THE SERUM LEVEL OF XENOANTIBODIES, AND hDAF OR αGAL EXPRESSION ON PIG CELLS, MODULATE THE PROTECTION GIVEN BY hDAF TO COMPLEMENT MEDIATED DAMAGE IN PIG-TO-BABOON XENOTRASPLANTATION MODEL.

Abstract

P1054 Aims: Acute humoral xenograft rejection (AHXR), the leading cause of xenograft failure after hDAF pig-to-baboon xenotransplantation, is mainly mediated by xenoreactive antibodies and complement. The uselessness of hDAF to prevent AHXR contrasts with the efficacy demonstrated to prevent hyperacute rejection of pig organs, which is triggered by the same mediators (xenoantibodies and complement). This study investigated whether the serum level of xenoantibodies, and hDAF or Gal α1-3 Gal (αGal) expression on pig cells, modulate the protection given by the transgene to complement-mediated-damage. Methods: The protective effect of hDAF was studied by a flow cytometry complement–mediated cytotoxicity assay (FCCA) using peripheral blood lymphocytes (PBLs) from hDAF transgenic pig as target cells. The cytotoxicity of baboon’s sera, individually or in a pool (NBS), undergoing pig heart xenotransplantation and a pool of normal human sera (NHS), was evaluated. Also, the levels of anti- αGal antibodies in sera and the cell surface expression of hDAF molecules and αGal antigens on the PBL used as targets were analysed by a specific ELISA assay and immunofluorescence with specific antibodies, respectively. Results: Transgenic hDAF expression was capable of protecting pig cells against injury produced by baboon and human serum. However, the hDAF molecule was more efficient against NHS (median of cytotoxicity 0.265 SRLU, rank 0.149-0.522), than against baboon’s sera (median of cytotoxicity 0.517 SRLU, rank 0.300-0.938). The humoral cytotoxicity capacity correlated with the level of anti-αGal antibodies (for IgG Spearman correlation coefficient was 0.736 p=0.003 and for IgM was 0.833 p<0.000, n=14). Also, inhibition of complement mediated cytotoxicity of hDAF pig cells correlated with the expression of hDAF and αGal molecules on target cells (Positive Spearman correlation coefficient 0.855 p=0.001 and negative Spearman correlation coefficient −0.709 p=0.011, n=10, respectively). Conclusions These results confirm “in vitro” the protective role of hDAF in pig cells to heterologus complement mediated damage. However, they also suggest that protection decreases in the presence of high levels of anti-porcine antibodies in serum, and low expression of hDAF or high expression of αGal on pig cells, which could explain the AHXR of hDAF organs despite the lack of HAR.