TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells.

We previously reported a method to generate dendritic cell (DC)-like antigen-presenting cells (APC) from human induced pluripotent stem (iPS) cells. However, the method is relatively complicated and laborious. In the current study, we attempted to establish a method through which we could obtain a large number of functional APC with a simple procedure. We transduced iPS cell-derived CD11b(+) myeloid cells with genes associated with proliferative or anti-senescence effects, enabling the cells to propagate for more than 4 months in a macrophage colony-stimulating factor (M-CSF)-dependent manner while retaining their capacity to differentiate into functional APC. We named these iPS cell-derived proliferating myeloid cells 'iPS-ML', and the iPS-ML-derived APC 'ML-DC'. In addition, we generated TAP2-deficient iPS cell clones by zinc finger nuclease-aided targeted gene disruption. TAP2-deficient iPS cells and iPS-ML avoided recognition by pre-activated allo-reactive CD8(+) T cells. TAP2-deficient ML-DC expressing exogenously introduced HLA-A2 genes stimulated HLA-A2-restricted MART-1-specific CD8(+) T cells obtained from HLA-A2-positive allogeneic donors, resulting in generation of MART-1-specific cytotoxic T lymphocyte (CTL) lines. TAP-deficient iPS-ML introduced with various HLA class I genes may serve as an unlimited source of APC for vaccination therapy. If administered into allogeneic patients, ML-DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. Based on the present study, we propose an APC-producing system that is simple, safe and applicable to all patients irrespective of their HLA types.

Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy.

This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid ß (Aß), the causal protein of Alzheimer's disease. The scFv-transfectant iPS-MP exhibited efficient Aß-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

Identification of HLA-A2-restricted CTL epitopes of a novel tumour-associated antigen, KIF20A, overexpressed in pancreatic cancer.

BACKGROUND: Identification of tumour-associated antigens (TAAs) that induce cytotoxic T lymphocytes (CTLs) specific to cancer cells is critical for the development of anticancer immunotherapy. In this study, we aimed at identifying a novel TAA of pancreatic cancer for immunotherapy. METHODS: On the basis of the genome-wide cDNA microarray analysis, we focused on KIF20A (also known as RAB6KIFL/MKlp2) as a candidate TAA in pancreatic cancer cells. The HLA-A2 (A*02:01)-restricted CTL epitopes of KIF20A were identified using HLA-A2 transgenic mice (Tgm) and the peptides were examined to check whether they could generate human CTLs exhibiting cytotoxic responses against KIF20A(+), HLA-A2(+) tumour cells in vitro. RESULTS: KIF20A was overexpressed in pancreatic cancer and in some other malignancies, but not in their non-cancerous counterparts and many normal adult tissues. We found that KIF20A-2 (p12-20, LLSDDDVVV), KIF20A-8 (p809-817, CIAEQYHTV), and KIF20A-28 (p284-293, AQPDTAPLPV) peptides could induce HLA-A2-restricted CTLs in HLA-A2 Tgm without causing autoimmunity. Peptide-reactive human CTLs were generated from peripheral blood mononuclear cells of HLA-A2(+) healthy donors by in vitro stimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2. CONCLUSION: KIF20A is a novel promising candidate for anticancer immunotherapeutic target for pancreatic cancers.

Identification of SARS-COV spike protein-derived and HLA-A2-restricted human CTL epitopes by using a new muramyl dipeptidederivative adjuvant.

Severe acute respiratory syndrome (SARS) spread during the winter of 2003, and attempts have been made to develop vaccines against SARS corona virus (SARS-CoV). The present study provides a strategy to rapidly identify SARS-CoV-derived antigenic peptides recognized by HLA-A2-restricted cytotoxic T lymphocytes (CTLs). Forty-three candidate peptides having HLA-A2-binding motifs were selected in silico and HLA-A2/Db chimeric MHC class I-transgenic mice were immunized with these peptides and a new derivative of muramyl dipeptide that can induce upregulation of HLA-DR, CD80, CD86, and CD40 in human CD14+ antigen presenting cells, was administered as an adjuvant. Six HLAA2-restricted mouse CTL epitopes were identified, including two new epitopes which have never been reported before. One of the novel peptides was naturally processed and successfully induced HLAA2-restricted specific CTLs in both HLA transgenic mice and healthy donors. The method was useful, convenient and efficient for rapid identification of CTL epitopes derived from SARS-CoV proteins and will be possibly applicable for other pathogens to develop a peptide-based vaccine.

