ROLE OF FUCOSYLTRANSEFERASE IN ANTIVIRAL CYTOTOXICITY

细胞毒性T淋巴细胞(CTL)是控制病毒感染和肿瘤的关键免疫效应细胞。然而，对它们在体内的分化只有部分了解。我们发现，APC-CD8 T细胞通过α(1，3)岩藻糖化的P-选择素糖蛋白-1(PSGL-1)相互作用产生效应性CTL。在缺乏它的情况下，痘苗病毒感染的缺乏α(1，3)岩藻糖基转移酶IV+VIII(ft-/-)的小鼠不会产生效应性CTL，尽管它们会产生激活的CD8T细胞，能够进行病毒特异性增殖。在目标1中，我们将培育转淋巴细胞性脉络膜脑膜炎(LCMV)糖蛋白TCR的FT-/-小鼠。这个系统将使我们能够研究FT在LCMV攻击的抗原特异性CTL反应中的作用。我们将在过继转移实验中确定APC或CD8T细胞上是否需要FT。白细胞黏附缺陷II型(LADII)是一种由于岩藻糖代谢缺陷而引起的人类疾病，它使我们能够研究岩藻糖基化PSGL-1在人类中的作用。我们还培育出了T细胞表达水母衍生绿色荧光蛋白(GFP-TG)的转基因小鼠。当这些小鼠被免疫时，效应CTL失去了GFP的表达，而幼稚和激活的前效应T细胞继续表达GFP。因此，GFP-TG小鼠为分离分化的效应器和前效应器CTL提供了有力的工具。利用这个系统，我们已经确定了在效应器分化过程中发生的几个以前未被识别的分子变化。因此，为了研究在缺乏T的情况下缺陷的分子机制，在目标2中，我们将培育FT-/-小鼠到GFP-TG小鼠，并分析它们的抗原激活的CD8T细胞与FT+/+CD8T细胞的分子差异。由于选择素配体缺陷的FT-/-小鼠，而不是选择素缺陷的小鼠，CTL的生成是有缺陷的，因此岩藻糖基化的PSGL-1可能存在非选择素受体。因此，在目标3中，我们将确定FT-/-小鼠的缺陷是否通过调节抗原递呈而间接发生，或者是否涉及与新分子的直接相互作用。如果存在这样的分子，我们将分离、克隆和鉴定该分子。

Cytotoxic T lymphocytes (CTL) are key immune effector cells in controlling viral infections and tumors. However, their differentiation in vivo is only partially understood. We have found that a novel APC-CD8 T cell interactions through an alpha (1,3) fucosylated P-selectin glycoprotein-1 (PSGL-1) is required to generate effector CTL. In its absence, vaccinia virus-infected mice deficient in alpha (1,3) fucosyl transferase IV+VIII (ft-/-) do not generate effector CTL, although they generate activated CD8 T cells capable of viral specific proliferation. In Aim 1, we will breed FT-/- mice to mice transgenic (Tg) to lymphocytic choriomeningitis (LCMV) glycoprotein TCR. This system will enable us to study the role of FT in antigen-specific CTL response that LCMV challenge. We will determine if FT is required on APC or on CD8 T cells in adoptive transfer experiments. Leukocyte adhesion deficiency type II (LADII), a human disease due to defective fucose metabolism allows us to study the role of fucosylated PSGL-1 in humans We have also produced transgenic mice whose T cells express the Jellyfish Victoria acquoria-derived green fluorescent protein (GFP-Tg). When these mice are immunized, the effector CTL lose GFP expression, whereas naive and activated pre-effector T cells continue to express GFP. Thus, GFP-Tg mice provide a powerful tool to separate differentiated effector from pre-effector CTL. Using this system, we have identified several previously unrecognized molecular changes that occur during effector differentiation. Thus, to study the molecular mechanism that are defective in the absence of T, in aim 2 we will breed FT-/- mice to GFP- Tg mice and analyze their antigen activated CD8 T cells for molecular differences from FT+/+ CD8 T cells. Since selectin-ligand deficient FT-/- mice, but not selectin-deficient mice are defective for CTL generation, a non-selectin receptor may exist for the fucosylated PSGL-1. Thus, in aim 3, we will determine if the defect in FT-/- mice is indirect through modulation of antigen presentation, or if a direct interaction with a novel molecule is involved. If such molecule exists, we will isolate, clone, and characterize the molecule.