In vitro analysis of the antileukemic effect of tumor necrosis factor-alpha gene therapy with myeloid progenitor cells: the role of dendritic cells

We have previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) gene therapy with transgene-expressing myeloid progenitor cells (32DTNF-alpha) is effective in inhibiting the progression of leukemia with a lethal dose of murine 32Dp210 myeloid leukemia cells. Because TNF-alpha has been shown to induce the activation and maturation of dendritic cells (DCs), we investigated the effect of TNF-alpha secreted by transduced cells (32DTNF-alpha cells) on the activation of DCs and their role in the production of antileukemic cytotoxic T lymphocytes (CTLs). We demonstrate that administration of 32DTNF-alpha cells to the mice enhances the allo-stimulatory capacity of the splenic (CD11c+) and bone marrow-derived DCs in both mixed leukocyte response and CTL development. The enhanced allo-stimulatory capacity of splenic DCs from mice injected with 32DTNF-alpha cells correlated with increase in the cell-surface expression of the costimulatory molecules CD40, CD80, CD86, and major histocompatibility complex (MHC) class II molecules (I-Ak), and production of interleukin-12 (IL-12). Furthermore, administration of 32DTNF-alpha cells during immunization with irradiated 32Dp210 leukemia cells augmented the capacity of splenic DCs to stimulate antileukemic CTL response in spleen cells. Collectively, these data suggest that in vivo production of TNF-alpha by transduced cells enhances the phenotypic and functional activation of DCs, resulting in induction of a stronger antileukemic cytotoxic T-cell immune response.     

局部注射小鼠白细胞介素12逆转录病毒包装细胞对大鼠肝癌的治疗

目的:研究低剂量环磷酸胺(Cy)联合MHC Ⅰ类限制性肿瘤抗原多肽Mutl致敏、白细胞介素2(IL-2)基因修饰的树突状细胞(DCs)对转移性肺癌小鼠的治疗作用及其免疫学机理.方法:制备小鼠骨髓来源的DCs,用转移性Lewis肺癌特异性多肽Mutl预激经IL-2基因修饰的DCs联合低剂量Cy治疗转移性肺癌小鼠.通过FACS分析其脾细胞内T淋巴细胞比例的变化,~51Cr释放法检测CTL和NK细胞杀伤活性.结果:肿瘤抗原多肽致敏、IL-2基因修饰的DCs与小剂量Cy联合后,能比单用DCs更有效地治疗转移性肺癌,小鼠脾细胞中CD8~ T细胞和NK1.1~ 细胞明显比例升高,联合治疗组诱导出的CTL杀伤活性最高.结论:以肿瘤抗原多肽冲击致敏的IL-2基因修饰的DCs联合小剂量Cy能更有效地促进荷瘤宿主免疫应答,具有显著地体内抑制肺癌转移的效果.     

低剂量环磷酰胺增强MHCⅠ类限制性肿瘤抗原多肽冲击致敏的树突状细胞的抗肿瘤转移效果

目的:研究低剂量环磷酸胺(Cy)联合MHC Ⅰ类限制性肿瘤抗原多肽Mutl致敏、白细胞介素2(IL-2)基因修饰的树突状细胞(DCs)对转移性肺癌小鼠的治疗作用及其免疫学机理.方法:制备小鼠骨髓来源的DCs,用转移性Lewis肺癌特异性多肽Mutl预激经IL-2基因修饰的DCs联合低剂量Cy治疗转移性肺癌小鼠.通过FACS分析其脾细胞内T淋巴细胞比例的变化,~51Cr释放法检测CTL和NK细胞杀伤活性.结果:肿瘤抗原多肽致敏、IL-2基因修饰的DCs与小剂量Cy联合后,能比单用DCs更有效地治疗转移性肺癌,小鼠脾细胞中CD8~+T细胞和NK1.1~+细胞明显比例升高,联合治疗组诱导出的CTL杀伤活性最高.结论:以肿瘤抗原多肽冲击致敏的IL-2基因修饰的DCs联合小剂量Cy能更有效地促进荷瘤宿主免疫应答,具有显著地体内抑制肺癌转移的效果.     

