Anti-tumor Effect Of Memory T Cell Induced By Allo-skin Transplantation

Cancer is one of the most important factors that threaten human health.Millions of people are killed by suffering from cancer worldwide each year.A global cancer report issued by the World Health Organization in February 2014 shows that 14 million new cancer cases and 8.2 million deaths occurred in 2012,of which China added 3.07 million new cases and 2.2 million cancer deaths.Currently,the traditional methods of treatment of tumors include surgery,radiotherapy,chemotherapy and Chinese medicine treatment,but these methods could hardly avoid the trauma,some of which might reduce the life quality of patients and cause other problems.Recently,immunotherapy has become a new method of treating cancer by stimulation of autologous immune cells in vitro and reinfusion to achieve the purpose of killing tumor cells.Lymphokine Activated Killer Cells(LAKs),Tumor-infiltration Lymphocytes(TILs)and Cytokine-induced killer(CIK)are able to be applied to cell immunotherapy of cancer.However,they lack the enduring anti-tumor effects.Besides,for the immunity of patients with cancer is usually inhibited,autoimmune cells would not have anti-tumor effects without in vitro activation.Therefore,people are attempt to find other autoimmune cells with anti-tumor effects.Previous studies indicated that allogeneic stem cell transplantation could cure multiple myeloma,which was also used to treat acute and chronic lymphocytic leukemia.Allotransplantation could reduce the recurrence rate of giant cell tumor.Since memory T cell is induced by allotransplantation,immune suppression measures might cause some malignant tumor like Non-melanoma skin cancer(NMSC).All of these suggest that memory T cells might have the potential of anti-tumor effects.Memory T cell is a subset of T lymphocytes.Naive T cells transform to effector T cells to clear antigen in the primary immune response.Some effector T cells become memory T cells and distributed in different parts of the body,with long-term stability.Once a small amount of the same antigen reappears,memory T cells can identify and remove the antigen quickly,and play a broad role in immune surveillance,which is an important factor in maintaining long-lasting immunity.Memory T cells contain many subsets like central memory T cells and effector memory T cells.It has been proved that the effector memory T cells have much more effects of clearing antigen than central memory T cell in peripheral tissues.Previous studies indicated that bone marrow-derived memory T cells have anti-tumor effects after in vitro stimulation.Therefore,this study is aimed at investigating the mechanism that the immune environment is transformed into suppression of tumor growth by stimulation of allo-skin transplantation,and the role that the Memory T cells play in the process of tumor suppression.Inducing the specificity and anti-tumor effects of memory T cells can be applied to adoptive immunotherapy of cancer.1.Part one anti-B16 melanoma effect of allo-skin transplantationPurpose:to investigate anti-B16melanoma effect of allo-skin transplantation.Methods:we used 6-week-old female Balb/c mice as donor to obtain skin graft,6-week-old female C57BL/6 mice as recipient to receive the skin graft on the back.Two months later B16 cells were injected subcutaneously in right groin of mice.We then evaluated the anti-tumor effects by tumor size curve and survival rate between the Skin-transplantation group(ST)and the Non-transplantation group(NT)C57BL/6 mice.The frequency of CD4+CD44+CD62L+T cells(CD4+ central memory T cells,CD4+TCM),CD8+CD44+CD62L+T cells(CD8+central memory T cells,CD8+TCM),CD4+ CD44+CD62L-T cells(CD4+effector memory T cells,CD4+TEM)and CD8+CD44+CD62L-T cells(CD8+effector memory T cells,CD8+TEM)in spleen,lymph nodes and tumor tissue of two groups of C57BL/6 mice was tested by flow cytometry,which was also used to test the frequency of Myeloid-Derived Suppressor Cells(CD 11 b+LY6G+)and Treg cells(CD4+CD25+ FOXP3+)in bone marrow and tumor tissue of the two groups.ELISA was used to test the level of INF-y,chemotactic factor CXCL9,and chemotactic factor CXCL10 in peripheral blood serum of ST group and NT group.Ki-67,CD34,CXCL9 and CXCL10 of tumor tissue were tested by Immunological Histological Chemistry(IHC).The apoptosis of tumor cells was measured by TUNEL.In order to assessment the cytotoxic of CD4+and CD8+effector memory T cells which were purified as effector cells,the