T cell receptor signaling is essential for the generation and maturation of T lymphocyte precursors. Here we identify the deubiquitinating enzyme CYLD as a positive regulator of proximal T cell receptor signaling in thymocytes. CYLD physically interacted with active Lck and promoted recruitment of active Lck to its substrate, Zap70. CYLD also removed both Lys 48- and Lys 63-linked polyubiquitin chains from Lck. Because of a cell-autonomous defect in T cell development, CYLD-deficient mice had substantially fewer mature CD4(+) and CD8(+) single-positive thymocytes and peripheral T cells.

The deubiquitinating enzyme CYLD has recently been implicated in the regulation of signal transduction, but its physiological function and mechanism of action are still elusive. In this study, we show that CYLD plays a pivotal role in regulating T cell activation and homeostasis. T cells derived from Cyld knockout mice display a hyperresponsive phenotype and mediate the spontaneous development of intestinal inflammation. Interestingly, CYLD targets a ubiquitin-dependent kinase, transforming growth factor–β-activated kinase 1 (Tak1), and inhibits its ubiquitination and autoactivation. Cyld-deficient T cells exhibit constitutively active Tak1 and its downstream kinases c-Jun N-terminal kinase and IκB kinase β. These results emphasize a critical role for CYLD in preventing spontaneous activation of the Tak1 axis of T cell signaling and, thereby, maintaining normal T cell function.

CD4 ؉ T cell responses to aerosol Mycobacterium tuberculosis (Mtb) infection are characterized by the relatively delayed appearance of effector T cells in the lungs. This delay in the adaptive response is likely critical in allowing the bacteria to establish persistent infection. Because of limitations associated with the detection of low frequencies of naïve T cells, it had not been possible to precisely determine when and where naïve antigen-specific T cells are first activated. We have addressed this problem by using early secreted antigenic target 6 (ESAT-6)-specific transgenic CD4 T cells to monitor early T cell activation in vivo. By using an adoptive transfer approach, we directly show that T cell priming to ESAT-6 occurs only after 10 days of infection, is initially restricted to the mediastinal lymph nodes, and does not involve other lymph nodes or the lungs. Primed CD4 T cells rapidly differentiated into proliferating effector cells and ultimately acquired the ability to produce IFN-␥ and TNF-␣ ex vivo. Initiation of T cell priming was enhanced by two full days depending on the magnitude of the challenge inoculum, which suggests that antigen availability is a factor limiting the early CD4 T cell response. These data define a key period in the adaptive immune response to Mtb infection.priming ͉ transgenic mice

The immune response elicited after Mycobacterium tuberculosis (Mtb) infection is critically dependent on CD4 T cells during both acute and chronic infection. How CD4 T-cell responses are maintained throughout infection is not well understood, and evidence from other infection models has suggested that, under conditions of chronic antigen stimulation, T cells can undergo replicative exhaustion. These findings led us to determine whether subpopulations of CD4 T cells existed that displayed markers of terminal differentiation or exhaustion during murine Mtb infection. Analysis of antigen-specific effector CD4 T cells revealed that programmed death-1 (PD-1) and the killer cell lectin-like receptor G1 (KLRG1) delineated subpopulations of T cells. PD-1-expressing CD4 T cells were highly proliferative, whereas KLRG1 cells exhibited a short lifespan and secreted the cytokines IFNγ and TNFα. Adoptive transfer studies demonstrated that proliferating PD-1-positive CD4 T cells differentiated into cytokine-secreting KLRG1-positive T cells, but not vice versa. Thus, proliferating PD-1-positive cells are not exhausted, but appear to be central to maintaining antigen-specific effector T cells during chronic Mtb infection. Our findings suggest that antigen-specific T-cell responses are maintained during chronic mycobacterial infection through the continual production of terminal effector cells from a proliferating precursor population.T uberculosis presents a challenging worldwide public heath problem. Infection with Mycobacterium tuberculosis (Mtb) elicits humoral and cellular immune responses that normally control bacterial burden. However, bacteria are seldom, if ever, eradicated, and control of the infection requires continual effector T-cell responses. Consequently, either the depletion or suppression of T-cell responses results in disease reactivation (1). In the mouse model, Mtb causes chronic infection and control of infection is maintained by T cells essentially indefinitely (2).Although a sustained T-cell response against Mtb infection is necessary, it is not understood how effector T cells are maintained. We have previously demonstrated that CD4 T-cell responses are associated with extensive proliferation throughout Mtb infection (3), suggesting that proliferation is a major mechanism required for the maintenance of T-cell responses. However, it is not clear how T-cell proliferation can be maintained during chronic infection, especially because effector T cells have been described to have a limited capacity for self-renewal (4).Although the numbers of antigen-specific T cells are relatively stable during chronic Mtb infection, CD4 T cells proliferate extensively, suggesting that a high turnover of effector CD4 T cells occurs. This high level of turnover suggests that effector T cells become exhausted or terminally differentiated, and that the maintenance of the T-cell response depends on the continual replacement of effector T cells. The expression of several cellsurface receptors has been correlated with functional e...

