Characterization of HLA-A2-restricted HPV-16 E7-specific CD8(+) T-cell immune responses induced by DNA vaccines in HLA-A2 transgenic mice

We have recently demonstrated that linkage of DNA-encoding calreticulin to DNA-encoding human papillomavirus-16 E7 antigen strongly enhances the efficacy of DNA vaccines against E7-expressing tumors in animal models. In this study, as a prelude to clinical translation, we characterized the ability of DNA-encoding calreticulin linked to DNA-encoding E7 antigen to generate HLA-A2-restricted E7-specific CD8(+) T-cell responses in HLA-A2 (AAD) transgenic mice, as well as antitumor effects against an E7(+) HLA-A2(+) tumor cell line, TC-1/A2. Our results show that while vaccination with CRT/E7 DNA generates strong H-2D(b)-restricted E7 (amino acid (aa)49-57)-specific CD8(+) T-cell immune responses in both C57BL/6 and HLA-A2 (AAD) transgenic mice, no such responses were generated to HLA-A2-restricted epitopes in either type of mouse. In contrast, vaccination with DNA-encoding calreticulin linked to DNA encoding a mutant version of E7 with a deleted aa49-57 epitope leads to the generation of an HLA-A2-restricted E7 (aa11-20)-specific CTL response in HLA-A2 (AAD) transgenic mice. More importantly, vaccination with CRT/mtE7 (del aa49-57) DNA protects against a lethal challenge with TC-1/A2 tumor cells in HLA-A2 (AAD) transgenic mice. Furthermore, our in vitro studies demonstrate that the presence of the E7 (aa49-57) epitope does not suppress presentation of the HLA-A2-restricted E7 (aa11-20) epitope through MHC class I molecules. Thus, the predominant E7 aa49-57-specific CD8+ T-cell immune response in HLA-A2 transgenic mice vaccinated with CRT/E7 is likely due to preferred expansion of E7 aa49-57-specific CD8(+) T cells in vaccinated mice. These results highlight the importance of epitope immunodominance in the evaluation of immune responses in HLA-A2 (AAD) transgenic mice.     

Targeted Coating With Antigenic Peptide Renders Tumor Cells Susceptible to CD8+ T Cell-mediated Killing

The potency of immunotherapies targeting endogenous tumor antigens is hindered by immune tolerance. We created a therapeutic agent comprised of a tumor-homing module fused to a functional domain capable of selectively rendering tumor cells sensitive to foreign antigen-specific CD8⁺ T cell-mediated immune attack, and thereby, circumventing concerns for immune tolerance. The tumor-homing module is comprised of a single-chain variable fragment (scFv) that specifically binds to mesothelin (Meso), which is commonly overexpressed in human cancers, including ovarian tumors. The functional domain is comprised of the Fc portion of IgG2a protein and foreign immunogenic CD8⁺ T cell epitope flanked by furin cleavage sites (R), which can be recognized and cleaved by furin that is highly expressed in the tumor microenvironment. We show that our therapeutic protein specifically loaded antigenic epitope onto the surface of mesothelin-expressing tumor cells, rendering tumors susceptible to antigen-specific cytotoxic CD8⁺ T lymphocytes (CTL)-mediated killing in vitro and in vivo. Our findings have important implications for bypassing immune tolerance to enhance cancer immunotherapy.     

Does B7-1 expression confer antigen-presenting cell capacity to tumors in vivo?

