Immunoglobulin gene usage and immunohistochemical characteristics of human monoclonal antibodies to the mitochondrial autoantigens of primary biliary cirrhosis induced in the XenoMouse

Abstract

Antimitochondrial antibodies (AMA) are detected in Ͼ95% of sera from patients with primary biliary cirrhosis (PBC) and recognize the highly conserved mitochondrial 2-oxo-acid dehydrogenase complex (OADC enzyme complex), including the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of the branched-chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), the E2 subunit of the 2-oxoglutarate dehydrogenase complex (OGDC-E2), and E3BP (E3-binding protein or protein X). [1][2][3][4] We hypothesized that utilization of specific members of the human immunoglobulin (Ig) repertoire plays a critical role in specific epitope recognition of PDC-E2 and initiates a cascade of mucosal autoimmune responses against mitochondrial autoantigens, as a result of specific structural features of the autoantigens or of specific oddities of the etiologic process. To address this issue, we have taken advantage of a unique transgenic animal, XenoMouse (Abgenix Inc., Fremont, CA), that contains the human heavy (H) and light chain loci cloned on yeast artificial chromosomes. 5,6 The yeast artificial chromosomes in XenoMouse include approximately 66 V H (ϳ80%) and 32 V (ϳ75%) gene regions, rendering these mice capable of producing high-affinity, fully functional IgM and IgG2 antibodies, which closely resemble those seen in humans, to multiple antigens. 7,8 Moreover, the Ig gene usage toward immunizing antigens has been shown to be quite diverse. 7 Therefore, the production of human antibodies in XenoMouse provides a valuable tool to investigate the nature and specificity of the human antibody response to autoantigens. Here, we report the successful generation and characterization of human mAbs (HmAbs) to the major mitochondrial autoantigens in PBC, together with a comparison of Ig gene usage with that found in patients with PBC. 9-11 Our data have implications for identifying the causative agent of PBC.Abbreviations: AMA, antimitochondrial antibody; PBC, primary biliary cirrhosis; OADC, the E2 component of the 2-oxo-acid dehydrogenase complex; PDC-E2, the E2 component of pyruvate dehydrogenase; BCOADC-E2, the E2 component of the branched-chain 2-oxo-acid dehydrogenase complex; OGDC-E2, the E2 component of the 2-oxoglutarate dehydrogenase complex; E3BP, E3-binding protein; Ig, immunoglobulin; HmAbs, human monoclonal antibody; r, recombinant; KLH, keyhole limpet hemocyanin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RT, room temperature; HRP, horseradish peroxidase; SDS, sodium dodecyl sulfate; ILD, inner lipoyl domain; PSC, primary sclerosing cholangitis; DIG, digoxigenin.From the

The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma

“…21 After transfer, the membranes were processed for immunoblotting as described previously. 21 The primary antibodies for Bmi1 and EZH2 are described above.…”

“…21 After transfer, the membranes were processed for immunoblotting as described previously. 21 The primary antibodies for Bmi1 and EZH2 are described above. a-Tubulin as an internal control was detected using mouse monoclonal anti-a-tubulin (clone TU-01, Zymed, South San Francisco, CA, USA).…”

Decreased Expression of Bmi1 Is Closely Associated with Cellular Senescence in Small Bile Ducts in Primary Biliary Cirrhosis

“…26 After transfer, the membranes were processed for immunoblotting as described previously. 26 The primary antibodies for bmi1 and p21 WAF1/Cip1 are described above.…”

“…26 After transfer, the membranes were processed for immunoblotting as described previously. 26 The primary antibodies for bmi1 and p21 WAF1/Cip1 are described above. P16 INK4a and ␣-tubulin were detected by using rabbit polyclonal antibody for p16 INK4a (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-␣-tubulin (clone TU-01; Zymed, South San Francisco, CA).…”

The Immunoglobulin genes and Chronic Lymphocytic Leukemia (CLL)

“…Considering the importance of the CDRs in the antigen binding site and in particular the CDR3, our results of restricted CDR3 features and predominant V 2-14 rearrangements indicated that the V H 3-21 + cases showed signs of a common antigen recognition site. The V H 3-21 gene has been shown to be utilized in certain autoantibodies produced in rheumatoid arthritis, Sjögren's syndrome and primary biliary cirrhosis and, similarly to the V H 1-69 gene, an association with autoreactivity specificities has been shown for this VH gene (60,74,75). However, the V H 3-21/ 2-14 antibody specificity and its role in the development of CLL have yet to be defined.…”