Study Of Antitumor Immunotherapy Effect Of Thymosin Alpha1 On DCs Or DC Vaccines In Vivo And In Vitro

Gastrointestinal carcinoma (GIC) is one of the most common malignant tumor in clinic. Conventional approaches, such as radical operation, chemotherapy, radiotherapy, offer little help to the recovery rate and have only slight impact on the their survival, specially in advanced patients. Recent insight in the field of immunotherapy, dendritic cell (DC)-based immunization has been considered as one of the most promising to date among the many developmental vaccination strategies. DCs are the most potent antigen presenting cells (APCs) with the ability to acquire, process, present antigens and the unique capability of initiating primary immune responses against tumor-associated antigens (TAA). However, immune escape is the most important biological characteristic in the development and progression of maglinancies. A significant reduction in functional mature DCs has been reported in GIC patients, suggesting that the block in immature myeloid cell development, may induce anergy T cells or regulatory T cells (Tregs) and result in tumoral tolerance and suppression. Many patients and animal experiments have confirmed that the suppressive status in tumor microenviroment of DCs could be recruited by culturing and priming DCs in vitro, retransfusion of loading tumor antigens DCs derived from peripheral blood mononuclear cells (PBMCs) could activate TAA-specific T cells immune response, and some also have clinical responses. Despite these encouraging results, the vaccine-induced immune responses achieved to date not yet sufficient to attain a robust and durable therapeutic effect in cancer patient. Therefore, further improvements are required to enhance vaccine potency and optimize the potential for clinical success.The strategies to further enhance the antigen-presenting function of DC-based vaccines include: (1) modulating the DC maturation process, (2) enhancing the CD4+ T cell arm of the immune system, (3) inhibiting regulatory pathways, (4) targeting antigens with critical role in oncogenesis. Successful therapy for complex diseases such as cancer will require a combination approach that includes multiple therapeutic steps. For example, DCs vaccine combinated by adjuvant therapy with CD40 ligand, IL-12, IL-2, and heat shock protein(Hsp70) can enhance and retain vaccine-mediated CD4+Th1 immune response and CTLs cytotoxicity, nevertheless, severe side effect often happened specially in using large dose of these cytokines. Thymosin alpha1 (Tα1) is a potential immune modulating agent which has been used in clinic many years with little side effect. It has been demonstrated that Tα1 can promote T cells differentiation, maturation, proliferation and increase cytokines IL-2, IL-6 and IFN-γsecretion. Recent further studies suggest that Tα1 also promote differentiation and maturation of DCs derived from mice bone marrow and normal healthy donors peripheral blood, stimulate DCs IL-12 secretion and enhance CD4+Th1 immune response.In most clinical trials, DCs vaccines were prepared by expanded autologous DCs loaded with tumor cell lysate or tumor-derived total RNA which contained tumor total antigens. These vaccination strategies were more likely to induce a polyclonal expansion of T cells and multiple TAA-specific CTLs. Furthermore, lysates or total mRNA from autologous tumor cells could be used without considering the HLA type and early translational trials using antigen-loaded DCs had established clear evidence of vaccine safety, relevant autoimmune responses were seldom detected in clinical trials. Successfully establishing animal model was a very important step in laboratory, in this article, humanized immune-reconstructed animal model bearing colon carcinoma was established by caudal intravenous injection of human peripheral blood T-lymphocytes in congenital immunodeficiency nude mice of T cells. We would be to investigate the effects of Tα1 on the differentiation, maturation and function of DCs or tumor lysate-pulsed dendritic cells (LyDCs) in vitro, and the therapeutic effects of Tα1 combined with LyDCs on humanized BALB/c nude mice bearing HT-29 colon carcinoma in vivo. The results maight be of benefit to explore