Expression and function of pT-alpha, a component of pre-TCR

在胸腺发育的早期，未成熟T淋巴细胞表达包括TCR-β链在内的前T细胞抗原受体复合物。通过前TCR的信号对于分化步骤成为表达成熟形式的TCR的成熟T细胞是至关重要的。与成熟TCR复合物不同，前TCR复合物的TCR-β链不与TCR-α链结合，而是与前TCR-α链（pTα）结合。有趣的是，pTα缺陷小鼠中的T细胞发育并没有完全停止，尽管这些小鼠中产生的T细胞数量严重受损。因此，pTα链如何参与T细胞发育尚不清楚。本研究的目的是建立一种特异性抗pTα链的单克隆抗体，以检测pTα的表达和功能。用含有小鼠pTα胞外区的重组硫氧还蛋白融合蛋白免疫亚美尼亚仓鼠。在几个候选克隆中，我们克隆了产生单克隆抗体的杂交瘤细胞， 关于我们 t与免疫的pTα融合蛋白结合，而不与单独的硫氧还蛋白结合。该抗体染色表达pTα的未成熟胸腺细胞克隆KKF和SCB.29的细胞表面，而不染色pTα阴性成熟T细胞克隆（包括2B 4）。此外，该抗体免疫沉淀TCR-β相关的33 kD表面蛋白，其类似于pTα，并染色小鼠pTα转染的COS 7细胞，而不染色模拟转染的COS 7细胞。结果表明，该抗体能与小鼠pTα胞外区特异性结合。事实上，这种单克隆抗体强烈染色约2%的成年小鼠胸腺细胞，这些染色的细胞主要局限于CD 4-CD 8-未成熟区室。因此，该单克隆抗体可能用于进一步分析pTα在胸腺和其他组织中的表达和功能。然而，我们最近的分析显示，该抗体也染色了完全不表达pTα mRNA水平的成熟B淋巴细胞亚群，这表明该抗体可能不完全特异于pTα蛋白。因此，我们目前正在筛选对pTα蛋白具有更严格特异性的新单克隆抗体。我们还旨在鉴定B细胞亚群表达的抗体检测分子，因为该抗体可能检测与pTα蛋白结构相关的新分子。少

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 … More 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