Three Distinct Subsets of Thymic Epithelial Cells in Rats and Mice Defined by Novel Antibodies

et al. (2014) Three Distinct Subsets of Thymic Epithelial Cells in Rats and Mice Defined by Novel Antibodies. PLoS ONE 9(10): e109995. doi:10.1371/journal.pone.0109995 Three Distinct Subsets of Thymic Epithelial Cells in Rats and Mice Defined by Novel Antibodies Yasushi Sawanobori 0 Hiashi Ueta 0 Christine D. Dijkstra 0 Chae Gyu Park 0 Motoyasu Satou 0 Yusuke Kitazawa 0 Kenjiro Matsuno 0 Taishin Akiyama, University of Tokyo, Japan 0 1 Department of Anatomy (Macro), Dokkyo Medical University , Tochigi , Japan , 2 Molecular Cell Biology and Immunology, VU University Medical Center Amsterdam , Amsterdam , Netherlands , 3 Laboratory of Immunology, Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea, 4 Department of Biochemistry, Dokkyo Medical University , Tochigi , Japan Aim: Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells. Results: Rat TECs were subdivided on the basis of phenotype into three subsets; ED18+ED19+/2keratin 5 (K5)+K8+CD205+ class II MHC (MHCII)+ cortical TECs (cTECs), ED18+ED212K52K8+Ulex europaeus lectin 1 (UEA-1)+CD2052 medullary TECs (mTEC1s), and ED18+ED21+K5+K8dullUEA-12CD2052 medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18+ED19+/2K5+K8+ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII+CD103+ but negative for TEC markers, including CD205. Those in the medulla were MHCII+CD103+ and CD205+ cells were found only in the TEC-free area. Conclusion: Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Japan Society for the Promotion of Science KAKENHI Grant Number 26860138 (http://www.jsps.go.jp/english/index.html) and by an official donation from Mr. Jin Sasanuma (no conflict of interest). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The thymus, a lymphoid organ with a lobular structure, is important for the development of T cells. Specifically, thymocytes (T cell precursors) are subjected to both negative and positive selection in the thymus. Each lobule of the thymus has a cortex that contains densely packed CD4 and CD8 double-positive thymocytes and a medulla that contains sparser CD4 or CD8 single-positive thymocytes. Mainly in the cortex, thymocytes are subjected to positive selection, in which precursors with low reactivity to the MHC complex are deleted/eliminated. Subsequently, the thymocytes are subjected to negative selection in the medulla, a process that deletes/eliminates cells that have reactivity against self antigens [1]. Thymic epithelial cells (TECs) and thymic dendritic cells (tDCs) are considered to be responsible for the positive and negative selection of thymocytes. In mice and humans, cortical and medullary TECs (cTECs and mTECs) can be distinguished by means of expression of certain keratins and specific cell-surface molecules, or selective binding of Ulex europaeus lectin 1 (UEA-1). For example, CD205 [23], and Ly51 [46] are used to identify cTECs, and UEA-1 [78] and keratin 5 (K5) [7,910] are recognized as mTEC markers. Keratin 8 (K8) [7,910] is expressed in both the cortex and the medulla. Numerous studies have used these markers to describe the development or function of the thymus in mice, but few such studies have been conducted in other animals or in humans. Thus, the distribution and specificity of these markers in species other than mice remain largely unknown. In addition to cTECs and mTECs, multinuclear cell structures called thymic nurse cells (TNCs) are found in isolated cell suspensions derived from the thymus [1116]. For many years, it was unclear whether TNCs were a type of TEC that holds numerous thymocytes, or if they were structures that were somehow created during the cell-isolation procedure. Recently, TNCs were observed in vivo, and their role in T cell development was reported in mice [17]. However, TNCs in other species have not been widely studied. The thymus also contains many dendritic cells (DCs) [1]. The role of thymic DCs (tDCs) in T cell development is still unclear, but studies on presentation of mTEC-derived antigens [18] have shown that tDCs are essential for the generation of naturally occurring regulatory T cells [19], although there is still room for debate. tDC subpopulations and the distribution of tDCs have not been reported in animals other than mice. Moreover, although the C-type lectin CD205 has been exploited as a marker of DC subsets [20], it is also expressed on cTECs [21]. Accordingly, mapping of tDCs remains incomplete. The aim of this study was to create a precise map of rat TECs and compare it with that of mice using multicolor immunostaining. To characterize rat TECs, we used the newly generated monoclonal antibodies ED18, ED19, and ED21 and HD83 (raised against rat CD205), as well as antibodies that were reported previously to be reactive to rat antigens. Our results show that there are three TEC subsets in both rats and mice that have somewhat similar phenotypes in terms of reactivity with known and new antibodies. We also identified two distinct TEC-free areas that are unique to the rat thymus, and we discuss their possible roles in thymocyte development. Materials and Methods Animals Inbred Lewis (RT1l), DA (RT1a) and PVG/c RT7b (RT1c) rats (812 wks old) of both sexes and 8-wk-old male C57BL/6 mice were purchased from SLC Company. (Shizuoka, Japan). Animal handling and care protocols were approved by the Dokkyo Medical University Animal Experiments Committee, and were in accordance with Dokkyo Universitys Regulations for Animal Experiments and with Japanese Governmental Law No. 105. To obtain the thymus, all animals (...truncated)