a new immune promoting pathway or target cell of Tα1 and offer a promising adjuvant candidate for DC-based immunotherapy of gastrointestinal carcinomas.This article is composed of four parts as follows:Part I Clinical significance of the dysfunction of peripheral blood dendritic cells and imbalance of CD4+CD25+T cells in patients with gastrointestinal carcinomaObjective: To investigate the immune function of peripheral blood monocyte-derived dendritic cells (DCs) and the proportion of CD4+CD25+T cells (Tregs) in CD4+T lymphocytes of the patients with gastrointestinal carcinoma (GIC), and their correlation with clinicopathological stage and the operation.Materials & Methods: PBMCs were obtained from normal healthy donors, patients with early GIC (stageⅠ/Ⅱ), including pre- or postoperative patients, and with advanced GIC (stageⅢ/Ⅳ), respectively. The proportion of Tregs in CD4+ T lymphocytes was calculated by flow cytometry. DCs were isolated from PBMCs of healthy donors and patients and cultured to d5 in the presence of rhGM-CSF and rhIL-4 to generate immature DCs (imDCs), and the expression of HLA-DR,CD80 and CD86 on the surface of imDCs was analyzed by flow cytometry. The imDCs were further matured with an extra incubation to d7 by adding rhTNF-α, and the capacity of matured DCs (mDCs) stimulating the proliferation of T lymphocytes and the IFN-γproduction in co-culturing mDCs and autologous T lymphocytes were measured by MTT assay and ELISA, respectively.Results: The expression levels of HLA-DR,CD80 and CD86 on imDCs from the patients with advanced GIC were significantly lower in comparison with those with preoperative early cancer and the healthy donors (P<0.01, respectively). The expression of HLA-DR and CD86 in postoperative patients with early GIC cancer was significantly down regulated in comparison with the healthy donors (P<0.05). In advanced GIC, T lymphocyte proliferation and IFN-γproduction induced by mDCs tend to be decreased in comparison with healthy donors(P=0.073,P=0.182, respectively). The proportion of Tregs in advanced GIC was significantly increased when compared to the early cancers or healthy donors (P<0.001, respectively), and similar tendency was also observed in postoperative patients with early cancer when compared to those preoperative patients (P=0.148) and healthy donors (P=0.068). A negative correlation was observed between the expression of HLA-DR and CD86 molecules on imDCs and the proportion of Tregs in CD4+T lymphocytes.Conclusions: The dysfunction of peripheral blood dendritic cells and imbalance of Tregs in patients with GIC, especially in the patients with advanced cancers and postoperative patients, may play an important role in immunotolerance of the patients with GIC.Part II Affects treated with thymosin alpha 1 on the dysfunction of peripheral blood dendritic cells and imbalance of CD4+CD25+T cells in patients with gastrointestinal carcinomaObjective: To investigate the immune function of peripheral blood monocyte-derived dendritic cells (DCs) and the proportion of CD4+CD25+ T cells (Tregs) in CD4+ T lymphocytes of the patients with gastrointestinal carcinoma (GIC) pre or post-treated by thymosin alpha1 (Tα1).Materials & Methods: Tα1 was used by subcutaneous injection for continous 6 days, 1.6mg/time, one time per day, in postoperative patients with early GIC (stageⅠ/Ⅱ) and patients with advanced GIC (stageⅢ/Ⅳ) . PBMCs were obtained from patients pre or post-treated by Tα1, respectively. The proportion of Tregs in CD4+ T lymphocytes was calculated by flow cytometry. DCs were isolated from PBMCs of patients and cultured to d5 in the presence of rhGM-CSF and rhIL-4 to generate immature DCs (imDCs), and the expression of HLA-DR,CD80 and CD86 on the surface of imDCs was analyzed by flow cytometry. The imDCs were further matured with an extra incubation to d7 by adding rhTNF-α, and the capacity of matured DCs (mDCs) stimulating the proliferation of T lymphocytes and the IFN-γproduction in co-culturing mDCs and autologous T lymphocytes were measured by MTT assay and ELISA, respectively.Results: The expression levels of HLA-DR,CD80 and CD86 on imDCs from the patients with advanced GIC post-treated by Tα1 were significantly higher in comparison with those pre-treated by Tα1 (P<0.01, respectively). The expression of HLA-DR and CD86 in postoperative patients with early GIC cancer post-treated by Tα1 was significantly up-regulated in comparison with those pre-treated by Tα1 (P<0.05). T lymphocyte proliferation and IFN-γproduction induced by mDCs post-treated by Tα1 were markedly enhanced in comparison with those pre-treated by Tα1 in advanced (P<0.01) and postoperative early patients (P<0.05) with GIC. The proportion of Tregs in advanced GIC tend to be decreased when compared to those pre-treated by Tα1 (P=0.065), and little changes was observed in postoperative patients with early cancer post-treated by Tα1 when compared to those pre-treated by Tα1.Conclusions: The dysfunction of peripheral blood dendritic cells and imbalance of Tregs in patients with GIC, especially in the patients with advanced cancers and postoperative patients, may play an important role in immunotolerance of the patients with GIC.Part III The anti-tumor efforts of thymosinα1 on tumor lysate-pulsed dendritic cells in colon cancer in vitroObjective: To investigate the effects of thymosin alpha1 (Tα1) on the differentiation, maturation and function of tumor lysate-pulsed dendritic cells (LyDCs) in vitro.Materials & Methods: Immature DCs (imDCs) were prepared routinely from human peripheral blood mononuclear cells. The lyDCs were prepared from the imDCs loaded with lysate of HT-29 tumor cell line. The phenotypes of imDCs and lyDCs pre- or post-stimulated by Tα1 were analyzed by flow cytometry. Autologous T cells were cocultured with LyDCs in the presence or absence of Tα1 2 days later. IL-12 secretion of LyDCs and IFN-γsecretion of the activated T cells in the supernatants were measured by ELISA. The in vitro cytotoxicity of antigen specific cytotoxic T lymphocytes (CTLs) induced by LyDCs which were treated with Tα1 was evaluated by MTT assay.Results: The expression levels of HLA-DR, CD80, CD86 and CD83 in imDCs and LyDCs were markedly up-regulated after the stimulation with Tα1 respectively (P﹤0.01). The levels of IL-12 and IFN-γwere also significantly increased in the presence of Tα1 (P﹤0.05 and P﹤0.01, respectively). Cytotoxicity induced by LyDCs treated with Tα1 was significantly enhanced (P﹤0.01) as compared with lyDCs in vitro.Conclusions: Tα1 can induce the functional maturation of DCs and enhance the immune response of CD4+Th1 arm and cytotoxicity induced by LyDCs.It might be a promising adjuvant candidate for DC-based immunotherapy of gastrointestinal carcinomas.Part IV In vivo Study of DC-based Immunotherapy Combined with Thymosin-alpha1 on Humanized Nude Mice with Colon CancerObjective: To investigate the therapeutic effects of thymosin alpha1 (Tα1) combined with tumor lysate-pulsed dendritic cells (LyDCs) on humanized BALB/c nude mice bearing HT-29 colon carcinoma.Materials & Methods: LyDCs were routinely prepared from immature DCs loaded with HT-29 tumor cell lysate. Humanized nude mice model bearing colon carcinoma was established by caudal intravenous injection of human peripheral blood T-lymphocytes, and 2 days later, the levels of CD4+ and CD8+ T cell subpopulations of human T-lymphocytes in peripheral blood were evaluated by flow cytometry. The mice were randomly divided into 3 groups, and immunized by subcutaneous injection of LyDCs plus Tα1, LyDCs or sodium alone, respectively. At day seven after the last immunization, cytokine release assay and cytotoxicity assay were performed by using spleen T-lymphocytes of the mice. At day 58 after inoculation of tumor cells, the in vivo immunotherapeutic effect on mice was observed. Results: Human T-lymphocyte subpopulations, CD4+ and CD8+ T cells, were detected out from peripheral blood of humanized nude mice. IFN-γrelease and cytotoxicity of CTLs from spleen T-lymphocytes of the mice were both significantly increased (P﹤0.01, respectively) in LyDCs+Tα1 group in comparison with those in LyDCs group. The inhibition rate of tumor growth was significantly higher in LyDCs+Tα1 group compared with that in LyDCs group (60.41% vs. 37.20%; P﹤0.01).Conclusions: Tα1 could strongly enhance CD4+Th1 immune response and the cytotoxicity effects of antigen specific CTLs induced by LyDCs, thus it may play a crucial role as an ideal adjuvant in DC-based immunotherapy.