Antitumor immune responses derived from transgenic expression of CD40 ligand in myeloma cells

Tumor cells engineered to express immunogenes have been used for cancer vaccines to induce the antitumor immunity and study the antitumor immune mechanisms derived from the immunogene expression. In the present study, we engineered a mouse myeloma cell line J558 with a cloned CD40 ligand (CD40L) gene. We demonstrated that (i) the engineered J558/CD40L tumor cells expressing the CD40 ligand molecule lost their tumorigenicity in syngeneic mice, and (ii) the inoculation of J558/CD40L tumor cells further lead to the protective immunity against wild-type J558 tumors. In animal studies using T-cell subset depleted mice, we further showed that the primary rejection of J558/CD40L tumors did not require T cells, but was mainly mediated by NK cells, whereas the effector phase of the protective immunity is mediated by CD8+ T cells. In addition, our data, for the first time, showed that the inoculation of engineered J558/CD40L tumor cells is able to stimulate stronger activation of dendritic cells with enhanced expression of B7-1 and ICAM-1 molecules than the wild-type J558 tumor cells Taken together, we demonstrated the antitumor effect of engineered J558/CD40L tumor cells that is mediated by the activation of the host dendritic cells in vivo. Our data indicate that the introduction of co-stimulatory CD40 ligand molecule will be useful as a new strategy of immunogene therapy against tumors.     

Treatment of hepatocellular carcinoma with adenoviral vector-mediated Flt3 ligand gene therapy

Fms-like tyrosine kinase 3 ligand (Flt3L) plays an important role in development and activation of dendritic cells (DCs) and natural killer cells (NK). It has been shown that administration of either tumor cells transfected in vitro with Flt3L vectors or soluble Flt3L fusion protein in a high dose can enhance host antitumor immunity in animal model systems. In this study, we developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter and investigated its biological efficacy in eliciting tumor-specific immune response against hepatocellular carcinoma in mouse hepatoma model. The constructed Ad-mFlt3L efficiently infected hepa 1-6 hepatoma cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCsNK cells and lymphocytes in local tumor tissues. Tumor cells infected with Ad-mFlt3L lost tumorigenicity and became more immunogenic in syngeniec animal models. Intratumoral injection of Ad-mFlt3L (10(9) expression-forming unit) x 3 significantly inhibited tumor growth with elicitation of long-lasting antitumor immunity, which is both preventive and curative. The tumor-specific immunity can be partially abrogated by depletion of either CD3+CD4+ T cells or NK cells and can be also re-established in na?ve animals by adoptive transfer of splenocytes from treated mice. The results suggest that adenovirus-mediated Flt3L gene therapy may provide a useful strategy for treatment of cancers.     

Induction of antitumor immunity by transduction of CD40 ligand gene and interferon-gamma gene into lung cancer

CD40-CD40 ligand (CD40L) interaction is an important costimulatory signaling pathway in the crosstalk between T cells and antigen-presenting cells. This receptor-ligand system is known to be essential in eliciting strong cellular immunity. Here we demonstrate that murine lung cancer cells (3LLSA) transduced with the CD40L gene (3LLSA-CD40L) were rejected in syngeneic C57BL/6 mice, but grew in CD40-deficient mice to the same extent as control tumor cells. Immunohistochemical study showed that inflammatory cells, including CD4+, CD8+ T cells and NK cells, infiltrated into the inoculated 3LLSA-CD40L tumor tissue. Inoculation of 3LLSA-CD40L cells into mice resulted in the induction of 3LLSA-specific cytotoxic T-cell immunity, and the growth of parental 3LLSA tumors was inhibited when 3LLSA cells were inoculated into C57BL/6 mice mixed with 3LLSA-CD40L cells or when they were rechallenged 4 weeks after 3LLSA-CD40L cells were rejected. Furthermore, co-inoculation of interferon (IFN)-gamma-transduced cells (3LLSA-IFNgamma) with 3LLSA-CD40L cells enhanced the antitumor immunity efficiently in vivo. These results indicate that the in vivo priming with CD40L- and IFN-gamma gene-transduced lung cancer cells is a promising strategy for inducing antitumor immunity in the treatment of lung cancer.     

