Takayuki Kanai

Characterization of self-glutamic acid decarboxylase 65-reactive CD4+ T-cell clones established from Japanese patients with insulin-dependent diabetes mellitus

To investigate autoimmunity to glutamic acid decarboxylase (GAD) 65 in Japanese patients with insulin-dependent diabetes mellitus (IDDM, type I diabetes), we established seven CD4+ T-cell clones, by stimulating peripheral blood mononuclear cells (PBMC) of six IDDM patients, using a mixture of overlapping human GAD65 peptides. No GAD65 autoreactive T-cell clones were evidenced in four healthy controls. Specificities of T-cell clones were as follows: (a) two clones specific to GAD65 p111-131 (residue 111 to 131) + DR53 (DRB4*0103); (b) one clone specific to GAD65 p413-433 + DR1 (DRB1*0101); (c) two clones specific to GAD65 p200-217 + either DR9 (DRB1*0901) or DR8 (DRB1*0802); and (d) two clones specific to GAD65 p368-388 + DP2 (DPA1*01 or 0201-DPB1*0201). Two DR53-restricted and one DR1-restricted T-cell clones, responded to a recombinant human GAD65 protein, and showed cytotoxicity against B lymphoblastoid cell lines pre-pulsed with the peptides. Six T-cell clones exhibited the Th1-like phenotype. Interestingly, two DR53-restricted T-cell clones killed a Fas-deficient B lymphoblastoid cell line, thereby indicating that cytotoxicity was not completely dependent on a Fas-Fas ligand interaction. Thus, the T-cell epitopes were mapped in a limited portion of GAD65 protein, with a tendency to be restricted by disease-associated HLA-DR, but not DQ molecules.

Peptide-Based Molecular Analyses of HLA class II-Associated Susceptibility to Autoimmune Diseases

Recent advances in knowledge of crystal structures of MHC class II molecules has advanced understanding of the molecular basis for interactions between peptides and HLA class II molecules. Polymorphism of HLA class II molecules influences structures of peptides bound to HLA class II molecules. To better understand mechanisms related to particular HLA class II alleles and autoimmune diseases, it is important to identify self-peptides presented by disease-susceptible HLA class II molecules and triggering disease-causative autoreactive T cells. Autoimmune diseases occur in Caucasians, Blacks and Asians, albeit with a different incidence. In some autoimmune diseases, disease-susceptible HLA class II alleles are closely related but different, and clinical manifestations of diseases differ among ethnic groups. These phenomena strongly suggest that difference in autoimmune self-peptide(s) in the context of disease-susceptible HLA class II molecules may explain the different clinical manifestations of diseases. Therefore, a comparison among disease-susceptible HLA class II alleles, autoimmune self-peptides and clinical manifestations of autoimmune diseases in different ethnic groups would be instructive. We directed efforts to determining: (1) HLA-class II alleles specific to Asian populations and which are associated with susceptibility to autoimmune diseases, (2) binding-peptide motifs for these HLA class II molecules, and (3) self-peptides presented by susceptible HLA class II molecules to stimulate autoreactive T cells related to the development of autoimmune diseases in Asians. In this review, our related recent investigations are described and the uniqueness of HLA class II-associated autoimmune diseases in Asians is given emphasis.

Immuno-suppressive Peptides for a Human T Cell Clone Autoreactive to a Unique Acetylcholine Receptor α Subunit Peptide Presented by the Disease-Susceptible HLA-DQ6 in Infant-Onset Myasthenia Gravis

Infant-onset myasthenia gravis, an autoimmune disease specific to Asians predominantly affects neuromuscular junctions in ocular muscles. An AChR alpha peptide (p71-91) specific autoreactive CD4+ alpha beta T cell clone was established by stimulating PBMC from a patient heterozygous for two disease-susceptible HLA-DR9-DQ9 and DR13-DQ6 haplotypes with a mixture of overlapping peptides covering AChR alpha. The T cell clone recognized the AChR alpha peptide in the context of the HLA-DQ6 molecule and produced a large amount of IFN-gamma and a trace amount of IL-4. A part (p75-83) of the core epitope of the autoantigenic peptide (p75-87) is encoded for by an exon P3A of the AChR alpha gene which can be alternatively spliced. The T cell clone responded to the recombinant AChR alpha protein with a P3A exon product, but not without a P3A exon product. We investigated responses of the T cell clone to 114 analogue peptides carrying single residue substitutions of the core AChR alpha peptide. The majority of analogues substituted at residues Phe-77, Leu-80 and Asn-82 stimulated proliferation of the T cell clone. Conversely, the majority of analogue peptides substituted at either Gln-81 or Glu-83 did not stimulate proliferative responses, and all exhibited strong or intermediate inhibitory effects on proliferative responses of the T cell clone to the wild type peptide, possibly by TCR antagonism. Thus, an HLA class II allele specific to Asians may directly control susceptibility to the Asian-specific type of myasthenia gravis. Analogues of the auto-antigenic AChR alpha peptide may prove effective for new immunosuppressive therapy.

