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958 PHENOTYPIC AND FUNCTIONAL SIGNATURE OF CD4POSCD25HIGHCD127LOW REGULATORY T-CELLS IN AUTOIMMUNE HEPATITIS
POSTERS 957 THE T-CELL EFFECTOR PHENOTYPE PROFILE IN AUTOIMMUNE SCLEROSING CHOLANGITIS DIFFERS FROM THAT OF AUTOIMMUNE HEPATITIS R. Liberal1,2 , C.R. Grant1 , B. Holder1 , Y. Ma1 , G. Mieli-Vergani1 , D. Vergani1 , M.S. Longhi1 . 1 Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, UK; 2 Faculdade de Medicina da Universidade do Porto, Porto, Portugal E-mail: [email protected] Background and Aim: Autoimmune hepatitis (AIH) and autoimmune hepatitis/sclerosing cholangitis overlap syndrome (autoimmune sclerosing cholangitis, AISC) are two forms of childhood autoimmune liver disease (AILD) characterised by hypergammaglobulinaemia, autoantibody seropositivity and interface hepatitis that, in AISC, are associated with bile duct abnormalities. Both CD4 and CD8 T-cell immune responses are involved in the pathogenesis of AILD. Whether the extent and the type of CD4 and CD8 effector immune responses differ between AIH and AISC remains untested. The aim of the current study was to determine the frequency and the functional phenotype of CD4 and CD8 effector cells in the two conditions. Methods: We studied 6 patients with ANA/SMA+ AIH (3 females, median age: 13.2 years), 6 with AISC (3 females, median age: 15.3 years), and 6 healthy subjects (HS, 4 females, median age: 27.5 years). The frequency and phenotype of T-cells was evaluated by cytofluorimetry using monoclonal antibodies to CD3, CD4 and CD8. Frequency of cells positive for T-bet, RORC, IFNg and IL-17 was determined by intracellular staining following cell fixation and permeabilization. Results: In AISC there was a lower frequency of CD4pos (36.5±3.6) and a higher frequency of CD8pos (31.8±2) cells compared to AIH(CD4: 47.2±2.7, P = 0.04; CD8: 21.8±2.5, P = 0.01) and HS (CD4:46.8±2.1, P = 0.04). Compared to HS: a) AIH and AISC displayed higher frequencies of CD4 cells positive for T-bet and IFNg (P < 0.05 for all comparisons) and higher numbers of CD8 cells positive for IFNg (AIH: P = 0.004; AISC: P = 0.08); b) AISC exhibited an increased percentage of IL-17-producing CD4 and CD8 T-cells (P = 0.02 and P = 0.1 respectively). Compared to AIH, AISC patients had higher frequencies of CD4 cells positive for RORC (P = 0.001) and IL-17 (P = 0.02) and higher numbers of CD8 cells positive for IL-17 (P = 0.05). In AISC frequencies of IL-17pos CD4 and CD8 cells correlated with levels of gamma-glutamyl-transpeptidase (CD4: R = 0.90, P = 0.01; CD8: R = 0.84, P = 0.04) and alkaline phosphatase (CD4: R = 0.75, P = 0.09; CD8: R = 0.80, P = 0.06). Conclusion: While IFNg-producing effectors are increased in both forms of AILD, IL-17pos cells predominate in AISC. The correlation between frequency of ’type 17’ immune responses and biochemical indices of cholestasis suggests that in AISC IL-17 may be involved in bile duct damage. 958 PHENOTYPIC AND FUNCTIONAL SIGNATURE OF CD4POS CD25HIGH CD127LOW REGULATORY T-CELLS IN AUTOIMMUNE HEPATITIS R. Liberal1,2 , C.R. Grant1 , G. Mieli-Vergani1 , D. Vergani1 , M.S. Longhi1 . 1 Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, UK; 2 Faculdade de Medicina da Universidade do Porto, Porto, Portugal E-mail: [email protected] Background and Aims: In autoimmune hepatitis (AIH) CD4pos CD25high regulatory T-cells (T-regs), a subset central to immune-tolerance, are numerically defective and impaired in their ability to control effector cell function. At variance with CD4 effectors, T-regs, classically known as CD25high and FOXP3pos , express low levels of the activation marker CD127. The aim of the current study was to provide a phenotypic and functional profile S374