Assessment of a combined, adenovirus-mediated oncolytic and immunostimulatory tumor therapy

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.t.) injection of adenovirus vectors into mice increases plasma levels of proinflammatory cytokines and chemokines, including TNF-alpha, INF-gamma, and MCP-1, which are potent inducers of dendritic cell maturation. Furthermore, we showed that in vivo expression of Flt3L from an adenovirus vector increases the number of CD11b+ and CD11c+ cells (populations that include dendritic cells) in the blood circulation. Based on these findings, we tested whether Ad.IR-E1A/TRAIL induced killing of tumor cells in combination with dendritic cell mobilization by Ad.Flt3L or, for comparison, Ad.GM-CSF would have an additive antitumor effect. As a model, we used immunocompetent C3H mice with syngeneic s.c. tumors derived from C3L5 cells. We found that vaccination of mice with C3L5 cells that underwent viral oncolysis in combination with Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression induces a systemic antitumor immune response. I.t. injection of the oncolytic and Flt3L expressing vectors into established tumors delayed tumor growth but did not cause efficient tumor elimination. This study shows the effectiveness of a combined oncolytic/immunostimulatory tumor therapy approach.     

Delivery of NKG2D ligand using an anti-HER2 antibody-NKG2D ligand fusion protein results in an enhanced innate and adaptive antitumor response

NKG2D ligands link the innate and adapative immune response by activating the receptors expressed on effector cells of both the innate (NK) and adaptive immune systems (CD8(+) T cells). In this study, we explored the potential therapeutic utility of this intersection by fusing the murine NKG2D ligand Rae-1β to the 3' end of an anti-HER2 IgG3 antibody containing an intact Fc domain (anti-HER2 IgG3-Rae-1β), thereby targeting an NK cell activation signal to HER2+ breast tumor cells. The antitumor efficacy of this anti-HER2-Rae-1β fusion protein was examined in a mouse mammary tumor model engineered to express HER2 (EMT6-HER2 cells). We observed an enhanced cytotoxic response of NK effectors against EMT-HER2 cells in vitro. Mice implanted on one flank with EMT6-HER2 cells and contralaterally with control EMT6 cells exhibited rapid regression of EMT6-HER2 tumors but delayed regression of contralateral EMT6 tumors. IFNγ was implicated, given a lack of antitumor efficacy in IFNγ(-/-) mice. Depletion of either NK cells or CD8(+) T cells abrogated tumor growth inhibition, suggesting essential roles for each in the observed antitumor activity. Mice rejecting EMT6-HER2 tumors after anti-HER2-Rae-1β treatment showed markedly decreased tumor growth when rechallenged with EMT6-HER2 or EMT6 cells, whereas both EMT6 and EMT6-HER2 cells grew in control mice, indicating the development of an adaptive memory response. Our findings demonstrate that administration of an antibody-NKG2D ligand fusion protein can enhance innate and adaptive immune antitumor responses, also evoking additional nontargeted antigens to enhance the potential clinical utility of this approach.     

Adenovirus vector-mediated in vivo gene transfer of OX40 ligand to tumor cells enhances antitumor immunity of tumor-bearing hosts

OX40 ligand (OX40L), the ligand for OX40 on activated CD4+ T cells, has adjuvant properties for establishing effective T-cell immunity, a potent effector arm of the immune system against cancer. The hypothesis of this study is that in vivo genetic engineering of tumor cells to express OX40L will stimulate tumor-specific T cells by the OX40L-OX40 engagement, leading to an induction of systemic antitumor immunity. To investigate this hypothesis, s.c. established tumors of three different mouse cancer cells (B16 melanoma, H-2b; Lewis lung carcinoma, H-2b; and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of a recombinant adenovirus vector expressing mouse OX40L (AdOX40L). In all tumor models tested, treatment of tumor-bearing mice with AdOX40L induced a significant suppression of tumor growth along with survival advantages in the treated mice. The in vivo AdOX40L modification of tumors evoked tumor-specific cytotoxic T lymphocytes in the treated host correlated with in vivo priming of T helper 1 immune responses in a tumor-specific manner. Consistent with the finding, the antitumor effect provided by intratumoral injection of AdOX40L was completely abrogated in a CD4+ T cell-deficient or CD8+ T cell-deficient condition. In addition, ex vivo AdOX40L-transduced B16 cells also elicited B16-specific cytotoxic T lymphocyte responses, and significantly suppressed the B16 tumor growth in the immunization-challenge experiment. All of these results support the concept that genetic modification of tumor cells with a recombinant OX40L adenovirus vector may be of benefit in cancer immunotherapy protocols.     