Tumor suppressor CYLD is a deubiquitinating enzyme (DUB) that inhibits the ubiquitination of key signaling molecules, including tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). However, how the function of CYLD is regulated remains unknown. Here we provide evidence that inducible phosphorylation of CYLD is an important mechanism of its regulation. Under normal conditions, CYLD dominantly suppresses the ubiquitination of TRAF2. In response to cellular stimuli, CYLD undergoes rapid and transient phosphorylation, which is required for signal-induced TRAF2 ubiquitination and activation of downstream signaling events. Interestingly, the CYLD phosphorylation requires IB kinase gamma (IKK␥) and can be induced by IKK catalytic subunits. These findings suggest that CYLD serves as a novel target of IKK and that the site-specific phosphorylation of CYLD regulates its signaling function.

Spermatogenesis involves an early wave of germ cell apoptosis, which is required for maintaining the balance between germ cells and supporting Sertoli cells. However, the signaling mechanism regulating this apoptotic event is poorly defined. Here we show that genetic deficiency of Cyld, a recently identified deubiquitinating enzyme, attenuates the early wave of germ cell apoptosis and causes impaired spermatogenesis in mice. Interestingly, the loss of CYLD in testicular cells leads to activation of the transcription factor NF-kappaB and aberrant expression of antiapoptotic genes. We further show that CYLD negatively regulates a ubiquitin-dependent NF-kappaB activator, RIP1. CYLD binds to RIP1 and inhibits its ubiquitination and signaling function. These findings establish CYLD as a pivotal deubiquitinating enzyme (DUB) that regulates germ cell apoptosis and spermatogenesis and suggest an essential role for CYLD in controlling the RIP1/NF-kappaB signaling axis in testis.

Osteoclastogenesis is a tightly regulated biological process, and deregulation can lead to severe bone disorders such as osteoporosis. The regulation of osteoclastic signaling is incompletely understood, but ubiquitination of TNF receptor-associated factor 6 (TRAF6) has recently been shown to be important in mediating this process. We therefore investigated the role of the recently identified deubiquitinating enzyme CYLD in osteoclastogenesis and found that mice with a genetic deficiency of CYLD had aberrant osteoclast differentiation and developed severe osteoporosis. Cultured osteoclast precursors derived from CYLD-deficient mice were hyperresponsive to RANKL-induced differentiation and produced more and larger osteoclasts than did controls upon stimulation. We assessed the expression pattern of CYLD and found that it was drastically upregulated during RANKL-induced differentiation of preosteoclasts. Furthermore, CYLD negatively regulated RANK signaling by inhibiting TRAF6 ubiquitination and activation of downstream signaling events. Interestingly, we found that CYLD interacted physically with the signaling adaptor p62 and thereby was recruited to TRAF6. These findings establish CYLD as a crucial negative regulator of osteoclastogenesis and suggest its involvement in the p62/TRAF6 signaling axis.

CD8+ T cells lacking CXCR3 and CCR5 expression have impaired contraction and generate an increased number of memory cells after virus infection.