of CD4pos CD25high CD127low T-regs (CD127low T-regs) in AIH and to explore to what extent absence or low levels of CD127 impact on T-reg ability to suppress. Methods: 20 ANA/SMA+ AIH patients and 12 healthy subjects (HS) were studied. T-reg phenotype was determined by flow cytometry using antibodies to CD4, CD25, CD127, CTLA-4 and Galectin-9, a molecule linked to T-reg ability to suppress. T-reg transcription factor and cytokine profile were assessed by intracellular staining. CD127low T-reg ability to suppress was evaluated in a proliferation assay following co-culture with CD25neg target cells. Results: In AIH CD4pos CD25high cells contained fewer CD127low cells than in HS. Compared to conventional CD4pos CD25high (cT-regs), CD127low T-regs from both AIH and HS had a) higher numbers of FOXP3pos , CTLA-4pos , Galectin-9pos and IL-10pos cells; b) lower numbers of T-betpos , RORCpos , IFNgpos and IL-17pos cells; and c) similar numbers of TGF-bpos cells. In AIH, CD127low T-regs contained fewer FOXP3pos , CTLA-4pos , Galectin-9pos , IL-10pos and TGF-bpos cells and higher frequencies of T-betpos , RORCpos , IFNgpos and IL-17pos cells than in HS. CD127low T-regs inhibited CD25neg cell proliferation more effectively than cT-regs, though less markedly in AIH than in HS. In AIH, treatment with anti-IFNg and anti-IL-17 neutralizing antibodies ameliorated the suppressive ability of cT-regs, while leaving unchanged that of CD127low T-regs; exposure to anti-IL-10 neutralizing antibodies reduced cT-reg suppression in HS, but not in AIH. Conclusion: CD127low T-regs bear the phenotypic and functional signature of ’true T-regs’. Low numbers and reduced suppressive function of CD127low T-regs in AIH may contribute to breakdown of immune-tolerance by permitting effector cells to perpetrate hepatocyte damage. 959 REDUCED EXPRESSION OF INHIBITORY MOLECULES CHARACTERISES CD4 AND CD8 EFFECTOR CELLS IN AUTOIMMUNE HEPATITIS R. Liberal1,2 , C.R. Grant1 , B. Holder1 , Y. Ma1 , G. Mieli-Vergani1 , D. Vergani1 , M.S. Longhi1 . 1 Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, UK; 2 Faculdade de Medicina da Universidade do Porto, Porto, Portugal E-mail: [email protected] Background: Autoimmune hepatitis (AIH) is a severe hepatopathy often progressing to end-stage liver disease despite immunosuppression. There is evidence that both CD4 and CD8 effector cells are involved in AIH liver damage. T-cell receptor signalling can be modulated following engagement of inhibitory molecules, namely cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and T-cell immunoglobulin and mucin domain-3 (Tim-3). Whether in AIH decreased expression of these molecules accounts for effector cell over-activation is unclear. Aims: To determine the expression of CTLA-4, PD-1, and Tim-3 in CD4 and CD8 T-cells in AIH. Methods: 12 ANA/SMA+ AIH patients (6 females, median age: 14 years) and 6 healthy subjects (HS, 4 females, median age: 26.4 years) were studied. Phenotype of CD4 and CD8 T-cells was determined by flow cytometry using monoclonal antibodies to CD4, CD8, PD-1 and Tim-3. Expression of CTLA-4 was determined by intracellular staining. Results: The frequency of Tim-3pos and PD-1pos cells within CD4 and CD8 T lymphocytes was lower in AIH (CD4pos Tim-3pos : 1.6±0.3; CD4pos PD-1pos : 4.8±0.5; CD8pos Tim-3pos : 9.6±1.6; CD8pos PD-1pos : 6.7±0.7) than in HS (CD4pos Tim-3pos : 6.2±0.8, P < 0.001; CD4pos PD1pos : 8.1±1.9, P = 0.04; CD8pos Tim-3pos : 15.8±1.8, P = 0.04; CD8pos PD1pos : 12.6±1.4 6.7±0.7, P < 0.001), while no difference was noted in the frequency of CTLA-4pos cells between the two groups of subjects. Tim-3/PD-1 double positive cells within CD4 and CD8 subsets were fewer in AIH (CD4pos Tim-3pos PD-1pos : 0.4±0.1; CD8pos Tim-3pos PD-
Journal of Hepatology 2012 vol. 56 | S225–S388
