- Email: [email protected]
[25] IDENTIFICATION OF HLA-DR3 RESTRICTED CD4 T-CELL EPITOPES ON SOLUBLE LIVER ANTIGEN IN AUTOIMMUNE HEPATITIS TYPE 1
Parallel Session 3: LIVER IMMUNOLOGY chemokines. MPA-treated DC expressed lower levels of CD40, CD54, CD62L, CD80, CD83 and CD86, down-regulated CCR3, CCR7 and upregulated CCRl , The expression of CCR5 didn’t change significantly. MPA-treated DC were more attracted to inflammatory chemokines CCL2, CCL3, CCL4, CCL7; whereas the migration towards chemokines expressed in secondary lymphoid organs (CCL19 and CCL21) didn’t change significantly. In addition, MPA-treated DC showed increased endocytotic capacity. Conclusion: Treatment of DC with MPA inhibits the maturation of DC and, therefore, may hinder the induction of the immune response. Moreover, MPA impairs the migration of DC from the periphery towards secondary lymphoid organs. Thus, MPA treatment of patients after solid organ transplantation may promote allograft tolerance.
IDENTIFICATION OF HLA-DR3 RESTRICTED CD4 T-CELL EPITOPES ON SOLUBLE LIVER ANTIGEN IN AUTOIMMUNE HEPATITIS TYPE 1 F. Meda, P. Wang, M.S. Longhi, D.P. Bogdanos, G. Mieli-Vergani, D. Vergani, Y. Ma. Institute of’ Liver Studies, Kings College London School of Medicine at Kings College Hospitul, London, London, UK E-mail: [email protected] Background: Antibodies to soluble liver antigen (anti-SLA) are present in 50% of patients with autoimmune hepatitis type 1 (AIH-1, ANA/SMA+ve), being associated to a worse prognosis. Humoral immunodominant regions within SLA are SLA 281-300, 3 8 5 4 0 4 and 40 1 4 4 1 . 70% ATH-I patients possess HLA DRBI*03 (DR3). Aim: To identify HLA-DR3 restricted CD4 T-cell epitopes on SLA in AIH-I. Methods: 54 overlapping icosameric peptides, spanning the 441 aa of the full-length SLA molecule, were constructed using fmoc solid phase chemistry (Mimotopes, Australia). T-cell reactivity was assessed by proliferation assay in 15 AIH-1 patients (median age 12.5, 9 females) and in 6 healthy subjects (33 years, 5 females). Four patients were investigated at diagnosis/relapse, the remaining during remission. Twelve were DR3+ve. Peripheral blood mononuclear cells (PBMCs) ( 1 00,00O/well) were cultured for 7 days in the presence of 10 mM individual peptides in a 96-well plate. A stimulation index (S1) >2 was considered positive. Anti-SLA was detected using an in-house radioligand assay. The RANKPEP and SYFPETTHT algorithm binding prediction programs were used to identify SLA sequences containing DR3 binding motifs. Results: Amongst 12 DR3+ve patients, PBMCs from 10 anti-SLA+ve patients recognized 3 to 19 SLA peptides (median 7) with S1 up to 9 (median 3). Eight antigenic regions, SLA 49-68, 129-148, 161-196, 201-220, 281-316, 361-380, 3 8 5 4 0 4 and 409-441, were identified, 3 overlapping with the R cell epitopes SLA 281-300, 3 8 5 4 0 4 and 401441 and 5 containing HLA DR3 binding motifs. PBMCs from two of the three anti-SLA+ve non-DR3 patients (1 DR2 and 2 DR7) reacted with 2 different SLA peptides (41-60, 57-76). There was no difference in SLA peptide reactivity between patients with normal or abnormal transaminases in terms of ST and number ofrecognised peptides. PBMCs from 3 healthy controls did not react with SLA peptides, while 3 reacted with 3 peptides (SLA 97-1 16, 113-121, 201-220; STs 2.7, 2.3 and 2.1). Summary and Conclusion: 1) We have identified 8 antigenic regions on SLA that are targets of T-cell immune responses in HLA DR3+ve AIH-1 patients; 2) five of these peptides contain predicted DR3 binding motifs; 3) humoral and cellular immune responses to SLA partially overlap.