Tumors engineered to express the costimulatory molecule B7-1 can elicit CD8+ cytotoxic T lymphocyte (CTL)-dependent antitumor responses in immunocompetent mice. It has been postulated that this result reflects direct priming of CTL by the modified tumor in vivo. Previous studies of the immune response to a B7-1- murine colon carcinoma expressing influenza nucleoprotein (NP) as a model tumor antigen have demonstrated the crucial role of bone marrow-derived antigen-presenting cells (APCs) in the priming of NP-specific CTL in vivo. In this system, no evidence of direct CTL priming by tumor was detected. We have performed a similar analysis to determine if B7-1 transfectant of this tumor results in the direct priming of CTL, and to compare this response to that primed by host APCs. When H-2b-->H-2bxd bone marrow chimeras were immunized with a single injection of CT26/NP/B7-1 (H-2d), NP-specific CTL were detected that were restricted to the bone marrow haplotype (H- 2b), but not to the tumor haplotype. In contrast, CTL recognizing the NP antigenic epitope in the context of the tumor's major histocompatibility complex were detectable only after multiple immunizations. These results suggest that whereas B7-1+ tumor vaccines result in some degree of direct presentation to CD8+ T cells, the dominant mechanism of CTL priming is through the uptake and presentation of tumor antigens by bone marrow-deprived APCs. However, repeated immunization with B7-1+ tumor cells can efficiently expand the directly primed CD8+ CTL population.     

Incomplete CD8(+) T lymphocyte differentiation as a mechanism for subdominant cytotoxic T lymphocyte responses to a viral antigen

CD8(+) cytotoxic T lymphocytes (CTLs) recognize antigen in the context of major histocompatibility complex (MHC) class I molecules. Class I epitopes have been classified as dominant or subdominant depending on the magnitude of the CTL response to the epitope. In this report, we have examined the in vitro memory CTL response of H-2(d) haplotype murine CD8(+) T lymphocytes specific for a dominant and subdominant epitope of influenza hemagglutinin using activation marker expression and staining with soluble tetrameric MHC-peptide complexes. Immune CD8(+) T lymphocytes specific for the dominant HA204-210 epitope give rise to CTL effectors that display activation markers, stain with the HA204 tetramer, and exhibit effector functions (i.e., cytolytic activity and cytokine synthesis). In contrast, stimulation of memory CD8(+) T lymphocytes directed to the subdominant HA210-219 epitope results in the generation of a large population of activated CD8(+) T cells that exhibit weak cytolytic activity and fail to stain with the HA210 tetramer. After additional rounds of restimulation with antigen, the HA210-219-specific subdominant CD8(+) T lymphocytes give rise to daughter cells that acquire antigen-specific CTL effector activity and transition from a HA210 tetramer-negative to a tetramer-positive phenotype. These results suggest a novel mechanism to account for weak CD8(+) CTL responses to subdominant epitopes at the level of CD8(+) T lymphocyte differentiation into effector CTL. The implications of these findings for CD8(+) T lymphocyte activation are discussed.     

Control of human mesothelin-expressing tumors by DNA vaccines

Mesothelin has been implicated as a potential ideal target antigen for the development of antigen-specific cancer immunotherapy for the control of mesothelin-expressing cancers such as ovarian cancer, mesothelioma and pancreatic adenocarcinoma. In the current study, we utilized a DNA vaccine encoding human mesothelin (pcDNA3-Hmeso) to treat C57BL/6 mice challenged with luciferase-expressing, Hmeso-expressing ovarian cancer cell line, Defb29 Vegf-luc/Hmeso. The therapeutic effect of the tumor-challenged mice was followed by noninvasive bioluminescence imaging systems. The mechanism of the antitumor effect was characterized by depletion of subsets of lymphocytes as well as adopted transfer of serum from pcDNA3-Hmeso-vaccinated mice. We found that vaccination with pcDNA3-Hmeso DNA vaccine generates a significant antitumor effect and promotes survival in mice challenged with Defb29 Vegf-luc/Hmeso. Furthermore, we found CD4+ and CD8+ T-cell immune responses as well as the humoral immune responses are important for the observed antitumor effects in vaccinated mice. Our data indicated that vaccination with DNA vaccine targeting Hmeso could generate potent antitumor effects against mesothelin-expressing tumors through both T cell-mediated immunity as well as antibody-mediated immunity.     

