NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti–PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9–10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.

Patients Who Respond to Donor Lymphocyte Infusion (DLI) Have an Antibody Response Against PDC-E2, the Immunodominant Autoantigens of Primary Biliary Cirrhosis (PBC), but with Different Specificity.

Although, the effectiveness of allogeneic hematopoietic stem cell transplantation (HSCT) is, in large part, due to the destruction of recipient malignant cells by donor immune cells, the antigenic targets of this response in most patients are not well defined. In previous studies we demonstrated that patients with multiple myeloma (MM) who achieved a complete response after HSCT and donor lymphocyte infusion (DLI) developed high titer antibodies to a variety of antigens expressed by myeloma cells. Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2) was one of the antigens identified in this screen. Importantly PDC-E2 is the immunodominant auto-antigen in primary biliary cirrhosis (PBC), a liver autoimmune disease in which greater than 95% of patients develop auto-antibodies against the inner lipoyl domain of this mitochondrial antigen. To further characterize the antibody response against PDC-E2 after allogeneic HSCT, we developed a sensitive ELISA to measure antibody responses to GST-PDC-E2 fusion protein. PDC-E2 antibodies following HSCT were compared to 52 normal donors and 50 patients with PBC. All samples were tested at 1:50 or 1:100 dilution. PDC-E2 antibodies were not detected in 20 patients with MM at the time of diagnosis, 10 patients with hematologic malignancies after allogeneic HSCT who did not receive DLI and 10 patients with chronic graft-versus-host disease (GVHD). However, when screening patients who achieved complete remission after DLI, PDC-E2 antibodies were detecting in 3 out of 10 patients with MM, 1 out of 5 patients with chronic myelocytic leukemia (CML) and 1 out of 2 patients with chronic lymphocytic leukemia (CLL). Although some of these patients developed GVHD, none developed chronic liver disease. In each case, PDC-E2 antibodies were only detectable after DLI and not pre-HSCT or pre-DLI. In 2 patients with particulary high titer antibodies (detectable down to 1:10,000 dilution) anti-PDC-E2 persisted for more than 2 years post-DLI and the dominant Ig subclasses at all time points were IgG1 and IgG2. The specificity of PDC-E2 antibodies post-DLI was also tested by ELISA against a panel of 85 overlapping peptides representing the entire amino acid sequence of PDC-E2 and by western blot against 2 common mitochondrial auto-antigens, branched-chain alpha-keto acid dehydrogenase (BCKD) and 2-oxoglutarate dehydrogenase (ODGC). Post-DLI sera were not reactive with BCKD or ODGC and most samples specifically recognized 2 peptides located in the E2 catalytic domain of PDC-E2. In contrast, serum from PBC patients had a different pattern of reactivity and were primarily directed against peptides in the inner lipoyl domain of PDC-E2. In conclusion, we demonstrate that patients with hematologic malignancies who achieve complete remission following DLI frequently develop allogeneic antibody responses directed against one of the most common auto-antigens in PBC. However, the epitope specificity of the antibody response following allogeneic HSCT is distinct and is not associated with chronic liver disease.

PDC-E2, a Common Auto Antigen in Primary Biliary Cirrhosis (PBC) Is Also a Target of an Antibody Response in Patients Who Achieve Complete Remission after Donor Lymphocyte Infusion.

The ability of donor lymphocyte infusions (DLI) to induce complete responses (CR) in patients with relapsed multiple myeloma (MM) following allogeneic bone marrow transplantation provides clear evidence of graft-versus-myeloma (GVM) immunity. To identify GVM associated target antigens, we previously screened an MM cDNA expression library with post DLI serum from 5 MM patients who achieved CR after DLI. One of the antigens identified in the screening was dihydrolipoamide acetyltranferase the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Using a phage plate assay, antibody reactivity against PDC-E2 was found in 2 of 9 MM and 1 of 5 chronic myeloid leukemia (CML) patients who achieved a CR after DLI. No antibodies were found before DLI, in 20 normal donors, 10 patients who underwent T-cell depleted allogeneic BMT, 6 MM DLI non-responders and 20 patients with chronic GVHD. Primary biliary cirrhosis (PBC) is an autoimmune liver disease in which more than 80% of patients have autoantibodies against PDC-E2. In patients with PBC the major immunogenic epitope of PDC-E2 has been mapped to the region associated with the inner lipoyl domain. To better characterize the antibody response in MM patients who had a CR after DLI a GST-PDC-E2 fusion protein was purified and used to quantify the antibody response by ELISA. Using this sensitive assay we found 1 additional MM DLI responder who had antibody reactivity against PDC-E2. Analysis performed at serial time points after DLI showed that reactivity persisted for 1.5 and 3 years after DLI in two MM patients and 3 years after DLI in the CML DLI responder. To map the antibody response after DLI and compare this to antibody reactivity in patients with PBC, we synthesized a series of 85 overlapping peptides covering the entire length of PDC-E2 and analyzed the specificity of the antibody response by ELISA. Using this assay, post-DLI serum was found to be reactive against 4 peptides located in the E2 catalytic domain of PDC-E2 but no reactivity was detected against peptides located in the inner lipoyl domain of the protein, the region commonly recognized by auto-antibodies in PBC. Analysis of serial samples showed that the antibody response persisted against these peptides up to 2–3 years after DLI but no reactivity was found pre-DLI and in normal serum. In conclusion we show that PDC-E2, a common auto-antigen in the autoimmune disease PBC, is also the target of an antibody response in patients with MM and CML who achieve a CR after DLI. The antibody response found after DLI is directed against a different region of the protein. Further studies characterizing T cell epitopes in these patients are underway and they will help to better characterize the immune response against PDC-E2 in patients after DLI.

Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mitochondrion, is diminished in animal models of diabetes. Studies with purified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydrolipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdomain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inactivation of the complex. By combining molecular modelling, site-directed mutagenesis and biophysical characterizations, we have also identified two amino acid residues in this subdomain (Ile229 and Phe231) that largely determine the magnitude of this effect.

Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.