Spleen-derived dendritic cells engineered to enhance interleukin-12 production elicit therapeutic antitumor immune responses

The major goal in cancer immunotherapy is the induction of tumor-specific T lymphocytes capable of killing tumor cells. As both dendritic cells (DCs) and interleukin-12 (IL-12) can play immunostimulatory roles in vivo, the use of a combination of these has become a promising approach. In the present study, we used a murine tumor model to examine whether spleen-derived DCs transduced with the IL-12 gene could elicit tumor-specific immune responses. BALB/c mice injected peritumorally with adenovirus-mediated IL-12 gene-transduced antigen-unpulsed DCs inhibited the growth of day 5-established subcutaneous CT26 tumors. Splenocytes from treated mice responded specifically to parental tumor cells and showed increased production of interferon gamma (IFN-gamma) and antitumor cytotoxic T-lymphocyte (CTL) activity. Increased numbers of both CD4(+) and CD8(+) T cells were detected in the treated tumors. The inhibition of tumor growth was significantly greater in mice injected with IL-12 gene-transduced DCs than in those injected with IL-12 gene-transduced fibroblasts or the IL-12 gene-encoding adenovirus itself. Taken together, these results indicate that DCs transduced with the IL-12 gene by a recombinant adenovirus are effective in inducing tumor-specific Th1 and CTL responses that inhibit the growth of established subcutaneous tumors.     

ANTITUMOR EFFECTS OF HUMAN IL-15 GENE MODIFIED LUNG CANCER CELL LINE

ＡＮＴＩＴＵＭＯＲＥＦＦＥＣＴＳＯＦＨＵＭＡＮＩＬ－１５ＧＥＮＥＭＯＤＩＦＩＥＤＬＵＮＧＣＡＮＣＥＲＣＥＬＬＬＩＮＥＳｈｅｎＹｏｎｇｑｕａｎ１沈永泉ＣｕｉＬｉａｎｘｉａｎ２崔莲仙ＨｅＷｅｉ１何维ＸｕｅＬｉ１薛莉ＢａＤｅｎｉａｎ１巴德年１Ｉｎｓｔ...     

A retrogen strategy for presentation of an intracellular tumor antigen as an exogenous antigen by dendritic cells induces potent antitumor T helper and CTL responses

Induction of an effective antitumor response requires CD4+ helper T (Th) cells to recognize antigens on the same dendritic cells (DCs) that cross-present CTL antigens. Such cross-presentation is difficult to achieve by current tumor vaccine strategies. Here, we develop a novel "Retrogen" strategy for DCs to efficiently cross-present an intracellular tumor antigen, MAGE-3, to both MHC class I and MHC class II in a cognate manner. Specifically, the MAGE-3 gene was linked to a leader sequence at its NH2 terminus for secretion and to a cell-binding domain at its COOH terminus for receptor-mediated internalization. DCs transduced with the modified MAGE-3 gene produced and secreted MAGE-3 proteins, which were efficiently taken up by DCs via receptor-mediated internalization and presented as exogenous antigens to class I and class II molecules. Immunization of mice with the transduced DCs expressing the MAGE-3 fusion protein, termed "Retrogen" for its retrograde transport/internalization after secretion, efficiently induced all arms of the adaptive antitumor immune responses. Thus, this retrogen strategy of using a unifying mechanism for DCs to cross-present an intracellular tumor antigen in a cognate manner could be generally used to improve the efficacy of tumor vaccines and immunotherapies.     

In vitro activation of cancer patient-derived dendritic cells by tumor cells genetically modified to express CD154