Treatment With Cyclooxygenase-2 Inhibitors Enables Repeated Administration of Vaccinia Virus for Control of Ovarian Cancer

Metastatic ovarian cancer is the leading cause of death among women with gynecologic malignancies in the United States. The lack of effective treatment for patients with advanced ovarian cancer warrants development of innovative therapies. Cancer therapy using oncolytic viruses represents a promising new approach for controlling tumors. Vaccinia virus has been shown to preferentially infect tumor cells but not normal tissue. However, oncolytic therapy using recombinant viruses faces the limitation of viral clearance due to generation of neutralizing antibodies. In the current study, we found that cyclooxygenase-2 (Cox-2) inhibitors circumvented this limitation, enabling repeated administration of vaccinia virus without losing infectivity. We quantified the antivaccinia antibody response using enzyme-linked immunosorbent assay (ELISA) and neutralization assays to show that treatment of Cox-2 inhibitors inhibited the generation of neutralizing antibodies. Furthermore, we showed that combination treatment of Cox-2 inhibitors with vaccinia virus was more effective that either treatment alone in treating MOSEC/luc tumor-bearing mice. Thus, the combination of Cox-2 inhibitors and vaccinia virus represents a potential innovative approach to controlling ovarian tumors.     

Mesothelin-specific CD8(+) T cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients

Tumor-specific CD8(+) T cells can potentially be activated by two distinct mechanisms of major histocompatibility complex class I-restricted antigen presentation as follows: direct presentation by tumor cells themselves or indirect presentation by professional antigen-presenting cells (APCs). However, controversy still exists as to whether indirect presentation (the cross-priming mechanism) can contribute to effective in vivo priming of tumor-specific CD8(+) T cells that are capable of eradicating cancer in patients. A clinical trial of vaccination with granulocyte macrophage-colony stimulating factor-transduced pancreatic cancer lines was designed to test whether cross-presentation by locally recruited APCs can activate pancreatic tumor-specific CD8(+) T cells. Previously, we reported postvaccination delayed-type hypersensitivity (DTH) responses to autologous tumor in 3 out of 14 treated patients. Mesothelin is an antigen demonstrated previously by gene expression profiling to be up-regulated in most pancreatic cancers. We report here the consistent induction of CD8(+) T cell responses to multiple HLA-A2, A3, and A24-restricted mesothelin epitopes exclusively in the three patients with vaccine-induced DTH responses. Importantly, neither of the vaccinating pancreatic cancer cell lines expressed HLA-A2, A3, or A24. These results provide the first direct evidence that CD8 T cell responses can be generated via cross-presentation by an immunotherapy approach designed to recruit APCs to the vaccination site.     

Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines

Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA- A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. E75- pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA- A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens.     

B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8(+) T lymphocytes in vivo.

B7H/B7RP (hereby called B7H) is a new member of the B7 family of costimulatory molecules and interacts with inducible costimulatory molecule (ICOS). Its function for CD8 T cells has not been reported. We report here that expression of B7H on the tumor cells reduced tumorigenicity and induced immunity to subsequent challenge with parental tumor cells. The immune protection correlates with an enhanced cytotoxic T lymphocyte (CTL) response against P1A, the major tumor antigen expressed in the J558 tumor. To understand the mechanism of immune protection, we adoptively transferred transgenic T cells specific for tumor antigen P1A into mice that bore P1A-expressing tumors. We found that while the transgenic T cells divided faster in mice bearing the B7H(+) tumors, optimal B7H-induced clonal expansion of P1CTL required costimulation by B7-1 and B7-2 on the endogenous host antigen-presenting cells (APCs). Interestingly, when B7H(+) and B7H(-) tumors were coinjected, P1CTL selectively eliminated the B7H(+) tumor cells. Moreover, B7H expressed on the tumor cells made them highly susceptible to destruction by CTL in vivo, even if the CTL was administrated into mice with large tumor burdens. Tumors that recurred in the P1CTL-treated mice lost transfected B7H and/or H-2L(d), the class I molecule that presents the P1A peptide. Taken together, our results reveal that B7H costimulates clonal expansion of, and cognate destruction by CD8(+) T lymphocytes in vivo.     

