**, miceA. such as conventional TCR T (cT) cells, NKT cells, regulatory T cells (Tregs), and TCR T (T) cells are generated in the thymus; some acquire effector function during intrathymic development (1, 2). A normal thymic environment is crucial to ensure that these T cell lineages develop properly and establish a repertoire of T cells that are functional but also self-tolerant (3). The thymus comprises many cell lineages of both hematopoietic and non-hematopoietic origin. Thymic (R)-3-Hydroxyisobutyric acid epithelial cells (TECs) are essential for thymopoiesis. Defects (R)-3-Hydroxyisobutyric acid in TECs can block thymus development, as athymus nude mice exemplify, because of SPRY2 a loss-of-function mutation in that results in the absence of T cells (4C6). TECs are defined into cortical (c) and medullary (m) TECs that reside in the cortex and medullar regions of the thymus, respectively. After early T cell progenitors seed in the thymus, they develop sequentially from the CD4?CD8? double negative (DN) to the CD4+CD8+ double positive (DP) and the CD4+CD8? and CD4?CD8+ single positive (SP) stages. SP thymocytes eventually migrate from the thymus to populate peripheral lymphoid organs (2). cTECs present self-peptide MHC complexes to the TCR expressed on DP thymocytes to ensure that these cells survive, a process also called positive selection (7C10). mTECs promiscuously express tissue-restricted antigens (TRAs) to trigger the death of (R)-3-Hydroxyisobutyric acid highly self-reactive CD4+ or CD8+ SP thymocytes that migrate from the cortex, a process called negative selection, and to induce Treg generation (7C9). Promiscuous expression of TRAs in mTECs, maturation of mTECs, and establishment of central tolerance depends on Aire (11), a deficiency of which impairs mTEC maturation and function, resulting in multi-organ autoimmune diseases (4C6). The mammalian or mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that integrates multiple signals to control cell growth, proliferation, survival, and metabolism. It signals through two complexes: mTORC1 and mTORC2. mTORC1 contains a crucial and unique adaptor molecule called Raptor and is sensitive to acute rapamycin inhibition, while mTORC2 contains Rictor and is resistant to acute rapamycin inhibition (12, 13). Many studies have demonstrated that mTOR is activated in both thymocytes and peripheral T cells following TCR engagement and intrinsically controls the development and/or function of cT-cells, mice (23) were obtained from the Jackson Laboratory and further backcrossed to C57Bl/6J background for at least four generations. mice (24) were gifts from Dr. Nancy Manley (University of Georgia). Mice were all housed under specific pathogen-free conditions and experiments described were carried out under the approval of the Institutional Animal Care and Use Committee of Duke University. TEC Preparation Thymic single-cell suspension as previously described with modifications (22, 25, 26). In brief, thymi were cut into small pieces (about 2mm), which were directly digested in FBS-free RPMI-1640 containing 10mg/ml collagenase type IV (Worthington) and 50mg/ml DNase I (Worthington) at 37 C with constant orbital shaking at 100C150 rpm for 10 minutes. After vortex, fragments were allowed to settle down; the supernatants were collected, filtered through a 70m nylon mesh, and kept on ice; settled remains were digested similarly twice and repeated a third time if necessary. After the last digestion, cells were combined and filtered. After centrifuging the pellets at 472g for 5 minutes, pellets were washed with 10ml RPMI-containing 10% FBS (RPMI-10) and resuspended in either cold FACS buffer (5Mm EDTA, 2%FBS in PBS) or RPMI-10. Antibodies and flow cytometry The FITC-conjugated TCR-V usage kit, including anti-TCR2 (clone B20.6), 3 (clone (R)-3-Hydroxyisobutyric acid KJ25), 4 (clone KT4), 5.1/5.2 (clone MR9-4), 6 (clone RR4-7), 7 (clone TR310), 8.1/8.2 (clone MR5-2), 8.3 (clone IB3.3), 9 (clone MR10-2), 10b (clone B21.5), 11 (clone RR3-15), 12 (clone MR11-1), 13 (clone MR12-3), 14 (clone (R)-3-Hydroxyisobutyric acid 14-2), and 17a (clone KJ23), was.