Humoral immune response directed against LEDGF in patients with VKH.

Vogt-Koyanagi-Harada disease is an autoimmune systemic disorder. In Vogt-Koyanagi-Harada disease, inflammatory disorders occur in multiple organs containing melanocytes, including uvea (resulting in acute bilateral panuveitis), skin (resulting in vitiligo and alopecia), central nervous system (resulting in meningitis) and inner ears (resulting in hearing loss and tinnitus). These inflammatory aspects are attributed to the destruction of melanocytes through immunological mechanisms. Studies have been carried out to elucidate the exact etiology and target autoantigen in Vogt-Koyanagi-Harada disease, but much remains to be investigated. Identification of target autoantigen is important to understand the etiology of autoimmune diseases, and for development of antigen-specific immuno-modulation therapy. To identify the target autoantigens in Vogt-Koyanagi-Harada disease, we made use of an immunoscreening of a bovine uveal cDNA expression library with serum samples obtained from patients with Vogt-Koyanagi-Harada disease. We identified an immunoreactive cDNA clone that encodes bovine lens epithelium derived growth factor. mRNA of human lens epithelium derived growth factor was determined by reverse transcription-polymerase chain reaction and it was expressed in human uvea, retina and melanocytes. Immunoglobulin G (IgG) autoantibodies were quantitated in an enzyme-linked immunosorbent assay, using recombinant human lens epithelium derived growth factor. The prevalence of IgG anti-lens epithelium derived growth factor autoantibodies in patients with Vogt-Koyanagi-Harada disease was significantly higher than that in healthy controls (66.7% versus 21.6%, P<0.001). On the other hand, the prevalence of the autoantibody in patients with panuveitis of other etiology, Behçet's disease and sarcoidosis, was almost same as that in healthy controls. These results suggest that the humoral immune response agonist lens epithelium derived growth factor is not a mere secondary phenomena caused by uveal tissue damage.

Establishment of an expression cloning system for CD4+ T cell epitopes.

We previously reported an epitope presenting vector, pCI, a derivative of a human invariant chain (Ii) expression vector, in which the class II associated invariant chain peptide (CLIP, Ii p89-101) could be substituted with antigenic peptides. In the current study, we used this vector to develop a new expression cloning system to identify CD4+ T cell epitopes. We inserted double-stranded oligo DNAs of randomized sequences into this vector and prepared an epitope-presenting library which loads randomized 13-mer peptides onto HLA class II molecules coexpressed in COS-7 cells. Utilizing this library, we isolated a cross-reactive epitope recognized by a glutamic acid decarboxylase (GAD) 65-autoreactive T cell clone established from a patient with insulin-dependent diabetes mellitus. Although the newly identified epitope (PVQLSNQWHVVGATF) was far different from the original epitope, GAD65 p116-128 (NILLQYVVKSFDR), it did have the capacity to stimulate the T cell clone comparable to that of the original GAD epitope. Our system may be applicable not only for identifying of cross-reactive epitopes for CD4+ T cells of known specificity, but also for detection of epitopes stimulatory for CD4+ T cells the epitopes of which are unknown.

Gene cloning of immunogenic antigens overexpressed in pancreatic cancer.

The serological analysis of recombinant cDNA expression libraries (SEREX) by utilizing a library derived from a human pancreatic adenocarcinoma cell line and IgG antibodies from an allogeneic patient serum led to the identification of 18 genes: 13 of these were known genes, and 5 were unknown genes. In Northern and RT-PCR analyses, we found that the expression of mRNA of 14 genes was elevated in pancreatic cancer cell lines compared with the levels in normal pancreatic tissues. In addition, the expression of mRNA of hsp105 in colon cancer was greater than that in normal colon tissue. Immunohistochemical analysis using anti-hsp105 antibody revealed that an increased expression of hsp105 is a characteristic feature of pancreatic ductal and colon adenocarcinoma. Furthermore, hsp105 immunoreactivity in some cases of gastric, esophageal, and hepatocellular carcinoma was much stronger than that in normal corresponding tissues. These molecules identified may provide good diagnostic markers for cancer cells.

Identification of a novel autoantigen UACA in patients with panuveitis.

