The Effect Of Regulatory T Cells On The Cytotoxic Activity Of Natural Killer Cell

Allogeneic hematopietic stem cell transplantation ( Allo-HSCT ) is a potentially curative treatment for both hematologic and nonhematologic disease,and Graft-versus-host disease(GVHD) is a major complication after Allogenic hematopietic stem cell transplantation (HSCT) .The wider use of Allo-HSCT is limited by the frequent and sebere outcome of GVHD. Drugs and T-cell depletion of the grafe to avoid GVHD place patients in danger of leukemic relapse,graft failure,and infectious complications. We have to find a way that can induce the tolerance to alloantigen to prevent GVHD.Recently,published data indicate that infusion donor-derived CD4~+CD25~+ regulatory T cells(Treg) with ordinary graft can markedly prevent GVHD but maintain Graft-versus-leukemic (GVL) effect,and promote engraftment. These finding open a door for preventing GVHD. We haven' t found other differential sign expressed on Treg cell' s surface except CD25.But,CD62L has been found required by the inhibitory effect of Treg cell,and CD45RO has been found related with activity of Treg cell. We have known Treg suppress CD4~+,CD8~+ T cells,But the effect of Treg cell on the cytotoxic activity of NK cell is not clear. NK cells show two different action in Allo-HSCT. Some is beneficial to recipient ,and some will do harm to recipient.So,the effect of Treg cell on the cytotoxic activity of NK cell may influence the application of Treg. We experiment ex vivo to find what is the effect of Treg cell on the cytotoxic activity of NK cell and how Treg cell effect NK cell.Treg cells and NK cells were sorted from health adult peripheral blood mononuclear cells by Mini-MACS. The purity of CD4~+CD25~+ T cells was of 88.7- 93.6%,and the NK cells' was of 85.6-89.2%. The purity is satisfying. The expression of CD62L,CD45RO on Treg cell's surface was analysed by Flow cytometry(FCM).We found 87.2-90.3% Treg cell express CD62L, 85.4-88.6% Treg cell express CD45RO.This finding accord with datas published. To find the effect of Treg cell on the cytotoxic activity of NK cell,We culture CD4~+CD25~+/CD4~+CD25~- cells together with NK cells and K562 cells in different ratio. In this part,we find CD4~+CD25~+ cells but not CD4~+CD25~- cells suppress activity of NK cell,and this effect is in dose-dependent manner. To determine how Treg cell suppress NK cell,we put Treg cell in transwell chambers to separate Treg cell and NK cell. The semipermeable membrane of transwell chambers' allow only free passage of soluble factors but not direct cell contact.So,Treg cell and NK cell cann't contact eachother directly,but the cytokines excreted by Treg cell can pass the semipermeable membrane and effect NK cell. We also make use of anti-IL-10,anti-TGF-βto block IL-10 and TGF-β.When Treg cell and NK cell was separated by transwell,Treg cell almost have no effect on cytotoxic activity of NK cell;when anti-IL-10,anti-TGF-βwas used to block IL-10 and TGF-β,Treg still suppress cytotoxic activity of NK cell.From above,we can draw a conclusion, the inhibitory effect of Treg cell on NK cell is in direct and contact dependent.In this experiment, we discuss what Treg cell do with the cytotoxic activity of NK cell and how. From this experiment we reach conclusions: Treg cell can inhibit the cytotoxic activity of NK cell,this effect is in dose-dependent manner and in direct and contact dependent. These conclusions provide helpful information for useing Treg cell to protect patient received Allo-HSCT from GVHD,avoiding inhibitory effect of Treg on beneficial activity of NK cell in Allo-HSCT.