CD45 ISOFORM REGULATION OF T CELL ACTIVATION

T细胞抗原受体（TcR）的连接不会导致单个T细胞的免疫应答。 一系列的事件，而是可以导致各种不同的 结果包括增殖、细胞毒性、凋亡、不同的 淋巴因子分泌模式和无反应性。精确的机制 负责调节这些不同的结果仍然不清楚。一 在调节T细胞活化中重要的分子是CD45，一个家族， 高分子量跨膜糖蛋白，由 含有两个蛋白酪氨酸磷酸酶结构域的胞质尾区 活性，和一个可变的外部结构域编码的三个外显子， 由于转录后替代RNA的差异表达 拼接这产生了多种不同的同种型， 在T细胞发育过程中和T细胞增殖后， 活化，并且与不同的T细胞效应子功能相关。 CD45的胞质酪氨酸磷酸酶结构域似乎是关键的 因为缺乏CD45的突变型T细胞是难治性的， T细胞受体信号，由于信号事件恢复， 在用CD45或用 缺乏外部结构域但保留酪氨酸磷酸酶结构域。 这些数据表明，CD45的共同胞质结构域直接与CD45结合。 参与信号传导后第二信使产生的调节 然而，通过TcR，CD45的可变亚型在 调节T细胞活化是不确定的。有两个主要的假设 对于这个角色（不相互排斥），这将在此探讨 提议第一个假设是，CD45的不同亚型可能 通过优先与其他细胞表面结合来调节信号传导 在同一个细胞上的分子（如CD4）。第二个假设是， 不同的亚型可能参与调节细胞间粘附， 相互作用，并可能影响T细胞与不同 抗原呈递细胞群。为了检验这些假设， 将制备一系列表达不同CD45的T细胞转染子 具有确定的T细胞受体的细胞上的同种型。个人的影响 将确定CD45同种型对抗原特异性T细胞活化的影响。 还将构建表达不同的 CD45的细胞外亚型通过糖基连接到膜 磷脂酰肌醇连接的锚。这些细胞将用于确定 细胞外结构域的作用，在缺乏细胞质 磷酸酶结构域，并产生可溶性CD45同种型，其将 用作探针以检测同种型特异性CD45配体，并评估 调节抗原特异性T细胞活化的能力。这些研究 对调节T细胞活化具有广泛的适用性领域 癌症发病机理、自身免疫和传染病的研究。

Ligation of the T cell antigen receptor (TcR) does not result in a single series of events, but rather can lead to a wide variety of different outcomes including proliferation, cytotoxicity, apoptosis, distinct patterns of lymphokine secretion, and anergy. The precise mechanisms responsible for regulating these different outcomes remains unclear. One molecule important in regulating T cell activation is CD45, a family of high molecular weight transmembrane glycoproteins consisting of a cytoplasmic tail containing two domains with protein tyrosine phosphatase activity, and a variable external domain encoded in three exons which are differentially expressed due to post-transcriptional alternative RNA splicing. This gives rise to a variety of different isoforms whose expression is regulated during T cell development and following T cell activation, and is correlated with different T cell effector functions. The cytoplasmic tyrosine phosphatase domains of CD45 appear to be critical for T cell activation since mutant T cells which lack CD45 are refractory to T cell receptor signaling, and since signaling events are restored following transfection with CD45, or with heterochimeric molecules which lack the external domains but retain the tyrosine phosphatase domains. These data indicate that the common cytoplasmic domain of CD45 is directly involved in the regulation of second messenger generation after signaling through the TcR, however, the role of the variable isoforms of CD45 in regulating T cell activation is uncertain. There are two main hypotheses for this role (not mutually exclusive) which are to be explored in this proposal. The first hypothesis is that the different isoforms of CD45 may regulate signaling by preferentially associating with other cell surface molecules on the same cell (such as CD4). A second hypothesis is that the different isoforms may be involved in regulating adhesion during cell-cell interactions and perhaps influence T cell interactions with different antigen presenting cell populations. In order to test these hypotheses, a series of T cell transfectants will be made which express different CD45 isoforms on cells with a defined T cell receptor. The effect of individual CD45 isoforms on antigen-specific T cell activation will be determined. Transfectants will also be constructed which express different extracellular isoforms of CD45 linked to the membrane by a glycolsyl phosphatidyl inositol linked anchor. These cells will be used to determine the role of the extracellular domains in the absence of the cytoplasmic phosphatase domains, and to produce soluble CD45 isoforms which will be used as a probe to detect isoform specific CD45 ligands, and evaluated for the ability to modulate antigen-specific T cell activation. These studies on the regulation of T cell activation have broad applicability to fields of cancer pathogenesis, autoimmunity, and infectious disease.