4B, compare lanes 2, 3 and 4). On the other hand, the elevated basal activity of JNK in thymocytes from LckCre-Cyldflx9/flx9 mice was Liproxstatin-1 not reduced by the concomitant

inactivation of Ikk2 (Supporting Information Fig. 3). These findings indicate that the developmental defect of CyldΔ9 thymocytes is due to excessive activation of IKK2-dependent NF-κB activity. One of the striking observations in LckCre-Cyldflx9/flx9 mice was the dramatic reduction of CD4+ and CD8+ T cells in the periphery as assessed by their enumeration in mesenteric lymph nodes and spleen. LckCre-Cyld+-Ikk2flx/flx mice showed a 20% reduction in peripheral CD4 cells and a 50% reduction in peripheral CD8 cells in accordance with previous observations (Fig. 5A–D). Surprisingly, LckCre-Cyldflx9/flx9-Ikk2flx/flx mice showed a severe reduction in both CD4 and CD8 peripheral, which exceeded the defect seen in LckCre-Cyld+-Ikk2flx/flx peripheral T cells. Most of the remnant peripheral T cells in LckCre-Cyldflx9/flx9-Ikk2+/+ mice possessed CD44hiCD62Llo effector-like phenotype (Fig. 5E), which is consistent with lymphopenia-induced expansion as described in other lymphopenic states 24, 25. Interestingly, while the peripheral T cells isolated from LckCre-Cyld+-Ikk2flx/flx mice showed reduced expression of CD44 as previously reported

19, the peripheral T cells isolated from the LckCre-Cyldflx9/flx9-Ikk2flx/flx mice showed an intermediate phenotype since they have almost 50% more CD44hiCD62Llo T cells when compared with control mice buy PLX-4720 and 50% less CD44hiCD62Llo T cells when compared with LckCre-Cyldflx9/flx9-Ikk2fl+/+ (Fig. 5E). These findings are consistent with a function of CYLD in the establishment of physiological peripheral T-cell populations which is IKK2 independent. Oxaprozin The implication of the deubiquitinating activity of CYLD in the regulation of thymocyte positive selection in an NEMO-dependent manner

and the ambiguity that surrounds the role of NF-κB in this process prompted an investigation into the specific function of IKK2-dependent NF-κB activity in Cyld-dependent regulation of thymocyte development. For this purpose, a conditional gene targeting approach was employed which permitted the concomitant inactivation of CYLD’s activity and IKK2 from the early stages of thymocyte development by crossing LckCre-Cyldflx9/flx9 to Ikk2flx/flx mice. Thymocyte-specific ablation of IKK2 does not affect the development of thymocytes but results in a mild phenotype in the periphery, which is manifested by a small reduction of CD4+ peripheral T cells and a 50% reduction of CD8+ peripheral T cells (19 and Fig. 5). The observation that the concomitant inactivation of IKK2 and CYLD leads to normal thymocyte development establishes the improper regulation of NF-κB activity as the main cause of defective development of thymocytes with inactive CYLD.