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Identification of HLA-A2-restricted CD8(+) cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross-presented by dendritic cells.

Kita H, Lian ZX, Van de Water J, He XS, Matsumura S, Kaplan M, Luketic V, Coppel RL, Ansari AA, Gershwin ME - J. Exp. Med. (2002)

Bottom Line: Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses.Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone.The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA 95616, USA.

ABSTRACT
Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4(+) and CD8(+) T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2-restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159-167 of PDC-E2, induces specific MHC class I-restricted CD8(+) CTL lines from 10/12 HLA-A2(+) PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2-specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.

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Cytotoxicity of PD5-induced CTL lines. PD5-specific CTL lines, induced by culturing PBMCs with PD5-loaded APCs for 12–14 d, were tested for their cytotoxicity against PD5 or control peptide-loaded autologous BCL targets at different effector/target (E/T) ratios. HBc18–27, an HLA-A2-restricted irrelevant epitope, was used as control. Displayed are mean specific lysis of triplicate cultures. (A) PBC patients PBC9 and PBC11. (B) Control donors CL2 (granulomatous liver disease) and H2 (healthy donor). (C) Cytotoxic activity of a PD5-induced CTL line against target cells presenting endogenously processed PDC-E2 Ag. The CTL line from patient PBC1 was tested for cytotoxicity against autologous BCL targets infected with a PDC-E2–expressing vaccinia vector (VVrPDC-E2), wild-type vaccinia virus (VVwild) alone, loaded with PD5 or a control peptide HBc18–27. Displayed are mean specific lysis of triplicate testing at an E/T ratio of 40:1. (D) Phenotype analysis of PD5-induced CTLs. CD4+ cell or CD8+ cells were depleted respectively from a CTL line derived from patient PBC2 using anti-CD4 or anti-CD8–coated magnetic beads before testing for cytotoxic activity with PD5 or control peptide-loaded autologous BCL targets. Unfractionated cells were also tested in parallel. Specific cytotoxicity was calculated by subtracting the cytotoxicity of the effector cells against control peptide-pulsed BCLs from that against PD5 peptide-pulsed BCLs. Displayed are mean specific lysis of triplicate testing at an E/T ratio of 40:1 according to the cell counts before depletion.
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fig2: Cytotoxicity of PD5-induced CTL lines. PD5-specific CTL lines, induced by culturing PBMCs with PD5-loaded APCs for 12–14 d, were tested for their cytotoxicity against PD5 or control peptide-loaded autologous BCL targets at different effector/target (E/T) ratios. HBc18–27, an HLA-A2-restricted irrelevant epitope, was used as control. Displayed are mean specific lysis of triplicate cultures. (A) PBC patients PBC9 and PBC11. (B) Control donors CL2 (granulomatous liver disease) and H2 (healthy donor). (C) Cytotoxic activity of a PD5-induced CTL line against target cells presenting endogenously processed PDC-E2 Ag. The CTL line from patient PBC1 was tested for cytotoxicity against autologous BCL targets infected with a PDC-E2–expressing vaccinia vector (VVrPDC-E2), wild-type vaccinia virus (VVwild) alone, loaded with PD5 or a control peptide HBc18–27. Displayed are mean specific lysis of triplicate testing at an E/T ratio of 40:1. (D) Phenotype analysis of PD5-induced CTLs. CD4+ cell or CD8+ cells were depleted respectively from a CTL line derived from patient PBC2 using anti-CD4 or anti-CD8–coated magnetic beads before testing for cytotoxic activity with PD5 or control peptide-loaded autologous BCL targets. Unfractionated cells were also tested in parallel. Specific cytotoxicity was calculated by subtracting the cytotoxicity of the effector cells against control peptide-pulsed BCLs from that against PD5 peptide-pulsed BCLs. Displayed are mean specific lysis of triplicate testing at an E/T ratio of 40:1 according to the cell counts before depletion.

Mentions: To determine if the IFN-γ synthesizing CD8+ T cell response corresponded with PD5 peptide specific cytotoxic activity, the PD5-stimulated PBMC cultures were assessed in 14 HLA-A2+ individuals: eight patients with PBC (PBC1, PBC2, PBC3, PBC4, PBC5, PBC9, PBC10, and PBC11); three patients with other chronic liver diseases (CL1, CL2, and CL3); and three healthy controls (H1, H2, and H3). The PBMCs were stimulated and expanded with PD5-loaded autologous APCs for 12–14 d and used as effector cells in a conventional CTL assay against autologous BCLs loaded with the PD5 peptide or a control HLA-A2–specific hepatitis B peptide. Targets loaded with PD5, but not with the control peptide, were lysed by the effector cells derived from each of the eight PBC patients. In contrast, PBMCs derived from the control individuals did not show significant cytotoxicity toward the PD5-loaded target cells. A representative CTL assay for two PBC patients (PBC9 and PBC11) is shown in Fig. 2 A along with two control individuals (CL2 and H2) in Fig. 2 B. These results indicate that PD5-specific CTL lines, inducible from PBC patients but not from other chronic liver diseases or healthy individuals, were indeed cytolytic for the targets presenting the PD5 epitope.


