馆, post office, bookstore, cinema, hospital ，tasty, buy, London Eye伦敦银, stomach胃,crossing十字路口, turn left, turn right, go straight=walk straight直走.next to紧挨着/与。。。相邻, far from(离。。。远), near在。。。附近,behind（在。。。后面）, in front of（在。。。前面）, between…and…（在。。。和。。。之间） 二：按要求写单词： hot(反义词)cold , cool(反义词)warm，too（同音词）to/two can not(缩写)can’t right(反义词)left/wrong buy(同音词)by/bye sea(同音 词)see first（基数词）one four(序数词)fourth did (原形)do /does three(序数词)third give(过去式)gave 三：重点句型分析 1. Where is the museum shop? 此问句是由特殊疑问词where 引导的一个特殊疑问句，where意为“在哪里，到哪里”，用来询问地点，放在句子的开头。询问“某人或某物在哪里”的基本句型是：“ Where +is/are+ 主语？”，where is 后接名词或代词的单数形式，where are 后接名词或代词的复数形式。表示地点的词：museum博物馆, post office邮局, bookstore书店, cinema电影院, hospital医院restaurant餐馆bank银行bus stop 公交车站lake湖library图书馆zoo动物园school学校park公园garden花园hotel旅馆 2. It’s nea r the door. 此句中near是表示位置的介词，意为“旁边，附近” ，其同义句是：It’s next to the door.它在门的旁边。表示位置的短语：next to the bookstore挨着书店near the hospital在医院附近near the post office在邮局附近over there 在那边on Dongfang Street在东方大街上in front of the school在学校前面 3. How can we get there? 此句用来询问“怎样去某地”，后面直接跟地点。回答时，可以用“ Turn left, turn right, go straight.”等句来回答。 同义句：1.Can you tell me the way to+地点？ 2.Where is the +地点？ 3.Which is the way to +地点 4. Turn left at the bookstore. Then turn right at the hospital. 此句是指路的句型。常用到的句型有：turn left, 向左转turn right, 向右转go straight直着走。同时表示在某处的介词用at. 5.Is the Thames far from here? No, it isn’t. 此句是个be动词开头的一般疑问句，其回答要用Yes或No. 句中的far from意为“离……远”。反义词组为next to.
人教版小学六年级上册英语时态专项习题 一般现在时专项练习 一．写出下列各词的复数 I _________him _________this ___________her ______ watch _______child _______photo ________diary ______ day________ foot________ book_______ dress ________ tooth_______ sheep ______box_______ strawberry _____ thief _______yo-yo ______ peach______ sandwich ______ man______ woman_______ paper_______ juice___________ water________ milk________ rice__________ tea__________ 二、写出下列动词的第三人称单数 drink ________ go _______ stay ________ make ________ look _________ have_______ pass_______ carry ____ come________ watch______ plant_______ fly ________ study_______ brush________ do_________ teach_______ 三、用括号内动词的适当形式填空。 1. He often ________(have) dinner at home. 2. Daniel and Tommy _______(be) in Class One. 3. We _______(not watch) TV on Monday. 4. Nick _______(not go) to the zoo on Sunday. 5. ______ they ________(like) the World Cup? 6. What _______they often _______(do) on Saturdays? 7. _______ your parents _______(read) newspapers every day? 8. The girl _______(teach) us English on Sundays. 9. She and I ________(take) a walk together every evening. 10. There ________(be) some water in the bottle. 11. Mike _______(like) cooking. 12. They _______(have) the same hobby. 13. My aunt _______(look) after her baby carefully. 14. You always _______(do) your homework well. 15. I _______(be) ill. I’m staying in bed. 16. She _______(go) to school from Monday to Friday. 17. Liu Tao _______(do) not like PE. 18. The child often _______(watch) TV in the evening. 19. Su Hai and Su Yang _______(have) eight lessons this term. 20. －What day _______(be) it today? It’s Saturday 四、按照要求改写句子 1. Daniel watches TV every evening.(改为否定句) ___________________________________________________ 2. I do my homework every day.(改为一般疑问句，作否定回答) ________________________________________________________ 3. She likes milk.(改为一般疑问句，作肯定回答)