To identify the target autoantigens in Vogt-Koyanagi-Harada disease, we made use of an immunoscreening of a bovine uveal cDNA expression library with serum samples obtained from patients with Vogt-Koyanagi-Harada disease. We identified a novel bovine antigen and homologous human autoantigen and designated it as UACA (uveal autoantigen with coiled coil domains and ankyrin repeats). mRNA of human UACA is expressed most abundantly in skeletal muscles and in various human tissues, including choroid, retina, and epidermal melanocytes. IgG autoantibodies were quantitated in an ELISA, using recombinant C-terminal 18.0% fragment of human UACA. The prevalence of IgG anti-UACA autoantibodies in patients with panuveitis (Vogt-Koyanagi-Harada disease, Behçet's disease, sarcoidosis) was significantly higher than that in healthy controls (19.6-28.1% vs 0%, P < 0.05) indicating that autoimmunity directed against UACA is a common phenomenon in these diseases.

Molecular and cellular analyses of HLA class II-associated susceptibility to autoimmune diseases in the Japanese population.

Abstract It is well known that individuals who are positive for particular HLA class II alleles show a high risk of developing autoimmune diseases. HLA class II molecules expressed on antigen-presenting cells 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. In order to gain a better understanding of mechanisms of the 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 of the autoimmune diseases are observed in all ethnic groups, whereas the incidence 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, autoantigenic peptides, and clinical manifestations of autoimmune diseases in different ethnic groups would be helpful in elucidating the pathogenesis of the diseases. In this review, we describe our recent findings on (1) the uniqueness of both clinical manifestations and the HLA-linked genetic background of Asian-type (opticospinal form) multiple sclerosis, (2) the characteristics of glutamic acid decarboxylase 65 (GAD65) or ß2-glycoprotein I (ß2-GPI) autoreactive T cells in Japanese patients with insulin-dependent diabetes mellitus (IDDM) or anti-ß2-GPI antibody-associated autoimmunity, respectively, and (3) the generation of an efficient delivery system of peptides to the HLA class II-restricted antigen presentation path-way by utilizing a class II-associated invariant chain peptide (CLIP)-substituted invariant chain, which may be applicable to an evaluation of the "molecular mimicry hypothesis" for the activation of autoreactive T cells.

Four cases of bronchiolitis obliterans organizing pneumonia associated with thyroid disease.

We present 4 cases of bronchiolitis obliterans organizing pneumonia (BOOP) associated with thyroid diseases. Case 1 had previously undergone surgery for thyroid cancer, and had a secondary hypothyroidism at the onset of BOOP. Case 2 developed Basedow's disease 3 months after the onset of BOOP and BOOP relapsed 21 months after the onset of Basedow's disease. Case 3 developed subacute thyroiditis 33 months after the onset of BOOP, and has had no relapse of BOOP for 7 years. Case 4 had hypothyroidism at the time of diagnosis of BOOP, and her BOOP relapsed 3 months after the initial onset of BOOP. Two of these 4 cases of BOOP with thyroid diseases relapsed, and thyroid dysfunction could modify the pathophysiology of BOOP.

Immunocytochemical analyses and targeted gene disruption of GTPBP1.

We previously identified a gene encoding a putative GTPase, GTPBP1, which is structurally related to elongation factor 1alpha, a key component of protein biosynthesis machinery. The primary structure of GTPBP1 is highly conserved between human and mouse (97% identical at the amino acid level). Expression of this gene is enhanced by gamma interferon in a monocytic cell line, THP-1. Although counterparts of this molecule in Caenorhabditis elegans and Ascaris suum have also been identified, the function of this molecule remains to be clarified. In the present study, our immunohistochemical analyses on mouse tissues revealed that GTPBP1 is expressed in some neurons and smooth muscle cells of various organs as well as macrophages. Immunofluorescence analyses revealed that GTPBP1 is localized exclusively in cytoplasm and shows a diffuse granular network forming a gradient from the nucleus to the periphery of the cells in smooth muscle cell lines and macrophages. To investigate the physiological role of GTPBP1, we used targeted gene disruption in embryonic stem cells to generate GTPBP1-deficient mice. The mutant mice were born at the expected Mendelian frequency, developed normally, and were fertile. No manifest anatomical or behavioral abnormality was observed in the mutant mice. Functions of macrophages, including chemotaxis, phagocytosis, and nitric oxide production, in mutant mice were equivalent to those seen in wild-type mice. No significant difference was observed in the immune response to protein antigen between mutant mice and wild-type mice, suggesting normal function of antigen-presenting cells of the mutant mice. The absence of an eminent phenotype in GTPBP1-deficient mice may be due to functional compensation by GTPBP2, a molecule we recently identified which is similar to GTPBP1 in structure and tissue distribution.

