Functional Characterization of Two Distinct TCR Beta Genes of Teleosts

斑点叉尾鱼和双色斑点叉尾鱼的TCRβ链常数(TCRBC)区域由两个非常不同的基因编码，这与哺乳动物中这两个基因极其相似的情况形成了鲜明对比。这个项目的目的是验证这样一种假设，即鱼类TCRBC中的这些主要差异对免疫功能很重要。对这两个TCRBC的V区利用和表达的测定将表明这两个基因是否使用相同的V区谱系，以及表达这些基因的细胞是否在宿主组织中被区隔。针对这两种TCRBC基因产物以及针对鲶鱼CD3 epsilon和Zeta家族成员(Zeta和Fc-Gamma)的抗体的开发将用于免疫沉淀和蛋白质印迹分析，以区分TCR受体复合体的成员。克隆表达不同TCRBC区域基因的鲶鱼淋巴细胞系将允许使用统一的细胞群体在体外比较抗原结合和受体信号传递过程。为了确定携带不同TCR受体的细胞所使用的膜相关辅助分子，并确定结扎TCR后的初始信号事件，将进行两种类型的实验。首先，免疫突触的组成将在受体连接后使用与TCR复合体成员或霍乱毒素反应的抗体来确定。其次，将测量细胞内信号的产生，包括酪氨酸磷酸化和钙动员。如果观察到辅助分子或信号能力的差异，由两个TCRB的C区外显子形成的杂交基因将被用来区分C区的哪一部分影响所观察到的差异。因此，这些实验将确定不同的TCRBC片段是否有能力招募或激活不同的信号肽。无论结果如何，这些实验都将定义用于叉尾鱼的TCR复合体。两个非常不同的TCRBC基因的系统发育意义尚不清楚，但对于理解所有脊椎动物的免疫反应性进化具有潜在的重要意义。本项目将确定这两个TCRB蛋白的C区是具有不同的调节功能，还是仅仅作为组装TCRα/CD3/Zeta和保持膜远端可变区的支架。某些鱼类的淋巴细胞使用两个本质上不同的T细胞受体，而哺乳动物细胞使用的T细胞受体基本上是相同的。这项研究的目的是了解这些鱼类T细胞受体差异对免疫和宿主防御功能的影响。该项目将确定不同的T细胞受体是否能够使用相同的抗原结合片段，从而将免疫功能引导到类似的病原体。这项研究还将确定这两种不同的T细胞受体是否使用相同的机制与细胞内部进行通信，从而影响后续反应的类型或幅度。目前，这些鱼类T细胞受体差异的意义尚不清楚，但可能对了解进化过程中宿主防御能力的变化很重要。

The TCR beta chain constant (TCRBC) regions of both channel catfish and bicolor damselfish are encoded by two substantially different genes in contrast to the situation in mammals where these two genes are extremely similar. The objective of this project is to test the hypothesis that these major differences in fish TCRBC are important for immune functions. Determination of V region usage and expression of the two TCRBC in catfish will indicate whether the two genes use the same V region repertoire and whether cells expressing these genes are compartmentalized within host tissues. Development of monoclonal antibodies directed toward the two TCRBC gene products as well as antibodies to catfish CD3 epsilon and zeta family members (zeta and Fc-gamma) will be used in immunoprecipitation and Western blot analyses to differentiate members of the TCR receptor complex. Cloning of catfish lymphoid cells lines expressing different TCRBC region genes will allow in vitro comparisons of antigen-binding and receptor signaling processes using uniform cell populations. To identify the membrane associated accessory molecules used by cells bearing the different TCR receptors and to determine the initial signaling events following ligation of the TCR, two types of experiments will be done. First, the composition of immune synapses will be defined following receptor ligation using antibody reactive with TCR complex members or cholera toxin. Secondly, the generation of intracellular signals including tyrosine phosphorylation and calcium mobilization will be measured. If differences in accessory molecules or signaling capacities are observed, hybrid genes formed from C region exons of both TCRB will be used to distinguish which portion of the C region influences the observed differences. Thus, these experiments will determine whether divergent TCRBC segments have the ability to recruit or activate different signaling peptides. Regardless of the outcome, these experiments will define the TCR complex used in catfish. The phylogenetic significance of two very divergent TCRBC genes is unknown, but is potentially important for understanding the evolution of immune reactivity in all vertebrates. This project will determine whether the C regions of the two TCRB proteins have different regulatory functions or merely serve as scaffolds for assembly of TCR alpha/CD3/zeta and for holding the variable region distal to the membrane. Two substantially different T cell receptors are used by lymphocytes from certain fish species whereas those used by mammalian cells are essentially identical. The goal of this research is to understand the impact of these fish T cell receptor differences on immune and host defense functions. This project will determine whether or not the different T cell receptors are capable of using the same antigen-binding segments, and thus directing immune function toward similar pathogens. The research will also determine whether the two different T cell receptors use the same mechanisms to communicate with the inside of the cell, thus influencing the type or magnitude of ensuing response. At present, the significance of these fish T cell receptor differences is not known, but may be important for understanding changes in host defense capabilities during evolution.