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Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2′-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.

Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptor–associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies. Mutations in the RNA-binding proteins Roquin-1 or Regnase-1 cause systemic autoimmunity. Heissmeyer and colleagues show that Roquin-1 and Regnase-1 physically interact and thereby regulate CD4+ and CD8+ T cell metabolism and functionality.

von Gamm et al. demonstrate that mice deficient for the RNase Regnase-3 (Zc3h12c) develop hypertrophic lymph nodes and a systemic interferon response. Regnase-3 is a functional RNase that acts in myeloid cells upon IRF signaling, suggesting it to be an evolutionary counterpart to Regnase-1., The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed Regnase-3–deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell–specific deletions of Regnase-3 to demonstrate that Regnase-3 acts specifically within myeloid cells. Regnase-3 deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as Zc3h12a (Regnase-1), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages., Graphical Abstract

BackgroundSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induced Coronavirus Disease 2019 (COVID-19) has posed a global threat to public health. The immune system is crucial in defending and eliminating the virus and infected cells. However, immune dysregulation may result in the rapid progression of COVID-19. Here, we evaluated the subsets, phenotypic and functional characteristics of natural killer (NK) and T cells in patients with COVID-19 and their associations with disease severity.MethodsDemographic and clinical data of COVID-19 patients enrolled in Wuhan Union Hospital from February 25 to February 27, 2020, were collected and analyzed. The phenotypic and functional characteristics of NK cells and T cells subsets in circulating blood and serum levels of cytokines were analyzed via flow cytometry. Then the LASSO logistic regression model was employed to predict risk factors for the severity of COVID-19.ResultsThe counts and percentages of NK cells, CD4(+) T cells, CD8(+) T cells and NKT cells were significantly reduced in patients with severe symptoms. The cytotoxic CD3(-)CD56(dim)CD16(+) cell population significantly decreased, while the CD3(-)CD56(dim)CD16(-) part significantly increased in severe COVID-19 patients. More importantly, elevated expression of regulatory molecules, such as CD244 and programmed death-1 (PD-1), on NK cells and T cells, as well as decreased serum cytotoxic effector molecules including perforin and granzyme A, were detected in patients with COVID-19. The serum IL-6, IL-10, and TNF-alpha were significantly increased in severe patients. Moreover, the CD3(-)CD56(dim)CD16(-) cells were screened out as an influential factor in severe cases by LASSO logistic regression.ConclusionsThe functional exhaustion and other subset alteration of NK and T cells may contribute to the progression and improve the prognosis of COVID-19. Surveillance of lymphocyte subsets may in the future enable early screening for signs of critical illness and understanding the pathogenesis of this disease.

Post-transcriptional gene regulation is complex, dynamic and ensures proper T cell function. The targeted transcripts can simultaneously respond to various factors as evident for Icos, an mRNA regulated by several RNA binding proteins (RBPs), including Roquin. However, fundamental information about the entire RBPome involved in post-transcriptional gene regulation in T cells is lacking. Here, we applied global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS) to human and mouse primary T cells and identified the core T cell RBPome. This defined 798 mouse and 801 human proteins as RBPs, unexpectedly containing signaling proteins like Stat1, Stat4 and Vav1. Based on the vicinity to Roquin-1 in proximity labeling experiments, we selected ∼50 RBPs for testing coregulation of Roquin targets. Induced expression of these candidate RBPs in wildtype and Roquin-deficient T cells unraveled several Roquin-independent contributions, but also revealed Celf1 as a new Roquin-1-dependent and target-specific coregulator of Icos.One sentence statementWe provide an atlas of RNA-binding proteins in human and mouse T helper cells as a resource for studying higher order post-transcriptional gene regulation.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17 similar to 92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

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Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation., Roquin-1 is a posttranscriptional regulator that controls the expression of many immune-related genes such as ICOS and TNFA. Here, the authors report a homozygous R688* loss of function mutation in Roquin-1 in a patient with syndromic uncontrolled hyperinflammation associated with immune cell activation and hypercytokinemia.