T-cell-dependent antitumor effects produced by CD40 ligand expressed on mouse lung carcinoma cells are linked with the maturation of dendritic cells and secretion of a variety of cytokines

CD40/CD40 ligand (CD40L) interaction plays an essential role in cell-mediated immune responses. We examined whether expression of CD40L in murine lung carcinoma (A11) cells could produce antitumor effects. The proliferation rate in vitro of A11 cells transfected with the murine CD40L gene (A11/CD40L) was not different from that of parent cells; however, half of the immunocompetent mice inoculated with A11/CD40L cells did not form tumors and the growth of A11/CD40L tumors developed in the rest of mice was significantly retarded compared with that of parent tumors. Protective immunity was also induced in the mice that had rejected A11/CD40L cells. In T-cell-defective nude mice, these antitumor effects were not observed. Bone-marrow-derived dendritic cells (DCs), when cultured with A11/CD40L cells, formed clusters with the tumors and showed upregulated CD86 expression. Expression of the interleukin-23 (IL-23) p19, IL-12p35, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with A11/CD40L but not with A11 cells, and P40, the subunit of both IL-12 and IL-23, was secreted from the cocultured DCs. These data directly showed that the expression of CD40L in tumors facilitated the interaction between DCs and the tumors, enhanced the maturation of DCs, induced secretion of cytokines, and consequently produced T-cell-dependent systemic immunity.     

Antitumor immunity against bladder cancer induced by ex vivo expression of CD40 ligand gene using retrovirus vector

The interaction between CD40 ligand (CD40L) and CD40 on antigen-presenting cells is essential for the initiation of antigen-specific T-cell responses. In order to clarify whether the expression of CD40L in tumor cells might be useful as a systemic therapy against bladder cancer, we investigated the antitumor immunity induced by CD40L in the mouse bladder cancer cell line MBT2. MBT2 was transduced by the retroviral vector expressing CD40L (MBT2-CD40L). Mouse bone marrow-derived dendritic cells cocultured with MBT2-CD40L cells produced eight times more IL-12 than those cocultured with parental MBT2 cells. In animal studies, subcutaneously inoculated MBT2-CD40L cells were rejected promptly. The vaccination of MBT2-CD40L cells induced antitumor immunity against parental tumors at a distant site. However, the antitumor effect of MBT2-CD40L inoculation was insufficient against pre-existing tumors. In the vaccination model, antibody ablation studies revealed that CD4(+) T cells were required for antitumor immunity, and tumor-specific cytotoxicity of sera was demonstrated. These data demonstrated that the antitumor immunity induced by CD40L was effective in the vaccination model and suggested that immunogene therapy using CD40L may be a new strategy of systemic therapy against bladder cancer.     

Adenovirus‐mediated CD40L gene therapy induced both humoral and cellular immunity against rat model of hepatocellular carcinoma

Adenoviral‐vector expressing CD40L (AxCAmCD40L)‐mediated gene therapy was studied for treatment of hepatocellular carcinoma (HCC) using CD40 ligand (CD40L) complementary DNA in rats. The particular focus was whether humoral immunity took part in antitumor effect. When tumor cells transduced by AxCAmCD40L were implanted into the subcutaneous tissues of syngeneic rats, the tumor growth was suppressed. Intratumoral injection of AxCAmCD40L to pre‐existing tumor in rats also led to significant reduction of tumor size. When tumor cells were re‐implanted to prevention model rats and treatment model rats, no tumor growth was observed. Many studies to date have reported that cellular immunity induces antitumor immunity. However, the present study demonstrated that not only cellular immunity but also humoral immunity plays an essential role in a HCC model. These observations suggested that CD40L‐mediated immune gene therapy for HCC was very effective treatment by activation of both cellular and humoral immune system. (Cancer Sci 2008; 99: 2097–2103)     