of CD4pos CD25high CD127low T-regs (CD127low T-regs) in AIH and to explore to what extent absence or low levels of CD127 impact on T-reg ability to suppress. Methods: 20 ANA/SMA+ AIH patients and 12 healthy subjects (HS) were studied. T-reg phenotype was determined by flow cytometry using antibodies to CD4, CD25, CD127, CTLA-4 and Galectin-9, a molecule linked to T-reg ability to suppress. T-reg transcription factor and cytokine profile were assessed by intracellular staining. CD127low T-reg ability to suppress was evaluated in a proliferation assay following co-culture with CD25neg target cells. Results: In AIH CD4pos CD25high cells contained fewer CD127low cells than in HS. Compared to conventional CD4pos CD25high (cT-regs), CD127low T-regs from both AIH and HS had a) higher numbers of FOXP3pos , CTLA-4pos , Galectin-9pos and IL-10pos cells; b) lower numbers of T-betpos , RORCpos , IFNgpos and IL-17pos cells; and c) similar numbers of TGF-bpos cells. In AIH, CD127low T-regs contained fewer FOXP3pos , CTLA-4pos , Galectin-9pos , IL-10pos and TGF-bpos cells and higher frequencies of T-betpos , RORCpos , IFNgpos and IL-17pos cells than in HS. CD127low T-regs inhibited CD25neg cell proliferation more effectively than cT-regs, though less markedly in AIH than in HS. In AIH, treatment with anti-IFNg and anti-IL-17 neutralizing antibodies ameliorated the suppressive ability of cT-regs, while leaving unchanged that of CD127low T-regs; exposure to anti-IL-10 neutralizing antibodies reduced cT-reg suppression in HS, but not in AIH. Conclusion: CD127low T-regs bear the phenotypic and functional signature of ’true T-regs’. Low numbers and reduced suppressive function of CD127low T-regs in AIH may contribute to breakdown of immune-tolerance by permitting effector cells to perpetrate hepatocyte damage. 959 REDUCED EXPRESSION OF INHIBITORY MOLECULES CHARACTERISES CD4 AND CD8 EFFECTOR CELLS IN AUTOIMMUNE HEPATITIS R. Liberal1,2 , C.R. Grant1 , B. Holder1 , Y. Ma1 , G. Mieli-Vergani1 , D. Vergani1 , M.S. Longhi1 . 1 Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, UK; 2 Faculdade de Medicina da Universidade do Porto, Porto, Portugal E-mail: [email protected] Background: Autoimmune hepatitis (AIH) is a severe hepatopathy often progressing to end-stage liver disease despite immunosuppression. There is evidence that both CD4 and CD8 effector cells are involved in AIH liver damage. T-cell receptor signalling can be modulated following engagement of inhibitory molecules, namely cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and T-cell immunoglobulin and mucin domain-3 (Tim-3). Whether in AIH decreased expression of these molecules accounts for effector cell over-activation is unclear. Aims: To determine the expression of CTLA-4, PD-1, and Tim-3 in CD4 and CD8 T-cells in AIH. Methods: 12 ANA/SMA+ AIH patients (6 females, median age: 14 years) and 6 healthy subjects (HS, 4 females, median age: 26.4 years) were studied. Phenotype of CD4 and CD8 T-cells was determined by flow cytometry using monoclonal antibodies to CD4, CD8, PD-1 and Tim-3. Expression of CTLA-4 was determined by intracellular staining. Results: The frequency of Tim-3pos and PD-1pos cells within CD4 and CD8 T lymphocytes was lower in AIH (CD4pos Tim-3pos : 1.6±0.3; CD4pos PD-1pos : 4.8±0.5; CD8pos Tim-3pos : 9.6±1.6; CD8pos PD-1pos : 6.7±0.7) than in HS (CD4pos Tim-3pos : 6.2±0.8, P < 0.001; CD4pos PD1pos : 8.1±1.9, P = 0.04; CD8pos Tim-3pos : 15.8±1.8, P = 0.04; CD8pos PD1pos : 12.6±1.4 6.7±0.7, P < 0.001), while no difference was noted in the frequency of CTLA-4pos cells between the two groups of subjects. Tim-3/PD-1 double positive cells within CD4 and CD8 subsets were fewer in AIH (CD4pos Tim-3pos PD-1pos : 0.4±0.1; CD8pos Tim-3pos PD-
Journal of Hepatology 2012 vol. 56 | S225–S388