During persistent antigen stimulation, CD8(+) Tcells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of Tcell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished Tcell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) Tcells to protect against Listeria infection and attenuate tumor growth invivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) Tcells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) Tcells invivo. Our data show that NFAT promotes Tcell anergy and exhaustion by binding at sites that do not require cooperation with AP-1.

CD4+ T cell activation–induced Argonaute degradation and global miRNA downregulation promotes acquisition of helper T cell effector functions., Activation induces extensive changes in the gene expression program of naive CD4+ T cells, promoting their differentiation into helper T cells that coordinate immune responses. MicroRNAs (miRNAs) play a critical role in this process, and miRNA expression also changes dramatically during T cell differentiation. Quantitative analyses revealed that T cell activation induces global posttranscriptional miRNA down-regulation in vitro and in vivo. Argonaute (Ago) proteins, the core effector proteins of the miRNA-induced silencing complex (miRISC), were also posttranscriptionally down-regulated during T cell activation. Ago2 was inducibly ubiquitinated in activated T cells and its down-regulation was inhibited by the proteasome inhibitor MG132. Therefore, activation-induced miRNA down-regulation likely occurs at the level of miRISC turnover. Measurements of miRNA-processing intermediates uncovered an additional layer of activation-induced, miRNA-specific transcriptional regulation. Thus, transcriptional and posttranscriptional mechanisms cooperate to rapidly reprogram the miRNA repertoire in differentiating T cells. Altering Ago2 expression in T cells revealed that Ago proteins are limiting factors that determine miRNA abundance. Naive T cells with reduced Ago2 and miRNA expression differentiated more readily into cytokine-producing helper T cells, suggesting that activation-induced miRNA down-regulation promotes acquisition of helper T cell effector functions by relaxing the repression of genes that direct T cell differentiation.

Autoreactive T cells can infiltrate the CNS to cause disorders such as multiple sclerosis. In order to visualize T cell activation in the CNS, we introduced a truncated fluorescent derivative of nuclear factor of activated T cells (NFAT) as a real-time T cell activation indicator. In experimental autoimmune encephalomyelitis, a rat model of multiple sclerosis, we tracked T cells interacting with structures of the vascular blood-brain barrier (BBB). 2-photon imaging documented the cytoplasmic-nuclear translocation of fluorescent NFAT, indicative of calcium-dependent activation of the T cells in the perivascular space, but not within the vascular lumen. The activation was related to contacts with the local antigen-presenting phagocytes and was noted only in T cells with a high pathogenic potential. T cell activation implied the presentation of an autoantigen, as the weakly pathogenic T cells, which remained silent in the untreated hosts, were activated upon instillation of exogenous autoantigen. Activation did not cogently signal long-lasting arrest, as individual T cells were able to sequentially contact fresh APCs. We propose that the presentation of local autoantigen by BBB-associated APCs provides stimuli that guide autoimmune T cells to the CNS destination, enabling them to attack the target tissue.

The Roquin-1 protein binds to messenger RNAs (mRNAs) and regulates gene expression posttranscriptionally. A single point mutation in Roquin-1, but not gene ablation, increases follicular helper T (Tfh) cell numbers and causes lupus-like autoimmune disease in mice. In Tcells, we did not identify a unique role for the much lower expressed paralog Roquin-2. However, combined ablation of both genes induced accumulation of Tcells with an effector and follicular helper phenotype. We showed that Roquin-1 and Roquin-2 proteins redundantly repressed the mRNA of inducible costimulator (Icos) and identified the Ox40 costimulatory receptor as another shared mRNA target. Combined acute deletion increased Ox40 signaling, as well as Irf4 expression, and imposed Tfh differentiation on CD4+ Tcells. These data imply that both proteins maintain tolerance by preventing inappropriate Tcell activation and Tfh cell differentiation, and that Roquin-2 compensates in the absence of Roquin-1, but not in the presence of its mutated form.

