Antitumor Effects Of Adenovirus-mediated Expression Of LIGHT In A Murine B-cell Lymphoma Model

Malignant lymphoma ( ML ) originates from lymph node or outside tissue of lymph node or apparatus, and results from maliganant changing of lymphocyte or tissue cells. According to the difference between clinical and pathological characteristic, ML divides into two categories, which are Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL), the tumor originating from B cell is B cell lymphoma. At presnt, chemotherapy and radiotherapy are mainly therapys to B cell lymphoma.Although patients with B cell non-Hodgkin's lymphoma respond well to treatment with chemotherapy and radiotherapy, they are rarely cured due to the relapses that occur after a period of months or years with loss of response to the treatment. High dose chemo-radiotherapy can induce longer remissions, but unfortunately carry a high early mortality due to their substantial toxicity. The need for new treatment modalities has driven evaluation of various immunotherapies including recombinant human interferon-α (IFN-α), monoclonal antibody targeting (CD20, CD19, CD22) or anti-tumor vaccines. The existence of B cell lymphoma's surface antigen makes the immune therapy of B cell lymphoma come true.One of the reasons why tumor B cells evade the immune system is because of the lack of expression of important accessory molecules necessary for an efficient activation of antigenspecific T cells. Recent efforts have focused on changing the phenotype of tumor B cells so that tumor-specific T cells can better recognize them. One of the most interesting approaches for doing so has been the genetic modification of tumor B cells with the CD40L molecule. Briones J et al have showed that geneticmodification of tumor B cells with CD40L can be a useful strategy to promote systemic immunity against B-cell malignancies and provide an in vivo system to allow for additional evaluation and refinement of this approach.They found that after transduction with an adenovirus encoding CD40L (AdvCD40L), tumor cells abundantly express costimulatory molecules, and when used as a vaccine, they induce a protective systemic immune response in vivo against unmodified tumor cells. LIGHT is a novel member of tumour necrosis factor family, belongs to the same family with CD40L, so may has the similar effects to CD40L. Studies have found that LIGHT can induce the expression of some accessory molecules on human B cell leucocythemia cell, then can enhance antigen-presenting capacity.LIGHT(also known as TNFSF14, which is an acronym for homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D fro HVEM, a receptor expressed by T lymphocyte) is a member of the TNF superfamily that was maily expressed on activated T cells , NK cells, immature dendritic cells (iDCs) and some tumor cell lines. LIGHT has three receptors, HVEM, LTβRand TR6. Early studies have found that LIGHT can selectively induce the apoptosis of some tumor cells as other TNF superfamily members, Following studies have proved that it can provide costimulatory signals to T cells and induce the maturation of DCs. Tamada K et al reported that LIGHT was a co-stimualtory molecular for CD28-independent T cell activation and preferentially induced the IFN-7 and GM-CSF production. In a fibrosarcoma animal model, Yu P and coworkers revealed that constitutive expression of LIGHT upregulated the production of chemokines and the expression of adhension molecules in tumor tissues, resulting in the recruitment of naive T cells that were then effciently activated and expanded inside the tumor, which greatly enhanced the host's resistance to progressive tumorigenesis and further led to the rejection of well-established parental tumors at local and distant sites. Taking together, LIGHT can be a promising candidate forcancer gene therapy and the underlying mechanisms need to be extensively investigated in the future.In the present study, it suggested that adenovirus-mediated LIGHT could effectively prevent and resist B-cell lymphoma. Firstly, we studied antitumor effect of LIGHT-transduced A20 cells as a vaccine for B-cell lymphoma; furthermore, we studied the antitumor effects by adenovirus-mediated LIGHT in a murine B-cell lymphoma model.So, the study can be divided into two parts:Part I: Characterization of Ad LIGHT in vitro.We used Ad LIGHT to infect A20 cells, RT-PCR,Western Blot and flow cytometry analysis were performed to prove that LIGHT can be effectively expressed in A20 cells both in mRNA and protein level. To test whether the adenovirus mediated expression of LIGHT were bioactive, we observed its ability to stimulate the proliferation of T lymphocytes. The results show that, compared with the negative control, A20 cells that infected Ad LIGHT can effectively stimulate the proliferation of T lymphocytes and IFN-7 production, indicated that LIGHT may have synergistic costimulation activity on T cells. Then we used flow cytometry to test whether LIGHT can induce the expression of costimulatory molecules on A20 cells and apoptosis of A20 cell, the results indicated that LIGHT can effectively induce the apotosis and expression of Fas and ICAM-1 which may enhance B-cell lymphoma cell's antigen-presenting capacity.Part II: The antitumor effects by adenovirus-mediated LIGHT in a murine B-cell lymphoma