1 一、 找出划线部分读音与其他三个不同的单词。 （ ）1.A.how B.know C.yellow D.window （ ）2. A.apple B.cat C.many D.mango （ ）3. A.not B.doctor C.kilo D.orange （ ）4. A.pears B.bananas C.apples D.grapes （ ）5. A.boxes B.peaches C.buses D.apples （ ）6. A.these B.thirsty C.those D.they 一、 选出划线部分读音与所给单词划线部分读音相同的一项。 （ ）1.baseball A.table B.father C.banana （ ）2. brother A.nose B.doctor C.son （ ）3. which A.knife B.with C.white （ ）4. this A.mouth B.three C.with （ ）5. room A.too B.book C.good （ ）6. ear A.pear B.bear C.hear 二、 找出划线部分读音与其他三个不相同的单词。 ( )1.A.hurry B.student C.puppet D.bus ( )2. A.that B.think C.mouth D.thank ( )3. A.guess B.she C.egg D.watermelon ( )4. A.right B.climb C.this D.pineapple ( )5. A.mother B.today https://www.doczj.com/doc/d6185685.html,e D.some 三、 找出与所给单词划线部分读音相同的单词。 1.( )father A.mouth B.brother 2. ( )nose A.close B.seven 3. ( )cake https://www.doczj.com/doc/d6185685.html,te B.father 4. ( )sister A.bike B.is 5. ( )boy A.climb B.brother 6. ( )mother A.sister B.her 四、 判断下列划线部分读音是否相同， ( )1.driver write ( )driver teacher ( )2.farmer her ( )knife big ( )3.cook good ( )drink dress ( )4.tree trousers ( )look school ( )5.their hair ( )where here ( )6.that mouth ( )they those 五、 将下列单词按th 的两种发音分类。 that mouth this three think thin thank thirsty those there these they [that] th [ ] mouth 六、 单词辨音，用“√”或“×”表示。 student ( ) bus ( ) bananas pear grapes ( ) ear ( ) teacher nose worker ( ) orange ( ) tree ( ) dress ( ) 八． 判断下列划线部分读音是否相同，用“√”或“×”表示。 1.hair airport ( ) 2.ship shop( ) 3.where who ( ) 4.good school ( ) 5.minibus student( ) 6.station library( ) 九．找出划线部分读音与其他不相同的单词。 ( )1.A.move B.noodle C.who D.coffee ()2. A.sweets B.friends C.cats D.biscuits ( )3. A.apple B.water C.at D.bag ( )4. A.juice B.cup C.bus D.much ( )5. https://www.doczj.com/doc/d6185685.html,k B.it C.kite D.his ( )6. A.classroom B.bedroom C.room D.bathroom 十一、找出与所给单词划线部分读音相同的单词。 1.music A.bus B.student C.cup D.buy 2.chocolate A.goB.open C.too D.office 3.teacher A.school https://www.doczj.com/doc/d6185685.html,e C.lunch D.cake 4.bookcase https://www.doczj.com/doc/d6185685.html,te B.cap C.apple D.snack 5.desk A.grape B.bed C.she D.he 6.kilo A.library B.like C.tiger D.hospital 7.oranges A.booksB.cats C.teachers D.maps 8.chair A.pear B.here C.ear D.dear 十二、判断下列划线部分读音是否相同，用“√”或“×”表示。 1.bread egg ( ) 2.plate snack ( ) 3.where whose ( ) 4.try right ( ) 5.bowl how ( ) 6.chopstick school( )