Identification and characterization of a WT1 (Wilms Tumor Gene) protein-derived HLA-DRB1*0405-restricted 16-mer helper peptide that promotes the induction and activation of WT1-specific cytotoxic T lymphocytes

Effective tumor vaccine may be required to induce both cytotoxic T lymphocyte (CTL) and CD4+ helper T-cell responses against tumor-associated antigens. CD4+ helper T cells that recognize HLA class II-restricted epitopes play a central role in the initiation and maintenance of antitumor immune responses. The Wilms tumor gene WT1 is overexpressed in both leukemias and solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for cancer immunotherapy. In this study, we identified a WT1 protein-derived 16-mer peptide, WT1(332)(KRYFKLSHLQMHSRKH), which was restricted with HLA-DRB1*0405, one of the most common HLA class II types in Japanese, as a helper epitope that could elicit WT1-specific CD4+ T-cell responses. We established a WT1(332)-specific CD4+ helper T-cell clone (E04.1), which could respond to both HLA-DRB1*0405-positive, WT1-expressing transformed hematopoietic cells and autologous dendritic cells pulsed with apoptosis-induced WT1-expressing cells, indicating that the WT1(332) was a naturally processed helper epitope. Stimulation of peripheral blood mononuclear cells with both the CTL epitope (WT1(235)) and the helper epitope (WT1(332)) in the presence of WT1(332)-specific TH1-type CD4+ T cell clone strikingly enhanced the induction and the functional activity of WT1(235)-specific CTLs compared with that of peripheral blood mononuclear cells with the WT1(235) alone. These results indicated that a helper epitope, WT1(332) should be useful for improvement of the efficacy of CTL epitope-based cancer vaccine targeting WT1 in the clinical setting.     

Mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer

Given the high fatality rate of pancreatic cancer, an effective treatment for this devastating disease is urgently needed. We have shown that mesothelin expression was higher in human pancreatic cancer cells than in human pancreatic duct epithelial cells, and mesothelin mRNA was substantially overexpressed in 18 of 21 (86%) clinical pancreatic adenocarcinoma specimens when compared with the surrounding normal tissues. However, the biological functions of mesothelin in tumor progression are not clearly understood. Here we studied the effects of mesothelin overexpression in pancreatic cancer cell proliferation and migration in vitro and pancreatic cancer progression in vivo. We found that forced expression of mesothelin significantly increased tumor cell proliferation and migration by 90% and 300%, respectively, and increased tumor volume by 4-fold in the nude mice xenograft model when compared with the vector control cell line. Silencing of mesothelin inhibited cell proliferation and migration in pancreatic cancer cells and ablated tumor progression in vivo. Vaccination with chimeric virus-like particles that contain human mesothelin substantially inhibited tumor progression in C57BL/6J mice. The increases in mesothelin-specific antibodies and CTL activity and the decrease in regulatory T cells correlated with reduced tumor progression and prolonged survival. This study revealed novel functions of mesothelin and suggested a new therapeutic vaccine strategy whereby mesothelin is targeted to control pancreatic cancer progression.     

Generation of tumor-associated cytotoxic T lymphocytes requires interleukin 4 from CD8(+) T cells.

Activation of tumor-associated CD8(+) cytotoxic T lymphocytes (CTLs) often requires antigen representation, e.g., by dendritic cells (DCs), and CD4(+) T cell help. Previously, we showed that CTL-mediated tumor immunity required interleukin 4 (IL-4) during the immunization but not effector phase. To determine the source and target cells of IL-4, we performed adoptive T cell transfers using CD4(+) and CD8(+) T cells from IL-4(-/-) and IL-4R(-/-) mice and analyzed CTL generation. Even though necessary for CTL generation, CD4(+) T cells did not need to express IL-4 or IL-4R. Surprisingly, CTL generation required IL-4 but not IL-4R expression by CD8(+) T cells. As IL-4 (a) was expressed by naive CD8(+) T cells within 24 h after antigen encounter, (b) IL-4 induced DC maturation, and (c) CTL development was impaired in T cell-reconstituted IL-4R(-/-) mice, CD8(+) T cell-derived IL-4 appears to act on DCs. We conclude that CD4(+) and CD8(+) T cells provide different signals for DC activation during CTL generation.     