PURPOSE: Triggering of CD40 on antigen-presenting cells via its ligand CD154 is an important event in the initial phase of an immune response against cancer cells. In this study, we investigated the effects of adenoviral CD154 immunomodulatory gene therapy on the activation of human dendritic cells (DCs) in a well-defined in vitro system. EXPERIMENTAL DESIGN: Human bladder cancer cell lines and tumor cells from patients with renal cell carcinoma (RCC) were transduced with Ad-CD154 vectors or control vectors. Activation of human in vitro generated DCs after coculture with transduced tumor cells was analyzed. Therapeutic efficacy and cytotoxic T-lymphocyte (CTL) activity were assessed in a subcutaneous (s.c.) murine bladder cancer model. RESULTS: Human bladder cancer cell lines expressing CD154 showed a decreased growth rate, increased apoptosis, and modulated expression of molecules important for recognition by cytotoxic lymphocytes. Further, CD154-expressing allogeneic bladder tumor cell lines and autologous tumor cells from patients with renal cell cancer induced maturation of DCs and stimulated IFN-gamma production from lymphocytes cocultured with mature DCs. In vivo studies showed that CD154 gene therapy was highly effective in wild-type mice but only minimally effective in nude mice. Consequently, strong tumor-specific CTL activity was detected in mice vaccinated with tumor cells expressing CD154. CONCLUSIONS: Using tumor cell lines as well as patient-derived material, we could show that tumor cells expressing CD154 efficiently induce maturation and activation of DCs as well as activation of lymphocytes. Our murine in vivo studies demonstrate that lymphocytes contribute to the observed antitumor effect in a s.c. bladder tumor model. These studies should stimulate CD154 gene therapy approaches for the treatment of urologic malignancies.     

The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen

The T-helper 1 (Th1) immune reaction is most important in dendritic cell (DC)-based immunotherapy. Interleukin 12 (IL-12) and granulocyte macrophage colony-stimulating factor (GM-CSF) play a pivotal role in inducing Th1 and cytotoxic T lymphocyte (CTL) responses. In this study, DCs expressing the natural tumor antigen gp70 of BALB/c-derived CT26 were adenovirally transduced with the IL-12 gene and/or GM-CSF gene, and it was examined whether vaccinations using these genetically engineered DCs can induce strong therapeutic antitumor immunity. Mice were immunized once by subcutaneous (s.c.) injection with genetically modified DCs. The cytotoxic activity of splenocytes against CT26 was assayed in a 51Cr-release assay 14 days after immunization. The therapeutic efficacy of the vaccination was examined in s.c. tumor models. The cytotoxic activity of CTLs against CT26 in mice immunized with DCs expressing gp70 (DC-AxCAgp70) was significantly augmented by co-transduction with the GM-CSF/IL-12 gene (p<0.0001) and remarkably reduced by the depletion of CD4+ or CD8+ cells (p<0.01). The cytotoxic activity against CT26 of the plain spleen cells in mice immunized with DC-AxCAgp70/GM-CSF/IL-12 was significantly higher than that in mice immunized with DC-AxCAgp70 (p<0.0001), and this activity decreased to almost 50% upon the depletion of NK cells. Vaccinations using DC-AxCAgp70/GM-CSF/IL-12 or DC-AxCAgp70/IL-12 could elicit potent therapeutic immunity in s.c. tumor models; tumor-free mice were observed in these vaccination groups. However, there was no significant difference between these two groups. A vaccination therapy using DCs co-transduced with the TAA gene and Th 1-type cytokine genes, especially the IL-12 gene, is ideal for immunotherapy in terms of the activation of DCs, NK cells, CD4+ T cells and CD8+ T cells, and may be useful in the clinical application of a cancer vaccine therapy.     

Efficient antitumor immunity derived from maturation of dendritic cells that had phagocytosed apoptotic/necrotic tumor cells

Dendritic cells (DCs) that acquired antigen from apoptotic tumor cells are able to induce major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes and antitumor immunity. In the present study, we investigated the efficiency of antitumor immunity derived from DCs that had phagocytosed apoptotic/necrotic BL6-10 melanoma cells compared with that of DCs pulsed with the tumor mTRP2 peptide. Our data showed that phagocytosis of apoptotic/necrotic tumor cells resulted in maturation of DCs with up-regulated expression of proinflammatory cytokines [interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, interferon-gamma and granulocyte-macrophage colony-stimulating factor], chemokines (MIP-1alpha, MIP-1beta and MIP-2), the CC chemokine receptor CCR7 and the cell surface molecules (MHC class II, CD11b, CD40 and CD86), and down-regulated expression of the CC chemokine receptors CCR2 and CCR5. These mature DCs displayed enhanced migration toward the CC chemokine MIP-3beta in a chemotaxis assay in vitro and to the regional lymph nodes in an animal model in vivo. Our data also showed that vaccination with DCs that had phagocytosed apoptotic/necrotic BL6-10 cells was able to (i) more strongly stimulate allogeneic T-cell proliferation in vitro, (ii) induce an in vivo Th1-type immune response leading to more efficient tumor-specific cytotoxic CD8(+) T-cell-mediated immunity and (iii) eradicate lung metastases in all 6 vaccinated mice compared with mice vaccinated with DCs pulsed with the tumor mTRP2 peptide, in which lung metastases were reduced (mean number of 16 per mouse) but not completely eradicated. Therefore, DCs that had phagocytosed apoptotic/necrotic tumor cells appear to offer new strategies in DC cancer vaccines.     