六年级上复习资料 Unit1 How Do You Go There? (你怎样去那里？) 重点单词： on foot= walk走路by bike骑自行车by bus坐公车by train坐火车by plane坐飞机by ship坐轮船by subway坐地铁near近的far远的usually通常sometimes有时候easy简单的the fifth floor第五层楼traffic lights交通灯traffic rules交通规则stop停止wait等待get to到达same相同的mean意思是driver司机right 右边left左边must必须know知道 重点句子： 1. How do you go to school? 你是怎样去上学的？ 2. I go to school by bus.我是坐公交车去上学的。 3. Usually I go to school on foot. 我通常走路去上学。 4. Sometimes I go by bike. 有时候我骑自行车去。、 5. My home is near/ far. 我的家是近的。/ 远的。 6. Look at the traffic lights, remember the traffic rules. 看着交通灯，记住交通规则。 7. Stop at a red light. Wait at a yellow light. Go at a green light. 红灯停。黄灯等一等。绿灯行。 8. Red means stop, yellow means wait, green means go. 红色的意思是停止，黄色的意思是等待，绿色的意思是通行。 9. How can I get to the park? 我该怎样到达公园呢？ 10. You can go by the No. 15 bus. 你可以坐15路公交车去。 重点知识： 1.坐某种交通工具用by，例如：by bike, by train。而走路用“on”例如on foot. 2.国家名字，地方名字第一个字母要大些：例如：Canada加拿大, China中国, America美 国，England英国，Australia澳大利亚 3.国家名缩写前面加the，缩写字母都要大写。例如：the USA=the US美国，the UK英 国，the CAN加拿大，the PRC中国。 4.频度副词是表示做的次数多少的词语。从多到少依次排列为：always总是，usually通 常，often经常，sometimes有时候，never从不。频度副词可以放在句首，也可以放在人称后面。例如：Usually I go to school by bus. = I usually go to school by bus. 5.near近的，far远的。这两个词是一对反义词。注意：not near= far, not far = near. 6.时间前面用at. 例如：在三点钟：at 3 o’ clock. 一段时间前面用for 7.表达第几层楼的时候我们要用序数词，前面还要有the。例如：第一、二、三、四、五 层楼分别是：the first floor. the second floor. the third floor. the fourth floor. the fifth floor. 8.交通灯traffic lights，交通规则：traffic rules。这两个词后面都一定要加s, 绝对不能少。 因为交通灯有红黄绿三盏，一定是复数，交通规则不可能只有一条，所以都一定要加s，考试的时候千万别忘了加s哦！ 9.大部分的国家都是靠右行驶的：drivers drive on the right side of the road. 记住England and Australia, drivers drive on the left side of the road.英国和澳大利亚，司机是靠左行驶的。
一、阅读理解。 Jenny is from New York, America.She is twelve.Now she is in Shanghai with her parents.She can speak English very well and a little Chinese. Her father is a teacher.He works in a university.And her mother is a doctor.They go to work by subway.Jenny is studying in a school near her home.She walks to school from Monday to Friday.Her classmates are friendly to her.Her family are going to take a trip to Xi’an next summer holiday.She is very excited,because she loves China. （）1.Jenny is an girl. A.English B.American C.Australian （）2.Jenny’s father is a . A.teacher B.doctor C.coach （）3.Jenny goes to school . A.on foot B.by bike C.by subway （）4.Jenny goes to school days a week. A.three B.four C.five （）5.Jenny is going to next summer holiday. A.Shanghai B.Xi’an C.Beijing 二、阅读短文，判断正（T）误（F）。 Ben is my pen pal.He is tall and strong.He likes playing basketball and swimming.Ben is going to have a picnic next weekend.So he plans to(打算) go shopping tomorrow.He wants to buy a pair of sunglasses and a big water bottle.The supermarket is next to the museum.It’s very far.So he is going to take the No.19 bus to get there.After that,Ben is going to visit his uncle.His uncle’s home is near the supermarket.They will have dinner together.They Ben is going to play games with his cousin. （）1.Ben is tall and strong. （）2.Ben is going to have a picnic next weekend. （）3.The supermarket is next to the school. （）4.Ben is going to the supermarket by bike. （）5.Ben will play games with his cousin. 