Leukocyte immune type receptors: gene structure and function

水产养殖是世界上发展最快的食品生产部门，鲶鱼是美国主要的淡水养殖品种。由于鲶鱼对美国水产养殖业的经济重要性，鲶鱼免疫系统的功能特征一直是该实验室的长期重点。在人类中，白细胞在对抗感染方面发挥着重要作用。然而，人们对鱼类中的这些细胞知之甚少。目前研究的目标是更好地了解鲶鱼中的白细胞如何识别和杀死细菌和病毒感染的细胞。最近的研究表明，一个被称为白细胞免疫型受体（LITRs）的蛋白家族在鲶鱼白细胞表面表达。这些表达LITR的细胞能够杀死病毒感染的细胞。该项目将确定在感染过程中不同LITR基因在免疫器官和白细胞中的表达模式如何变化，并提供LITR识别的蛋白质或分子结构类型的信息。这项研究还将为研究生、本科生和高中生提供独特的研究培训机会，包括来自科学领域代表性不足群体的学生。学生将学习基础免疫学，并获得各种分子和细胞技术的专业知识。研究生和本科生将有自己的研究项目，学习如何设计实验，解释数据，并在地区或国家会议上展示他们的发现。硬骨鱼免疫反应的研究很重要，因为它们提供了不同动物群体适应的各种免疫策略的信息。本项目重点研究通道鲶鱼和其他硬骨鱼中发现的LITR免疫调节受体家族。litr包括一个高度多样化的多基因家族，许多已经测序的litr在结构上与哺乳动物NK受体编码在白细胞受体复合体中相关。此外，某些litr，根据其与单克隆抗体CC41的反应性，在鲶鱼细胞毒性细胞上表达，通过分泌-穿孔素颗粒酶途径识别并杀死病毒感染的细胞。该项目将首先定义鲶鱼中完整的LITR基因库，并使用PacBio单分子长读测序协议检测病毒和细菌攻击后LITR的表达。其次，选择cc41反应性litr的配体将使用重组融合蛋白（以四聚体或免疫球蛋白融合蛋白的形式）来确定。分离litr -配体复合物，用LS-MS/MS分析免疫选择蛋白。第三，由于初步测序已经鉴定出少量类似于哺乳动物fc受体的litr，因此将对该组代表进行免疫球蛋白结合分析。该项目的成功完成将定义litr的功能特性，并为先天免疫调节受体的进化提供见解。此外，该项目将提供关于变温脊椎动物如何维持自我耐受性和识别改变自我的新信息。

Aquaculture is the fastest growing food producing sector in the world, and catfish represent the major fresh water farmed species in the US. Because of the economic importance of catfish to the U.S. aquaculture industry, it has been a long-term focus of this laboratory to functionally characterize the catfish immune system. In humans, white blood cells play important roles in fighting infection. However, very little is known about these cells in fish. The goal of the current study is to better understand how white blood cells in the catfish recognize and kill bacteria and virus-infected cells. Recent studies have shown that a family of proteins termed Leukocyte Immune Type Receptors (LITRs) is expressed on the surface of catfish white blood cells. These LITR expressing cells are capable of killing virus infected cells. This project will determine how the expression patterns of different LITR genes change in immune organs and white blood cells during the course of an infection and also provide information on the types of proteins or molecular structures LITRs recognize. This study will also provide unique research training opportunities for graduate, undergraduate and high school students, including students from groups under-represented in science. The students will learn basic immunology and gain expertise in a variety of molecular and cellular techniques. Graduate and undergraduate students will have their own research projects, learn how to design experiments, interpret data and present their findings at regional or national meetings. Studies of teleost immune responses are important because they provide information about the various immune strategies adapted by different animal groups. This project focuses on the LITR immunoregulatory receptor family that is found in channel catfish and other bony fish. LITRs comprise a highly diverse multigene family and many of the LITRs that have been sequenced are structurally related to mammalian NK receptors encoded within the leukocyte receptor complex. Additionally, certain LITRs, as defined by their reactivity with monoclonal antibody CC41, are expressed on catfish cytotoxic cells that recognize and kill virus infected cells via the secretory-perforin granzyme pathway. This project will first define the complete LITR gene repertoire in catfish and examine LITR expression after viral and bacterial challenge using PacBio single molecule long-read sequencing protocols. Second, the ligands for selected CC41-reactive LITRs will be determined using recombinant fusion proteins either in the form of tetramers or immunoglobulin-fusion proteins. The LITR-ligand complexes will be isolated and immunoselected proteins will be analyzed by LS-MS/MS. Third, since preliminary sequencing has identified a small number of LITRs that resemble mammalian Fc-receptors, representatives from this group will be analyzed for immunoglobulin binding. The successful completion of this project will define the functional properties of LITRs and provide insights into the evolution of innate immune regulatory receptors. Moreover, this project will provide novel information about how ectothermic vertebrates maintain self-tolerance and recognize altered-self.