| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
During early development in the thymus, immature T lymphocytes express pre-TCR (pre-T cell antigen-receptor) complex including TCR-β_chain. The signals through pre-TCR are crucial for a differentiation step to become mature T cells expressing mature form of TCR. Unlike mature TCR complex, TCR-β chain of pre-TCR complex is not associated with TCR-α chain but is associated with pre-TCR-α chain (pTα). Interestingly, T cell development in pTα-deficient mice is not completely arrested, despite that the number of T cells generated in these mice is severely impaired. Thus, it is unclear how pTα chain is involved in T cell development. The present study aims to establish a monoclonal antibody specific for surface pTα chain, in order to examine the expression and function of pTα. By immunizing Armenian hamsters with a recombinant thioredoxin-fusion protein containing extracellular region of mouse pTα. Among several candidate clones, we cloned a hybridoma cell producing a monoclonal antibody tha
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t bound to the immunized pTα-fusion protein, not to thioredoxin alone. This antibody stained cell-surface of pTα-expressing immature thymocyte clones, KKF and SCB.29, without staining pTα-negative mature T cell clones including 2B4. Inaddition, this antibody immunoprecipitated a TCR-β-associated 33kD-surface protein, which resembled pTα, and stained mouse pTα-transfected COS7 cells without staining mock-transfected COS7 cells. These results suggest that this novel antibody can specifically bind to extracellular region of mouse pTα. Indeed, this monoclonal antibody strongly stained approximately 2% of adult mouse thymocytes, and these stained cells were mostly confined in CD4-CD8-immature compartment. Thus, this monoclonal antibody is likely useful in further analysis of expression and function of pTα, not only in the thymus but also in other tissues. However, our recent analysis has revealed that this antibody also stains a subpopulation of mature B lymphocyies, which do not express pTα mRNA levels at all, suggesting a possibility that this antibody may not be exclusively specific for pTα protein. Consequently, we are currently screening for a new monoclonal antibody exhibiting more rigid specificity to pTα protein. We also aims to identity the antibody-detected molecule expressed by B cell subpopulation, since this antibody may detect a novel molecule which is structurally related to pTα protein. Less
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