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

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in their activation and is essential for induction of antigen-specific T-cell responses. In the present study, we investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express CD40L and pulsed with Mut1 tumor peptide. Our data show that transfection of DCs with recombinant adenovirus AdV-CD40L resulted in activation of DCs with up-regulated expression of proinflammatory cytokines (IL-1beta and IL-12), chemokines (RANTES, IP-10, and MIP-1alpha), and immunologically important cell surface molecules (CD54, CD80, and CD86). Our data also demonstrate that DCs transfected with AdV-CD40L (DC(CD40L)) are able to stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8(+) cytotoxic T-cell responses in vitro. Vaccination of mice with Mut1 peptide-pulsed control virus-transfected DC (DC(pLpA)) could only protect mice from challenge of a low dose (0.5 x 10(5) cells per mouse, 8/8 mice), but not a high dose (3 x 10(5) cells per mouse, 0/8 mice) of 3LL tumor cells. However, vaccination of Mut1 peptide-pulsed AdV-CD40L-transfected DC(CD40L) induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of 3LL tumor cells. Thus, DCs engineered to express CD40L by adenovirus-mediated CD40 ligand gene transfer may offer a new strategy in production of DC cancer vaccines.     

Combinational FLt3 ligand and granulocyte macrophage colony-stimulating factor treatment promotes enhanced tumor infiltration by dendritic cells and antitumor CD8(+) T-cell cross-priming but is ineffective as a therapy

Dendritic cells play significant roles in the development and maintenance of antitumor immune responses. Therapeutic recruitment of dendritic cells into the tumor microenvironment has the potential to result in enhanced antitumor T-cell cross-priming against a broad array of naturally processed and presented tumor-associated antigens. We have observed that the treatment of BALB/c mice bearing syngeneic CMS4 sarcomas with the combination of recombinant Flt3 ligand and recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) for five sequential days is sufficient to optimize the number of tumor-infiltrating dendritic cells (TIDC). However, despite the significant increase in the number of TIDCs, the therapeutic benefit of Flt3 ligand and GM-CSF treatment is minimal. Therapy-associated TIDCs do not exhibit a "suppressed" or "suppressor" phenotype in vitro, and their enhanced numbers in cytokine-treated mice were associated with increased levels of peripheral antitumor CD8(+) T effector cells and with an augmented population of CD8(+) tumor-infiltrating lymphocytes (TIL). These data suggest that Flt3 ligand + GM-CSF therapy of murine tumors fails at a mechanistic point that is downstream of specific T-cell priming by therapy-induced TIDCs and the recruitment of these T cells into the tumor microenvironment. Based on the enhanced infiltration of tumors by CD4(+)CD25(+) TIL in Flt3 ligand + GM-CSF-treated mice, this could reflect the dominant influence of regulatory T cells in situ.     

Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long- term remission of liver metastases in a mouse model

BACKGROUND: The success of immunomodulatory cancer therapy is frequently hampered by the transient nature of the antitumor immune response. We have shown previously in a mouse model that interleukin 12 (IL-12) generates a strong natural killer (NK) cell-mediated antitumor response and reduces liver metastases induced by a colon carcinoma cell line. However, only a small percentage of the treated animals developed the cytotoxic T-lymphocytic response required for a long-term systemic antitumor immunity. 4-1BB is a co-stimulatory molecule expressed on the surface of activated T cells. Interaction of 4-1BB with its natural ligand (4-1BBL) has been shown to amplify T-cell (especially CD8+)-mediated immunity. In this study, we investigated the effects of adenovirus-mediated gene therapy delivering both IL-12 and 4-1BBL genes on mice with hepatic metastases induced by colon cancer cells. METHODS: Syngeneic BALB/c mice received intrahepatic injection of poorly immunogenic MCA26 colon cancer cells. Various combinations of replication-defective adenoviruses expressing IL-12 and 4-1BBL genes were injected into the established liver tumors. Changes in tumor size and animal survival were then monitored. All statistical tests were two-sided. RESULTS: The long-term survival rate of mice treated with the combination of IL-12 and 4-1BBL was significantly improved over that of animals in the control group (P =.0001). In vivo depletion of NK cells or CD8+ T cells completely abolished the long-term survival advantage of the IL-12 plus 4-1BBL-treated animals (P<.002). Moreover, the systemic immunity induced by this combination treatment protected these animals against a subcutaneous challenge with parental MCA26 cells. CONCLUSION: Adenovirus-mediated transfer of IL-12 and 4-1BBL genes directly into liver tumors resulted in tumor regression that required both NK and CD8+ T cells and generated a potent, long-lasting antitumor immunity.     