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

Treatment for fibrosis represents a critical unmet need, because fibrosis is the leading cause of death in industrialized countries, and there is no effective therapy to counteract the fibrotic process. The development of fibrosis relates to the interplay between vessel injury, immune cell activation, and fibroblast stimulation, which can occur in various tissues. Immunotherapies have provided a breakthrough in the treatment of immune diseases. The glycoprotein OX40-OX40 ligand (OX40L) axis offers the advantage of a targeted approach to costimulatory signals with limited impact on the whole immune response. Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. OX40L is overexpressed in the fibrotic skin and serum of patients with SSc, particularly in patients with diffuse cutaneous forms. Soluble OX40L was identified as a promising serum biomarker to predict the worsening of lung and skin fibrosis, highlighting the role of this pathway in fibrosis. In vivo, OX40L blockade prevents inflammation-driven skin, lung, and vessel fibrosis and induces the regression of established dermal fibrosis in different complementary mouse models. OX40L exerts potent profibrotic effects by promoting the infiltration of inflammatory cells into lesional tissues and therefore the release of proinflammatory mediators, thereafter leading to fibroblast activation.

The Carma1–Bcl10–Malt1 (CBM) complex bridges T-cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF-κB pathway. NF-κB activation is triggered by PKCθ-dependent phosphorylation of Carma1 after TCR/CD28 co-stimulation. PKCθ-phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10–Malt1 complexes to the membrane. We have identified the serine–threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA-mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ-dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T-cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF-κB activation and IL-2 or IFN-γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A-mediated dephosphorylation of Carma1 as a critical step to limit T-cell activation and effector cytokine production.

Summary According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity., Graphical Abstract, Highlights • Two-photon imaging indicates that CTLs kill 2–16 virus-infected cells per day • CTLs form kinapses rather than stable synapses when killing virus-infected cells • Some CTL contacts trigger long-lasting calcium fluxes in virus-infected cells • CTLs can cooperate during killing of virus-infected cells, According to in vitro assays, T cells are thought to kill rapidly and efficiently. Using two-photon microscopy, Forster and colleagues have found that killing capacities of single cytotoxic T lymphocytes (CTLs) in vivo are heterogeneous and limited. Quantification of target-cell-death probabilities identified efficient cooperative killing when multiple CTLs attacked a virus-infected cell.

Post-transcriptional gene regulation by RNA-binding proteins controls mRNA half-life and efficiency of translation. Recently, the RNA-binding proteins Roquin and Regnase-1 have been shown to play pivotal roles in T lymphocytes by preventing inflammatory and autoimmune disease. These factors share an overlapping set of target mRNAs and are both regulated by proteolytic cleavage through the paracaspase MALT1. This review discusses the mouse models of inactivation or deregulation and how these trans-acting factors recognize target mRNAs. Based on different affinities of cis-elements in target mRNAs and regulation of the trans-acting factors, we propose the following model: Increasing TCR signal strength will gradually inactivate Roquin and Regnase-1 causing differential target mRNA derepression that specifies cell fate decisions and effector functions of T cells.