Fine dissection of a nine amino acid glycoprotein epitope, a major determinant recognized by lymphocytic choriomeningitis virus-specific class I-restricted H-2Db cytotoxic T lymphocytes

We define a nine-amino acid (aa) sequence of VAL-GLU-ASN-PRO-GLY-GLY-TYR-CYS-LEU as a major epitope for immunologic recognition of lymphocytic choriomeningitis virus (LCMV) by H-2b-restricted CTL. The epitope was characterized using molecular genetics to bracket broadly and chemistry to precisely identify aa residues 278-286 of the viral glycoprotein. The epitope's composition is characteristic of a reverse (beta turn) but not an amphipathic alpha helix. A series of peptides with a single aa substitution in position 278 of VAL with other nonpolar (hydrophobic) amino acids (LEU, ILE, ALA, or GLY) coat targets that are recognized and lysed by CTL clones recognizing this epitope. In contrast, substitution of VAL with either large aromatic amino acids (that add bulk: PHE, TYR) or polar side chains (SER, THR) segregates CTL clones normally recognizing aa 278-286 into two groups, one that remains lytic (permissive) despite these changes and another that fails to lyse, indicating CTL can discriminate at a single aa. A change in charge at this position (VAL----ASP or GLU), in general, reduces CTL lysis while a change of VAL to LYS or ASN has minimal affect for four of the five CTL clones analyzed. CTL reactivity with the viral epitope is restricted by the Db but not the Kb of the murine MHC haplotype. A 16-aa peptide of Db that spans alpha 1 residues 37-52 blocks CTL lysis, whereas the corresponding Kb peptide that differs from Db in a single aa in position 50 does not.     

Vaccines targeting the cancer-testis antigen SSX-2 elicit HLA-A2 epitope-specific cytolytic T cells

The cancer-testis antigen synovial sarcoma X breakpoint-2 (SSX-2) is a potentially attractive target for tumor immunotherapy based upon its tissue-restricted expression to germline cells and its frequent expression in malignancies. The goal of this study was to evaluate genetic vaccine encoding SSX-2 to prioritize human leukocyte antigen (HLA)-A2-specific epitopes and determine if a DNA vaccine can elicit SSX-2-specific cytotoxic T lymphocytes (CTLs) capable of lysing prostate cancer cells. HLA-A2-restricted epitopes were identified based on their in vitro binding affinity for HLA-A2 and by the ability of a genetic vaccine to elicit peptide-specific CTL in A2/DR1 (HLA-A2.1+/HLA-DR1+/H-2 class I-/class II-knockout) transgenic mice. We found that SSX-2 peptides p41-49 (KASEKIFYV) and p103-111 (RLQGISPKI) had high affinity for HLA-A2 and were immunogenic in vivo; however, peptide p103-111 was immunodominant with robust peptide-specific immune responses elicited in mice vaccinated with a plasmid DNA vaccine encoding SSX-2. Furthermore, p103-111-specific CTLs were able to lyse an HLA-A2+ prostate cancer cell line. The immunodominance of this epitope was found not to be due to a putative HLA-DR1 epitope (p98-112) flanking p103-111. Finally, we demonstrated that SSX-2 epitope-specific CTLs could be detected and cultured from the peripheral blood of HLA-A2+ prostate cancer patients, notably patients with advanced prostate cancer. Overall, we conclude that SSX-2 peptide p103-111 is an immunodominant HLA-A2-restricted epitope, and epitope-specific CD8 T cells can be detected in patients with prostate cancer, suggesting that tolerance to SSX-2 can be circumvented in vivo. Together, these findings suggest that SSX-2 may be a relevant target antigen for prostate cancer vaccine approaches.     

