Hiroto Kita

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

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.

Quantitative and functional analysis of PDC-E2–specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis

While the pathologic mechanisms responsible for organ-specific tissue damage in primary biliary cirrhosis (PBC) remain an enigma, it has been suggested that the pathology is mediated by autoreactive T cells infiltrating the intrahepatic bile ducts. Previously, we have documented that there is 100-fold enrichment in the frequency of CD4(+) autoreactive T cells in the liver that are specific for peptides encoded by the E2 components of the pyruvate dehydrogenase complexes (PDC-E2). We have also recently characterized the first MHC class I-restricted epitope for PDC-E2, namely amino acid 159-167, a region very similar to the epitope recognized by MHC class II-restricted CD4(+) cells and by autoantibodies. The effector functions of these PDC-E2(159-167)-specific CD8(+) cytotoxic T lymphocytes (CTLs) are not well understood. We have taken advantage of tetramer technology and report herein that there is tenfold increase in the frequency of PDC-E2(159-167)-specific CTLs in the liver as compared with the blood in PBC. In addition, the precursor frequency of the CTLs in blood was significantly higher in early-stage PBC. Of interest was the fact that, upon stimulation with the peptide, the response of PDC-E2(159-167) tetramer-positive cells is heterogeneous with respect to IFN-gamma synthesis. These data, we believe for the first time, document the enrichment of autoantigen-specific CD8(+) T cells in the PBC liver, suggesting that CD8(+) T cells play a significant role in the immunopathogenesis of PBC.

Heterogeneity of dendritic cells in the mouse liver: Identification and characterization of four distinct populations

Liver dendritic cells (DC) are believed to play important roles in liver immunity, autoimmunity, and in the regulation of hepatic allograft acceptance. However, limited information is available on the phenotypes and functions of DC in the liver. To address this issue, we isolated DC from murine liver using procedures that do not involve collagenase, and characterized the freshly isolated DC population that had not been subjected to in vitro expansion. Thence, based on the expression of CD4, B220, and CD11b, four subsets or groups of hepatic NK1.1−CD11c+ DC were identified with the following phenotypes: B220+CD4+, B220+CD4−, B220−CD11b+, and B220−CD11b−. Each subset was further characterized both phenotypically and functionally. In addition to unique phenotypic expression, each subset displayed different allostimulation capability in mixed lymphocyte reaction assays. All four groups developed DC morphology following in vitro culture with activation agents and synthesized distinct patterns of cytokines in response to different stimuli. Taken together, our results suggest that groups I and II are IFN-α-producing plasmacytoid DC, group III cells are myeloid-related DC, while group IV is a heterogenous population containing both myeloid- and lymphoid-related DC. Our results demonstrate the highly heterogeneous nature of hepatic DC, which is in agreement with the unique requirements for APC in the complex liver environment.

Pathogenesis of primary biliary cirrhosis

Primary biliary cirrhosis is an enigmatic autoimmune disease that predominantly affects women. The serologic signatures of PBC are high titer antimitochondrial antibodies that are directed at the inner lipoyl domains of the 2-oxo-dehydrogenase enzymes, particularly PDC-E2. Of note, is that the antibody response and the CD4 and CD8 response, are all directed at a similar epitope, the inner lipoyl domain. This unique immunologic response suggests that modification of the inner lipoyl domain is associated with the immunogenetic basis of disease.

Application of tetramer technology in studies on autoimmune diseases

Autoreactive T cells are thought to play a role in the immunopathogenesis of autoimmune diseases. Analysis of such cells had long been hampered by lack of suitable assays. Recently developed tetramer technology is based on the recognition of specific peptide-MHC complex by T cell receptor and on the increased binding affinity of multimerized peptide-MHC complex. MHC class I and class II tetramers can be used to detect autoreactive CD4(+) and CD8(+) T cells, while nonclassical MHC (such as CD1d) tetramer can be used to detect other T cell groups, for example natural killer T cells. Tetramer technologies enable direct quantitation of autoreactive T cells in blood and affected tissues. It is also possible to carry out phenotypic and functional characterization of specific T cells on a single cell basis by using tetramers. Of special interest, in situ tetramer staining has the great potential of analyzing autoreactive T cells in their cellular environments. Utilization of tetramers in studies of autoreactive T cells is expected to generate important information regarding the role of such cells in the underlying mechanisms of autoimmune diseases.