三、阅读理解。 Dear Frank, I’m very happy to be your new pen pal.I live in Guangzhou,China.It’s very hot in summer.So i like swimming very much.It’s cool to swim in the pool or in the sea in summer.I also like playing the piano. It’s interesting.My parents are scientists.They work in the same university.They go to work on foot.They think that’s good exercise.We often go hiking on the weekend. Tell me something about you,please. Yours, Fang Yan （）1.Fang Yan lives in . A.Shanghai B.Shenzhen C.Guangzhou （）2.Fang Yan likes . A.dancing and reading B.swimming and playing the piano
PEP新版六年级上册英语知识点归纳总结 目录 Unit1How can I get there? (2) Unit2Ways to go to school (3) Unit3My weekend plan (4) Unit4I have a pen pal (5) Unit5What does he do? (6) Unit6How do you feel? (7)
Unit1How can I get there? library图书馆north(北) post office邮局 hospital医院turn left左转turn right右转places:cinema电影院 (地点)bookstore书店(东)east west(西) science museum科学博物馆 pet hospital宠物医院crossing十字路口 school学校south(南) shoe store/shop鞋店 supermarket超市go straight直行 一、问路 1．Where is the cinema,please?请问电影院在哪儿？ next to the hospital.在医院的旁边。 in front of the school.在学校的前面. behind the park在公园的后面 It’s near the zoo.在动物园的附近. on the right/left of the bookstore.在书店的左/右边. east of the bank.在银行的东边. far from here.离这儿很远. 2.Excuse me,is there a cinema near here请问这附近有电影院吗？ Yes,there is./No,there isn’t.有./没有。 3.How can I get to the hospital?我该怎样到达医院呢？ Take the No.57bus.乘坐57路公汽。 二、指引路 1．You can take the No.312bus.你可乘坐312路公交车去那儿. 2.Go straight for three minutes.向前直走3分钟. 3.Turn right/left at the…在…地方向右/左转. 4.Walk east/west/south/north for…minutes.朝东/西/南/北/走…分钟. 三、Is it far from here?离这儿远吗? Yes,it is./No,it isn’t.是的，很远/不是，很远。
2016人教版小学六年级上册英语时态专项习题 look , read and write 1. There ( is / are ) some bananas ( on / near ) the table . 2. There ( is / are ) a football (in / under ) the bed . 3. There (is / are ) some tall buildings in the ( village /city ). 4. My teacher is ( strong / thin ) . And she’s(old / young ) . 5. I have ( onions / mutton ) and cabbage for lunch . 6. Mike ( has / have ) English and ( science / math ) on _________________ ( Friday / Sunday ) . When , Where , What , Who , How , Why 1. ---- Can I get to the zoo ?---- You can ride a bike there . 2. ---- do you go to school on foot ?---- Because my home is near . 3. ---- are you going after lunch ?---- I am going to the bookstore . 4. ---- are you going ? ---- I am going at 4 o’clock . 5. ---- are you going to do ? ---- I am going to play football . 6. ---- are you going to play with ? ---- My brother . 一般现在时专项练习 一．