Ivana Munitic

Regulation of Constitutive TCR Internalization by the ζ-Chain

The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the ζ-chain in this process. We found that cell surface TCRs lacking ζ were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length ζ led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4+CD8+ thymocytes from ζ-deficient mice, whereas TCR internalization on thymocytes from CD3-δ deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for ζ in the regulation of constitutive receptor internalization. Furthermore, chimeric ζ molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular ζ-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the ζ-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.

The CD8+ memory T-cell state of readiness is actively maintained and reversible

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.

Optineurin Insufficiency Impairs IRF3 but Not NF-κB Activation in Immune Cells

Optineurin is a widely expressed polyubiquitin-binding protein that has been implicated in regulating cell signaling via its NF-κB essential modulator–homologous C-terminal ubiquitin (Ub)-binding region. Its functions are controversial, with in vitro studies finding that optineurin suppressed TNF-mediated NF-κB activation and virus-induced activation of IFN regulatory factor 3 (IRF3), whereas bone marrow–derived macrophages (BMDMs) from mice carrying an optineurin Ub-binding point mutation had normal TLR-mediated NF-κB activation and diminished IRF3 activation. We have generated a mouse model in which the entire Ub-binding C-terminal region is deleted (Optn470T). Akin to C-terminal optineurin mutations found in patients with certain neurodegenerative diseases, Optn470T was expressed at substantially lower levels than the native protein, allowing assessment not only of the lack of Ub binding, but also of protein insufficiency. Embryonic lethality with incomplete penetrance was observed for 129 × C57BL/6 Optn470T/470T mice, but after further backcrossing to C57BL/6, offspring viability was restored. Moreover, the mice that survived were indistinguishable from wild type littermates and had normal immune cell distributions. Activation of NF-κB in Optn470T BMDM and BM-derived dendritic cells with TNF or via TLR4, T cells via the TCR, and B cells with LPS or anti-CD40 was normal. In contrast, optineurin and/or its Ub-binding function was necessary for optimal TANK binding kinase 1 and IRF3 activation, and both Optn470T BMDMs and bone marrow–derived dendritic cells had diminished IFN-β production upon LPS stimulation. Importantly, Optn470T mice produced less IFN-β upon LPS challenge. Therefore, endogenous optineurin is dispensable for NF-κB activation but necessary for optimal IRF3 activation in immune cells.

Suppression of Dendritic Cell-Derived IL-12 by Endogenous Glucocorticoids Is Protective in LPS-Induced Sepsis

Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GRCD11c-cre) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GRCD11c-cre mice, CD8+ DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c+ cells rescued LPS-induced loss of CD8+ DCs but not other DC subsets. Unlike wild-type animals, exposure of GRCD11c-cre mice to low-dose LPS did not induce CD8+ DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.

CD70 Deficiency Impairs Effector CD8 T Cell Generation and Viral Clearance but Is Dispensable for the Recall Response to Lymphocytic Choriomeningitis Virus

CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memory adaptive immune responses to a variety of pathogens. Thus far, all studies addressing the function of the CD27–CD70 axis have been performed in mice lacking CD27, in those overexpressing CD70, or in those in which these molecules were blocked or mimicked by Abs or recombinant soluble CD70. Because these methods have in some cases led to divergent results, we generated CD70-deficient mice to directly assess its role in vivo. We find that lack of CD70-mediated stimulation during primary responses to lymphocytic choriomeningitis virus lowered the magnitude of CD8 Ag-specific T cell response, resulting in impaired viral clearance, without affecting CD4 T cell responses. Unexpectedly, CD70–CD27 costimulation was not needed for memory CD8 T cell generation or the ability to mount a recall response to lymphocytic choriomeningitis virus. Adoptive transfers of wild-type memory T cells into CD70−/− or wild-type hosts also showed no need for CD70-mediated stimulation during the course of the recall response. Moreover, CD70 expression by CD8 T cells could not rescue endogenous CD70−/− cells from defective expansion, arguing against a role for CD70-mediated T:T help in this model. Therefore, CD70 appears to be an important factor in the initiation of a robust and effective primary response but dispensable for CD8 T cell memory responses.

T Cells in G1 Provide a Memory-Like Response to Secondary Stimulation

The commitment of naive T cells to proliferate is a function of the strength and duration of stimuli mediated by the TCR and coreceptors. Ranges of 2–20 h of stimulation have been reported as necessary in vitro. Whether T cells actually experience uninterrupted stimulation for such long periods under physiological conditions is controversial. Here we ask whether commitment to proliferate requires continuous stimulation, or can T cells integrate intermittent periods of stimulation. T cells were stimulated for two short-term (subthreshold) periods (5–7 h) either sequentially or separated by an interval of rest. Naive lymph node T cells were able to integrate interrupted stimulation, even when the duration of rest was as long as 2 days. Furthermore, when short-term-stimulated T cells were separated by density, three populations were observed: low density blasts, intermediate density G1 cells, and high density G0 cells. Low density cells progressed to division without further stimulation, whereas G0 and G1 cells remained undivided. However, after a period of rest, a second subthreshold stimulation caused the G1 but not the G0 fraction to quickly proceed through the cell cycle. We conclude that noncycling T cells in the G1 phase of the cell cycle remain in a state of readiness for prolonged periods of time, and may represent a population of memory-like effectors capable of responding rapidly to antigenic challenge.

