e expression of orphan nuclear receptor Nur77 and the proapoptotic protein Bim, which are crucial mediators of negative selection, by using the same subsets as used in the above experiment. Interestingly, Immunoblot revealed that the expression of Nur77 and Bim was up-regulated in CkbTg AZD-2281 biological activity thymocytes and greater difference was found between CD69high DP thymocytes than between CD692/low DP thymocytes, indicating that more TCR signaled thymocytes were undergoing negative selection in transgenic mice. We then asked whether the enhanced negative selection of CkbTg thymocytes is mediated by the altered TCR signal strength, to address this issue, we stimulated thymocytes with plate-bound anti-TCR and anti-CD28 to mimic negative selection in vitro. Ckb Modulates TCR Signaling AND TCR transgenic thymocytes express the same MHC restricted TCR and undergo positive selection of CD8 and CD4 T cells respectively. Intriguingly, on Ckb transgenic background, positive selection of TCR transgenic thymocytes was greatly impaired as evidenced by the dramatically decreased number of both DP and SP thymocytes. Notably, the Ckb and TCR double transgenic DP thymocytes expressed CD5 and CD69 at slightly lower levels, which might reflect quick death of CD5high and CD69high cells, they had higher CD25 expression compared with their counterparts from single TCR Ckb Modulates TCR Signaling transgenic mice. And more, the expression of Nur77 and Bim was up-regulated in the Ckb and TCR double transgenic thymocytes. These data reveal that transgenic expression of Ckb promoted TCR-mediated apoptosis and thus contributed to increased premature thymocyte death in CkbTg thymus. Ectopic Ckb expression augments T cell activation In contrast to the population variation found in the thymus, the relative proportions and absolute numbers of peripheral CD4 and CD8 T cell were similar 12504917 between transgenic and littermate mice. Given the positive impact of Ckb on TCR signal transduction, we next examined whether Ckb participates in the regulation of T cell activation. As shown in 7 Ckb Modulates TCR Signaling Down-regulation of Ckb reduces T cell sensitivity to TCR stimulation Finally, we examined whether Ckb is necessary for T cells to response properly. To achieve this, T cells were stimulated with anti-TCR and anti-CD28 antibodies in the absence or presence of cyclocreatine, one of the most kinetically active analog of creatine. Both cCr and creatine are substrates for CK and have been shown to modulate rates of ATP production, cCr was widely Ckb Modulates TCR Signaling used as specific inhibitor to block the enzymatic activity of CK. Cell viability was not affected in the presence of cCr; however, the CD25 expression and proliferation of T cells 
were greatly impaired. In addition, by using the same approach described in Discussion It is well known that CKs play key roles in energy metabolism in high energy-consuming 20237073 tissues like brain and skeletal muscle through providing a temporal and spatial ATP buffer to maintain cellular energy homeostasis. While cellular ATP is used not only for fitting the fluctuating energy demands, but also for many other biological events, such as protein phosphorylation, cAMP generation and so on. Tissue specific and developmentally regulated CKs may integrate metabolic and apoptotic signals to mount other cellular response besides muscle contraction and neuron function. In this study, we have demonstrated an unexpected role of Ckb in regulating thymocyte