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ANTIGEN PRESENTATION BY BONE-MARROW DERIVED ANTIGEN PRESENTING CELLS IS REQUIRED FOR EFFECTIVE T-CELL PRIMING IN AN ANIMAL MODEL OF AUTOIMMUNE LIVER DISEASE K. Derlcow’, N. Kruse’, K. Klugewitz’, B. Wiedenmann’, E. Schott’ . ‘Department of‘ Hepatology and ~a,stroenterol~)gy, Charite, CVK, Berlin, Germany; 2Depnmtent of‘ Gustroenterology, Charite, CBE Berlin, Germany E-mail: [email protected] Background: In autoimmune liver disease, the liver is damaged by autoreative T-cells with specificity for liver antigens. It is unclear, where the priming of these autoreactive T-cells occurs. We have generated transgenic mice expressing the model antigen ovalbumin in hepatocytes or cholangiocytes to investigate the mechanisms involved in priming of autoreactive T-cells. Methods: Transgenic mice were generated that express ovalbumin under the transferrin-promoter in hepatocytes (TF-OVA mice) or under the apical sodium-dependent bile transporter-promoter in cholangiocytes (ASBTOVA mice). Naive transgenic CD8 or CD4 T-cells were isolated from OT-T or OT-TI mice and transferred intravenously to induce priming by the hepatic transgenes. Bone-marrow chimeras were generated by lethal irradiation of TF-OVA and ASBT-OVA mice and reconstitution with MHC-1- or MHC-IT-deficient bone marrow. Priming of T-cells was assessed by CFSEdilution, the degree of hepatitis caused was determined by histology and measurement of the serum-alanin-aminotransferase. Results: Transfer of naive transgenic CD8 T-cells resulted in intrahepatic priming in both ASBT-OVA and TF-OVA mice as determined by CFSEdilution. CD4 T-cells were primed in spleen and liver-draining lymph nodes of TF-OVA mice but no priming was observed in ASBT-OVA mice. The administration of OT-1 T-cells led to a transient hepatitis as detected by an increase in serum-ALT values, whereas transfer of naive CD4 T-cells bone-marrow chimeras, intrahepatic did not cause hepatitis. In MHC-T priming of CD8 T-cells still occurred in both ASBT-OVA and TF-OVA mice despite the lack of professional antigen-presenting cells capable of presenting the OT-1 epitope. However, the degree of hepatitis caused by the T-cells was greatly diminished. In MHC-TI bone-marrow chimeras, OT-11 CD4 T-cells were no longer primed in TF-OVA mice. Conclusions: Autoantigens expressed in the liver are presented by a variety of professional and non-professional antigen-presenting cells to CD8 T-cells in vivo but induction of autoimmunity is triggered only if professional antigen-presenting cells are involved. CD4 T-cells require the presence of bone-marrow derived professional antigen-presenting cells for priming by an antigen expressed in the liver.
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DIFFERENTIAL CYTOKINE PATTERN OF HDV-SPECIFIC CELLULAR IMMUNE RESPONSES DISTINGUISHES TREATMENT RESPONDER AND NONRESPONDER TO PEG-IFNa-2a TREATMENT: RESULTS FROM THE HEP-NETIINTERNATIONAL HIDIT-I STUDY H. Wedemeyer’, A. Ciner’, C. Yurdaydin’, K. Zachou3, N. Aslan’, S. Meyer’ , B. Heidrich’, M.P. Manns’ . ‘Medizinische Hochschub Hunnouer, Hunnouer, Gerntuny; ’Ankuru lJniuersity, Ankara, Turkey: Larissa Uniwrsity, Larissa, Greece E-mail: [email protected]
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Introduction: Delta hepatitis is the most severe form of chronic viral hepatitis with limited treatment options available. We recently reported data of a randomised study investigating pegylated-interferon-alpha-2a with or without adefovir vs. adefovir alone for the treatment of chronic delta hepatitis. Cellular immune responses in hepatitis D virus (HDV) infection are largely undefined and HDV-specific T cell responses during antiviral therapy of hepatitis D have not been studied at all.
IDENTIFICATION OF HLA-DR3 RESTRICTED CD4 T-CELL EPITOPES ON SOLUBLE LIVER ANTIGEN IN AUTOIMMUNE HEPATITIS TYPE 1 F. Meda, P. Wang, M.S. Longhi, D.P. Bogdanos, G. Mieli-Vergani, D. Vergani, Y. Ma. Institute of’ Liver Studies, Kings College London School of Medicine at Kings College Hospitul, London, London, UK E-mail: [email protected] Background: Antibodies to soluble liver antigen (anti-SLA) are present in 50% of patients with autoimmune hepatitis type 1 (AIH-1, ANA/SMA+ve), being associated to a worse prognosis. Humoral immunodominant regions within SLA are SLA 281-300, 3 8 5 4 0 4 and 40 1 4 4 1 . 70% ATH-I patients possess HLA DRBI*03 (DR3). Aim: To identify HLA-DR3 restricted CD4 T-cell epitopes on SLA in AIH-I. Methods: 54 overlapping icosameric peptides, spanning the 441 aa of the full-length SLA molecule, were constructed using fmoc solid phase chemistry (Mimotopes, Australia). T-cell reactivity was assessed by proliferation assay in 15 AIH-1 patients (median age 12.5, 9 females) and in 6 healthy subjects (33 years, 5 females). Four patients were investigated at diagnosis/relapse, the remaining during remission. Twelve were DR3+ve. Peripheral blood mononuclear cells (PBMCs) ( 1 00,00O/well) were cultured for 7 days in the presence of 10 mM individual peptides in a 96-well plate. A stimulation index (S1) >2 was considered positive. Anti-SLA was detected using an in-house radioligand assay. The RANKPEP and SYFPETTHT algorithm binding prediction programs were used to identify SLA sequences containing DR3 binding motifs. Results: Amongst 12 DR3+ve patients, PBMCs from 10 anti-SLA+ve patients recognized 3 to 19 SLA peptides (median 7) with S1 up to 9 (median 3). Eight antigenic regions, SLA 49-68, 129-148, 161-196, 201-220, 281-316, 361-380, 3 8 5 4 0 4 and 409-441, were identified, 3 overlapping with the R cell epitopes SLA 281-300, 3 8 5 4 0 4 and 401441 and 5 containing HLA DR3 binding motifs. PBMCs from two of the three anti-SLA+ve non-DR3 patients (1 DR2 and 2 DR7) reacted with 2 different SLA peptides (41-60, 57-76). There was no difference in SLA peptide reactivity between patients with normal or abnormal transaminases in terms of ST and number ofrecognised peptides. PBMCs from 3 healthy controls did not react with SLA peptides, while 3 reacted with 3 peptides (SLA 97-1 16, 113-121, 201-220; STs 2.7, 2.3 and 2.1). Summary and Conclusion: 1) We have identified 8 antigenic regions on SLA that are targets of T-cell immune responses in HLA DR3+ve AIH-1 patients; 2) five of these peptides contain predicted DR3 binding motifs; 3) humoral and cellular immune responses to SLA partially overlap.
