Introduction:

Relevant alloantibodies in kidney transplantation comprise anti-HLA antibodies, blood group antibodies (ABO incompatible) and anti-endothelial cell antibodies (AECA) and can initiate antibody-mediated rejection (ABMR). ABMR is characterized by complement activation products, such as C4d, on the endothelium of the microvasculature in the kidneys at the time of rejection. However, endothelium directed cytotoxicity of the various antibodies and the role of complement (regulation) are not fully elucidated.

Methods:

In this study, we set up a flow cytometry method and an endothelial complement dependent cytotoxicity test (EC-CDC) to evaluate the involvement of various transplant related antibodies in the process of complement mediated endothelial damage. We used primary endothelial cells (EC) cultured from donor kidney perfusate after machine perfusion, circulating human EC progenitors or conditionally immortalized human glomerular EC. Antibody binding and complement activation was evaluated by FACS analysis and compared to classical donor lymphocyte CDC (L-CDC) and EC-CDC.

Results:

First, ABO incompatible serum caused complement mediated cell cytotoxicity in the EC-CDC. Second, sera containing high titer HLA-DSA that tested negative in the L-CDC, caused complement dependent cytotoxicity in the EC-CDC. This correlated with increased IgG binding and activation of C3 by flow cytometry. Serum, suspected to contain AECA, caused abundant cell death in the EC-CDC whereas no cytotoxicity was seen in the L-CDC.

Conclusion:

We successfully developed an EC-CDC and flow cytometry method to assess complement dependent endothelial cell damage and thereby show DSA mediated cytotoxicity and cytotoxicity of allo-antibodies that were undetectable by the classical L-CDC method, confirming the potential value of endothelial cell based cross-matching in transplantation.

CITATION INFORMATION: Lammerts R., van den Born J., Dam W., Seelen M., Hepkema B., Kroesen B., Daha M., Pol R., Berger S., On behalf of the COMBAT Consortium A Novel Endothelial Cell Based Complement Dependent Cytotoxicity Test in Kidney Transplantation Am J Transplant. 2017;17 (suppl 3).

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