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase.

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.

Augmentation of immune response by altered peptide ligands of the antigenic peptide in a human CD4+ T-cell clone reacting to TEL/AML1 fusion protein.

The 12;21 chromosomal translocation occurs in leukemic cells from 20(30% of patients with B-lineage childhood acute lymphoblastic leukemia, the result being the TEL/AML1 fusion gene carrying a sequence different from TEL or AML1. Because the protein newly formed by TEL/ AML1 fusion is probably not tolerated by human immune system, the fusion region is a good candidate for tumor antigen expressed only in TEL/ AML1-positive leukemic cells. We established two human CD4+ alphabeta T-cell clones (T31.1 and Y41.2) reacting to the TEL/AML1 fusion region, from two unrelated healthy donors. In order to do this, we stimulated peripheral blood mononuclear cells with synthetic peptides corresponding to the TEL/ AML1 fusion region. Both T31.1 and Y41.2 proliferated in response to TEL/ AML1 fusion protein as well as to a peptide IGRIAECILGMNPSR, in the context of HLA-DP5 and DP17, respectively, and killed B lymphoblastoid cells pulsed with the peptide. Furthermore, these T-cell clones proliferated in response to several altered peptide ligands carrying a single residue substitution in the TEL/AML1 peptide, and some induced augmentation of proliferation and production of Th1-type cytokines. These superagonistic altered peptide ligands can be given consideration for anti-leukemic immunotherapy.

The CLIP-substituted invariant chain efficiently targets an antigenic peptide to HLA class II pathway in L cells.

The presentation of antigenic peptides by major histocompatibility complex (MHC) class II to CD4+ T cells is crucial to initiate immune responses. We developed a new system for delivery of an antigenic peptide to the MHC class II pathway, using the invariant chain (Ii). We designed a mutated human p33-form Ii, CLIP-substituted Ii, in which streptococcal M12p55-68 (RDLEQAYNELSGEA) was substituted for CLIP (class II associated invariant chain peptide). We examined the peptide presenting function of this construct, in comparison with the previously reported C-terminal fused Ii, in which a cathepsin cleavage site and M12p54-68 was ligated to the C-terminus of Ii. Mouse L cell transfectants expressing either of these two mutated Ii along with HLA-DR4 could process and present M12p55-68 to the peptide specific and DR4-restricted CD4+ T cell clone. CLIP-substituted Ii was much more efficient in antigen presentation than was the C-terminal fused Ii. Similar to the wild-type Ii, the CLIP-substituted Ii was associated intracellularly with DR4 molecules. These results indicate that the peptide substituted for CLIP of Ii p33 bound to the groove of DR molecules in the same manner as CLIP and it was preferentially presented to the CD4+ T cell clone in the absence of HLA-DM molecules. This system may prove useful for immunotherapy with DNA vaccines or for construction of an antigen presenting cell library with diverse peptides.

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms.

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.

Factors related to the relapse of bronchiolitis obliterans organizing pneumonia.

