4-1BB Signal In The Immune Regulation Function Of Treg And The Preliminary Studies Of Allogeneic Treg’s Effect On Tumor Cells

Part1 The expression level in patients with multiple myeloma Treg and4-1BB in the immune regulation function of TregObjective:(1) Study the expression level of Treg in multiple myeloma patients;(2) Explore 4-1 bb signal effect on promoting proliferation of Treg cells.Methods:(1) Using immunofluorescence and flow cytometry test the expression level of Treg in peripheral blood and bone marrow of multiple myeloma patients;(2)Separating CD4 + CD25 + CD127 low /- cells by using immune magnetic bead.Using flow cytometry test the expression level of Treg. Observing the changes of the expression level of Treg after adding IL-2;(3)Primary myeloma cells co-culture with homologous PBMC,and use CFSE measure CD4 + CD25 high proliferation after blocking 4-1BB/4-1BBL signaling pathwaysResult:(1) The Treg levels of peripheral blood in patients with Multiple myeloma is higher than normal control group, the level of Treg in patients with multiple myeloma has no obvious correlation between clinical features and prognosis.Conclusion:In patients with multiple myeloma, treg is higher expression than normal control. 4-1BB molecule is constructively expressed on the surface of Treg cells.Engagement of 4-1BB/4-1BBL promotes Treg cell proliferation. These mechanisms now give us novel targets for intervention.Part 2 The preliminary studies of allogeneic Treg’s effect on tumor cellsObjective:Explore the direct effect of antigen specific Treg cells on tumor cellsMethods :(1) Separate normal donor monocyte, culture with granulocyte-macrophage colony stimulating factor(GM-CSF) and IL-4 to induce for immature dendritic cells;(2) use dendritic cells loaded with tumor antigen to induce antigen specific Treg generation and amplification in vitro;(3)The antigen specific Treg cultures with myeloma cell line in vitro,using Annexin V/PI staining method to measure the change of myeloma cells apoptosis level, using CFSE to determine proliferation inhibition of myeloma cells, and using tracellular cytokine staining to test synthesis changes of IL-6 level;(4) Measure if there is up-regualted expression of CD95 L on the surface of Treg after activation.Result:(1)By morphology and phenotype characteristics we could identify the immature dendritic cells;(2)The immature dendritic cells can induce Treg amplify 10 times in 7 days in vitro;(3) the antigen specific Treg can induce tumor cell apoptosis and inhibit the proliferation, and deprive promotion myeloma cells autocrine IL- 6;(4)After activation CD95 L isn’t up-regualted on the surface of Treg.Conclusion:We set up the model of dendritic cells inducing antigen specific Treg expansion,and we clear about the antigen specific Treg can inhibit tumor cell proliferation and induce its apoptosis,independent of CD95 / CD95 L pathway.