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.

Abstract 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.

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

Journal of Experimental Medicine ◽

10.1084/jem.20051911 ◽

2006 ◽

Vol 203 (5) ◽

pp. 1209-1219 ◽

Cited By ~ 138

Author(s):

Junichiro Irie ◽

Yuehong Wu ◽

Linda S. Wicker ◽

Daniel Rainbow ◽

Michael A. Nalesnik ◽

...

Keyword(s):

T Cells ◽

Primary Biliary Cirrhosis ◽

Pyruvate Dehydrogenase Complex ◽

Biliary Cirrhosis ◽

Biliary Disease ◽

Nonobese Diabetic ◽

Biliary Pathology ◽

Biliary Ducts ◽

Inner Lipoyl Domain ◽

Lipoyl Domain

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.

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HLA DRB4 0101-restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis: evidence of molecular mimicry in human autoimmune diseases.

Journal of Experimental Medicine ◽

10.1084/jem.181.5.1835 ◽

1995 ◽

Vol 181 (5) ◽

pp. 1835-1845 ◽

Cited By ~ 210

Author(s):

S Shimoda ◽

M Nakamura ◽

H Ishibashi ◽

K Hayashida ◽

Y Niho

Keyword(s):

T Cell ◽

Primary Biliary Cirrhosis ◽

Molecular Mimicry ◽

Cell Clone ◽

Pyruvate Dehydrogenase Complex ◽

Biliary Cirrhosis ◽

T Cell Clones ◽

Cell Clones ◽

T Cell Clone ◽

Lipoyl Domain

We established six T cell clones specific for pyruvate dehydrogenase complex (PDC)-E2 peptides from four different patients with primary biliary cirrhosis using 33 different peptides of 17-20 amino acid residues corresponding to human PDC-E2 as stimulating antigens. The minimal T cell epitopes of these six T cell clones were all mapped to the same region of the PDC-E2 peptide 163-176 (GDLLAEIETDKATI), which corresponds to the inner lipoyl domain of PDC-E2. The HLA restriction molecules for this epitope were all identified as HLA DRB4 0101. The common essential amino acids of this epitope for these T cell clones were E, D, and K at positions 170, 172, and 173, respectively; other crucial amino acids for this epitope differed in each T cell clone. In addition, the alanine-substituted peptides at positions 170 and 173, but not 172, inhibited the proliferation of all T cell clones induced by the original peptide of human PDC-E2 163-176, indicating that amino acid D at position 172 is a critical MHC-binding site for all T cell clones tested. Interestingly, all T cell clones reacted to PDC-E2 peptide 36-49 (GDLIAEVETDKATV), which corresponds to the outer lipoyl domain of human PDC-E2. Furthermore, one T cell clone cross-reacted with exogenous antigens such as Escherichia coli PDC-E2 peptide 31-44/134-147/235-248 (EQSLITVEGDKASM), which has an EXDK sequence. This is a definite demonstration of the presence of molecular mimicry at the T cell clonal level in human autoimmune diseases. It is also considered possible to design peptide-specific immunotherapy based on the findings of T cell autoepitopes in primary biliary cirrhosis.

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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.

Blood ◽

10.1182/blood.v106.11.3105.3105 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3105-3105

Author(s):

Roberto Bellucci ◽

Meagan Gallagher ◽

Sabine Oertelt ◽

Emmanuel Zorn ◽

Edwin P. Alyea ◽

...

Keyword(s):

Liver Disease ◽

Antibody Response ◽

High Titer ◽

Hematologic Malignancies ◽

Donor Lymphocyte Infusion ◽

Antibody Responses ◽

Biliary Cirrhosis ◽

Allogeneic Hsct ◽

Inner Lipoyl Domain ◽

Lipoyl Domain

Abstract 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.

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T-cell responses to the components of pyruvate dehydrogenase complex in primary biliary cirrhosis*1

Hepatology ◽

10.1016/0270-9139(95)90246-5 ◽

1995 ◽

Vol 21 (4) ◽

pp. 995-1002 ◽

Cited By ~ 5

Author(s):

D JONES

Keyword(s):

T Cell ◽

Primary Biliary Cirrhosis ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

T Cell Responses ◽

Biliary Cirrhosis ◽

Cell Responses ◽

Dehydrogenase Complex

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Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis.

Journal of Experimental Medicine ◽

10.1084/jem.181.2.723 ◽

1995 ◽

Vol 181 (2) ◽

pp. 723-733 ◽

Cited By ~ 87

Author(s):

J Van de Water ◽

A Ansari ◽

T Prindiville ◽

R L Coppel ◽

N Ricalton ◽

...

Keyword(s):

T Cell ◽

Primary Biliary Cirrhosis ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Autoimmune Response ◽

Biliary Cirrhosis ◽

Human Organ ◽

T Cell Clones ◽

Autoreactive T Cell ◽

Cell Clones

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen-specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous.

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