STUDY OBJECTIVE: The purpose of this study is to determine factors, including laboratory data, related to the relapse of bronchiolitis obliterans organizing pneumonia (BOOP). DESIGN: Retrospective study. SETTING AND PATIENTS: The medical files of Fukuoka University Hospital and Nishi Fukuoka Hospital patients from 1984 to 1996 were reviewed, and 18 cases of BOOP that had been diagnosed using transbronchial or open lung biopsy were selected for evaluation. MEASUREMENTS: The 18 cases were put into two groups composed of 7 patients who relapsed and 11 who did not relapse. Their clinical symptoms and laboratory data at first admission, including hemograms, blood chemistry tests, and pulmonary function tests were compared. Patients with or without associated diseases, such as collagen vascular diseases, were compared using the same parameters in order to examine the relationship between the associated diseases and BOOP relapse. RESULTS: The serum levels of total protein and albumin in patients who relapsed were significantly lower than in patients who did not relapse, respectively: 5.8 (range, 4.4 to 6.2) vs 6.3 (range, 4.5 to 6.8) g/dL, p < 0.05; and 2.9 (range, 2.5 to 3.4) vs 3.7 (range, 2.8 to 4.3) g/dL, p < 0.01. Levels of serum albumin in BOOP patients with associated diseases, however, were significantly lower than in those without associated diseases, respectively: 2.95 (range, 2.5 to 3.9) vs 3.65 (range, 2.8 to 4.3) mg/dL, p < 0.05. The fall in serum albumin levels in patients who relapsed, therefore, was probably due to associated diseases. The fact that 5 of 8 patients with associated diseases relapsed but only 2 of 10 without associated diseases relapsed suggests that a relationship exists between associated diseases and the prognosis of BOOP, although this finding was not statistically significant because of the small number of cases and the heterogeneity of the associated diseases. The most striking observation was that PaO2 levels in patients who relapsed were significantly lower than in those who did not, respectively: 55.4 (range, 39.9 to 73.2) vs 78.0 (range, 48.4 to 89.4) mm Hg, p < 0.05. However, PaO2 levels were not statistically different between patients with and without associated diseases, respectively: 66.0 (range, 45.4 to 78.8) vs 71.4 (range, 39.9 to 89.4) mm Hg. CONCLUSIONS: The severity of hypoxemia at first medical examination may be an important determinant for the subsequent BOOP relapse.

Long-term continuous intravenous infusion of prostacyclin for severe primary pulmonary hypertension.

A patient suffering from severe symptomatic primary pulmonary hypertension (PPH) underwent long-term intravenous prostacyclin therapy; the first time for such treatment in Japan. A 26-year-old male had experienced gradually progressive dyspnea for about one year. Despite conventional therapy he suffered repeated syncopal attacks. However, after receiving a permanent central venous access device and a portable infusion pump, he recovered fully and was discharged. This remedy seems to be promising for PPH as has already been proven in Europe and North Americas, although in Japan it is not as yet commercially available and some problems still need to be resolved.

Thickness of the basement membrane of bronchial epithelial cells in lung diseases as determined by transbronchial biopsy.

The thickness of the basement membranes of bronchial epithelial cells varies under various pathological conditions. It has been reported that this membrane is thickened in patients with bronchial asthma. By light microscopy, this parameter was measured in biopsy specimens of bronchial mucosa obtained by fibre-optic bronchoscopy. These specimens were obtained from 171 patients who had undergone bronchial biopsy between 1984 and 1994. It was demonstrated that the thickness of the basement membrane of bronchial epithelial cells was weakly correlated with the patient's age, when thickness was examined in patients with lung cancer (r = 0.242, P = 0.0268). The basement membranes in patients with bronchial asthma (8.193 +/- 1.362 mu, mean +/- SEM) were significantly thicker than those without bronchial asthma (5.145 +/- 0.233 mu) (P = 0.0180, Mann-Whitney's U-test). In addition, it is noteworthy that the basement membranes in patients with diabetes mellitus (7.217 +/- 0.753 mu) were also significantly thicker than those without diabetes mellitus (4.968 +/- 0.235 mu) (P = 0.0038, Mann-Whitney's U-test). The background or underlying pathophysiology in such patients should be studied further, with attention directed towards the thickness of the bronchial basement membrane in bronchial biopsy specimens.

Immuno-suppressive peptides for a human T cell clone autoreactive to a unique acetylcholine receptor alpha 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.

Identification of human and mouse GP-1, a putative member of a novel G-protein family.

To identify genes induced in monocytes by interferon-gamma, we carried out PCR-based cDNA subtraction and subsequent differential display on mRNA isolated from a human monocytic leukemia cell line, THP-1. We detected a novel gene encoding a protein bearing GTP-binding motifs, the characteristics of GTP-binding proteins (G-proteins). We also identified the mouse homologue of this gene and designated the gene GP-1. The amino acid sequence of GP-1 deduced from the nucleotide sequence is highly conserved in human and mouse (97% identical over the entire protein), suggesting a fundamental physiological role for this molecule. As amino acid sequences of GTP-binding motifs of human and mouse GP-1 are practically identical to those of recently identified putative G-proteins of nematode, AGP-1 and CGP-1, these proteins are likely to be members of the same, novel G-protein family. GP-1 mRNA was readily detected in mouse brain, thymus, lung, and kidney, while GP-1 mRNA is rarely expressed in liver.