|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1993 : ¥1,200,000 (Direct Cost : ¥1,200,000)
A.Analysis of human TSHR specific T cell : (1) Expression of extracellular domain of human TSHR cDNA fragment encoded extracellular domain of human TSHR (1.2kb) was amplified by polymerase chain reaction (PCR) and the protein correspond to the sequence was expressed in Sf9 cells using vaculo virus system. The protein was migrated as 50kDa by SDS PAGE and recognized with an antibody against specific sequence of extracellular domain of human TSHR.After gel permeation chromatography, the purified protein was injected to rabbits to raise an antibody. The raised antibody blocked binding between TSHR and 125I-TSH to show that the antibody was against for TSHR.From experiments described above, it was clear that the extracellular domain of TSHR expressed in Sf9 cell was suitable for antigen to raise for an antibody. (2) Analysis of TSHR specific T cell in model mouse
It has not been successful to isolate the specific T cell clone for TSHR from patients of Basedow's disease. Therefore, first we
tried to immunize mouse with TSHR.Human TSHR cDNA (full length) was transfected to mouse fibroblast (RT,H-2k haplotype) with lipofectin to get cell line (RT-TSHR) stably expressed human TSHR.After immunizing RT-TSHR to Akl/N mouse (H-2k haplotype) , we found blast formation for TSHR in spleen cells. It was concluded that, at least, k haplotype in H-2 (mouse MHC) was able to present TSHR molecule to T cell as an antigen.
B.Analysis of epitopes for the antibodies against human TSH-receptor : The correlation of the epitopes between the specific T-cell and B-cell, which recognize TSH-receptor as an anto-antigen, is thought to be very important. Therefore, we also studied the epitopes for specific B-cell against TSH-receptor (namely, the epitope for the antibody). At first, we made the chimera-receptors of human TSH partly substituted with leutenizing hormone-receptor (LH/CGR). In order to clarify the epitopes, we checked the accumulation of cAMP in the chimera-receptors induced by the stimulating antibody of TSH-receptor. In conclusions, those were clarified that 1) the epitopes of the stimulating antibody were mainly located within the 145 amino acids residues of N-terminal of TSH-receptor and, 2) the each epitope was different and dependent on the each stimulating antibody.
C.Future of this study : The cause of Basedow' disease is the production of autoantibodies that bind the TSH receptor (TSHR) and act as TSH.This is one of the unique model in autoimmnodisease. To elucidate the pothogenesis of this disease, we have to make clear all factors which participate in the immune-response of this disease, especially response through the TSHR.In this study, we induced a specific T-cell against TSHR in vivo. By developping this techniqe, we could get a model system which induce antibodies against TSHR like Basedow' disease. Using this excerent system, we could clarify not only epitopes of T-cell or B-cell but also antigen-presentating cells. This is the real pathway to elucidatethe pothogenesis of Basedow' disease. The analysis of epitopes for the stimulating antibodies against TSH-receptor using the chimera-receptors has been on the way to clarify and these in vivo models should give some suggestions to recognize the correlation of the epitopes between the specific T-cell and B-cell against TSH-receptor. However, the epitopes for the stimulating antibody have not been completely clarified yet because the epitope for the antibody, which recognize the architecture of three-dimentions, is different form the epitope for the specific T-cell, the epitopes of the antibody so varied in the Basedow's diseases. Moreover, the cautions comparison of the data between in vitro and in vivo model is most important since both always have benefit and deficiet. Less