In the past years we identified immunogenic amino acid sequences within the liver soluble liver antigen/liver-pancreas antigen (SLA/LP) in humanized mice that are transgenic for HLA-DRB1*0301 and negative for murine MHC class II and were immunized with human SLA/LP. We tested antibody and T cell responses of the immunized mice and established T cell hybridoma to identify the minimal optimal DRB1*0301-restricted peptides (epitopes) within SLA/LP. These epitopes were then used to generate fluorescently labeled tetrameric complexes of DRB1*0301 and epitope peptides for ex vivo detection of murine autoantigen-specific T cells by flow cytometry.? ? In the past 12 months we continued these studies and used the identified T cell epitopes and tetramers to analyze the immune response of patients with anti-SLA/LP+ AIH. As specificity controls, PBMC from an HLA-DRB1*0301- patient with anti-SLA/LP+ AIH and HLA-DRB1*0301+, anti-SLA/LP- patients with past history of hepatitis B were studied in parallel. Overall, tetramer+ CD4 T cells were detected in peripheral blood mononuclear cells (PBMC) of 7/9 AIH patients after enrichment with anti-PE-labeled magnetic beads as compared to 0/3 (0%) control patients (p=0.045, Fishers exact test). When PBMC were stimulated with their cognate SLA/LP peptide more tetramer+ cells than tetramer- cells proliferated (47.0% versus 6.2%). In contrast, only 3.7% tetramer+ cells proliferated upon stimulation with the unrelated, DRB1*0301-restricted HCV peptide which was used as control. ? ? In addition, the newly identified CD4 T cell epitopes were tested in IFN-γ ELISpot assays. Tetramer-enriched CD4+ T cells of HLA-DR1B1*0301-positive AIH patients recognized both epitopes and produced IFN-γ in ELISpot assays, but tetramer-enriched CD4 T cells of HLA-DR1B1*0301-negative AIH patients or DR1B1*0301-positive controls without AIH did not. In these experiments CD4 T cell cells were tested ex vivo and after several weeks of antigen-nonspecific in vitro expansion with 40 ng/ml anti-CD3, 200 U/ml IL-2, 10 ng/ml IL-7 and 100 ng/ml IL-15. The rapid, antigen-nonspecific expansion protocol was chosen over a peptide-specific expansion protocol because of the very low number (< 105) of tetramer-positive cells with which the expansion was started. ELISpot assays with the nonspecifically expanded T cell lines confirmed the ex vivo ELISpot results.? Overall, the frequency of tetramer-positive antigen-specific, CD4+ T cells detectable by Elispot and tetramer analysis in peripheral blood mononuclear cells of patients with anti-SLA/LP+ AIH was in the same range as frequencies of antigen-specific CD4+ T cells reported for other autoimmune diseases such as type I diabetes (Oling et al., J Autoimmun 2005) and chronic infectious diseases such as mycobacterium tuberculosis (Hohn et al., Scand J Immunol 2007), borrelia burgdorferi (Meyer et al., Proc Natl Acad Sci U S A 2000) and HCV infection (Day et al., J Clin Invest 2003; Ulsenheimer et al., J Viral Hepat 2006). The identified SLA/LP-specific T cell epitopes and their corresponding tetramers should therefore be useful to monitor AIH-specific T cells during acute AIH and active disease progression and to study their role in the liver, the site of this disease.
| Yoon, Joo Chun; Rehermann, Barbara (2009) Determination of HCV-specific T-cell activity. Methods Mol Biol 510:403-13 |
| Shiina, Masaaki; Rehermann, Barbara (2009) Analysis of HCV-specific T cells by flow cytometry. Methods Mol Biol 510:415-26 |
| Racanelli, Vito; Rehermann, Barbara (2006) The liver as an immunological organ. Hepatology 43:S54-62 |