Antitumor Activity of Lentivirus-mediated Interleukin -12 Gene Modified Dendritic Cells in Human Lung Cancer in Vitro

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associatedantigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whethertransduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigenspecificcytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocytederivedDCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated targetof the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCswas measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry.DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag).Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulatedby LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methylthiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfullyconstructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstratedgood stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumoreffects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have anenhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.     

Tumor cell lysate-pulsed dendritic cells induce a T cell response against colon cancer in vitro and in vivo

To investigate whether tumor cell lysate-pulsed (TP) dendritic cells (DCs) induce cytotoxic T lymphocyte (CTL) activity against colon cancer in vitro and in vivo. Hematopoietic progenitor cells were magnetically isolated from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analyzed by morphological observation and phenotype analysis. DCs were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. CTL activity and interferon gamma (IFNγ) secretion was evaluated ex vivo. In order to determine whether or not vaccination with CT26 TP DCs induce the therapeutic potential in the established colon tumor model, CT26 colon tumor cells were implanted subcutaneously (s.c.) in the midflank of naïve BALB/c mice. Tumor-bearing mice were injected with vaccination with CT26 TP DCs on days 3 and 10. Tumor growth was assessed every 2–3 days. Finally, CTL activity and IFNγ secretion were evaluated in immunized mice. Hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 days showed the character of typical mature DCs. Morphologically, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed high levels of MHC II, CD11b, CD80, and CD86 antigen, and were negative for CD8α. However, immature DCs cultured with cytokines for 5 days did not have typical DCs phenotypic markers. Ex vivo primed T cells with CT26 TP DCs were able to induce effective CTL activity against CT26 tumor cells, but not B16 tumor cells (E:T = 100:1, 60.36 ± 7.11% specific lysis in CT26 group vs. 17.36 ± 4.10% specific lysis in B16 group), and produced higher levels of IFNγ when stimulated with CT26 tumor cells but not when stimulated with B16 tumor cells (1210.33 ± 72.15 pg/ml in CT26 group vs. 182.25 ± 25.51 pg/ml in B16 group, P < 0.01). Vaccination with CT26 TP DCs could induce anti-tumor immunity against CT26 colon tumor in murine therapeutic models (tumor volume on day 19: CT26 TP DCs 342 ± 55 mm³ vs. the other control groups, P < 0.05). In addition, all splenic CD3⁺ T cells obtained from mice vaccinated with CT26 TP DCs produced high levels of IFNγ and shown specific cytotoxic activity against CT26 tumor cells, but no cytotoxic activity when stimulated with B16 tumor cells. Tumor cell lysate-pulsed DCs can induce tumor-specific CTL activity against colon cancer in vitro and in vivo.     

Dendritic cells transduced with tumor-associated antigen gene elicit potent therapeutic antitumor immunity: comparison with immunodominant peptide-pulsed DCs