The NF-κB and Rel transcription factors regulate expression of genes involved in such diverse processes as inflammation, immune response, differentiation, proliferation, and apoptosis (23, 32). NF-κB plays a central role in innate immunity and is activated by signals initiated by tumor necrosis factor receptor, interleukin-1 receptor, and Toll-like receptors. NF-κB is also activated in T and B cells during the adaptive immune response (26, 37, 55). Activation of T lymphocytes requires interaction of the T-cell antigen receptor (TCR) with an antigen peptide presented by a major histocompatibility complex. While TCR engagement is essential for T-cell activation, productive T-cell activation requires additional costimulatory receptors, of which CD28 is the most prominent (2, 54). TCR/CD28 costimulation initiates a series of signal transduction events which modulate the activity of several nuclear transcription factors, including NF-κB, AP-1, and NF-AT, ultimately leading to the activation, differentiation, and proliferation of T lymphocytes (42). In both B and T cells, signal transduction from antigen receptors to NF-κB requires protein kinase C (PKC) isoforms. In B lymphocytes, PKCβ appears to be the major isoform that transduces signals from the B-cell receptor to NF-κB (53, 59). A great body of biochemical, pharmacological, and genetic evidence supports a crucial role of PKCθ for activation of NF-κB and interleukin-2 expression in response to TCR stimulation in T cells (29). Nevertheless, conflicting results have been obtained regarding the extent of PKCθ contribution to NF-κB activation in peripheral T cells (45, 60). The protein Bcl10, originally cloned from the chromosomal translocation t(1;14) (p22;q32) found in mucosa-associated lymphoid tissue (MALT) B-cell lymphomas (69, 73), is also essential for antigen receptor-induced NF-κB signaling in B and T cells (51). Lack of Bcl10 selectively blocks antigen receptor-mediated NF-κB activation without affecting AP-1 activation, suggesting that Bcl10 acts downstream of PKCs in an NF-κB-specific pathway. Recently, it was demonstrated that Bcl10 controls the development and function of mature B cells (70). Overexpression of Bcl10 activates NF-κB (33, 69), but the mechanism by which Bcl10 promotes NF-κB activation is not fully understood. Transfected Bcl10 is phosphorylated under certain conditions (20, 62), but the responsible protein kinase has not been identified, nor has the function of this phosphorylation been elucidated. Bcl10 contains an N-terminal caspase recruitment domain (CARD), which mediates self-oligomerization and is necessary and sufficient for NF-κB activation (33, 58). Bcl10 has been shown to associate with the paracaspase Malt1 (39), and target disruption in mice reveals that Malt1 operates downstream of the TCR, PKCθ, and Bcl10 in the IKK/NF-κB signaling cascade in T lymphocytes (50, 52). Furthermore, Bcl10 binds to proteins of the membrane-associated guanylate kinase (MAGUK) family, which include CARD9, CARD10 (Bimp1 or CARMA3), CARD11 (Bimp3 or CARMA1), and CARD14 (Bimp2 or CARMA2) via CARD-CARD interactions (9, 10, 20, 66). Recent experiments have demonstrated that the Bcl10 binding protein CARMA1 is bridging antigen receptor proximal signaling in both B and T cells to JNK activation and Bcl10-mediated NF-κB induction (19, 25, 30, 47, 65). CARMA1 probably acts through coupling Bcl10 and PKCθ and other potential regulators to the membrane. Congruently, PKCθ, CARMA1, Bcl10, and IKKs are recruited to the membrane and into lipid raft microdomains in response to TCR ligation (13, 19, 67). In these lipid rafts, IKKs are most probably activated either through direct phosphorylation by an unknown IKK kinase or through the induction of structural changes in the IKK complex, which is subsequently activated through enhanced autophosphorylation of IKKβ/α (61). Much genetic and biochemical evidence indicates that the RING finger-containing ubiquitin ligases c-Cbl and Cbl-b and the HECT (homology to the E6-AP carboxyl terminus) domain ubiquitin ligase Itch are critical negative regulators of T-cell activation (8, 18). Cbl-b-deficient mice show increased susceptibility to spontaneous or induced autoimmune disease (7, 11, 17), while mutation or lack of Itch results in aberrant activation of the immune response (16, 44). T cells from mice engineered to lack both c-Cbl and Cbl-b did not efficiently downmodulate surface TCR due to a defect in trafficking of the internalized TCR to the lysosomal compartment, resulting in sustained TCR signaling (41). Ubiquitination may target cellular proteins for destruction by the 26S proteasome (27) or direct trafficking of membrane-anchored proteins to lysosomal vesicles for degradation (31, 40). Even though it is evident that ubiquitin ligases play an important role in negatively regulating T-cell activation, considerably less is known about the signaling molecules that are substrates for degradation or the mechanism through which degradation occurs. Here we demonstrate that, in T cells, Bcl10 is posttranslationally modified by phosphorylation and subsequently degraded in response to phorbol myristate acetate (PMA) stimulation or CD3/CD28 coligation. Degradation of Bcl10 involves activation of PKCs and is accompanied by ubiquitination and trafficking to lysosomal vesicles. The CARD of Bcl10 serves as a recognition motif for degradation. We show that Bcl10 is a substrate for ubiquitination by the HECT-type ubiquitin ligases NEDD4 and Itch, and loss of Bcl10 mediated by these E3 enzymes interferes with NF-κB activation. Depletion of Bcl10 correlates with cessation of TCR-induced NF-κB and IKK activation without affecting signaling to JNK. These results define a new negative-feedback mechanism in which downregulation of Bcl10 through degradation might selectively terminate induction of the NF-κB signaling pathway in response to TCR activation.

Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (T(FH) cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the T(H)17 subset of helper T cells in the lungs. Roquin inhibited T(H)17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the T(H)17 cell-promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance T(H)17 differentiation.

The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.

Both activation and termination of transcription factor NF-κB signaling require ubiquitin modification of pathway components. The E3 ligase Itch teams up with the NF-κB inhibitor A20 to edit the composition of ubiquitin chains on the signaling adaptor RIP, thereby limiting inflammatory responses.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGFβ and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown. Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation. &nbsp

Post-transcriptional gene regulation by RNA-binding proteins is a fast and effective way to adapt gene expression and change cellular responses. These trans-acting factors have been involved in a number of cell fate decisions, and their mutation is often associated with the development of disease. The RNA-binding protein Roquin-1 has been found to be crucial in the maintenance of peripheral tolerance and the prevention of autoimmune disease. This review describes the molecular role of Roquin family proteins in the control of follicular T-helper cell differentiation. Here, we discuss the redundant regulation of Icos and Ox40 costimulatory receptor mRNAs by Roquin-1 and Roquin-2 proteins. A major focus is placed on the distinct activity of Roquin-1 or Roquin-2 proteins in the mouse models of conditional gene targeting. These recent data are then integrated into an interpretation of altered Roquin protein function in the sanroque mouse that expresses the Roquin-1 protein with just one amino acid substitution and, different from the Roquin-1-deficient mouse, develops lupus-like autoimmune disease.

Background The pentaspan protein CD133 (Prominin-1) is a predictive marker and part of the signature of tumour-initiating cells (TICs) for various cancer entities. Methods The correlation of CD133 expression with clinical parameters was assessed in primary samples of head and neck squamous cell carcinomas (n=98) and normal mucosas (n=24). Results A gradual and inversely proportional correlation between CD133 expression in primary tumours and decreased overall survival was observed, along with a positive correlation with the presence of lymph node metastases. Conclusions CD133 has the potential of being a novel clinically relevant prognostic marker for head and neck malignancies, which is possibly involved in regulation of tumourigenicity.

Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here, we show that mice deficient in Eri1, a conserved 3′-to- 5′ exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK-cell development and maturation. Eri1 -/- NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow (BM) and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse CMV (MCMV) infection. Ly49H + NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1 -/- T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1 -/- T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK-cell development and antiviral immunity. © 2012 by The American Society of Hematology.

