Cell-based BSEP trans-inhibition: A novel, non-invasive test for diagnosis of antibody-induced BSEP deficiency

Abstract

<h2>Abstract</h2><h3>Background&Aims</h3> Antibody-induced bile salt export pump deficiency (AIBD) is an acquired form of intrahepatic cholestasis, which may develop following orthotopic liver transplantation (OLT) for progressive familial intrahepatic cholestasis type 2 (PFIC-2). Approximately 8-33% of transplanted PFIC-2 patients develop bile salt export pump (BSEP) antibodies which trans-inhibit this bile salt transporter from the extracellular, biliary side. AIBD is diagnosed by demonstration of BSEP-reactive and -inhibitory antibodies in patient serum. We developed a cell-based test directly measuring BSEP trans-inhibition by antibodies in serum samples to confirm AIBD diagnosis. <h3>Methods</h3> Sera from healthy controls and cholestatic Non-AIBD or AIBD cases were tested I) for anti-canalicular reactivity by immunofluorescence (IF) staining of human liver cryosections, II) for anti-BSEP reactivity by IF staining of HEK293 cells expressing BSEP-EYFP and immunodetection of BSEP-EYFP on Western blot, and III) for BSEP trans-inhibition using HEK293 cells stably expressing NTCP-mCherry and BSEP-EYFP. The trans-inhibition test uses [³H]-taurocholate as substrate and is divided into an uptake phase dominated by NTCP followed by BSEP-mediated export. For functional analysis, sera were bile salt depleted. <h3>Results</h3> We found BSEP trans-inhibition by seven sera containing anti-BSEP antibodies, but not by five cholestatic or nine control sera, all lacking BSEP reactivity. Prospective screening of a post-OLT PFIC-2 patient showed seroconversion to AIBD and the novel test method allowed monitoring of treatment response. Notably, we identified a post-OLT PFIC-2 patient with anti-BSEP antibodies yet without BSEP trans-inhibition activity, in line with asymptomatic presentation at serum sampling. <h3>Conclusions</h3> Our cell-based assay is the first direct functional test for AIBD and allows confirmation of diagnosis as well as monitoring under therapy. We propose an updated workflow for AIBD diagnosis including this functional assay. <h3>Lay Summary</h3> Antibody-induced BSEP deficiency (AIBD) is a potentially serious complication that may affect PFIC-2 patients after liver transplantation. To improve its early diagnosis and thus immediate treatment, we developed a novel functional assay to confirm AIBD diagnosis using patient's serum and propose an updated diagnostic algorithm for AIBD.