Several studies have shown that vaccine therapy using dendritic cells (DCs) pulsed with specific tumor antigen peptides can effectively induce antitumor immunity. Peptide-pulsed DC therapy is reported to be effective against melanoma, while it is still not sufficient to show the antitumor therapeutic effect against epithelial solid tumors such as gastrointestinal malignancies. Recently, it has been reported that vaccine therapy using DCs transduced with a surrogate tumor antigen gene can elicit a potent therapeutic antitumor immunity. In this study, we investigated the efficacy of vaccine therapy using DCs transduced with the natural tumor antigen in comparison with peptide-pulsed DCs. DCs derived from murine bone marrow were adenovirally transduced with murine endogenous tumor antigen gp70 gene, which is expressed in CT26 cells, or DCs were pulsed with the immunodominant peptide AH-1 derived from gp70. We compared these two cancer vaccines in terms of induction of antigen-specific cytotoxic T lymphocyte (CTL) responses, CD4+ T cell response against tumor cells, migratory capacity of DCs and therapeutic immunity in vivo. The cytotoxic activity of splenocytes against CT26 and Meth-A pulsed with AH-1 in mice immunized with gp70 gene-transduced DCs was higher than that with AH-1-pulsed DCs. CD4+ T cells induced from mice immunized with gp70 gene-transduced DCs produced higher levels of IFN-gamma by stimulation with CT26 than those from mice immunized with AH-1-pulsed DCs (p < 0.0001), and it was suggested that DCs transduced with tumor-associated antigen (TAA) gene induced tumor-specific CD4+ T cells, and those CD4+ T cells played a critical role in the priming phase of the CD8+ T cell response for the induction of CD8+ CTL. Furthermore, DCs adenovirally transduced with TAA gene showed an enhancement of expression of CC chemokine receptor 7 and improved the migratory capacity to draining lymph nodes. In subcutaneous models, the vaccination using gp70 gene-transduced DCs provided a remarkably higher therapeutic efficacy than that using AH-1-pulsed DCs. These results suggested that vaccine therapy using DCs adenovirally transduced with TAA gene can elicit potent antitumor immunity, and may be useful for clinical application.     

Induction of potent antitumor immunity by intratumoral injection of interleukin 23-transduced dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in priming immune responses to tumor. Interleukin (IL)-23 can act directly on DC to promote immunogenic presentation of tumor peptide in vitro. Here, we evaluated the combination of bone marrow-derived DC and IL-23 on the induction of antitumor immunity in a mouse intracranial glioma model. DCs can be transduced by an adenoviral vector coding single-chain mouse IL-23 to express high levels of bioactive IL-23. Intratumoral implantation of IL-23-expressing DCs produced a protective effect on intracranial tumor-bearing mice. The mice consequently gained systemic immunity against the same tumor rechallenge. The protective effect of IL-23-expressing DCs was comparable with or even better than that of IL-12-expressing DCs. IL-23-transduced DC (DC-IL-23) treatment resulted in robust intratumoral CD8(+) and CD4(+) T-cell infiltration and induced a specific TH1-type response to the tumor in regional lymph nodes and spleen at levels greater than those of nontransduced DCs. Moreover, splenocytes from animals treated with DC-IL-23 showed heightened levels of specific CTL activity. In vivo lymphocyte depletion experiments showed that the antitumor immunity induced by DC-IL-23 was mainly dependent on CD8(+) T cells and that CD4(+) T cells and natural killer cells were also involved. In summary, i.t. injection of DC-IL-23 resulted in significant and effective systemic antitumor immunity in intracranial tumor-bearing mice. These findings suggest a new approach to induce potent tumor-specific immunity to intracranial tumors. This approach may have therapeutic potential for treating human glioma.     

Mice vaccination with interleukin 12-transduced colon cancer cells potentiates rejection of syngeneic non-organ-related tumor cells

Cell-based gene therapy after cytokine gene transfer is being investigated for autologous and allogeneic vaccination in cancer therapy. Here we show that mice vaccinated with 3-5 x 10(6) interleukin 12 (IL-12) gene-transduced CT26 colon cancer cells developed a long-lasting antitumor immune memory able to reject not only parental cells but also syngeneic, LM3 mammary, and MCE fibrosarcoma tumorigenic cells. In contrast, mice vaccinated with 0.5-1 x 10(6) CT26 cells transduced with pBabe neo IL-12 retrovirus cells (CT26-IL12) were only able to reject parental cells. An increase in the total circulating levels of IgG2a and a clear shift toward a systemic Th1 response developed, regardless of the amount of injected CT26-IL12 cells. On the contrary, a strong increase in anti-CT26-specific IgG2a levels was observed only when 3-5 x 10(6) CT26-IL12 cells were injected. Immunocompetent mice vaccinated with 3-5 x 10(6) CT26-IL12 cells developed local nodules for a few days, which then ceased growing. These nodules comprised mainly blood vessels, suggesting that an angiogenic process was taking place. CD8+ T cells were responsible for the anti-LM3 tumor cell memory, whereas CD4+ T cells were not involved. Splenocytes and lymphocytes obtained from mice immunized against CT26 cells were able to kill LM3 cells in vitro. Adoptive transfer of lymphocytes obtained from animals immunized against CT26 colon cancer cells suppressed LM3 mammary tumor growth in tumor-bearing mice. The present studies raised the possibility of isolating CTL clones and identifying CTL epitopes shared by different tumor cell types, which can be a target for cancer therapy.     

