Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Inhibition of CTL activity by HLA class I and HLA class II (DR) mAbs. A CTL line derived from patient PBC5 was tested for cytotoxicity against PD5 or control peptide-loaded autologous BCL targets in the presence of predetermined optimal concentrations of Abs against different HLA molecules. Ab against HLA class I (W6/32), HLA class II DR (TAL.1B5; Dako) or a control Ab (mouse IgG2a; Caltag) were added respectively to the peptide-loaded targets and incubated for 30 min at 4°C. After the incubation the target cells were mixed with effector cells for EuTDA release assay. The CTL assays were performed at an E/T ratio of 40:1. 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. (B) HLA restriction of the PD5 epitope. CTL lines derived from three PBC patients were tested for cytotoxicity against PD5 or control peptide-loaded allogeneic BCLs derived from five individuals with distinct combinations of HLA haplotypes. The CTL assays were performed at an E/T ratio of 40:1. 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196012&req=5

fig3: (A) Inhibition of CTL activity by HLA class I and HLA class II (DR) mAbs. A CTL line derived from patient PBC5 was tested for cytotoxicity against PD5 or control peptide-loaded autologous BCL targets in the presence of predetermined optimal concentrations of Abs against different HLA molecules. Ab against HLA class I (W6/32), HLA class II DR (TAL.1B5; Dako) or a control Ab (mouse IgG2a; Caltag) were added respectively to the peptide-loaded targets and incubated for 30 min at 4°C. After the incubation the target cells were mixed with effector cells for EuTDA release assay. The CTL assays were performed at an E/T ratio of 40:1. 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. (B) HLA restriction of the PD5 epitope. CTL lines derived from three PBC patients were tested for cytotoxicity against PD5 or control peptide-loaded allogeneic BCLs derived from five individuals with distinct combinations of HLA haplotypes. The CTL assays were performed at an E/T ratio of 40:1. 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.

Mentions: The CD8+ phenotype of PD5-induced CTLs from six PBC patients (PBC2, PBC3, PBC4, PB5, PBC10, and PBC11) was also confirmed by a cytotoxicity assay after depletion of different T cell subsets. Depletion of the CD4 or CD8 fraction was performed after the in vitro priming and expansion of the CTLs and before the cytotoxic assay. In all patients tested, the CTL activity was abrogated by depletion of CD8+ cells, but not by depletion of CD4+ cells, consistent with the view that CTL activity was mediated by CD8+ T cells, which likely synthesize IFN-γ when restimulated with the specific peptide. Representative data from a PBC patient (PBC2) are shown in Fig. 3 D.


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)

(A) Inhibition of CTL activity by HLA class I and HLA class II (DR) mAbs. A CTL line derived from patient PBC5 was tested for cytotoxicity against PD5 or control peptide-loaded autologous BCL targets in the presence of predetermined optimal concentrations of Abs against different HLA molecules. Ab against HLA class I (W6/32), HLA class II DR (TAL.1B5; Dako) or a control Ab (mouse IgG2a; Caltag) were added respectively to the peptide-loaded targets and incubated for 30 min at 4°C. After the incubation the target cells were mixed with effector cells for EuTDA release assay. The CTL assays were performed at an E/T ratio of 40:1. 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. (B) HLA restriction of the PD5 epitope. CTL lines derived from three PBC patients were tested for cytotoxicity against PD5 or control peptide-loaded allogeneic BCLs derived from five individuals with distinct combinations of HLA haplotypes. The CTL assays were performed at an E/T ratio of 40:1. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196012&req=5

fig3: (A) Inhibition of CTL activity by HLA class I and HLA class II (DR) mAbs. A CTL line derived from patient PBC5 was tested for cytotoxicity against PD5 or control peptide-loaded autologous BCL targets in the presence of predetermined optimal concentrations of Abs against different HLA molecules. Ab against HLA class I (W6/32), HLA class II DR (TAL.1B5; Dako) or a control Ab (mouse IgG2a; Caltag) were added respectively to the peptide-loaded targets and incubated for 30 min at 4°C. After the incubation the target cells were mixed with effector cells for EuTDA release assay. The CTL assays were performed at an E/T ratio of 40:1. 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. (B) HLA restriction of the PD5 epitope. CTL lines derived from three PBC patients were tested for cytotoxicity against PD5 or control peptide-loaded allogeneic BCLs derived from five individuals with distinct combinations of HLA haplotypes. The CTL assays were performed at an E/T ratio of 40:1. 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.
Mentions: The CD8+ phenotype of PD5-induced CTLs from six PBC patients (PBC2, PBC3, PBC4, PB5, PBC10, and PBC11) was also confirmed by a cytotoxicity assay after depletion of different T cell subsets. Depletion of the CD4 or CD8 fraction was performed after the in vitro priming and expansion of the CTLs and before the cytotoxic assay. In all patients tested, the CTL activity was abrogated by depletion of CD8+ cells, but not by depletion of CD4+ cells, consistent with the view that CTL activity was mediated by CD8+ T cells, which likely synthesize IFN-γ when restimulated with the specific peptide. Representative data from a PBC patient (PBC2) are shown in Fig. 3 D.

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