S13
ANTIGEN PRESENTATION BY BONE-MARROW DERIVED ANTIGEN PRESENTING CELLS IS REQUIRED FOR EFFECTIVE T-CELL PRIMING IN AN ANIMAL MODEL OF AUTOIMMUNE LIVER DISEASE K. Derlcow’, N. Kruse’, K. Klugewitz’, B. Wiedenmann’, E. Schott’ . ‘Department of‘ Hepatology and ~a,stroenterol~)gy, Charite, CVK, Berlin, Germany; 2Depnmtent of‘ Gustroenterology, Charite, CBE Berlin, Germany E-mail: [email protected] Background: In autoimmune liver disease, the liver is damaged by autoreative T-cells with specificity for liver antigens. It is unclear, where the priming of these autoreactive T-cells occurs. We have generated transgenic mice expressing the model antigen ovalbumin in hepatocytes or cholangiocytes to investigate the mechanisms involved in priming of autoreactive T-cells. Methods: Transgenic mice were generated that express ovalbumin under the transferrin-promoter in hepatocytes (TF-OVA mice) or under the apical sodium-dependent bile transporter-promoter in cholangiocytes (ASBTOVA mice). Naive transgenic CD8 or CD4 T-cells were isolated from OT-T or OT-TI mice and transferred intravenously to induce priming by the hepatic transgenes. Bone-marrow chimeras were generated by lethal irradiation of TF-OVA and ASBT-OVA mice and reconstitution with MHC-1- or MHC-IT-deficient bone marrow. Priming of T-cells was assessed by CFSEdilution, the degree of hepatitis caused was determined by histology and measurement of the serum-alanin-aminotransferase. Results: Transfer of naive transgenic CD8 T-cells resulted in intrahepatic priming in both ASBT-OVA and TF-OVA mice as determined by CFSEdilution. CD4 T-cells were primed in spleen and liver-draining lymph nodes of TF-OVA mice but no priming was observed in ASBT-OVA mice. The administration of OT-1 T-cells led to a transient hepatitis as detected by an increase in serum-ALT values, whereas transfer of naive CD4 T-cells bone-marrow chimeras, intrahepatic did not cause hepatitis. In MHC-T priming of CD8 T-cells still occurred in both ASBT-OVA and TF-OVA mice despite the lack of professional antigen-presenting cells capable of presenting the OT-1 epitope. However, the degree of hepatitis caused by the T-cells was greatly diminished. In MHC-TI bone-marrow chimeras, OT-11 CD4 T-cells were no longer primed in TF-OVA mice. Conclusions: Autoantigens expressed in the liver are presented by a variety of professional and non-professional antigen-presenting cells to CD8 T-cells in vivo but induction of autoimmunity is triggered only if professional antigen-presenting cells are involved. CD4 T-cells require the presence of bone-marrow derived professional antigen-presenting cells for priming by an antigen expressed in the liver.
-’-
-’-
DIFFERENTIAL CYTOKINE PATTERN OF HDV-SPECIFIC CELLULAR IMMUNE RESPONSES DISTINGUISHES TREATMENT RESPONDER AND NONRESPONDER TO PEG-IFNa-2a TREATMENT: RESULTS FROM THE HEP-NETIINTERNATIONAL HIDIT-I STUDY H. Wedemeyer’, A. Ciner’, C. Yurdaydin’, K. Zachou3, N. Aslan’, S. Meyer’ , B. Heidrich’, M.P. Manns’ . ‘Medizinische Hochschub Hunnouer, Hunnouer, Gerntuny; ’Ankuru lJniuersity, Ankara, Turkey: Larissa Uniwrsity, Larissa, Greece E-mail: [email protected]
’
Introduction: Delta hepatitis is the most severe form of chronic viral hepatitis with limited treatment options available. We recently reported data of a randomised study investigating pegylated-interferon-alpha-2a with or without adefovir vs. adefovir alone for the treatment of chronic delta hepatitis. Cellular immune responses in hepatitis D virus (HDV) infection are largely undefined and HDV-specific T cell responses during antiviral therapy of hepatitis D have not been studied at all.