Flt3 ligand delivered in a pluronic formulation prolongs the survival of mice with orthotopic pancreatic adenocarcinoma

Pancreatic adenocarcinoma is a devastating disease, characterized by asymptomatic development and extremely poor prognosis. Given the resistance of pancreatic cancer to standard chemo- and radiotherapy, we have focused on the development of immunotherapies for this disease. The number of dendritic cells (DCs), natural killer (NK) cells, and T-cells in the blood and secondary lymphoid organs is regulated by a group of hematopoietic growth factors, which includes fms-like tyrosine kinase-3 ligand (Flt3L). We have demonstrated previously that the bioavailability and in vivo half-life of Flt3L are increased by Flt3L formulation in the pluronic ProGelzx. In this study, we first examined the effectiveness of Flt3L delivered in ProGelz against subcutaneous (s.c.) pancreatic adenocarcinomas in mice. We found that an intramuscular (i.m.) injection of Flt3L in ProGelz significantly increased the survival of mice bearing s.c. pancreatic tumors, compared to the administration of phosphate-buffered saline (PBS) in ProGelz. We then tested Flt3L in ProGelz in an orthotopic pancreatic tumor model, and demonstrated that it significantly enhanced the survival of tumor-bearing mice, compared to PBS in ProGelz. Overall, these observations suggest that Flt3L formulated in ProGelz may have potential clinical utility as a treatment for pancreatic cancer.     

Coexpression of Flt3 ligand and GM-CSF genes modulates immune responses induced by HER2/neu DNA vaccine

DNA vaccine and dendritic cells (DCs)-based vaccine have emerged as promising strategies for cancer immunotherapy. Fms-like tyrosine kinase 3-ligand (Flt3L) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) have been exploited for the expansion of DC. It was reported previously that combination of plasmid encoding GM-CSF with HER2/neu DNA vaccine induced predominantly CD4(+) T-cell-mediated antitumor immune response. In this study, we investigated the modulation of immune responses by murine Flt3L and GM-CSF, which acted as genetic adjuvants in the forms of bicistronic (pFLAG) and monocistronic (pFL and pGM) plasmids for HER2/neu DNA vaccine (pN-neu). Coexpression of Flt3L and GM-CSF significantly enhanced maturation and antigen-presentation abilities of splenic DC. Increased numbers of infiltrating DC at the immunization site, higher interferon-gamma production, and enhanced cytolytic activities by splenocytes were prominent in mice vaccinated with pN-neu in conjunction with pFLAG. Importantly, a potent CD8(+) T-cell-mediated antitumor immunity against bladder tumors naturally overexpressing HER2/neu was induced in the vaccinated mice. Collectively, our results indicate that murine Flt3L and GM-CSF genes coexpressed by a bicistronic plasmid modulate the class of immune responses and may be superior to those codelivered by two separate monocistronic plasmids as the genetic adjuvants for HER2/neu DNA vaccine.     

Tumor regression induced by intratumor administration of adenovirus vector expressing CD40 ligand and naive dendritic cells

We have previously shown that in vivo genetic modification of tumors with an adenovirus (Ad) vector engineered to express CD40 ligand (AdmCD40L) induces tumor-specific CTLs and suppresses tumor growth in vivo. In the present study, we investigate the hypothesis that this treatment can be made more efficient with 10(2)-fold less Ad vector by also administering bone marrow-generated dendritic cells (DCs) to the tumor. Using AdmCD40L and the number of DCs that alone had no effect on tumor growth, the data show that the growth of CT26 (colon adenocarcinoma; H-2d) and B16 (melanoma; H-2b) murine s.c. tumors is significantly suppressed by direct administration of DCs into s.c. established tumors that had been pretreated with AdmCD40L 2 days previously. The antitumor effect produced by the combination therapy AdmCD40L + DCs correlated with in vivo priming of tumor-specific CTLs. The antitumor cell-mediated immunity was transferable to naive mice by spleen cells from AdmCD40L + DC-treated animals. The interactions between CD40L and CD40 within treated tumors were critical because tumor suppression was abrogated by coadministration to the tumors of neutralizing monoclonal antibody against CD40L along with the DCs. Finally, in vivo depletion and knockout mice experiments demonstrated that tumor regression produced by this combination therapy depends on CD8+ T cells, but not on CD4+ T cells. These findings should be useful in designing strategies for use of DCs and AdmCD40L in cancer immunotherapy.     