Fusion hybrid of dendritic cells and engineered tumor cells expressing interleukin-12 induces type 1 immune responses against tumor

AIMS AND BACKGROUND: Dendritic cell (DC)-tumor fusion hybrid vaccinees that facilitate antigen presentation represent a novel powerful strategy in cancer immunotherapy. Preclinical studies have demonstrated that IL-12 promotes specific antitumor immunity mediated by T cells in several types of tumors. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between DCs and engineered J558/IL-12 myeloma cells secreting Th1 cytokine IL-12. METHODS: The expression vector pcDNA-IL-12 was generated and transfected into J558 myeloma cells and then bone marrow-derived DCs were fused with engineered J558/IL-12 cells. The antitumor immunity derived from vaccination of the fusion hybrid DC/J558/IL-12 was evaluated in vitro and in vivo. RESULTS: DC/J558/IL-12 cells secreted recombinant IL-12 (1.6 ng/mL), and inoculation of BALB/c mice with DC/J558/IL-12 hybrid induced a Th1 dominant immune response and resulted in tumor regression. Immunization of mice with engineered DC/J558/IL-12 hybrid elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro as well as more potent protective immunity against J558 tumor challenge in vivo than immunization with the mixture of DCs and J558/IL-12, J558/IL-12 and J558, respectively. Furthermore, the anti-tumor immunity mediated by DC/J558/IL-12 tumor cell vaccination in vivo appeared to be dependent on CD8+ CTL. CONCLUSIONS: These results demonstrate that the engineered fusion hybrid vaccines that combine Th1 cytokine gene-modified tumor cells with DCs may be an attractive strategy for cancer immunotherapy.     

凋亡肿瘤细胞致敏的树突状细胞疫苗治疗肺癌的实验研究

目的：用凋亡肿瘤细胞致敏的树突状细胞（dendritic cells，DCs）激发肿瘤抗原特异性的细胞毒T细胞（cytotoxic T lymphocytes，CTL）活性，观察其体内外抗肺癌的特性。方法：常规方法从健康人外周血单个核细胞中诱导DCs，采用或不采用凋亡肿瘤细胞负载DCs，并利用激发型CD40单克隆抗体（CD40mAb）诱导DCs成熟；成熟DCs与自体T细胞共育，分别获得Ag-CTL及non-Ag-CTL，流式细胞仪检测Ag-CTL细胞表型的变化；^3H-TdR掺入法测定DNA片段形成率；建立人肺癌细胞株A549荷瘤裸鼠模型，过继回输Ag-CTL和non-Ag-CTL，评价其在体内的抗肿瘤活性。结果：CD40mAb激发可使DCs上调CD1a、CD80、CD86、CD83、HLR-DR的表达；凋亡肿瘤细胞负载联合CD40mAb激发可进一步促进DCs的成熟；成熟DCs和自体的T细胞共育活化后CD8^＋T细胞明显上调；Ag—CTL对A549具有高效特异的杀伤力，明显强干Ag-CTL对肝癌细胞株HepG2的作用（P〈0．01），且Ag-CTL对A549的杀伤力明显强于non—Ag-CTL（P〈0．01），而non-Ag-CTL对A549及HepG2细胞的杀伤力无显著性差异；体内实验表明，Ag-CTL可有效抑制裸鼠皮下移植瘤的生长，与生理盐水组（NS组）、non-Ag-CTL组相比在统计学意义上有显著差异（P〈0．05），non-Ag-CTL组与NS组相比在统计学意义上有差异（P〈0．05）。结论：凋亡肿瘤细胞致敏的树突状细胞疫苗激发的Ag—CTL在体内外均呈现抑制肺癌细胞的特性。     

Immune deviation and Fas-mediated deletion limit antitumor activity after multiple dendritic cell vaccinations in mice