CD70 Is Downregulated by Interaction with CD27

Engagement of the receptor CD27 by CD70 affects the magnitude and quality of T cell responses in a variety of infection models, and exaggerated signaling via this pathway results in enhanced immune responses and autoimmunity. One means by which signaling is regulated is tight control of cell surface CD70, which is expressed on dendritic cells (DCs), T cells, and B cells only upon activation. In this article, we show that a second level of regulation also is present. First, although undetectable on the cell surface by flow cytometry, immature DCs have a small pool of CD70 that continuously recycles from the plasma membrane. In addition, surface levels of CD70 on DCs and T cells were higher in mice deficient in CD27, or on DCs for which the interaction between CD70 and CD27 was precluded by blocking Abs. Binding of CD70 by its receptor resulted in downregulation of CD70 transcription and protein levels, suggesting that CD70-mediated “reverse signals” regulate its own levels. Therefore, the ability of CD70 to trigger costimulation is self-regulated when it binds its complementary receptor.

Optineurin in amyotrophic lateral sclerosis: Multifunctional adaptor protein at the crossroads of different neuroprotective mechanisms

When optineurin mutations showed up on the amyotrophic lateral sclerosis (ALS) landscape in 2010, they differed from most other ALS-causing genes. They seemed to act by loss- rather than gain-of-function, and it was unclear how a polyubiquitin-binding adaptor protein, which was proposed to regulate a variety of cellular functions including cell signaling and vesicle trafficking, could mediate neuroprotection. This review discusses the considerable progress that has been made since then. A large number of mutations in optineurin and optineurin-interacting proteins TANK-binding kinase (TBK1) and p62/SQSTM-1 have been found in the ALS patients, suggesting a common neuroprotective pathway. Moreover, functional studies of the ALS-causing optineurin mutations and the recently established optineurin ubiquitin-binding deficient and knockout mouse models helped identify three major mechanisms likely to mediate neuroprotection: regulation of autophagy, mitigation of (chronic) inflammatory signaling, and blockade of necroptosis. These three processes crosstalk, and require multiple levels of control, many of which can be mediated by optineurin. Based on the role of optineurin in multiple processes and the unexpected finding that targeted optineurin deletion in microglia and oligodendrocytes ultimately leads to the same phenotype of axonal degeneration despite different initial defects, we propose that the failure of the weakest link in the optineurin neuroprotective network is sufficient to disturb homeostasis and set-off the domino effect that could ultimately lead to neurodegeneration.

The TBK1-binding domain of optineurin promotes type I interferon responses.

Pathogen‐associated molecular pattern (PAMP) recognition leads to TANK‐binding kinase (TBK1) polyubiquitination and activation by transautophosphorylation, resulting in IFN‐β production. Here, we describe a mouse model of optineurin insufficiency (OptnΔ157) in which the TBK1‐interacting N‐terminus of optineurin was deleted. PAMP‐stimulated cells from OptnΔ157 mice had reduced TBK1 activity, no phosphorylation of optineurin Ser187, and mounted low IFN‐β responses. In contrast to pull‐down assays where the presence of N‐terminus was sufficient for TBK1 binding, both the N‐terminal and the ubiquitin‐binding regions of optineurin were needed for PAMP‐induced binding. This report establishes optineurin as a positive regulator TBK1 via a bipartite interaction between these molecules.

The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

BackgroundAfter viral infection and the stimulation of some pattern-recognition receptors, TANK-binding kinase I (TBK1) is activated by K63-linked polyubiquitination followed by trans-autophosphorylation. While the activated TBK1 induces type I interferon production by phosphorylating the transcription factor IRF3, the precise molecular mechanisms underlying TBK1 activation remain unclear.ResultsWe report here the localization of the ubiquitinated and phosphorylated active form of TBK1 to the Golgi apparatus after the stimulation of RIG-I-like receptors (RLRs) or Toll-like receptor-3 (TLR3), due to TBK1 K63-linked ubiquitination on lysine residues 30 and 401. The ubiquitin-binding protein optineurin (OPTN) recruits ubiquitinated TBK1 to the Golgi apparatus, leading to the formation of complexes in which TBK1 is activated by trans-autophosphorylation. Indeed, OPTN deficiency in various cell lines and primary cells impairs TBK1 targeting to the Golgi apparatus and its activation following RLR or TLR3 stimulation. Interestingly, the Bluetongue virus NS3 protein binds OPTN at the Golgi apparatus, neutralizing its activity and thereby decreasing TBK1 activation and downstream signaling.ConclusionsOur results highlight an unexpected role of the Golgi apparatus in innate immunity as a key subcellular gateway for TBK1 activation after RNA virus infection.