T Cell Immunotherapy Against Glioblastoma With Mouse Model

Malignant glioma is one of the most common brain tumors and the main treatments including surgery, radiotherapy and chemotherapy, but even if the application of these treatment methods were combined, the prognosis of patients with glioma is still poor. Newly emerged therapies, such as immunotherapy, molecular targeted therapy and gene therapy, etc. provides a bright prospect for the treatment of gliomas. Especially the cellular immunothreapy which developed rapidly in recent years. In the year of 1985, Rosenberg applicated autologous lymphokine activated killer cells(LAK) to treat melanoma for the first time, since then cellular immunothreapy has been gradually used in chronic leukemia, kidney cancer and breast cancer, etc. and turned out to works well. Some researchers also attempt to using this method in the treatment of glioma, but due to the particularity immune invironment of the central nervous system and other side effects such as brain edema, we didn’t find obviously improvement in the patient’s overall survival.In recent years, cells immunotherapy of glioma ushered in the new opportunities with the constant progress of immunotechnology and the update of the central nervous system concept. T cells refer to thymus dependent lymphocytes which derived from pluripotent stem cells in the bone marrow and play a crutial role in the immune responsibility. Matured T cells recycled through lymphatic vessels, peripheral blood and tissue fluid and also play an important role in the process of development of tumor. T cells adoptive immunotherapy constantly updating in recent years. This study discusses the method of T cells adoptive immunotherapy of glioma. First of all, GL261 cells lysates was used to sensitize dentric cell(DC). Then, it is the matured DC to stimulate and amplifie peripheral T cells in vitro. Finally, the T cells were transfused into glioma-burdened model to verify its feasibility and effectiveness and provide the basis for cellular adoptive immunotherapy in future clinical application.Objective: Investigating the feasibility, safety and efficiency of specific T cell immunotherapy against glioblastoma.Methods: Total tumor antigens(TTA) were extracted from GL261 cells after three freeze-thaw cycles. Immatured DC was drived from healthy C57BL/6 mice bone marrow mononuclear cells and induced with GM-CSF and IL-4 in vitro. TTA were used to sensitize immatured DC to obtain matured DC. Healthy C57BL/6 mice spleen was extracted under sterile environment and was digested by trypsin into single cell suspension. Red cells lysis is needed.To get specific T cells, the single spleen cell suspension was sorted through nylon fiber column and co-cultured with maturated DC while adding IL-2 and anti-CD3 e m Ab. T cell proliferation and change of T lymphocyte subsets were investigated by flow cytometry. Cytotoxicity of T cells against glioma cells were detected by MTT analysis.To build a GL261-C57BL/6 mouse glioma model, ALC-H type mouse brain sterotactic instrument was used to fix mouse head, the GL261 glioma cell was injected into healthy C57BL/6 mice. 16 of GL261-C57BL/6 mouse glioma model were randomly divided into treatment group and control group, 8 mice in each group. The treatment group were treated with glioma specific T cells via intravenous, while the control group was injected with PBS. A consecutive monitoring was conducted and change of T lymphocyte subsets was tested by flow cytometry.Results: ① The ratio of CD11c+ lymphocyte was 87±3.47 %. ② Expression of CD80 and CD86 was increased after pulsed with TTA(P<0.05) ③ After stimulated by matured DC, T cells division index is up to 0.56 ± 0.01, while with the immatured DC stimulated, T cells division index is just 0.28±0.02. Flowcytometric analysis showed that T cells had better proliferation(P<0.05)after co-cultured with maturated DC. ④ The ratio of CD8 a / CD4 in T cells with the immatured DC stimulated was also increased(P<0.05). ⑤ The cytotoxicity of T cells against GL261 cells was gradually improved as the increase of effector:target(E:T) ratio. When the E:T ratio was 60:1, effector cells loaded with maturated DC killed(62.67±2.52)% of GL261 cells while effector cells loaded with immaturated DC eliminated only(26.33±2.08)% of GL261 cells(P<0.05). ⑥ Successful mouse orthotopic tumor models was confirmed by MRI. ⑦ Flowcytometric analysis of peripheral blood after T cell immunotherapy suggested that CD8 a / CD4 ratio was 0.69±0.09 in control group, while the treatment group is up to 1.02±0.28(P<0.05). ⑧ Cell adoptive immunotherapy in mice can also prolong survival time(P<0.05).Conclusions: TTA have good immunogenicity and specific T cells induced by DCs pulsed with TTA showed significant anti-glioma effect both in vitro and in vivo. It provided robust applications for clinical trials on T cell adoptive immunotherapy.