写出下列各词的复数 I _________him _________this ___________her ______ watch _______child _______photo ________diary ______ day________ foot________ book_______ dress ________ tooth_______ sheep ______box_______ strawberry _____ thief _______yo-yo ______ peach______ sandwich ______ man______ woman_______ paper_______ juice___________ water________ milk________ rice__________ tea__________ 二、写出下列动词的第三人称单数 drink ________ go _______ stay ________ make ________ look _________ have_______ pass_______ carry ____ come________ watch______ plant_______ fly ________ study_______ brush________ do_________ teach_______ 三、用括号内动词的适当形式填空。 1. He often ________(have) dinner at home. 2. Daniel and Tommy _______(be) in Class One.
PEP7最新六年级下册Unit4-Unit6英语课文翻译 Unit4 I have a pen pal.单词表：学习的三单形式，迷，远足，笔友，业余爱好，茉莉， 想法主意，堪培拉，令人惊奇的，表示征求意见，射门，加入，俱乐部，分享 P38Let’s talk奥利弗：彼得的爱好是什么？ 张鹏：他喜欢读故事。他住在一个农场上，所以有时他读给那些奶牛！ 奥利弗：那是有趣的。张鹏：他喜欢练功夫和游泳。奥利弗：真的吗？我也是。张鹏：他也喜欢唱歌。奥利弗：哦，你也喜欢唱歌。张鹏：是的。我打算教他中国歌曲《茉莉花》！奥利弗：好主意！P39Let’s learn彼得：嘿，张鹏，你的爱好是什么？张鹏：我喜欢读故事。我也喜欢唱歌和练功夫。 跳舞唱歌读故事踢足球练功夫 P40Let’s talk约翰：嘿，一。你正在干什么？（现在进行时）吴一凡：我正在写一份电子邮件 给我的朋友在澳大利亚。约翰：他住在悉尼吗？吴一凡：不，他不是。他住在堪培拉。他的名字是约翰，也。约翰：真的吗？他喜欢猜字谜和去远足吗？吴一凡:是的，他是。约翰：太神奇了！我也喜欢这些。我也能成为他的笔友吗？吴一凡：当然。为什么不呢？约翰：酷！ P41Let’s learn约翰：过来并且看看我的新的笔友。他的名字也是约翰！兄弟：真的吗？他也住在中国吗？约翰：不，他不在。他住在澳大利亚，但是他学语文。烧中国食物学语文猜字谜去远足 P42let’s read布告栏：我们能跳舞吗？有一个跳舞课在星期天在下午一点。我喜欢跳舞,兵器我需要一个搭档。打电话给艾米：3345567射门！射门！射门让我们一起读。你的爱好是什么？你喜欢阅读吗？我有很棒的书。我们能分享！打电话给迈克：4435678 科学俱乐部，你的俱乐部。你想学习关于机器人吗？来到科学房间。见见罗宾。他教学生制作机器人。robin Unit 5 What does he do?单词表：工厂，工人，邮递员，商人企业家，警察，渔民科学家， 飞行员，教练，国家，校长，大海，保持，大学，体育馆，如果，记者，使用，打字，快速地， 秘书 P48Let’s talk莎拉：你的爸爸是在这里今天吗？奥利弗：不，他在澳大利亚。莎拉：他是干什么的？奥利弗：他是一个商人。他经常去其它的城市。莎拉：并且你的妈妈是干什么的？奥利弗：她是一个班主任。莎拉：那是好的。奥利弗：是的，她将要是在这里今天。莎拉：你想成为一个班主任吗？奥利弗：不，我想成为一个出租车司机。 P49Let’s learn职业日工厂工人邮递员商人警察张鹏：你的爸爸是一个邮递员吗？奥利弗：不，他不是。张鹏：他是干什么的？：奥利弗：他是一个商人。 P50Let’s talk迈克：我的叔叔是一个渔夫。小雨：他在哪里工作？迈克：他工作在海上。他看 到很多鱼每天。小雨：我知道了。他怎么去工作的？乘小船？迈克：不，他工作在小船上。他骑自行车去工作。小雨：他有一个很健康的生活。迈克：是的。他工作非常努力并且保持健康。小雨：我们也应该努力学习并且保持健康。 P51Let’s learn渔民科学家飞行员教练奥利弗：我的叔叔是科学家。吴一凡：他在哪里工作？奥利弗：他工作在一个大学。 P52let’s read胡斌喜欢运动。他擅长足球，乒乓球和篮球。他经常去跑步放学后。他想工作在一个体育馆。提示：
人教版六年级英语上册期末重点知识复习资料 六年级上册复习要点 Unit1 How can I get there? 一、重点单词： 地点：science museum科学博物馆post office 邮局bookstore 书店 cinema 电影院hospital 医院 动作：go straight 直走turn left/right 左转、右转 方位：in front of :在···前面behind 在···后面near在…旁边next to 紧挨着beside 在旁边 over 在…上方on the left 在左边on the right 在右边 二、重点句型： （1）Is / Are there…？某处有某物吗？ 肯定回答：Yes, there is/are. 否定回答：No, there isn’t/aren’t. （2）Where is the + 地点？... ... 在哪里？ It’s + 表示地点的名词. 它... ... 例句：Where is the cinema? 电影院在哪？ It’s next to the bookstore. 在书店的旁边。 （3）How can + 主语+get（to）+ 地点? ... ...怎么到... ...? （如果get后面接的词为副词，则要省略介词to.） 例句：我们怎么到那儿？ 同义句型：Can you tell me the way to + 地点？ ( 4 )Where is + 地点？Which is the way to + 地点？ 到书店左转。 Unit 2 Ways to go to school? 一、重点单词/短语： 交通方式：by bike /bus /plane /subway /train /ship /taxi /ferry 骑自行车/乘公共汽车/飞机/地铁/火车/船/出租汽车/轮渡take the No.57 bus 乘57路公共汽车 on foot 步行 其他：slow down慢下来pay attention to 注意traffic lights 交通信号灯look right 向右看cross the road 横穿马路at home 在家 二、重点句型： （1）How do you come (to) + 地点？你们怎么来... 的？ 我通常/经常/有时... How do you go(to) + 地点？你们怎么去...的？ How do you get (to) + 地点？你们怎么到达...的？