Identification of HLA-A2-restricted CD8(+) cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross-presented by dendritic cells.

Kita H, Lian ZX, Van de Water J, He XS, Matsumura S, Kaplan M, Luketic V, Coppel RL, Ansari AA, Gershwin ME - J. Exp. Med. (2002)

Cytotoxicity of PD5-induced CTL lines. PD5-specific CTL lines, induced by culturing PBMCs with PD5-loaded APCs for 12–14 d, were tested for their cytotoxicity against PD5 or control peptide-loaded autologous BCL targets at different effector/target (E/T) ratios. HBc18–27, an HLA-A2-restricted irrelevant epitope, was used as control. Displayed are mean specific lysis of triplicate cultures. (A) PBC patients PBC9 and PBC11. (B) Control donors CL2 (granulomatous liver disease) and H2 (healthy donor). (C) Cytotoxic activity of a PD5-induced CTL line against target cells presenting endogenously processed PDC-E2 Ag. The CTL line from patient PBC1 was tested for cytotoxicity against autologous BCL targets infected with a PDC-E2–expressing vaccinia vector (VVrPDC-E2), wild-type vaccinia virus (VVwild) alone, loaded with PD5 or a control peptide HBc18–27. Displayed are mean specific lysis of triplicate testing at an E/T ratio of 40:1. (D) Phenotype analysis of PD5-induced CTLs. CD4+ cell or CD8+ cells were depleted respectively from a CTL line derived from patient PBC2 using anti-CD4 or anti-CD8–coated magnetic beads before testing for cytotoxic activity with PD5 or control peptide-loaded autologous BCL targets. Unfractionated cells were also tested in parallel. Specific cytotoxicity was calculated by subtracting the cytotoxicity of the effector cells against control peptide-pulsed BCLs from that against PD5 peptide-pulsed BCLs. Displayed are mean specific lysis of triplicate testing at an E/T ratio of 40:1 according to the cell counts before depletion.
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Related In: Results  -  Collection

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fig2: Cytotoxicity of PD5-induced CTL lines. PD5-specific CTL lines, induced by culturing PBMCs with PD5-loaded APCs for 12–14 d, were tested for their cytotoxicity against PD5 or control peptide-loaded autologous BCL targets at different effector/target (E/T) ratios. HBc18–27, an HLA-A2-restricted irrelevant epitope, was used as control. Displayed are mean specific lysis of triplicate cultures. (A) PBC patients PBC9 and PBC11. (B) Control donors CL2 (granulomatous liver disease) and H2 (healthy donor). (C) Cytotoxic activity of a PD5-induced CTL line against target cells presenting endogenously processed PDC-E2 Ag. The CTL line from patient PBC1 was tested for cytotoxicity against autologous BCL targets infected with a PDC-E2–expressing vaccinia vector (VVrPDC-E2), wild-type vaccinia virus (VVwild) alone, loaded with PD5 or a control peptide HBc18–27. Displayed are mean specific lysis of triplicate testing at an E/T ratio of 40:1. (D) Phenotype analysis of PD5-induced CTLs. CD4+ cell or CD8+ cells were depleted respectively from a CTL line derived from patient PBC2 using anti-CD4 or anti-CD8–coated magnetic beads before testing for cytotoxic activity with PD5 or control peptide-loaded autologous BCL targets. Unfractionated cells were also tested in parallel. Specific cytotoxicity was calculated by subtracting the cytotoxicity of the effector cells against control peptide-pulsed BCLs from that against PD5 peptide-pulsed BCLs. Displayed are mean specific lysis of triplicate testing at an E/T ratio of 40:1 according to the cell counts before depletion.
Mentions: To determine if the IFN-γ synthesizing CD8+ T cell response corresponded with PD5 peptide specific cytotoxic activity, the PD5-stimulated PBMC cultures were assessed in 14 HLA-A2+ individuals: eight patients with PBC (PBC1, PBC2, PBC3, PBC4, PBC5, PBC9, PBC10, and PBC11); three patients with other chronic liver diseases (CL1, CL2, and CL3); and three healthy controls (H1, H2, and H3). The PBMCs were stimulated and expanded with PD5-loaded autologous APCs for 12–14 d and used as effector cells in a conventional CTL assay against autologous BCLs loaded with the PD5 peptide or a control HLA-A2–specific hepatitis B peptide. Targets loaded with PD5, but not with the control peptide, were lysed by the effector cells derived from each of the eight PBC patients. In contrast, PBMCs derived from the control individuals did not show significant cytotoxicity toward the PD5-loaded target cells. A representative CTL assay for two PBC patients (PBC9 and PBC11) is shown in Fig. 2 A along with two control individuals (CL2 and H2) in Fig. 2 B. These results indicate that PD5-specific CTL lines, inducible from PBC patients but not from other chronic liver diseases or healthy individuals, were indeed cytolytic for the targets presenting the PD5 epitope.

Bottom Line: Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses.Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone.The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA 95616, USA.

ABSTRACT
Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4(+) and CD8(+) T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2-restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159-167 of PDC-E2, induces specific MHC class I-restricted CD8(+) CTL lines from 10/12 HLA-A2(+) PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2-specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.

Show MeSH
Related in: MedlinePlus