Phenotyping and auto-antibody production by liver-infiltrating B cells in primary sclerosing cholangitis and primary biliary cholangitis

Abstract

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are immune-mediated biliary diseases that demonstrate prominent and restricted genetic association with human leukocyte antigen (HLA) alleles. In PBC, anti-mitochondrial antibodies (AMA) are specific and used as diagnostic biomarkers. PSC-relevant auto-antibodies remain controversial despite a distinct HLA association that mirrors archetypical auto-antigen driven disorders. Herein, we compared antibody-secreting B cells (ASCs) in PSC and PBC liver explants to determine if liver-infiltrating ASCs represent an opportune and novel source of disease-relevant auto-antibodies. Using enzymatic digestion and mechanical disruption, liver mononuclear cells (LIMCs) were isolated from fresh PSC and PBC explants and plasmablast (CD19+CD27+CD38CD138-) and plasma cell (CD19+CD27+CD38CD138+) ASCs were enumerated by flow cytometry. We observed 45-fold fewer plasma cells in PSC explants (n = 9) compared to PBC samples (n = 5, p < 0.01) and 10-fold fewer IgA-, IgG- and IgM-positive ASCs (p < 0.05). Liver-infiltrating ASCs from PSC and PBC explants were functional and produced similar concentrations of IgA, IgG and IgM following 2 weeks of culture. Antibody production by PBC ASCs (n = 3) was disease-specific as AMA to pyruvate dehydrogenase complex E2 subunit (PDC-E2) was detected by immunostaining, immunoblotting and ELISA. Antibody profiling of PSC supernatants (n = 9) using full-length recombinant human protein arrays (Cambridge Protein Arrays) revealed reactivities to nucleolar protein 3 (5/9) and hematopoietic cell-specific Lyn substrate 1 (3/9). Array analysis of PBC supernatants (n = 3) detected reactivities to PDC-E2 and hexokinase 1 (3/3). In conclusion, we detected unique frequencies of liver-infiltrating ASCs in PSC and PBC and in so doing, highlight a feasible approach for understanding disease-relevant antibodies in PSC.

Clonal characteristics of paired infiltrating and circulating B lymphocyte repertoire in patients with primary biliary cholangitis

“…An insignificantly abundant clonal diversity in the infiltrating B cell repertoire of PBC relative to ALD patients was observed from all perspectives (Fig. ), which conforms with more infiltrating B lymphoplasmacytes in the end‐stage PBC patients …”

Gut and Liver B Cells of Common Clonal Origin in Primary Sclerosing Cholangitis–Inflammatory Bowel Disease

“…The pathogenic role of B cells in PSC‐IBD is poorly established as a broad spectrum of non‐specific autoantibodies to biliary and colonic epithelial antigens, neutrophil granulocytes (perinuclear anti‐neutrophil cytoplasmic antibodies [pANCA]), and several ubiquitous self‐proteins appear in affected individuals . Although disease‐specific autoantibodies have not been identified in sera, we have demonstrated that B cells infiltrating liver explants of patients with PSC‐IBD can be isolated and cultured to produce a diverse repertoire of autoantibodies whose specificities may point toward relevant self‐antigens or cross‐reactive exogenous targets . Thus, a better appreciation of B‐cell reactivities in PSC‐IBD may uncover useful biomarkers similar to patients with primary biliary cholangitis (PBC) where liver‐infiltrating B cells produce disease‐associated anti‐mitochondrial antibodies to pyruvate dehydrogenase complex‐E2 .…”

“…Although disease‐specific autoantibodies have not been identified in sera, we have demonstrated that B cells infiltrating liver explants of patients with PSC‐IBD can be isolated and cultured to produce a diverse repertoire of autoantibodies whose specificities may point toward relevant self‐antigens or cross‐reactive exogenous targets . Thus, a better appreciation of B‐cell reactivities in PSC‐IBD may uncover useful biomarkers similar to patients with primary biliary cholangitis (PBC) where liver‐infiltrating B cells produce disease‐associated anti‐mitochondrial antibodies to pyruvate dehydrogenase complex‐E2 . A better understanding of B‐cell reactivities in PSC‐IBD may also help define patient subgroups, including specific genetic and disease characteristics of pANCA‐positive versus seronegative patients …”

Genetic Discoveries Highlight Environmental Factors as Key Drivers of Liver Disease

“…Recent efforts of characterizing disease-associated antibodies using liver-resident B cells from PSC patients could lead to the identification of relevant autoantibodies, but further work is needed to validate such methodologies [85] . In 30-80% of cases, PSC strongly associates with inflammatory bowel disease (PSC-IBD) suggesting that the gut-liver axis contributes to the pathogenesis of PSC, including lymphocyte migration and the exposure to shared antigens between the gut and liver [86] .…”