Amanda J. Robe

Investigation of a Mechanism for Accelerated Breakdown of Immune Tolerance to the Primary Biliary Cirrhosis–Associated Autoantigen, Pyruvate Dehydrogenase Complex

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by autoreactive T- and B-cell responses to the highly conserved enzyme pyruvate dehydrogenase complex (PDC). In this study we have examined the breakdown of T-cell tolerance to self-PDC using a mouse model. Female SJL/J mice were sensitized intraperitoneally with foreign-PDC (bovine) and/or self-PDC (murine) in complete Freunds adjuvant, and serum, spleen, and liver tissue was taken 8 weeks later. Animals sensitized with foreign-PDC produced IgG antibodies that were reactive with both foreign and self-PDC, but splenic T cells from these animals only responded to stimulation with foreign PDC. Sensitization with self-PDC elicited neither antibodies nor reactive T cells. Significantly, cosensitization with mixed self-PDC and foreign-PDC resulted in a full breakdown of self-tolerance, with generation of both antibody and T-cell responses to self-PDC of the type seen exclusively in human PBC patients. Mild bile duct lesions deficient in CD8+ T cells were seen 8 weeks after sensitization with either foreign or self-PDC. However, after sensitization with mixed self-PDC and foreign-PDC, these lesions were significantly larger and heavily infiltrated by CD8+ T cells. Liver-infiltrating T cells derived from the self-PDC and foreign-PDC cosensitized but not from control animals showed reactivity with self-PDC, suggesting a possible role for autoreactive PDC-specific T-cell responses in the pathogenesis of the observed histologic changes. It is likely that B-cell cross-reactivity between foreign and self-PDC enhances the potential for breakdown of T-cell self-tolerance by allowing efficient presentation of self-antigens in the inoculum. This model may provide a useful system for investigating the etiology and treatment of PBC.

Covalent modification as a mechanism for the breakdown of immune tolerance to pyruvate dehydrogenase complex in the mouse

The autoimmune liver disease primary biliary cirrhosis (PBC) is characterized by the breakdown of normal immune self tolerance to pyruvate dehydrogenase complex (PDC). How tolerance is broken to such a central and highly conserved self antigen in the initiation of autoimmunity remains unclear. One postulated mechanism is that reactivity arises to an altered form of self antigen with subsequent cross‐reactivity to native self. In this murine study, we set out to examine whether sensitization with a covalently modified form of self PDC can give rise to the pattern of breakdown of B‐cell and T‐cell tolerance to self PDC seen in PBC patients. The notion that altered self can lead to tolerance breakdown was studied by sensitizing SJL/J mice with a covalently modified (biotinylated) preparation of self murine PDC (mP/O‐B). Subsequently, antibody and T‐cell reactivities to unmodified self mP/O were studied. Sensitization with mP/O‐B elicited high‐titre, high‐affinity antibody responses reactive with both the mP/O‐B immunogen and, importantly, native mP/O. In addition, significant MHC class II restricted splenic T‐cell responses to native mP/O (i.e., true autoimmune responses) were seen in mP/O‐B sensitized animals. The breakdown of T‐cell self tolerance to mP/O was not seen in animals sensitized with irrelevant biotinylated antigens. In conclusion, this study provides evidence to support the concept that exposure to covalently modified self PDC can, in the correct proimmune environment, replicate the full breakdown of B‐cell and T‐cell immune tolerance to PDC seen in PBC. One potential etiological pathway in PBC therefore could be the breakdown of tolerance to self PDC occurring after exposure to self antigen covalently modified in the metabolically active environment of the liver. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;39:1583–1592.)

A key role for autoreactive B cells in the breakdown of T-cell tolerance to pyruvate dehydrogenase complex in the mouse.

The key immunological event in the pathogenesis of the autoimmune liver disease primary biliary cirrhosis is breakdown of T‐cell self‐tolerance to pyruvate dehydrogenase complex (PDC). The mechanism resulting in this breakdown of tolerance remains unclear. Mice exposed to self‐PDC mount no immune response; however, animals coexposed to self‐PDC and PDC of foreign origin (which in isolation induces a cross‐reactive antibody but not an autoreactive T‐cell response) show breakdown of T‐cell as well as B‐cell tolerance. This observation raises the possibility that a cross‐reactive antibody response to self‐PDC can promote breakdown of T‐cell tolerance. The aim of this study was to address the hypothesis that breakdown of T‐cell tolerance to PDC can be driven by the presence of B cells and/or antibodies cross‐reactive with this self‐antigen. Naive female SJL/J mice were exposed to self‐PDC alone and in the presence of purified splenic B cells from animals primed with foreign PDC (or controls) or purified immunoglobulin (Ig) G from the same animals. Breakdown of T‐cell tolerance was assessed by splenic T‐cell proliferative response to antigen at 5 weeks. CD4+ T‐cell proliferative responses indicative of breakdown of T‐cell tolerance to self‐PDC were seen in the majority (7 of 9, 78%) of animals receiving self‐PDC together with purified PDC‐reactive B cells. Tolerance breakdown was not seen in animals receiving self‐PDC with purified anti‐PDC IgG or with B cells from animals sensitized with an irrelevant antigen. In conclusion, breakdown of T‐cell tolerance to the highly conserved self‐antigen PDC may be mediated by high‐level presentation of self‐derived epitopes by activated cross‐reactive B cells. (HEPATOLOGY 2005.)

Oral Tolerisation to Pyruvate Dehydrogenase Complex as a Potential Therapy for Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is characterised by immune-mediated damage to the intra-hepatic biliary epithelial cells (BEC). Immuno-modulatory/suppressive therapy represents, therefore, a logical approach to treatment in this disease. Conventional immuno-suppressive and immuno-modulatory agents suffer from the breadth of their action and/or excessive side effects. Autoreactive responses to pyruvate dehydrogenase complex (PDC) have been extensively characterised in PBC, and implicated in target cell damage. The aim of the current study was to study the potential efficacy of an antigen specific approach (oral tolerisation with autoantigen) to modulation of anti-PDC immune responses characteristic of PBC, utilising a mouse model of PDC immuno-reactivity. Groups of SJL/J mice were orally dosed with PDC alone, dosed with carrier only (saline) but systemically sensitised with PDC in adjuvant, or orally dosed with PDC at high (5 mg) or low (0.01 mg) dose and systemically sensitised with PDC. Oral dosing with PDC in isolation had no adverse effects on the animals and did not prime anti-PDC responses at doses below 1 mg. Pre-dosing with PDC at both high and low doses was effective at skewing the phenotype of the T-cell response to PDC induced by subsequent sensitisation away from the disease associated Th-1 phenotype (IL-2 and IFN- n secreting) and towards a theoretically protective Th-2 phenotype (IL-4 secreting) in a majority of, but not all, animals. No augmentation of Th-1 response was seen in any animal. Although the effects on liver histology remain to established oral tolerisation with PDC holds promise as a novel, antigen specific approach to therapy in PBC.

PDC-E3BP is not a dominant T-cell autoantigen in primary biliary cirrhosis

Abstract: Background: Autoantibody responses reactive with the E2 and E3BP components of pyruvate dehydrogenase complex (PDC), which characterise primary biliary cirrhosis (PBC) crossreact, precluding the identification, from serological studies, of the antigen to which the principal breakdown of tolerance occurs. Although autoreactive T‐cell responses to PDC‐E2 have been well characterised it is, at present, unclear whether T‐cell tolerance breakdown also occurs to PDC‐E3BP. The aims of this study were to characterise autoreactive T‐cell responses to PDC‐E3BP in PBC and potential factors regulating their expression.