Primary Biliary Cholangitis (PBC) is a chronic autoimmune liver disease characterized by the presence of anti-mitochondrial autoantibodies (AMAs), elevated serum IgM levels, and progressive destruction of intrahepatic bile ducts, ultimately leading to cholestasis and liver fibrosis [[1], [2], [3]]. Various mouse models have been extensively utilized to understand the early and late events of the human PBC pathogenesis [4,5]. These models, including both genetically modified spontaneous models and induced mouse models, have been instrumental in understanding PBC and the evaluation of therapeutics. Spontaneous autoimmune biliary disease mouse models, such as NOD.c3c4 [6,7], dominant negative TGFβ receptor II (dnTGFβRII) [8], IL-2Rα−/− [9], and IFNγ-ARE-Del−/− [7,10] mice, along with induced mouse models like E. coli infection [11] and 2-OA injection [12], have been widely accepted. These models share some of the key characteristics of human PBC, such as biliary leukocyte infiltration, higher serum IFNγ and TNF, elevated serum AMAs, and destruction of intrahepatic bile ducts [[13], [14], [15], [16]]. While no single mouse model fully recapitulates all human PBC phenotypes, they remain crucial tools for understanding the genetic, environmental, and immunological factors contributing to PBC development.

Koaroda S et al. [6] identified PBC-like features in NOD mice when replaced with B10 and B6 mice insulin-dependent diabetes (Idd) loci on chromosomes 3 and 4, (c3: Idd3, Idd10, Idd17 and Idd18 and c4: Idd9, Idd9.2 and Idd9.3), and is termed NOD.c3c4 mice. Studies that investigated the immunogenetics underlying autoimmune biliary disease of NOD.c3c4 mice unexpectedly found a novel disease-associated locus on chromosome 1, designated Abd3, which was present in all diseased congenic strains [17,18]. NOD mice carrying only the Abd3 interval demonstrated that this region alone was both necessary and sufficient to induce autoimmune biliary disease. Subsequently, studies identified a truncating mutation in Pkhd1 (Pkhd1Del 36–67), a gene encoding fibrocystin—a cilia-associated protein highly expressed in cholangiocytes [[19], [20], [21]]. Similar to other Pkhd1 mutations known to cause hepatobiliary pathology [[22], [23], [24]], this variant was associated with liver-restricted autoimmune features, including the production of anti-PDC-E2 autoantibodies, characteristic of PBC. Importantly, disease manifestation required both the Pkhd1 mutation and a permissive NOD genetic background with intact adaptive immunity, suggesting a critical interplay between cholangiocyte-intrinsic defects and immune dysregulation [19,25,26], supporting liver-specific autoimmunity can arise from epithelial cell abnormalities in genetically susceptible hosts.

NOD.c3c4 strain surprisingly developed several key features characteristics of human PBC, although it stopped developing diabetes. It develops PDC-E2 inner lipoyl domain reactive AMAs, higher titers of IgM, IgG against double stranded (ds)DNA, and single stranded (ss)DNA, as well as increased anti-nuclear autoantibodies (ANA) and serum bilirubin and transaminases [27,28]. This strain displayed infiltration of the disease-transferable CD3+, CD4+ and CD8+ T cells around the bile ducts and granuloma formation [6,25,27]. As liver inflammation progresses, NOD.c3c4 mice experience compromised biliary tree function, leading to ascites formation (enlarged abdomen phenotype) and liver failure. However, in-depth disease kinetics, immunological events and key pathogenic events like fibrosis are poorly understood in this model.

Previously, we demonstrated that the NOD.c3c4 mouse strain develops PBC with clinical and immunological features closely resembling the human condition [18,27]. These studies uncovered a previously underappreciated role for both polyclonal CD4+ and CD8+ T cells, alongside PDC-E2-specific autoantibody responses, in disease pathogenesis, laying foundational groundwork for mechanistic and therapeutic investigations. However, key aspects of disease progression remained unresolved, including the dynamics of antigen-specific T cell responses, the role of innate immunity, and the extent to which this model replicates fibrotic remodeling, a hallmark of advanced human PBC. Importantly, our recent work established that NOD.c3c4 mice also serve as an effective platform for evaluating antigen-specific immunotherapies, such as peptide-MHCII- and α-GalCer-loaded CD1d-coated nanomedicines [7,16,29,30], further implicating PDC-E2-specific autoreactive T cells in disease development.

The primary aim of this study was to systematically characterize disease kinetics, immunopathological features, and fibrotic progression in the NOD.c3c4 mouse model of PBC. While this model is known to exhibit key autoimmune features that mirror human PBC, detailed analyses of early versus late disease stages have been limited. To address this gap, we conducted a comprehensive evaluation of immune and fibrotic changes across disease progression, with a particular focus on the roles of innate immunity and autoreactive PDC-E2–specific CD4+ and CD8+ T cells. Our findings confirm that NOD.c3c4 mice exhibit complete disease penetrance, with both innate immune responses and adaptive immunity contributing to liver autoimmunity. Notably, CD4+ and CD8+ T cells targeting PDC-E2 were enriched in the liver, supporting previous reports that T cell depletion ameliorates disease and that adoptive transfer of polyclonal T cells from diseased mice induces PBC in NOD.c3c4.scid recipients [18,27]. While liver fibrosis has been previously observed in this model, particularly in specific environmental contexts, our study adds value by providing a systematic, time-resolved analysis of fibrotic progression in relation to immune activation and fibrotic gene expression. All affected mice in our colony developed progressive hepatic lesions and fibrosis with age, reinforcing the model's relevance for studying late-stage disease features. These findings establish NOD.c3c4 mice as a robust and translationally relevant system for dissecting immune-fibrotic mechanisms in PBC and for testing antigen-specific therapeutic strategies.