Cellular immune responses to human betaretrovirus in patients with primary biliary cholangitis

Abstract

Human betaretrovirus (HBRV) infection has been characterized in patients with primary biliary cholangitis (PBC). Our lab has documented HBRV proviral integrations in bile ducts of PBC patients. However, serological diagnostics cannot detect HBRV infection in the majority of PBC patients, limiting further confirmation of viral infection. In FACS studies using pooled 17-20 aa peptides derived from the HBRV Gag (n=58) and Env (n=85) proteins, 40% of PBC patients PBMCs were found to make proinflammatory cellular immune responses to HBRV. Then, to characterize immunodominant HBRV epitopes, we screened intra-hepatic lymphocytes (IHL) from PBC patients and control subjects for evidence of IFN-γ production. IHL isolated from liver transplant recipients with PBC (n=8) and other hepatic disorders (n=9) were individually stimulated with 18-mer peptides from HBRV Gag or Env proteins (n=143) or the characterized CD8+ reactive epitope derived from the mitochondrial autoantigen, pyruvate dehydrogenase-E2 (PDC-E2). ELISpot was used to measure spot forming colonies (SFC) producing IFN-γ. 10 HBRV Gag and 12 HBRV Env peptides were found to stimulate IHL. The mean number of SFC producing IFN-γ was higher in PBC patients versus control subjects. Using background cut off level of 1:100 SFC, the individual HBRV Gag and Env peptides provided a high specificity and sensitivity for detecting HBRV infection in PBC patients’ IHL. Notably, only one PBC patient had detectable IFN-γ producing IHL following stimulation with the characterized PDC-E2 peptide. These are the first data to demonstrate that the intrahepatic IFN-γ cellular immune responses to HBRV greatly exceed the autoimmune response, suggesting that HBRV infection plays an important role in mediating PBC. The identified HBRV peptides can be evaluated to measure the IFN-γ release in peripheral blood mononuclear cells and construct a diagnostic IFN-γ release assay