The Relationship Between Thymic Nurse Cells and Macrophages During MHC Restriction

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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.