Genetic immunization with a single injection of dendritic cells (DCs) expressing a model melanoma antigen generates antigen-specific, MHC-restricted, protective immune responses. After initiating the immune response, additional vaccinations may increase the protection or conversely downregulate the immune response. Groups of mice were vaccinated several times with DCs transduced with the MART-1 gene, and the anti-tumor protection was compared with that of mice receiving a single vaccination. C3H mice had poorer protection from a syngeneic MART-1-expressing tumor challenge with multiple vaccinations. This was accompanied by lower levels of splenic CTL effectors and a shift from a type 1 to a type 2 cytokine profile. On the contrary, multiple vaccinations in C57BL/6 mice generated greater in vivo antitumor protection with no decrease in splenic CTLs and no cytokine shift. Antiadenoviral humoral or cellular immune responses did not seem to contribute to these effects. When studies were performed in Fas receptor-negative C3H.(lpr) mice, the adverse effect of multiple vaccinations disappeared, and there was no cytokine shift pattern. In conclusion, C3H mice but not C57BL/6 mice receiving multiple vaccinations with DCs expressing the MART-1 tumor antigen show decreased protection associated with deviation from a type 1 to a type 2 cytokine response attributable to a Fas-receptor mediated clearance of antigen-specific IFN-gamma-producing cells.     

MIG (CXCL9) chemokine gene therapy combines with antibody-cytokine fusion protein to suppress growth and dissemination of murine colon carcinoma.

The induction of a CTL response capable of eradicating disseminated tumor metastases and the establishment of a persistent tumor-protective immunity remain major goals of cancer immunotherapy. Here, we demonstrate for the first time that the combination of interleukin 2 (IL-2) targeted to the tumor microenvironment by a recombinant antibody-IL-2 fusion protein (huKS1/4-IL-2) with gene therapy by the murine chemokine MIG (CXCL9) markedly reduced s.c. tumor burden and decisively suppressed dissemination of experimental lung metastases of CT26-KSA colon carcinoma in syngeneic BALB/c mice. This combined therapy significantly prolonged the life span of these mice 3-4-fold by concurrently delivering MIG and IL-2 to the tumor site and thereby achieving chemoattraction of T cells together with their activation. The antitumor effect obtained was mediated predominantly by MHC class I antigen-restricted CD8(+) T cells with help from MHC class II antigen-restricted CD4(+) T lymphocytes. In addition, the MIG chemokine also induced angiostatic effects in the tumor vasculature. Taken together, this combination of MIG chemokine gene therapy with tumor-targeted cytokine IL-2 provides an approach for the rational design of novel cancer immunotherapy modalities.     

Immunological properties and vaccine efficacy of murine dendritic cells simultaneously expressing melanoma-associated antigen and interleukin-12

Interleukin (IL)-12 is a key factor for inducing cellular immune responses, which play a central role in the eradication of cancer. In the present study, in order to create a dendritic cell (DC)-based vaccine capable of positively skewing immune response toward a cellular immunity-dominant state, we analyzed immunological characteristics and vaccine efficacy of DCs cotransduced with melanoma-associated antigen (gp100) and IL-12 gene (gp100+IL12/DCs) by using RGD fiber-mutant adenovirus vector (AdRGD), which enables highly efficient gene transduction into DCs. gp100+IL12/DCs could simultaneously express cytoplasmic gp100 and secretory IL-12 at levels comparable to DCs transduced with each gene alone. In comparison with DCs transduced with gp100 alone (gp100/DCs), upregulation of major histocompatibility complex class I, CD40, and CD86 molecules on the cell surface and more potent T-cell-stimulating ability for proliferation and interferon-gamma secretion were observed as characteristic changes in gp100+IL12/DCs. In addition, administration of gp100+IL12/DCs, which were prepared by a relatively low dose of AdRGD-IL12, could induce more potent tumor-specific cellular immunity in the murine B16BL6 melanoma model than vaccination with gp100/DCs. However, antitumor effect and B16BL6-specific cytotoxic T-lymphocyte activity in mice vaccinated with gp100+IL12/DCs diminished with increasing AdRGD-IL12 dose during gene transduction, and paralleled the decrease in presentation levels via MHC class I molecules for antigen transduced with another AdRGD. Collectively, our results suggested that optimization of combined vector dose was required for development of a more efficacious DC-based vaccine for cancer immunotherapy, which relied on genetic engineering to simultaneously express tumor-associated antigen and IL-12.