Intratumoral administration of immature dendritic cells following the adenovirus vector encoding CD40 ligand elicits significant regression of established myeloma

Our previous study showed that J558 myeloma cells engineered CD40L lost their tumorigenicity in syngeneic mice, and the inoculation of J558/CD40L tumor cells further led to the protective immunity against wild tumors. In the present study, we investigated whether the vaccine can exert more efficient antitumor immunity by combination with adenovirus mediated CD40L gene therapy and immature dendritic cells (iDCs). The results demonstrated that intratumoral administration of iDCs 2 days after AdVCD40L injection, not only significantly suppressed the tumor growth, but also eradiated the established tumors in 40% of the mice. The potent antitumor effect produced by the combination therapy correlated with high expression of MHC, costimulatory and Fas molecules on J558 cells, which was derived from CD40L transgene expression. In addition, transgene CD40L expression could dramatically induce J558 cell apoptosis. Effectively capturing apoptotic bodies by iDCs in vivo could induce DC maturation, prime tumor-specific CTLs and tend to Th1-type immune response. Finally, in vivo depletion experimentation suggested both CD4+ and CD8+ T cells were involved in mediating the antitumor immune responses of combined treatment of AdVCD40L and iDCs, with CD8+ T cells being the major effector. These findings could be beneficial for designing strategies of DCs vaccine and CD40L for anticancer immunotherapy.     

Flt3配体对多器官功能障碍综合征小鼠脏器功能和结构的保护作用

目的观察Flt3配体（Flt3L）对酵母多糖致多器官功能障碍综合征（multiple organ dysfunc-tion syndrome,MODS）小鼠多脏器功能和结构损伤的防护作用。方法采用腹腔注射酵母多糖的方法复制小鼠MODS模型。雄性C57BL/6小鼠随机分为正常组（n=10）,MODS组（n=30）和Flt3L治疗组（n=30）。MODS组在致伤后腹腔注入生理盐水; Flt3L治疗组在致伤后第5 d起腹腔注射Flt3配体（5μg/kg）,连续给药7 d。观察酵母多糖致伤12 d动物死亡率,检测各组血丙氨酸转氨酶活性（alanine aminotransforase activity,ALT）、尿素氮（blood urea nitrogen,BUN）和肌酐（cre-atinine,Cr）水平,外周血T淋巴细胞亚群变化,镜下观察致伤后12 d动物肝、肺、肾、心等脏器的组织病理学改变。结果在酵母多糖致伤后12 d,MODS组小鼠死亡率达18%,Flt3L治疗组的小鼠死亡率为7%。MODS组小鼠血浆AIT、BUN和Cr在伤后12 d升高,而同时间点经Flt3L治疗的小鼠血浆AIT、BUN和Cr趋于正常,明显低于MODS组。MODS组小鼠肝脏、肺脏、肾脏和心脏发生明显的病理改变,主要表现为脏器实质细胞多呈变性与灶状坏死改变,而Flt3L治疗组小鼠上述病变明显减轻。MODS组小鼠CD4＋T细胞数目减少,而CD8＋T细胞数目呈明显增高,CD4/CD8比值显著下降; 而Flt3L治疗组CD4＋T细胞数目增加恢复至正常水平,CD4/CD8比值较MODS组明显上调。结论给予Flt3L可以改善机体免疫状态,降低MODS期动物的死亡率,减轻脏器功能和结构的损伤,对MODS期的脏器损伤具有保护作用。     

Immunization with wild-type p53 gene sequences coadministered with Flt3 ligand induces an antigen-specific type 1 T-cell response.

