William W. Reiley

Regulation of T cell development by the deubiquitinating enzyme CYLD.

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.

ESAT-6-specific CD4 T cell responses to aerosol Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes

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-γ and TNF-α 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.

Regulation of the Deubiquitinating Enzyme CYLD by IκB Kinase Gamma-Dependent Phosphorylation

ABSTRACT 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 IκB kinase gamma (IKKγ) 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.

Negative Regulation of JNK Signaling by the Tumor Suppressor CYLD

CYLD is a tumor suppressor that is mutated in familial cylindromatosis, an autosomal dominant predisposition to multiple tumors of the skin appendages. Recent studies suggest that transfected CYLD has deubiquitinating enzyme activity and inhibits the activation of transcription factor NF-κB. However, the role of endogenous CYLD in regulating cell signaling remains poorly defined. Here we report a critical role for CYLD in negatively regulating the c-Jun NH2-terminal kinase (JNK). CYLD knockdown by RNA interference results in hyper-activation of JNK by diverse immune stimuli, including tumor necrosis factor-α, interleukin-1, lipopolysaccharide, and an agonistic anti-CD40 antibody. The JNK-inhibitory function of CYLD appears to be specific for immune receptors because the CYLD knockdown has no significant effect on stress-induced JNK activation. Consistently, CYLD negatively regulates the activation of MKK7, an upstream kinase known to mediate JNK activation by immune stimuli. We further demonstrate that CYLD also negatively regulates IκB kinase, although this function of CYLD is seen in a receptor-dependent manner. These findings identify the JNK signaling pathway as a major downstream target of CYLD and suggest a receptor-dependent role of CYLD in regulating the IκB kinase pathway.

Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection

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.

IκB Kinase Is an Essential Component of the Tpl2 Signaling Pathway

ABSTRACT IκB kinase (IKK), a key regulator of immune and inflammatory responses, is known as an effector kinase mediating activation of the transcription factor NF-κB. Whether IKK also participates in other signaling events is not known. Here we show that IKK serves as an essential component of a signaling pathway that involves activation of the Tpl2 kinase and its downstream targets, MEK1 and ERK. Inhibition of IKKβ in macrophages eliminates Tpl2 activation and ERK phosphorylation induced by lipopolysaccharide and tumor necrosis factor alpha. Using IKK-deficient murine fibroblasts, we further demonstrate that IKKβ, but not IKKα, is required for Tpl2 activation. Moreover, this novel function of IKKβ appears to involve phosphorylation and degradation of the Tpl2 inhibitor NF-κB1/p105. These findings suggest that IKKβ exerts its immune-regulatory functions by targeting different downstream signaling pathways.

An Atypical Tumor Necrosis Factor (TNF) Receptor-associated Factor-binding Motif of B Cell-activating Factor Belonging to the TNF Family (BAFF) Receptor Mediates Induction of the Noncanonical NF-κB Signaling Pathway

BAFF receptor (BAFFR) is a member of the TNF receptor (TNFR) superfamily that regulates the survival and maturation of B cells. BAFFR exerts its signaling function by inducing activation of NF-κB, although the underlying mechanism has not been well defined. By using a chimeric BAFFR, we show that BAFFR preferentially induces the noncanonical NF-κB signaling pathway. This specific function of BAFFR is mediated by a sequence motif, PVPAT, which is homologous to the TRAF-binding site (PVQET) present in CD40, a TNFR known to induce both the canonical and noncanonical NF-κB pathways. Mutation of this putative TRAF-binding motif within BAFFR abolishes its interaction with TRAF3 as well as its ability to induce noncanonical NF-κB. Interestingly, modification of the PVPAT sequence to the typical TRAF-binding sequence, PVQET, is sufficient to render the BAFFR capable of inducing strong canonical NF-κB signaling. Further, this functional acquisition of the modified BAFFR is associated with its stronger and more rapid association with TRAF3. These findings suggest that the PVPAT sequence of BAFFR not only functions as a key signaling motif of BAFFR but also determines its signaling specificity in the induction of the noncanonical NF-κB pathway.

In a Murine Tuberculosis Model, the Absence of Homeostatic Chemokines Delays Granuloma Formation and Protective Immunity

Mycobacterium tuberculosis infection (Mtb) results in the generation of protective cellular immunity and formation of granulomatous structures in the lung. CXCL13, CCL21, and CCL19 are constitutively expressed in the secondary lymphoid organs and play a dominant role in the homing of lymphocytes and dendritic cells. Although it is known that dendritic cell transport of Mtb from the lung to the draining lymph node is dependent on CCL19/CCL21, we show in this study that CCL19/CCL21 is also important for the accumulation of Ag-specific IFN-γ-producing T cells in the lung, development of the granuloma, and control of mycobacteria. Importantly, we also show that CXCL13 is not required for generation of IFN-γ responses, but is essential for the spatial arrangement of lymphocytes within granulomas, optimal activation of phagocytes, and subsequent control of mycobacterial growth. Furthermore, we show that these chemokines are also induced in the lung during the early immune responses following pulmonary Mtb infection. These results demonstrate that homeostatic chemokines perform distinct functions that cooperate to mediate effective expression of immunity against Mtb infection.

Cutting Edge: T-bet and IL-27R Are Critical for In Vivo IFN-γ Production by CD8 T Cells during Infection

CD8+ T cells are a major source of IFN-γ, a key effector cytokine in immune responses against many viruses and protozoa. Although the transcription factor T-bet is required for IFN-γ expression in CD4+ T cells, it is reportedly dispensable in CD8+ T cells, where the transcription factor Eomesodermin is thought to be sufficient. The diverse functions of IFN-γ are mediated through the IFN-γR and STAT1. In CD4+ T cells, STAT1 appears to be critical for the activation of T-bet and IFN-γ, suggesting an IFN-γ-dependent positive feedback loop. However, STAT1 can also be activated by other cytokines, including IL-27. In the present study we show that, in contrast to in vitro conditions and the prevailing paradigm, T-bet is critical for the in vivo IFN-γ production by CD8+ T cells upon infection of mice with diverse pathogens. Whereas IFN-γR signals are dispensable for the T-bet-dependent IFN-γ production, direct IL-27Rα signals are critical.

Early T‐cell responses in tuberculosis immunity

Summary: Tuberculosis (TB) has plagued mankind for millennia yet is classified as an emerging infectious disease, because its prevalence in the human population continues to increase. Immunity to TB depends critically on the generation of effective CD4+ T‐cell responses. Sterile immunity has not been achieved through vaccination, although early T‐cell responses are effective in controlling steady‐state infection in the lungs. Although such early T‐cell responses are clearly protective, the initiation of the Mycobacterium tuberculosis (Mtb) T‐cell response occurs much later than is the case following other aerogenic infections. This fact suggests that there is a critical period, before the activation of the T‐cell response, in which Mtb is able to establish infection. An understanding of the factors that regulate early T‐cell activation should, therefore, lead to better control of the disease. This review discusses recent work that has investigated the early development of T‐cell immunity following Mtb infection in the mouse.