Molecular analyses of HLA class II-associated susceptibility to subtypes of autoimmune diseases unique to Asians

It is well known that individuals positive for particular HLA-class II alleles show high risks for the development of Takayasu arteritis and other diseases caused by immunological disorders such as autoimmune diseases and allergies. HLA class II molecules present antigenic peptides to CD4+ T cells. Their extensive polymorphism affects the structures of peptides bound to HLA class II molecules to create individual differences in immune responses to antigenic peptides. To better understand the mechanisms for association between HLA class II alleles and susceptibility to autoimmune diseases, it is important to identify self-peptides presented by disease-susceptible HLA class II molecules and triggering disease-causative T cells. Many autoimmune diseases are observed in all ethnic groups, whereas the incidences of diseases, clinical manifestations and disease-susceptible HLA class II alleles are different among various ethnic groups for some autoimmune diseases. These phenomena suggest that differences in autoimmune self-peptide(s) in the context of disease-susceptible HLA class II molecules may cause these differences. Therefore, comparisons among disease-susceptible HLA class II alleles, autoimmune self-peptides and clinical manifestations of autoimmune diseases in different ethnic groups would be helpful in determining the pathogenesis of the diseases. In this paper, we describe our recent findings on: (1) the uniqueness of both clinical manifestations and HLA-linked genetic background of Asian-type (optico-spinal form) multiple sclerosis; (2) the structural characteristics of peptides bound to HLA-DQ molecules susceptible to insulin-dependent diabetes mellitus; (3) the identification of a disease-related autoantigenic peptide presented by disease-susceptible HLA-DQ molecules in Asians-specific infant onset myasthenia gravis; and (4) a manipulation of human T cell response by altered peptide ligands, as a possible candidate for new and antigen-specific immuno-suppressive therapy against autoimmune diseases.

A murine monoclonal antibody (928) recognizing a new epitope formed with a combination of HLA-DPA1*0201 and DPB1*0301 gene products.

A murine monoclonal antibody (mAb), 928, that recognizes a cell surface antigen (928 Ag) on a human Epstein-Barr virus-transformed fetal liver-derived lymphoid progenitor cell line (FL4.4) was generated. The 928 mAb reacted with only FL4.4; it did not react with any other 57 cell lines tested. Two color flowcytometry analysis of peripheral blood mononuclear cells (PBMC) revealed that the 928 mAb reacted with B cell and monocyte fractions from only two individuals out of 63 unrelated donors. Biochemical analyses showed that the 928 Ag composes of two molecules (33 and 34 Kd) and forms a SDS-resistant, noncovalently linked dimer conformation, the feature being similar to that of peptide-bound MHC class II molecules. Treatment of FL4.4 cells with the 928 mAb significantly facilitated homotypic cell aggregation. In addition, treatment of PBMC of the 928 Ag+ donor with recombinant IL-4 augmented the expression of the 928 Ag on CD64+ monocytes. Typing of HLA-DRB1, DPA1 and DPB1 alleles of the 928 Ag expressing and nonexpressing cells revealed that the 928 Ag is expressed only on PBMC of HLA-DPA1*0201 and DPB1*0301 positive donors. Finally, anti-DP antibody precleared 928 Ag from the cell lysate. These results demonstrate that the 928 mAb recognizes a polymorphic determinant of HLA-DPA1*0201-DPB1*0301 gene products. The possibility that amino acids in the groove of the peptide-binding site of HLA-DP molecules are critical for the 928 epitope is discussed.

A Case Report of Tuberculosis of the Cervical Spine with the Barré-Liéou Syndrome

Tuberculosis of the cervical spine is uncommon to arise in the whole apine. We report a case of tuberculous cervical spondylitis with Barre-Lieou syndrome.The case was a 61 year old female, who complained of mild muscle weakness and strong dizziness at motion, headache, nausea, so called Barre-Lieou syndrome. MRI showed rim enhancement of the retropharyngeal soft tissue and peridural abscess. We therefore performed the operation of curretage and anterior spinal body fusion by use of autogenous fibula bone, adding the chemotherapy of INH, RFP and SM. She gradually recovered from Barre-Lieou syndrome.The origin of Barre-Lieou syndrome in this case was unsidered the source of the irritation of the vertebral nerve by tuberculous abscess.

A Follow-up Study of Postoperative Cervical Laminoplasty

We studied 69 patients with cervical spondylotic myelopathy, and 33 patients with cervical ossification of the posterior longitudinal ligament with a mean age of 65.5 years and an average follow-up period of 4 years and 5 months. Clinical results were evaluated using the JOA score for cervical myelopathy and recovery rates using Hirabayashis method. The overall mean recovery rate was 58%. There was no significant difference between recovery rate and age, duration of symptoms or preoperative JOA score.Moreover, we radiographically investigated changes in cervical alignment and rate of enlargement, antero-posterior movement, and instability in 50 cases.