HLA-G Inhibit Xenotransplantation Rejection Mediate By Human NK And T Cells

Part I A Study of HLA-G1 Protection of Porcine Endothelial Cells Against Human NK Cell Cytotoxicity[Objective] To study the effect of protecting porcine aortic endothelial cells (PEC) transfected with HLA-G1 from human NK cell lysis.[ Methods ] The recombinant expression vector pcDNA3.0-HLA-G1 was transfected into primary cultured PEC by lipofection. Surface expression of HLA-G1 in transected PEC was confirmed by an immunofluoresence technique. Human peripheral blood mononuclear cells (hPBMC) and NK cell line (NK92) were used as NK effect cells with pcDNA3-HLA-G1-transfected PEC as targets in a MTT method using pcDNA3 transfection as a negative control.[Results] Expression of HLA-G1 on PEC conferred significant protection against NK-mediated lysis. The rate of NK92 cytotoxicity was reduced to 41.5% ± 14.0% from 75.3% ±10.5% in the control group (P<0.01). Similarly the rate of the hPBMC cytotoxicity among different donors (n =7) was reduced to 45.4% ± 12.1% in contrast to 74.6% ±11.2% in the control group (P<0.05).[Conclusion] HLA-G1 molecules can directly protect xenogeneic PEC against attack by human NK cells. These results indicate that the expression of HLA-G1 on the porcine cell surface may provide a new approach to overcome NK-mediated immunity to xenografts. Part II Establishment of cell line stably expressing soluble HLA-G1 and purification of soluble HLA-G1 protein[Objective] To establish the LCL721.221 cell line stably expressing sHLA-G1 and purify the sHLA-G1 protein.[Methods] The recombinant plasmid pcDNA3.0-sHLA-G1 was transfected by a novel nonviral, electroporation-based gene transfer method termed nucleofection into the host cell lymphoblastoid cell line LCL721.221 which does not express any HLA-classical I molecules. After selection by G418, the cell line stably expressing sHLA-G1 is identified by RT-PCR and Dot-ELISA with HLA-G specific monoclonal antibody MEM-G/9. The W6/32 antibody (anti HLA-G monoclonal) was produced by injecting the hybridoma cell line HB-95 cell into Balb/c mice. Then the sHLA-G1 protein is purified by affinity chromatograpHy using the monoclonal antibody.[Results] After analysis by RT-PCR and Dot-ELISA, it is confirmed that the eukaryotic cell line expressing sHLA-G1 has been established successfully. And about 1.3mg sHLA-G1 protein was purified by affinity chromatography using the monoclonal antibody W6/32.[Conclusion] In this study, we have established the LCL721.221 cell line expressing sHLA-G1 and get the sHLA-G1 protein successfully.Part III Soluble HLA-G1 inhibit the biological function of NK92[Objective] To study the function of sHLA-G1 inhibition the biological function of NK92.[Methods] The effect of sHLA-G1 on the interaction between SV-40-PED and NK92 cells was assessed in terms of adhesion and cytotoxicity. The inhibitive effect of the releasing of porforin and granzyme by NK92 was analyzed by the β-hexosaminidase release assay. The IFN-γ, TNF-α secretion by NK92 wsa detect by ELISA methods. And the cytotoxicity of NK92 was analyzed by MTT methods.[Results] sHLA-G1 conferred a significant degrading the role of NK92 to adhere to SV-40-PED (P<0.01) and degrading the releasing of porforin and granzyme by NK92, the IFN-γ, TNF-α secretion by NK92 was inhibition significant by sHLA-G1, and sHLA-G1 protected SV-40-PED against NK2 medicated lysis, the rate of NK92 cell cytotoxicity was reduced to 25.5±2.1% in contrast to 71.2±2.6% in the control group (P<0.05).[Conclusion] sHLA-G1 can inhibit the biological function of NK92. These results indicate that sHLA-G1 may be useful to prevent human NK cells to be responsed to porcine xenografts.Part IV Establishment A Xenograft Model for graft-versus-host Disease in SCID Mice Engrafted With Human Peripheral Blood Mononuclear Cells[Objective] To Establishment the xenograft model for graft-versus-host disease (XGVHD) by transfer of human peripheral blood mononuclear cells (hPBMCs) and NK92 cells into severe combined immunodeflcient (SCID) mice.[Methods] SCID mice were injected i.v 5×10~7hPBMCs and NK92 cells under sterile conditions. SCID mice were pretreated 1 day prior to hPBMCs and NK92 cells injection with 30μl anti-asialo GM1 antibodies. Immediately prior to hPBMCs and NK92 cells engraftment SCID mice were irradiated with a dose of γ-radiation (3.5 Gy). NK92 cells with 200u/ml of rIL-2 were injected into SCID mice in one of NK92 group. IFN-γ and TNF-α ELISA kits were used for IFN-γ and TNF-α assay.[Results] The high level of hPBMCs engraftment achieved in SCID mice induces severe XGVHD with concomitant weight loss, hunched back, and ruffled fur. Histological analysis of hPBMCs-SCID tissues showed massive leukomonocyte infiltration in liver. The IFN-γ, TNF-α secretion were increased significantly when XGVHD occurred. Almost 100% of the SCID mice engraft with hPBMCs died within 25 days postengraftment. Inversely, the XGVHD didn't occur in SCID mice in NK92 group.[Conclusion] Successfully development a human model of xenogeneic graft-versus-host disease in SCID mice engrafted with hPBMCs. Part V Soluble HLA-G1 inhibit the XGVHD disease induce by engraft Human Peripheral Blood mononuclear cells into SCID mice[Objective] To study the whether sHLA-G1 can inhibit the biological function of T cells in vivo, inhibit the XGVHD disease induce by engraft hPBMCs into SCID mice.[Methods] The effects of sHLA-G1 inhibition the proliferation of T cell was analyzed by mixed lymphocyte culture method. SCID mice were injected i.v 5×10~7 hPBMCs under sterile conditions. SCID mice were pretreated 1 day prior to hPBMCs injection with 30μl anti-asialo GM1 antibodies. Immediately prior to hPBMCs engraftment SCID mice were irradiated with a dose of γ-radiation (3.5 Gy). In the two experiment groups, SCID mice were injected i.v with a dose of 2ng and 4ng sHLA-G1 (0,3, 6, 9, 12and15d).[Results] sHLA-G1 can inhibitive the proliferation of T cell markedly. SCID mice in control group induce severe XGVHD with concomitant weight loss, hunched back, and ruffled fur. Histological analysis of hPBMCs-SCID tissues showed massive leukomonocyte infiltration in liver. The IFN-γ, TNF-α secretion were increased significantly when XGVHD occurred. Almost 100% of the SCID mice engraft with hPBMCs died within 25 days postengraftment. Inversely, SCID mice in experiment groups didn't suffer from XGVHD disease, The IFN-γ TNF-α secretion were kept in a low level. Histological analysis of hPBMCs-SCID- sHLA-G1 tissues showed only a few leukomonocyte infiltrated in liver.[Conclusion] sHLA-G1 can inhibit the biological function of T cells in vivo, and inhibit the XGVHD disease induce by engraft hPBMCs into SCID mice.