Cervical cancer cells induce apoptosis of cytotoxic T lymphocytes

The goal of immunotherapy is to eliminate tumors by generating tumor-specific cytotoxic T lymphocytes (CTLs) in patients or by adoptively transferring ex vivo-activated CTLs into patients. Clinical trials have shown that tumor-specific CTLs often disappear before tumors are completely eliminated. In this study, the authors show that CTLs specific for cervical tumor cells undergo apoptosis after they are co-cultured with cervical tumor cells. The established cervical tumor cell lines and cervical cancer tissues express CD95 (Fas/Apo-1) ligand. The tumor cell-induced T-cell apoptosis can be blocked by an inhibitory anti-CD95 (APO-1/Fas) antibody, indicating that tumor cells induce apoptosis of CTLs through CD95-CD95 ligand interaction. Addition of interleukin-2 (IL-2) and IL-7 into the culture rescues the CTL from tumor cell-induced apoptosis. The rescued T cells retain their full antitumor cytotoxicity. These data suggest that human cervical tumor cells might actively down-regulate a cellular immune response by inducing apoptosis of specific T cells during immunotherapy. Local use of IL-2 and IL-7 as adjuvants may promote survival of the CTL and, thus, enhance the efficacy of immunotherapy.     

Lymphotactin cotransfection enhances the therapeutic efficacy of dendritic cells genetically modified with melanoma antigen gp100

Lymphotactin (Lptn) is a C chemokine that attracts T cells and NK cells. Dendritic cells (DC) are highly efficient, specialized antigen-presenting cells and antigen-pulsed DC has been regarded as promising vaccines in cancer immunotherapy. The aim of our present study is to improve the therapeutic efficacy of DC-based tumor vaccine by increasing the preferential chemotaxis of DC to T cells. In this study, Lptn and/or melanoma-associated antigen gp100 were transfected into mouse bone marrow-derived DC, which were used as vaccines in B16 melanoma model. Immunization of C57BL/6 mice with DC adenovirally cotransfected with Lptn and gp100 (Lptn/gp100-DC) could enhance the cytotoxicities of CTL and NK cells, increase the production of IL-2 and interferon-gamma significantly, as compared with immunization with gp100-DC, Lptn-DC, LacZ-DC, DC or PBS counterparts. The Lptn/gp100-DC immunized mice exhibited resistance to tumor challenge most effectively. It was found that the tumor mass of mice vaccinated by Lptn/gp100-DC showed obvious necrosis and inflammatory cell infiltration. In vivo depletion analysis demonstrated that CD8(+) T cells are the predominant T cell subset responsible for the antitumor effect of Lptn/gp100-DC and CD4(+) T cells were necessary in the induction phase of tumor rejection, while NK cells were less important although they participated in the antitumor response either in the induction phase or in the effector phase. In the murine model with the pre-established subcutaneous B16 melanoma, immunization with Lptn/gp100-DC inhibited the tumor growth most significantly when compared with other counterparts. These findings provide a potential strategy to improve the efficacy of DC-based tumor vaccines.     

KLIANNTRV是结核抗原Ag85A的HLA-A*0201限制性CTL表位

目的鉴定结核杆菌抗原Ag85A的HLA-A*0201限制性CTL优势表位。方法采用数据库SYFPEITHI初步预测结核抗原Ag85A的HLA-A*0201限制性CTL表位;经流式细胞术分析抗原肽与HLA-A*0201的亲和力;经时间荧光分辨法检测患者外周血单个核细胞(PBMCs)对抗原肽的增殖反应;最后通过细胞毒实验逐步鉴定Ag85A的HLA-A*0201限制性CTL预测的表位。结果实验证明,预测的位于Ag85A氨基酸序列(242-250aa)的肽表位与HLA-A*0201具有较高的亲和力。结论筛选出的KLIANNTRV(242-250aa)是结核杆菌抗原Ag85A的HLA-A*0201限制性CTL优势表位。     