Increased Frequency of Pre- Pro B Cells in the Bone Marrow of New Zealand Black (NZB) Mice: Implications for a Developmental Block in B Cell Differentiation

Reductions in populations of both Pre-B cell (Hardy fractions D) and Pro-B cells (Hardy fractions B–C) have been described in association with murine lupus. Recent studies of B cell populations, based on evaluation of B cell differentiation markers, now allow the enumeration and enrichment of other stage specific precursor cells. In this study we report detailed analysis of the ontogeny of B cell lineage subsets in New Zealand black (NZB) and control strains of mice. Our data suggest that B cell development in NZB mice is partially arrested at the fraction A Pre–Pro B cell stage. This arrest at the Pre-Pro B cell stage is secondary to prolonged lifespan and greater resistance to spontaneous apoptosis. In addition, expression of the gene encoding the critical B cell development transcription factor BSAP is reduced in the Pre–Pro B cell stage in NZB mice. This impairment may influence subsequent B cell development to later stages, and thereby accounts for the down-regulation of the B cell receptor component Igα (mb-1). Furthermore, levels of expression of the Rug2, λ5 and Igβ (B29) genes are also reduced in Pre–Pro B cells of NZB mice. The decreased frequency of precursor B cells in the Pre–Pro B cell population occurs at the most primitive stage of B cell differentiation.

Proteasome is required for class I-restricted presentation by Fcγ receptor-mediated endocytosis in primary biliary cirrhosis

There is a considerable database on the effector mechanisms for CD8 recognition of PDC-E2 in primary biliary cirrhosis (PBC). In particular, the specific roles of MHC class I, the mitochondrial autoepitope, and the liver-specific T cell precursor frequency, are defined for HLA-A2.1 patients. There is evidence for a role of MHC class I-mediated presentation of exogenous antigens, or cross-presentation, in the development of the antimitochondrial response and a contributory role of Fcgamma receptor-mediated uptake of autoantigen-autoantibody complexes for the induction of a PDC-E2 specific autoreactive CTL response. Based on this background, we examined potential intracellular pathways for processing the immunodominant mitochondrial autoantigen, PDC-E2, by dendritic cells (DC). In particular, we studied the effects of the proteasome inhibitor lactacystin and the endosomal acidification inhibitor bafilomycin on the induction of PDC-E2-specific CTL response in PBC. Importantly, our data indicate that pre-treatment with either lactacystin or bafilomycin inhibits the PDC-E2 immune complex-induced CTL response. The processing and presentation of PDC-E2 by CD8(+)T cells is mediated by proteasomes and facilitated by Fcgamma receptor-mediated endocytosis. This data reflects another layer of interaction between components of the immune system in the development of autoimmunity. Further characterization of autoantigen uptake and processing may lead to potential therapeutic intervention.

Age‐Related Alterations in the Lymphohematopoietic and B‐Lineage Precursor Populations in NZB Mice

Significant disturbances in B lineage populations of New Zealand Black (NZB) mice have been reported, both with respect to their phenotypes as well as to their function. Notably, there is a profound age‐dependent decrease in B‐cell precursors in this strain of lupus prone mice. In efforts to characterize the impact of this disturbance in disease, we performed an intensive phenotypic and B‐cell population analysis in young and old NZB mice. Our results revealed that there was a significant age‐dependent decrease in B cell precursors at all levels of the B‐cell‐lineage developmental pathway. Analysis of the proliferative capacity of these cell populations showed a comparative decrease in cycling activity in the B‐cell‐lineage populations of old NZB mice. Furthermore, these cell subsets were much more susceptible to spontaneous apoptosis when compared with similar populations from age‐matched BALB/c or young NZB mice. Since the frequency of cells that express the interleukin‐7 receptor (IL‐7R) declines as NZB mice age, we hypothesize that impairment of IL‐7R signal transduction pathways could contribute to severe perturbations of B‐cell function in aged NZB mice.