Studies On HLA-G Lentivirus Transfection Induced Immune Tolerance At Rat And Cell Levels

Background:Human leukocyte antigen (HLA) class I molecule HLA-G has been verified in vitro and clinical studies to have tolerogenic properties. So far only one study reported that injection of HLA-G tetramer-coated beads into recipient mice before allogenic skin grafting induced suppressive T cells and resulted in a significant increase of skin allograft survival. We intend to use human HLA-G lentivirus to transfect renal transplant in rat rejection model to ensure a high amount of HLA-G expression and directly test its effectiveness as an interfering treatment. Previous studies have shown that HLA-G expression in target cells can effectively inhibite release of the cytotoxic target particles towards target cells through inhibition of receptor binding on NK cells. Membrane containing the HLA-G can also be transferred to the NK cells, temporarily transforms NK cells into suppressor cells inhibit other NK cells from kill target cells.Methods:We first successfully constructed a SD to Wistar renal rejection model. When the renal transplantation performed by renal artery, vein and ureter-bladder anastomosis, before reperfusion we injected0.5ml of saline containing10’pfu HLA-G lentivirus into renal artery.We designed a positive control group with regular dose of FK506to suppress rejection. In negative control group before reperfusion we injected0.5ml of saline containing10pfu blank lentivirus. We assessed the blood BUN and Cr values, HLA-G levels and histo-pathological grading of rejections in renal transplant of recipients in three groups on6d post-transplantation. In order to clarify the difference between HLA-G inhibition of NK cell cytotoxicity in regard of in mode of interaction and effects between rats and humans, we used rat endothelial cells and human endothelial cells transfected or untransfected by HLA-G lentiviruses as target cells for human NK cells or rat NK cells attacking, to measure the killing rate differences in the four combinational circumstances. Results:HLA-G lentivirus transfection turned out no transplant preserving effects comparing to negative control because there were no differences in BUN and Cr values and survival(p>0.05). Histo-pathological examination based on Banff schema demonstrated that7d post-transplantation there were severe rejections in control group and HLA-G group, while there was mild rejection in FK506group. For human NK cells attacking HLA unmatched human endothelial cells,before or after transfection,the killing rate reduced from74.8±14.1%to21.2±4.7%(p<0.05). For rat NK cells attacking human endothelial cells,before or after transfection,the killing rate was unchanged from67.7±11.6%to65.8±10.9%(p>0.05). For human NK cells attacking rat endothelial cells, before or after transfection, the killing rate was reduced from85.3±16.3%to31.7±7.8%. For rat NK cells attacking allogenic rat endothelial cells, before or after transfection, the killing rate was unchanged from25.8±6.5%to26.0±5.9%.Conclusion:Our results suggested that either the expressed HLA-G were not functional or the supposed receptors on rat blood cells could not bind with them. HLA-G transfection as a therapeutic method could not proceed in rat level and we shall have to retreat to cell level to clarify this unexpected irregularity. The inhibiting functions of HLA-G presuppose corresponding receptors on the attacking cells which are present on human NK cells but are not present on Rat NK cells. Figure19, Table10, Reference73.