Effectors and regulators of cellular immune response in autoimmune liver disease

[45] ABSTRACT Autoimmune hepatitis (AIH), an immune-mediated liver disorder with a strong female preponderance, is characterised by hypergammaglobulinaemia, seropositivity for autoantibodies and interface hepatitis on histology. The aetiology of AIH is unknown, though both genetic and environmental factors are involved. CD4 effector lymphocytes are the main orchestrators of liver damage, their proliferation and pro-inflammatory cytokine secretion being correlated with the activity and severity of liver disease. A wealth of data indicates that in AIH the extent of self-reactive CD4 T cell effector immune responses is associated with defective CD4 pos CD25 pos regulatory T cells (Tregs), a lymphocyte subset central to immune-tolerance maintenance. Whether impaired immune-regulation in AIH is due to a reduced number of Tregs, intrinsic defects in their function; or effector cell resistance to their suppression or even a combination of all them is unknown. Recent studies have shown that functional Tregs express very low levels of CD127, whereas activated T cells were characterised by the expression of this marker, indicating CD127 as a good phenotypic marker for the quantification of these cell population. On the other hand, murine studies have shown that galectin-9 (GAL-9), a member of the galectin family expressed by Tregs, inhibits Th1 effector immune responses after binding to the T-cell-imunoglobulin-and-mucindomain-3 (TIM-3), its receptor on CD4 effector cell. The aims of this Thesis were thus to investigate possible causes of functional Treg impairment by exploiting the role of CD127 and that of TIM-3/GAL-9 pathway in patients with autoimmune liver disease. Seventy-seven patients with anti-nuclear (ANA) and/or smooth-muscle antibody (SMA) positive AIH, including thirty nine patients diagnosed with AIH/sclerosing cholangitis (AISC) overlap syndrome, were studied. When considered together they are Effectors and regulators in autoimmune liver disease [46] referred to as autoimmune liver disease (AILD). Circulating CD127 neg and GAL9 pos CD4 pos CD25 pos and TIM-3 pos CD4 pos CD25 neg T cell phenotype was assessed by flow cytometry. CD127 neg Treg ability to suppress was evaluated in a proliferation assay following co-culture with CD25 neg responder cells. To determine whether GAL-9 expression is essential to Treg function, Tregs were treated with small-interfering-RNA to repress GAL-9 translation; Treg suppressor function was assessed by a reduction in target cell proliferation. To evaluate whether TIM-3 expression renders CD4 pos CD25 neg T cells amenable to Treg control, purified CD4 pos CD25 neg TIM-3 pos (TIM-3 pos ) and CD4 pos CD25 neg TIM-3 neg (TIM-3 neg ) cells were co-cultured with Tregs. The frequency of CD4 pos CD25 pos CD127 neg cells was reduced in AILD patients compared to HS. Compared to conventional CD4 pos CD25 pos (cTregs), CD127 neg Tregs from both AILD and HS had a) higher numbers of FOXP3 pos , CTLA-4 pos and IL-10 pos cells; b) lower numbers of T-bet pos , RORC pos , IFN pos and IL-17 pos cells; and c) similar numbers of TGF- pos cells. CD127 neg Tregs inhibited CD25 neg cell proliferation more effectively than cTregs, though less markedly in AILD than in HS. In AILD, treatment with anti-IFN and anti-IL-17 neutralising antibodies ameliorated the suppressive ability of cT-regs, while leaving unchanged that of CD127 neg Tregs; exposure to anti-IL10 neutralising antibodies reduced Treg suppression in HS, but not in AILD. In AILD the frequency of GAL-9 expressing Tregs was reduced, and inversely correlated with IgG levels and autoantibodies titres. Within GAL-9 pos cells the frequency of: a) FOXP3 pos cells was lower in AILD than in HS; b) Tbet pos , GATA-3 pos and RORC pos cells was similar in AILD and HS; c) IL-10-producing cells was lower in AILD than in HS but higher than in the GAL-9 neg Treg fraction for both; d) TGF-producing cells was lower in AILD than in HS; e) IFN-and IL-17-producing cells was