We examined the ability of immunization with sequential adenovirus/plasmid DNA vectors expressing human wild-type p53 to stimulate a type 1 T-cell response and induce protection against challenge from a metastatic tumor that expresses mutated murine p53. We found that tumor protection and an antigen (Ag)-specific immune response were enhanced by prior injection of Flt3 ligand (Flt3L) at a dose and schedule that significantly increased dendritic cell (DC) number and frequency. Preliminary studies using enzyme-linked immunospot and Winn assays suggested that Ag-specific CD8 cells, with their significant increase in IFN-gamma-secreting activity (Tc1 cells), were responsible for the tumor protection. The delayed-type hypersensitivity response to p53 was increased in mice immunized with p53 alone or p53 and Flt3L compared with a negative control. In contrast, spleen cells from mice immunized with p53 and Flt3L exhibited a higher Ag-specific proliferative response than mice immunized with p53 alone. The frequencies of Ag-specific IFN-gamma and interleukin (IL)-4-secreting cells were determined using an enzyme-linked immunospot assay, which demonstrated that the frequency of IFN-gamma-secreting cells was significantly higher in mice immunized with p53 and Flt3L than in mice receiving Flt3L, excipient, or p53 treatment alone. In contrast, the frequency of IL-4-secreting cells did not differ significantly among these groups. We also observed an increased frequency of IL-12 and IFN-gamma-secreting cells (but not IL-4 or IL-10) in the spleens of mice immediately after 10 days of Flt3L treatment, which was also the day of p53 priming. This observation supports the likelihood that there are multiple mechanisms of Flt3L adjuvant activity, including expansion of DC and type 1 T-cell number. Overall, these results suggest that immunization with p53 genetic sequences after in vivo expansion of DC, using Flt3L, provides a useful strategy to induce p53-specific, and protective, type 1 T-cell responses.     

Therapeutic vaccination against murine lymphoma by intratumoral injection of recombinant fowlpox virus encoding CD40 ligand

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 on antigen-presenting cells is essential for the initiation of cell-mediated and humoral immune responses. Malignant B cells also express CD40 and respond to CD40L by enhancing expression of costimulatory molecules. In this study, we investigated the therapeutic antitumor effect of intratumoral administration of recombinant fowlpox virus encoding murine CD40L (rF-mCD40L) in a murine B-cell lymphoma model. BALB/c mice with established s.c. and widely metastatic A20 lymphoma tumors were treated with intratumoral injections of rF-mCD40L together with systemic chemotherapy. This combined chemoimmunotherapy resulted in complete tumor regression and long-term survival of the mice. Some tumor cells in the injected sites expressed the CD40L transgene and had increased expression of the CD80 and CD86 costimulatory molecules. The therapeutic effect was dependent on CD8 but not on CD4 T cells. Moreover, there was a requirement that the recombinant CD40L virus be injected directly into the tumor, as opposed to peritumoral or distant sites. Thus, rF-mCD40L injected directly into the tumor microenvironment enhances the immunogenicity of tumor B cells. The results support future plans for intratumoral injection of rF-mCD40L in patients with lymphoma.     

Induction of protective immunity to RM-1 prostate cancer cells with ALVAC-IL-2/IL-12/TNF-alpha combination therapy

Human prostate cancers characteristically express low levels of major histocompatibility complex (MHC) Class I, which makes it challenging to induce protective antitumor responses involving T cells. Here we demonstrate that a whole cell tumor vaccine can induce protective T cell immunity to a low MHC Class I-expressing mouse prostate cancer cell line, RM-1. ALVAC recombinant canarypox viruses encoding interleukin-2, interleukin-12 and tumor necrosis factor-alpha were used to create therapeutic vaccines in 2 different ways. The RM-1 cells were pre-infected in vitro with the viruses prior to injection (pre-infection vaccine) or the RM-1 cells were injected alone, followed by the viruses (separate injection vaccine). The vaccines were each tested subcutaneously or intradermally. The pre-infection vaccine resulted in 100% clearance of primary tumors, whereas intradermal delivery of the separate injection vaccine cleared 40-60% of primary tumors. Despite the highly efficient primary tumor clearance by the pre-infection vaccine, only the separate injection vaccine generated protection upon rechallenge. Tumor-free survival induced by the separate injection vaccine required natural killer (NK) cells, CD4(+), and CD8(+) T cells. None of these cells alone were sufficient to induce tumor-free survival to the primary challenge, demonstrating an important cooperativity between NK cells and T cells. Secondary clearance of tumors also required NK and CD8(+) T cells, but not CD4(+) T cells. We report for the first time the generation of T cell immunity to the RM-1 prostate cancer cell line, demonstrating that it is possible to generate protective T cell immunity to a MHC I-low expressing tumor.