Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease, which has a prevalence of approximately 1/10,000 in the United States and Europe and accounts for 2-6% of all liver transplantations. AIH is diagnosed by elevated alanine amino transferase levels, an intrahepatic lymphocytic mononuclear cell infiltrate, hypergammaglobulinemia and autoantibodies, and by the absence of liver disease of viral, toxic or metabolic etiology.? ? Autoantibodies target nuclear antigens (antinuclear antibodies, ANA), smooth muscle antigens (SMA), liver-kidney microsomes (LKM-1) and/or soluble liver antigen/liver-pancreas antigen (SLA/LP). Autoantibodies to SLA/LP are detectable in about 20% of AIH cases, and are unique in that they are the only autoantibodies specific for AIH. The target of SLA/LP autoantibodies has been cloned and recently been termed SEPSECS by the Nomenclature Commission of the Human Genome Organization. However, even though autoantibodies are well accepted as serologic markers for diagnosis and classification of AIH, it is still unclear whether they play any role in disease pathogenesis.? ? In contrast, the intrahepatic T cell infiltrate, the histologic hallmark of AIH, correlates with disease progression and are therefore thought to contribute directly to disease pathogenesis. The clonal restriction of the intrahepatic T cell population and the observation that HLA-DRB1*0301 and HLA-DRB1*0401 predispose to AIH and influence disease severity, have led to the hypothesis that intrahepatic CD4+ T cells recognize self-antigens in the context of HLA-DRB1*0301 and HLA-DRB1*0401. HLA-DRB1*0301, the principal AIH susceptibility allele among white Europeans and Americans, and HLA-DRB1*0401 may even present the same autoantigen to CD4+ T cells because both alleles share an amino acid motif in their antigen binding groove (shared binding hypothesis). ? ? Despite much effort, it still remains unknown which immunogenic peptides HLA-DRB1*0301 and HLA-DRB1*0401-restricted CD4+ T cells recognize in AIH. This question has recently been declared a high priority investigational challenge, but proven difficult to answer for the following reasons. As in other autoimmune diseases, the frequency of autoreactive CD4+ T cells in the peripheral blood is too low to screen a large number of candidate peptides for T cell recognition. Although the relative frequency of autoreactive CD4+ T cells is presumably higher at the site of inflammation, only a small piece of tissue and thereby only a small number of lymphocytes can be obtained by liver biopsy. It remains necessary to expand and/or clone liver-biopsy-derived T cells in vitro before they can be studied in functional assays. This approach renders an ex vivo assessment of the number and function of autoantigen-specific T cells impossible. Furthermore, assays that solely rely on the function of autoantigen-specific T cells are often compromised due to immunosuppressive therapy of AIH. ? ? To identify T cell targets, we immunized 10 DRB1*0301-transgenic C57BL/6 mice that were negative for all murine class II alleles with human recombinant SLA/LP, a recently identified B cell antigen in autoimmune hepatitis. After 2 rounds of immunization, SLA/LP-specific IgG1 and IgG2a antibodies were detectable by ELISA. Importantly, antibodies against the immunodominant epitope described in patients with autoimmune hepatitis could only be induced in DR3-transgenic mice, but not in wildtype mice suggesting a role of HLA-DR3 restricted T cells in the induction of these autoantibodies. ? SLA-specific T cells were detectable in spleen and draining lymph nodes by proliferation and IL-2 ELISPOT assays. Three distinct immunogenic regions within SLA/LP that are recognized by T cells were identified using overlapping 20mer peptides. SLA/LP-specific T cell hybridoma were established and minimal optimal epitopes were mapped with truncated peptides. Notably, one of the T cell epitopes overlapped with the sequence of the dominant B cell epitope described in patients with autoimmune hepatitis. ? ? In summary, this study identified three T cell epitopes within SLA, a protein targeted by organ-specific B cells in autoimmune hepatitis. The data suggest that cellular immune responses contribute to the pathogenesis of autoimmune hepatitis in the context of an HLA DRB1*0301 haplotype. The identified peptides can now be used to monitor T cell responses of patients with AIH.
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