Differential epitope mapping of antibodies to PDC-E2 in patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation and primary biliary cirrhosis

Abstract

A unique characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC) is the presence of high-titer and extremely specific autoantibodies to the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Autoantibodies to PDC-E2 antigen have only been detected in patients with disease or in those who subsequently develop PBC. One exception has been a subgroup of patients with multiple myeloma (MM) who underwent allogeneic hematopoietic stem cell transplantation (HSCT) and received donor lymphocyte infusions (DLIs) after transplantation. These patients developed high-titer antibodies to a variety of myeloma-associated antigens, including PDC-E2, coincident with rejection of myeloma cells in vivo. To examine the specificity of autoantibodies to PDC in these patients, we screened sera from patients with MM, chronic leukemias, monoclonal gammopathy of unknown significance (MGUS), PBC, and healthy donors. Three of 11 patients with MM (27%) and 2 of 6 patients with chronic leukemias (33%) developed anti-PDC-E2 antibodies in association with DLI response; 2 of 12 (17%) patients in the MGUS pretreatment control population also had detectable anti-PDC responses. Interestingly, the epitope specificity of these PDC-E2 autoantibodies was distinctive, suggesting that the mechanisms leading to loss of tolerance in the transplantation patients are distinct from PBC.

Figure 1

Reactivity of patient serum with PDC-E2 demonstrated by Western blot. Purified recombinant PDC-E2–GST or GST proteins were loaded in different lanes and probed with patient serum at 1:500 dilution. Antibodies against GST or PDC-E2 were used as a control. Arrows indicate protein fragments recognized by anti–PDC-E2 antibody.

Figure 2

Indirect immunofluorescence analysis. Sera from MM after DLI (A), CML after DLI (B), and PBC (C) were diluted 1:40 in PBS and applied to human Hep-2 slides. FITC-conjugated secondary antibodies were used as detectors. All slides display the characteristic antimitochondrial immunofluorescence pattern.

Figure 3

Quantitative analysis of PDC-E2 antibodies in post-DLI, post-HSCT, MGUS, PBC, and MM patients compared with healthy donors. PDC-E2 antibodies were measured by ELISA in patient serum (1:100 dilution). Results are shown for 95 patient samples in 7 groups. The dashed line represents the cutoff limit based on the 95th percentile of reactivity in 52 healthy donors.

Figure 4

Serial analysis of post-DLI patient serum. PDC-E2 antibody reactivity was measured by ELISA in 5 patients before HSCT, before DLI, and at several times after DLI. GST background reactivity was subtracted for each sample. The dashed line represents the cutoff limit based on the 95th percentile of reactivity in 52 healthy donors.

Figure 5

IgG subclass analysis. IgG subclasses were measured by ELISA in post-DLI, MGUS, and 12 positive PBC samples. GST background reactivity was subtracted for each sample. Black bars represent the mean reactivity for each group.

Figure 6

Comparison of antibody specificity in post-DLI, MGUS, and PBC patient serum. Serum samples (1:100 dilution) were analyzed for peptide specificity using ELISA and a set of 85 overlapping peptides representing the entire amino acid sequence of PDC-E2. The graph shows the number of samples reactive with each peptide and the corresponding functional domain of the protein.

Figure 7

PDC-E2 peptide titers in post-DLI and MGUS patients. Serum samples were analyzed at serial dilutions against individual PDC-E2 peptides. Reactivity was measured by ELISA against each purified peptide. Reactivity with an irrelevant peptide was subtracted in each assay.