Investigation On Tolerogenic Dendritic Cell Immune Function Reversed By Cryoablated Glioma Lysates

Glioma is one of the most common malignant tumors, surgical resection and postoperative radiotherapy and chemotherapy are the most common performed procedures at present. Glioma is invasive growth, and surgical trauma and radiotherapy and chemotherapy suppress the systemic immunity, leading to the recurrence rate and mortality rate remains high. Argon-helium cryoablation technology is a new minimally invasive targeted tumor treatment in recent years, and allows precise cryoablation of a wide variety of neoplasms, and has been used in the clinical treatment in prostate cancer, lung cancer, liver cancer, breast cancer. Compared with surgical resection, cryoablation causes not only tumor cells destructions but also enhancement of the host immune function, which reduce tumor recurrence and improve the prognosis of patients.Dendritic cells (DCs) are the most powerful antigen-presenting cells (APC) in host and can present antigen to T cells, activate naive T cells, and then cause anti-tumor immune response. Depending on their phenotypic and functional states, DCs may promote immunogenic or tolerogenic responses. Studies have demonstrated that mature DCs (mDCs) are activators of naive T cells, while immature DCs (imDCs) have been applied for anergy induction. Moreover, an intermediate stage of maturation was described that the cells are referred to as semimature DCs. Although these DCs expressed moderate levels of co-stimulatory molecules CD80, CD86 and MHC-Ⅱ, they exhibited an IL-12lowIL-10high phenotype. And the semimature DCs can induce tolerance by generating regulatory T cells and/or T cell anergy.DCs have been shown to play a critical role in the induction of anti-tumor immune responses, but studies have demonstrated that tumor growth is closely related to impaired differentiation and maturation of DCs. Tumor cells and tumor-derived factors can form tumor microenvironment, in which bone marrow precursors can differentiate into tolerogenic semimature DCs and then do favor immune tolerance and tumor immune evasion.Den Brok MH et al. have reported that cryoablation of tumor could enhance systemic anti-tumor immune responses. When pulsed with cryoablated tumor lysates, DCs become maturation and activation and induce immunogenic responses. But whether the cryoablated tumor lysates can prevent the differentiation of tolerogenic semimature DCs from precursors? In this study, we use culture supernatants of GL261 glioma cell lines to mimic tumor microenvironment. First of all, tolerogenic semimature DCs are generated from precursor cells and their cell phenotype and function are identified. Then, in the process of tolerogenic semimature DCs induction, the cryoablated tumor lysates are added in the culture medium, and to observe the role of the cryoablated tumor lysates played in the process, the cells are collected and their function are identified.Chapter Ⅰ Culture and identification of C57BL/6 mice bone marrow-derived tolerogenic dendritic cellsObjective:To establish a method of culturing tolerogenic dendritic cells (TDCs) from bone marrow of C57BL/6 mouse, and observe their biological feature and immune function.Methods:The bone marrow-derived precursors were cultured in the RPMI1640 medium in the presence of recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin-4 (rmIL-4).24 hours before completion of cell culture, lipopolysaccharide (LPS) was added in the medium in one group (LPS-DC); in another group, tumor culture supernatants (TSN) from GL261 cell lines was added in medium at the beginning of culture (TSN-DC), the third group was cultured without LPS or TSN. The suspended cells were collected after 6 days, and the cells’morphology, phenotype and immune function such as mixed lymphocyte reaction, ctotoxicity assay were analyzed.Results:Three group cells all exhibited typical dendritic morphology and the expression of CD11c was more than 90%. The expression of MHC-II molecule and co-stimulatory molecule CD86 was low in imDCs group, while in LPS-DCs and TSN-DCs groups, the expressions were high and moderate. And the TSN-DCs could secrete high level of IL-10 and low level of IL-12 compared with imDCs and LPS-DCs (P<0.01). In the mixed lymphocyte reaction, the ability of T cell proliferation stimulated by the TSN-DCs was weak (P>0.05), and the induced cytotoxic T lymphocytes (CTL) showed weak killing effect on GL261 cells (P> 0.05)Conclusion:High purity of glioma tolerogenic dendritic cells could be generated from C57BL/6 mice bone marrow cells through simulation of tumor microenvironment.Chapter Ⅱ Investigation on tolerogenic dendritic cell immune function influenced by cryoablated glioma lysatesObjective:To explore the role in the process of tolerogenic dendritic cells culture played by cryoablated glioma lysates and effect on immune function.Method:On the first day, the third day, the fifth day of tolerogenic dendritic cells culture, cryoablated glioma lysates were added in the culture medium, termed as L-1-TDCs, L-3-TDCs, L-5-TDCs,6 days later, these nonadherent cells were harvested and their morphology, phenotype and immune function such as mixed lymphocyte reaction, ctotoxicity assay were analyzed.Result:Compared with L-3-TDCs, L-5-TDCs, L-1-TDCs exhibited a more typical dendritic morphology like mature dendritic cells, secreted higher levels of MHC-Ⅱ molecule and co-stimulatory molecule CD86, secrete low level of IL-10 and high level of IL-12 (P<0.01), induced T cells proliferation and then showed strong killing effect on GL261 cells (P>0.05)Conclusion:In the early time of tolerogenic dendritic cells culture, adding cryoablated glioma lysates into the culture medium could prevent differentiation and maturation of tolerogenic dendritic cells culture from bone marrow cells, and these cells demonstrated immunological characteristics just like mature dendritic cells.