The Relationship Between Thymic Nurse Cells and Macrophages During MHC Restriction

项目摘要：胸腺护理细胞(TnCs)是一种上皮细胞，具有将未成熟的胸腺细胞内化成特化的胞浆内空泡的能力。这种相互作用在胸腺细胞发育过程中的作用是这一提议的重点。研究表明，跨国公司可以结合和内化未成熟的abTCRlowCD69-双阳性胸腺细胞，b)将一部分内化的胸腺细胞从凋亡中解救出来，c)使一部分被解救的胸腺细胞成熟到abTCRhiCD69+的发育阶段。本项目的具体目标是：(1)为了确定MHC限制过程中巨噬细胞和TNCs之间的关系，我们将监测MHC限制过程中CFDA染色的巨噬细胞(从腹膜归巢到胸腺后)在胸腺和TNC内的位置。我们将使用Nomarski和荧光显微镜来确定巨噬细胞与TNC内胸腺细胞的时间关系。这些研究很重要，因为MHC限制的过程决定了在哺乳动物免疫系统中发挥作用的T细胞的特征。这种不同寻常的细胞排列方式与迄今报道的其他发育系统不同。未来对这种独特的细胞复合体的研究将对T细胞的发育产生前所未有的见解。(2)为了确定TNC/巨噬细胞相互作用在MHC限制过程中的作用，我们将使用H-Y转基因小鼠建立TNC和MHC限制过程之间的直接关联。在这些转基因动物中，每个发育中的胸腺细胞都会产生一个细胞表面abTCR，识别男性特有的H-Y抗原。绝大多数雄性转基因胸腺细胞通过负选择被删除，而在雌性转基因动物中检测到异常高的正选择比例。跨国公司直接参与了这些进程。雌性H-Y转基因小鼠有大的TNCs，其中含有5倍于细胞质的胸腺细胞。女性H-Y转基因患者中的跨国公司数量是男性的40倍。这与在雌性动物中发现的积极选择百分比的增加有很好的相关性。此外，雄性转基因动物的跨国公司很少。雄性TNCs很小，几乎一半的细胞质胸腺细胞是凋亡的，这可能是由于H-Y转基因小鼠中发生了高水平的负选择。这些数据意味着跨国公司直接参与了MHC限制的过程。利用CDFA染色技术，将从雄性H-Y转基因动物(表达驱动负选择的HY抗原)中获得巨噬细胞，并将其传递给雌性H-Y转基因小鼠。TNC复合体的数量和大小的变化将通过胸腺切片的免疫荧光染色以及胸腺的酶解离和显微镜分析来确定。注射胸腺半胱氨酸的发育中的动物的轮廓变化将使用FACS分析来确定。

Project Summary:Thymic nurse cells (TNCs) are epithelial cells with the ability to internalize immature thymocytes into specialized intra-cytoplasmic vacuoles. The function of this interaction during thymocyte development is the focus of this proposal. TNCs have been shown a) to bind and internalize immature abTCRlowCD69- double positive thymocytes, b) to rescue a subset of the internalized population from apoptosis, and c) to allow a subset of the rescued population to mature to the abTCRhiCD69+ stage of development.These data suggest that TNCs play a pivotal role in determining the fate of developing thymocytes. To specific aims of this project are:(1) To determine the relationship between macrophages and TNCs during MHC restriction, we will monitor the intra-thymic and intra-TNC location of CFDA-stained macrophages (after homing to the thymus from the peritoneum) during the process of MHC restriction. We will determine the temporal relationship of macrophages with intra-TNC thymocytes using Nomarski and fluorescence microscopy. These studies are important because the process of MHC restriction determines the profile of T cells that function within the mammalian immune system. This unusual arrangement of the cells is like no other developmental system reported to date. Future studies of this uniquecellular complex should yield unprecedented insights into T cell development.(2) To determine the role of the TNC/macrophage interaction during MHC restriction, we will use H-Y transgenic mice to establish a direct correlation between TNCs and the process of MHC restriction. Each developing thymocyte in these transgenic animals produces a cell surface abTCR that recognizes the male specific H-Y antigen. The vast majority of male transgenic thymocytes are deleted through negative selection while anabnormally high percentage of positive selection is detected in female transgenic animals. TNCs are directly involved in these processes. Female H-Y transgenic mice have large TNCs that contain 5 times as many cytoplasmic thymocytes. There are 40 times as many TNCs in female versus male H-Y transgenics. This correlates well with the increased percentage of positive selection found in female animals. Further, the male transgenic animals have very few TNCs. The male TNCs are small and almost half of their cytoplasmic thymocytes are apoptotic, which would result from the high level of negative selection reported to occur in the H-Y transgenic mouse. These data imply a direct involvement of TNCs in the process of MHC restriction. Using the CDFA-staining technique, macrophages will be obtained from male H-Y transgenics (which express the HY antigen that drives negative selection) and delivered into female H-Y transgenic mice. Changes in TNC complex numbers and sizes will be determined by immunofluorescence staining of thymic sections as well as by enzymatic dissociation of the thymus and microscopic analysis. Profile changes of developing thymocytesin injected animals will be determined using FACS analyses.