Testing for Antibody-Induced BSEP Deficiency (AIBD) using a Novel Cell-based Assay

Background Antibody-induced bile salt export pump (BSEP) deficiency (AIBD) is an aquired type of intrahepatic cholestasis that may arise after orthotopic liver transplantation for PFIC-2 in patients with compete absence of BSEP expression. IgG-type antibodies are formed against the transplanted alloantigen which bind to BSEP from the canalicular lumen, effectively causing recurrence of pre-transplant symptoms by BSEP trans-inhibition. We devised and validated a simple robust assay for measuring BSEP trans-inhibition by patients” serum antibodies to functionally confirm AIBD diagnosis.

Methods Human embryonal kidney (HEK) 293 cell lines stably expressing either human NTCP-mCherry, BSEP-EYFP, or both were generated by lentiviral transduction and clonal isolation. Patient sera were tested for BSEP antibodies by western blot and immunofluorescence staining of human liver and HEK293-BSEP-EYFP cells. Using our cell lines, a two-phase assay was devised consisting of preloading cells by NTCP with [³H]-Taurocholate (TC) followed by TC export into fresh medium by BSEP. To test for BSEP trans-inihibition, NTCP/BSEP-expressing cells were pre-incubated with either AIBD or control sera prior to the assay. Live cell staining was performed to demonstrate extracellular serum IgG binding to BSEP and exclude BSEP internalization upon IgG binding.

Results and Conclusion Comparison of the stable cell lines demonstrated specific [³H]-TC uptake during the import phase dominated by NTCP and excretion during the export phase by BSEP in which re-import of [³H]-TC by the sodium-dependent NTCP was prevented by substituting choline for extracellular sodium. Thus, BSEP inhibition by sera from an AIBD patient cohort as well as by purified AIBD antibodies, but not by control or non-AIBD cholestatisis cohorts could be demonstrated. Our data show that the assay readout results from direct functional BSEP inhibition and is not impaired by serum complement activation or BSEP internalization upon antibody binding. In contrast, excessive levels of serum bile salts need to be removed before the assay as they compete with the model substrate. Notably, we found high anti-BSEP titers in one asymptomatic post-OLT PFIC-2 patient, which showed no trans-inhibition in vitro. This clearly shows that detection of anti-BSEP antibodies alone is insufficient for AIBD diagnosis but needs to be complemented by a functional inhibition test.

Publikationsverlauf

Artikel online veröffentlicht:
04. Januar 2021