modelFirstly, we used a murine B-cell lymphoma model to analyze in vivo antitumor effect of A20 tumor cells transduced with LIGHT as a vaccine. A20 cells wereinfected with Ad LIGHT or Ad LacZ at a multiplicity of infection of 200 for 24 h, then collected, washed and inactivated with mitomycin C (50/μg/ml) . Four groups of mice were vaccinated twice (2 weeks apart) were subcutaneously in one flank with 1×10~6 inactivated A20 cells (A20/LIGHT,A20/LacZ,A20/PBS,and PBS as blank control). One week after the second vaccine, mice were challenged by subcutaneous injection in the other flank with 2×10~5 parental A20 tumor cells, the tumor occurrence and survial time were observed. The results showed that the mice of A20/LIGHT group slowed the growth of tumor, promoted the tumor's receding and prolonged survival time of tumor-bearing mice.To examine the generation of CTL responses against parental tumor cells, splenocytes from mice vaccinated with irradiated AdLIGHT-transduced tumor cells were cultured with inactivated parental A20 tumor cells for 6 days. The results showed that, effector cells from mice vaccinated with tumor cells transduced with Ad LIGHT were able to lyse parental A20 tumor cells. This lysis was tumor specific, because the same effector cells were not able to lyse the syngeneic tumor cell line P815. No significant CTL activity was detected in mice vaccinated with tumor cells infected with Ad-LacZ. Also, the results indicated that the activity of NK cells induced by Ad LIGHT was higher than PBS and Ad LacZ. In conclusion, LIGHT could strengthen the activity of NK and CTL.Furthermore, we studied the antitumor effects by adenovirus-mediated LIGHT in a murine B-cell lymphoma model. Female BALB/c mice with 6-8 week old were subcutaneously inoculated with A20 cells, when the tumor nodules were visible and palpable, 30 mice were randomly devided into three groups (each contained 10 mice) and treated with PBS (blank control), Ad LacZ (blank virus control) and Ad LIGHT respectively. The tumor size was measured every other day and survival period of tumor-bearing mice was observed. Five days after the last adenovirus injection,splenocytes were collected from tumor-bearing mice for testing the CTL and NK activity. The activity was determined by a standard LDH release assay. Tumors were removed for histological examination; semi-quantative RT-PCR was performed to characterize the CCL21, IP-10 and Mig expression in the tumor tissues. To test whether the therapy of Ad LIGHT can induce a protective immunity against the parent A20 cells, the tumor free mice that received the Ad LIGHT therapy were rechallenged with A20 cells. The tumor occurrence and survial time were observed.The results showed that the effetive inhibition of tumor growth was achieved after the therapy of Ad LIGHT compared with mice received therapy of Ad LacZ and PBS. In the negative control group, tumor grew progressively and the tumor-bearing mice eventually died around 40 days after tumor inoculation. Tumor-bearing mice that received injection of Ad LIGHT showed a significantly reduced tumor growth, and 71.4% of the mice turned tumor free and survived more than 90 days, and consequently acquired a strong protective immunity against parent A20 tumor cells. Lymphocytes from the mice with the therapy of Ad LIGHT showed a higher NK and CTL activity, suggesting that augmentation of tumor-specific and nonspecific immunity both participated in the enhanced antitumor immune respose induced by Ad LIGHT therapy. These results suggested that adenovirus mediated expression of LIGHT may elicit potent antitumor immune response in the murine B-cell lymphoma model.Histological examination of tumor tissues showed that necrosis was observed from the tumor-bearing mice that received the therapy of Ad LIGHT, accompaning with obvious inflitration of inflammatory cells (neutrophils, lymphocytes, monocytes) inside the tumors, whereas relatively less necrosis and infiltration were present in the tumors of Ad LacZ or PBS treated group.IP-10 and Mig are two important chemokines that have been reported to possess antiangiogenic and antineoplastic activity, they may also modulate a variety of otherfunctions such as lymphocytes activation, differentiation and effector function. CCL21 is also an important antitumor chemokine. We used semiquantative RT-PCR to examine the expression of the three chemokines in tumor tissues after Ad LIGHT injection. We found CCL21,IP-10 and Mig mRNA to be expressed in tumor tissues of Ad LIGHT treated group and little or no CCL21,IP-10 and Mig mRNA was detected from the tumor tissues of Ad LacZ or PBS treated group. Therefore, the expression of CCL21, IP-10 and Mig may contribute to the reduction of new blood vessels and further the retardation of tumor progression.In conclusion, here we demonstrated that, in a murine B-cell lymphoma model, the intratumoral therapy of Ad LIGHT led to significantly suppression of the tumor growth, increased tumor necrosis and inflammatory cell infiltration, and enhanced antitumor effects with a higher NK and CTL activity, as compared to either Ad Lacz or PBS. Besides, the therapy also induced a long protective immune response against parent tumor cells rechallenge. These collectively data suggested that the immunotherapy by Ad LIGHT can elicit a more potent antitumor immune response.