bcr-abl融合基因真核表达载体诱导小鼠特异性免疫应答

目的在小鼠体内外研究bcr-abl融合基因真核表达载体诱导的特异性免疫应答,探索肿瘤免疫治疗的新途径。方法构建表达bcr-abl融合基因cDNA片段的真核细胞质粒pVbcr-abl,将 pVbcr-abl质粒用纳米颗粒聚乙烯亚胺包裹后给BALB／c小鼠肌内注射,检测小鼠血清中bcr-abl特异性抗体水平。小鼠免疫后20 d皮下接种具有相同遗传背景的SP2／0／ber-abl细胞(H-2~d),观察小鼠生存情况、移植肿瘤的生长情况和肿瘤细胞浸润情况。利用免疫组织化学方法观察肿瘤组织中T淋巴细胞浸润情况;流式细胞术分析免疫小鼠脾脏T细胞亚群的变化;LDH释放法检测脾脏细胞毒性T淋巴细胞(CTL)活性。结果成功构建真核表达载体pVbcr-abl,bcr-abl融合基因cDNA片段在真核细胞中可得到高效表达。pVbcr-abl免疫BALB／c小鼠能激活机体免疫系统,诱导产生特异性抗体和特异性CTL活性,并形成特异性免疫保护力。免疫小鼠的移植肿瘤形成时间、表面出现破溃时间、生长速度明显降低,荷瘤生存时间明显延长。免疫小鼠移植肿瘤组织中有大量CD3~+T细胞浸润。免疫小鼠的脾细胞杀伤SP2／0／bcr-abl细胞的细胞毒活性明显升高。小鼠脾脏T细胞亚群发生改变,CIM~+／ CD8~+细胞比值升高到1．54±0．29。结论pVbcr-abl真核表达载体除能在小鼠体内诱导产生特异性抗体以外,还能诱导高水平特异性CTL活性,直接杀伤肿瘤细胞,抑制肿瘤细胞的生长速度。     

p53基因修饰树突细胞诱导特异性抗肿瘤免疫应答的实验研究

目的　探讨含p5 3基因的质粒pC5 3 SN3转染的树突细胞 (DC)体外诱导特异性抗肿瘤细胞毒性T淋巴细胞 (CTL)。方法　应用脂质体介导将质粒pC5 3 SN3转染肺癌患者单个核细胞 [HLA A2 + ]衍生的DC ,然后与未经纯化的自体T细胞混合培养以诱导CTL(T pC5 3 SN3) ,并通过乳酸脱氢酶(LDH)释放实验测定其对p5 3基因突变的HLA A2 + 人肺癌细胞系Calu 6的杀伤活性。结果　用质粒pC5 3 SN3转染DC后 ,发现CD1a、CD83显著升高 ,转染前表达率分别为 (5 .4 5± 0 .89) %、(3.2 6± 0 .4 7) % ,转染后分别为 (5 2 .15± 11.5 6 ) %、(2 5 .78± 12 .35 ) %。但CD86 、CD4 0 、HLA DR等DC相关标志与转染前无明显改变。LDH释放实验显示 ,以Calu 6作为靶细胞 ,T pC5 3 SN3诱导的杀伤作用显著高于T IL 2(IL 2 10 0U ml刺激外周血单个核细胞产生的CTL) ,效靶比为 10∶1时其杀伤活性分别为 (5 6 .79±15 .6 7) %和 (39.33± 9.88) %。同时细胞表面标记检测可见T pC5 3 SN3细胞中以CD8+ 细胞为主 ,且CD6 9、CD4 5RO CD8表达均显著升高。结论　p5 3基因修饰的DC对p5 3突变的人肺癌细胞系可有效诱导HLA A2限制的特异性CTL。