purity was over 95 percent.B16 cells and Hepal-6 cells in logarithmic growth phase were taken as target cells.Meanwhile,we used purified CD4+and CD8+effector memory T cells of the two groups to cure B16 tumor bearing C57BL/6 mice by tail vein injection on 7,14 and 21 day since the B16 cells was injected subcutaneously.The therapeutic effects were measured by tumor size curve and survival rate of each group of C57BL/6 mice.Results:the growth of B16 tumor was suppressed in C57BL/6 mice of Skin-transplantation group.Compared with C57BL/6 mice of Non-transplantation group,the frequency of CD8+effector memory T cells in B16 tumor tissue was significantly higher,so was the TUNEL,but the Ki-67 or CD34.The tumor tissue of Skin-transplantation group showed lower nuclear proliferation and average density of blood vessels.CXCL9 and CXCL10 of Skin-transplantation group were significantly higher than those of Non-transplantation group,no matter in peripheral blood serum or in tumor tissue,and so was INF-y in peripheral blood serum.The CD8+effector memory T cells from Skin-transplantation group showed higher cytotoxicity to B16 cells than that from Non-transplantation group.The growth of B16 tumor tissue of C57BL/6 mice which were cured by effector memory T cells from Skin-transplantation group was suppressed,with lower Ki-67,and higher TUNE.The adoptive immunotherapy of effector memory T cells from Skin-transplantation group prolonged lives of B16 tumor bearing C57BL/6 mice significantly.Conclusion:allo-skin transplantation has significant anti-B16 tumor effect.Effector memory T cells with anti-B16 tumor effect could be induced by allo-skin transplantation.2.Part two anti-B16 melanoma effect of effector memory T cell induced by allo-skin transplantation which were amplified by high hybrid rate DC/B16 fusion cell.Purpose:use high hybrid rate DC/B16 fusion cell to enhance the specific anti-melanoma effect of effector memory T cells induced by allo-skin transplantation.Methods:Bone marrow cells of 6-week-old female C57BL/6 mice were obtained and cultured with complete RPMI-1640 which contained 10 ng/mL mice granulocyte-macrophage colony-stimulating factor(GM-CSF)and 10 ng/mL mice Interleukin-4(IL-4)to induce mature dendritic cells(DC)for the preparation of cell fusion.B16 cells stained by PKH26 and the DC stained by CFSE were used to make fusion cells 5 days later and cultured another 5 days.Nuclear was stained by DAPI,and the fusion efficiency was measured by fluorescence microscopy.Then the DC/B16 fusion cells were co-cultured with effector memory T cells(CD4+ TEM and CD8+ TEM)purefied from B16 tumor bearing C57BL/6 mice with Skin transplantation.The effector memory T cell induced by fusion cells and non-induced effector memory T cells were used as effect cells in cytotoxicity test.B16 cells and Hepal-6 cells in logarithmic growth phase stained by PKH26 were taken as target cells.The E:T was set at 1:1,3:1 and 9:1.ELISA was used to detect IL-2,IL-4,IL-10,INF-γ and TNF-αin the supernatant.We used the effector memory T cells induced by fusion cells and non-induced T cells to treat B16 tumor bearing C57BL/6 mice by tail vein injection on 7,14 and 21 day since the B16 cells were injected subcutaneously.The therapeutic effects were measured by tumor size curve and survival rate of each group of C57BL/6 mice.Ki-67 of tumor tissue of each group was tested by IHC,and apoptosis was tested by TUNEL.Results:The fusion efficiency was about 80.1%.Compared with non-induced effector memory T cells,the effector memory T cells induced by fusion cells(FC-TEM)showed significantly higher cytotoxicity and specificity to B16 tumor cells.Concentration of IL-2,INF-γ and TNF-α of T cells induced group in the supernatant were higher than those of non-induced group.The growth of B16 tumor tissue of C57BL/6 mice in groups cured by effector memory T cells induced by fusion cells was suppressed,with lower Ki-67 but higher TUNEL.And the adoptive immunotherapy of effector memory T cells induced by fusion cells prolonged survival time of B16 tumor bearing C57BL/6 mice significantly.The median survival time of FC-CD4+TEM and FC-CD8+TEM mixed group was 82.7 percent longer when compaird with PBS group.Conclusion:B16 specific effector memory T cell induced by allo-skin transplantation could be stimulated by DC/B16 fusion cells,and those effector memory T cell had capacity of secreting IL-2,INF-y and TNF-α,and showed explicit effects of anti-B16 tumor in vitro and in vivo.