Effector CD4 and CD8 T cell immune responses to cytochrome P450IID6 (CYP2D6), the autoantigen of autoimmune hepatitis type 2 (AIH-2), are permitted by a numerical and functional impairment of CD4posCD25high regulatory T cells (T-regs). We aimed to investigate whether T-regs specific for CYP2D6 immunodominant regions and restricted by the appropriate human leukocyte antigen (HLA)-DR molecule can be generated in patients with AIH-2 and can control CD4 and CD8 T cell effectors targeting identical or overlapping CYP2D6 regions. CYP2D6-specific regulatory T cells (CYP2D6 T-regs) were obtained from peptide-pulsed monocyte-depleted peripheral blood mononuclear cells of 17 patients with AIH-2, who were positive for the predisposing HLA-DR7 and/or HLA-DR3 alleles. Their antigen specificity was assessed by cytofluorimetry using HLA class II tetramers and their cytokine profile by intracellular staining. T-reg ability to suppress was ascertained by measuring reduction of CD4posCD25neg cell proliferation/effector cytokine secretion and of CD8 T cell cytotoxicity. The most efficient suppression of effector T cell proliferation, inflammatory cytokine release, and cytotoxicity was obtained by coculturing T-regs with CYP2D6-peptide-loaded semimature dendritic cells (smDCs), and smDC-CYP2D6 T-regs also expressed high levels of FOXP3 (forkhead box P3). Possession of the appropriate HLA-DR molecule and recognition of the CYP2D6 autoantigenic sequence were critical to the synergistic smDC-CYP2D6 T-reg immunoregulatory functions, and lack of either element led to poor control of responder cell proliferation and cytokine secretion. Moreover, interferon-γ neutralization significantly boosted the suppressive ability of CYP2D6 T-regs. Conclusion: T-regs generated under CYP2D6-specific conditions and cocultured with smDCs are highly effective at controlling autoreactive T cells, thus providing the basis for a powerful and tailored form of immunotherapy for AIH-2. (HEPATOLOGY 2011;53:536-547)