Detection of complement‐fixing and non‐fixing antibodies specific for endothelial precursor cells and lymphocytes using flow cytometry

Abstract

Donor human leukocyte antigen (HLA)-specific antibodies (Abs) with the ability to activate complement are associated with an increased risk of early Ab-mediated rejection (AMR) of kidney allografts. In recent years, also non-HLA Abs-binding endothelial cells have been shown to elicit early AMR. Donor-specific anti-endothelial cell Abs escape detection in the pre-transplant evaluation if only lymphocytes are used as target cells in crossmatch tests. We addressed whether endothelial precursor cells (EPCs) could be used for detection of complement-fixing as well as non-fixing Abs and if complement factor and immunoglobulin G (IgG) deposition on co-purified T and B cells correlated to the outcome of the T- and B-cell complement-dependent cytotoxicity assay. Deposition of complement factors C3c and C3d, but not C1q nor C4d, were detected on EPCs and lymphocytes upon incubation with HLA Ab-positive sera. There was a correlation between the amount of C3c deposition and IgG binding on EPCs (R(2) = 0.71, P = 0.0012) and T cells (R(2) = 0.74, P = 0.0006) but not for B cells (R(2) = 0.34, P = 0.059). The specificity and sensitivity for C3d deposition on endothelial precursor cell crossmatch (EPCXM) T cells vs the T complement-dependent cytotoxicity (CDC) assay were 69% and 72%, respectively. The EPCXM B-cell C3d assay had considerably lower sensitivity (39%) than the B CDC assay. Altogether, this novel assay based on the detection of complements factors on EPCs and lymphocytes by flow cytometry may widen the diagnostic repertoire and thereby improve the clinical management of patients undergoing kidney transplantation.

Not All Antibodies Are Created Equal: Factors That Influence Antibody Mediated Rejection

“…Antibody affinity mediated by IgG hexamers has been shown to be more efficient than isolated IgG molecules at activating the complement cascade [91]. Complement binding is also increased when there is an increase in the amount of antibody bound to cells [92, 93]. High panel reactive antibodies (PRA) are associated with increased complement activation [92].…”

“…Complement binding is also increased when there is an increase in the amount of antibody bound to cells [92, 93]. High panel reactive antibodies (PRA) are associated with increased complement activation [92]. Lastly, polymorphisms within the complement genetic locus could potentially affect the degree of complement activation [94, 95], whereas differential expression of complement regulatory proteins by the donor tissue could also affect the response of endothelium to complement components [96].…”

Antibody-mediated graft injury

“…In recent years, new assays have been developed to characterize the functional deposition of complement components, including C1q, C3d and C4d, by HLA antibody in solid phase assays or cell-based protocols (1, 44-46). However, contradictory conclusions have been reached regarding the predictive significance of complement fixing DSA detected by these assays (47, 48), and the results often do not correlate with lymphocytotoxicity (CDC-XM) results (46).…”

“…Whereas HLA antibodies trigger complement-dependent cytotoxicity of target lymphocytes, endothelial cells—the primary target of antibody-mediated alloimmunity—are generally resistant to complement-mediated cell lysis in response to HLA antibodies. Despite evidence of terminal complement activation at the endothelial cell surface (44, 56), endothelial cells may upregulate cytoprotective factors and survival signaling to prevent HLA antibody-induced cytotoxicity (57, 58). Therefore, the physiological relevance of complement activating antibodies detected by C1q or C3d-fixing assays remains unclear, and the mechanisms of antibody-mediated graft injury in C4d negative AMR have yet to be fully elucidated.…”

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

“…Anti-endothelial cell antibodies and HLA antibodies cause generation of complement split products, including C5a, C3c and C3d, at the surface of endothelial cells 25,26 . C5a is a strong chemoattractant for monocytes and neutrophils 27,28 , promoting adhesion through increased expression of the Mac-1 (CD11b) β2 integrin 29–32 .…”