Thymocyte-Specific Truncation of the Deubiquitinating Domain of CYLD Impairs Positive Selection in a NF-κB Essential Modulator-Dependent Manner

Abstract

The cylindromatosis tumor suppressor gene (Cyld) encodes a deubiquitinating enzyme (CYLD) with immunoregulatory function. In this study, we evaluated the role of Cyld in T cell ontogeny by generating a mouse (CyldΔ9) with a thymocyte-restricted Cyld mutation that causes a C-terminal truncation of the protein and reciprocates catalytically inactive human mutations. Mutant mice had dramatically reduced single positive thymocytes and a substantial loss of peripheral T cells. The analyses of polyclonal and TCR-restricted thymocyte populations possessing the mutation revealed a significant block in positive selection and an increased occurrence of apoptosis at the double-positive stage. Interestingly, in the context of MHC class I and II restricted TCR transgenes, lack of functional CYLD caused massive deletion of thymocytes that would have been positively selected, which is consistent with an impairment of positive selection. Biochemical analysis revealed that CyldΔ9 thymocytes exhibit abnormally elevated basal activity of NF-κB and JNK. Most importantly, inactivation of NF-κB essential modulator fully restored the NF-κB activity of CyldΔ9 thymocytes to physiologic levels and rescued their developmental and survival defect. This study identifies a fundamental role for functional CYLD in establishing the proper threshold of activation for thymocyte selection by a mechanism dependent on NF-κB essential modulator.

Ubiquitin enzymes in the regulation of immune responses

“…2007, Tsagaratou et al. 2010). Two DUBs, cylindromatosis (CYLD) and USP18 are responsible for hydrolyzing ubiquitin chains from TAK1, and thereby represent two crucial negative regulators of the CBM signaling axis.…”

Emerging Role of Ubiquitination in Antiviral RIG-I Signaling

“…By deubiquitinating essential signaling molecules (TRAF2, TRAF6, RIP1, and NEMO), CYLD limits the NF-B activation induced by TNFRI, CD40, or TLR stimulation (82). In T cells, CYLD is required for positive selection of developing double-positive thymocytes, resulting in fewer peripheral T cells in CYLD knockout mice (114,147). However, CYLD-deficient peripheral T cells are hyperresponsive to T cell receptor (TCR) stimulation due to spontaneous TAK1 activity (113).…”

Truncation of the Deubiquitinating Domain of CYLD in Myelomonocytic Cells Attenuates Inflammatory Responses

“…M-Cyld Δ9 mice were viable and normal in appearance. Since CYLD has been implicated in T cell development [7], [11] the hematopoietic development in M-Cyld Δ9 mice was evaluated. No gross hematopoietic defect was detected by flow cytometric analysis of the combined expression of Ly6C and CD31 surface markers both in steady state conditions and after thioglycollate-induced peritonitis that was adopted in order to evaluate the hematopoietic capacity of M-Cyld Δ9 mice under stress (Fig.…”