The differential contribution of antimitochondrial antibodies and T cell subsets in the pathogenesis of autoimmune cholangitis in the murine models of primary biliary cirrhosis

Primary Biliary Cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial autoantibodies (AMA) and the progressive destruction of the intrahepatic bile ducts. Most, if not all PBC patients are characterized by three major preclinical serological and pathological observations: (1) early elevation of serum IgM, (2) presence of AMA, and (3) lymphocytic infiltration and granuloma formation in the hepatic lesions. The relative contribution and exact mechanism of how these three observations are triggered and lead to disease onset is unknown at this moment. We hypothesized that tyrosine sulfation, a common post-translational modification, may be involved in the generation of high affinity AMA and together with CD8 T cell portal tract infiltrates are directly responsible for the destruction of the small bile duct. In order to test this hypothesis, we have generated and characterized AMA hybridomas derived from various murine models. Both AMA positive IgM and IgG isotypes were adoptively transferred into C57BL/6 mice to elucidate their ability to transfer or accelerate disease. In a parallel study, we selectively depleted CD4 and CD8 T cells in IL-2Ralpha-/- mice to understand the role of T cells in the pathogenesis of autoimmune cholangitis. Using a position specific scoring matrix, 4 tyrosine sulfation sites were predicted to exist on the CDR3 region of PD2, a human AMA monoclonal antibody. Inhibition of tyrosine sulfation via sodium chlorate treatment did not alter antigen binding specificity and affinity of all antibodies tested. In search of additional monoclonal AMAs, we isolated 2 IgG and 20 IgM AMA hybridomas from 9 separate hybridoma fusions from 4 different PBC murine models: (1) non-obese diabetic c3c4 congenic, (2) dominant negative TGF-beta receptor type II, (3) IL-2 receptor alpha knockout, and (4) xenobiotic induced model. After selecting for specificity against the mitochondrial autoantigen, the lack of cross reactivity against control proteins, and stable antibody production, one clone each of IgM and IgG monoclonal antibodies (mAb) was used for transfer into C57BL/6 mice. AMA-IgM mAb transferred mice remained disease-free after 24 weeks. In an effort to accelerate disease onset, the AMA-IgG mAb were injected into the xenobiotic induced murine model. Surprisingly, after 12 weeks of high affinity AMA-IgG mAb transfer, the treated mice had reduced levels of bile duct damage compared to the controls that received either PBS or low affinity AMA-IgG mAb. In the T cell studies, the depletion of CD8 T cells prevented the development of autoimmune cholangitis as expected. Contrary to the current paradigm, these animal studies suggest AMA to play a protective role with CD8 T cells being the primary effector cells in the pathogenesis of PBC. Future studies with this panel of AMA hybridomas will confirm whether high affinity AMA are protective against the development of PBC and whether tyrosine sulfated AMA break tolerance against self antigens.