SPECIFIC AIMSAspirin inhibits IKKβ in vitro; however, the in vivo relevance of the phenomenon is unclear. We have developed a model of severe angiotensin (Ang) II-induced vascular injury and now tested the hypothesis that chronic aspirin treatment of transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) protects from Ang II-induced end organ damage by inhibiting NF-κB activation in vivo.PRINCIPAL FINDINGSHigh-dose aspirin reduces mortality and renal damage independent of blood pressureRenal glomerulus of untreated 7-wk-old dTGR shows mesangial expansion and podocyte damage. Renal dTGR arterioles exhibit severe changes in wall structure, including proliferation of smooth muscle cells and deposition of dense vacuoles. In contrast, nontransgenic Sprague-Dawley (SD) rats showed no vascular or glomerular damage. Untreated dTGR show signs of glomerular sclerosis and vascular damage with mortality > 50% at wk 7. High-dose aspirin reduced mortality to < 10% whereas low-dose aspirin produ...

MicroRNAs (miRNAs) are small noncoding RNAs of an average length of 22 nucleotides, which repress translation of a large number of target mRNAs. The particular importance of this group of small RNAs arises from the ever growing evidence that they control many biological processes, such as differentiation, proliferation, and apoptosis and that deregulation of individual miRNAs frequently results in cancer. The expression of miRNAs is spatially and temporarily fine-tuned and expression levels can reach more than 50,000 copies of one miRNA within a single cell. It is well documented that the comparison of miRNA signatures of normal and diseased tissues results in a small number of differentially expressed miRNAs, which are consequently of high diagnostic value. However, measuring miRNA expression can easily produce false-positive results, due to the high sequence similarity of the miRNAs within families and because biologically inactive pre-miRNAs as well as contaminating bystander cells may falsify the signal. The application of a quantitative PCR-based method is described here to specifically and reliably detect miRNA expression levels from as little as 50 cells. Pure cell populations were either derived from fluorescence-activated cell sorting (FACS) or laser capture microdissection (LCM). Importantly, a combination of quantitative PCR and LCM can also be applied to measure miRNA expression of cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissues, thereby giving experimental access to archives with large numbers of routinely collected normal and diseased tissue samples.

Publisher Summary Gene targeting in mice—inactivating single genomic loci at a time—has elucidated a predominant role for T cells in various autoimmune diseases. Mice carrying such mutations develop fatal systemic or organ-specific damage, the symptoms of which correlate well with many detrimental human autoimmune diseases. Genetic linkage analysis of human patients points to a complex multigenic basis for many autoimmune syndromes, and the associated genes or genetic regions show considerable overlap with genes linked to autoimmunity in mice. Although the knowledge of general “autoimmune factors” is limited at present, some of the genes identified could have diagnostic and therapeutic potential. Experimental access to cells and tissues in mice has allowed investigators to study perturbations in processes such as thymic selection, T cell homeostasis, differentiation, or activation that are shown to result in autoimmunity, and critical molecules and pathways of signal transduction have been identified.

SummaryAntigen stimulation of immune cells activates the transcription factor NFAT, a key regulator of T cell activation and anergy. NFAT forms cooperative complexes with the AP-1 family of transcription factors and regulates T cell activation-associated genes. Here we show that regulatory T cell (Treg) function is mediated by an analogous cooperative complex of NFAT with the forkhead transcription factor FOXP3, a lineage specification factor for Tregs. The crystal structure of an NFAT:FOXP2:DNA complex reveals an extensive protein-protein interaction interface between NFAT and FOXP2. Structure-guided mutations of FOXP3, predicted to progressively disrupt its interaction with NFAT, interfere in a graded manner with the ability of FOXP3 to repress expression of the cytokine IL2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function in a murine model of autoimmune diabetes. Thus by switching transcriptional partners, NFAT converts the acute T cell activation program into the suppressor program of Tregs.

Follicular helper T (Tfh) cells are required for the establishment of T-dependent B cell memory and high affinity antibody-secreting cells. We have revealed herein opposing roles for signal transducer and activator of transcription 3 (STAT3) and type I interferon (IFN) signaling in the differentiation of Tfh cells following viral infection. STAT3-deficient CD4+ T cells had a profound defect in Tfh cell differentiation, accompanied by decreased germinal center (GC) B cells and antigen-specific antibody production during acute infection with lymphocytic choriomeningitis virus. STAT3-deficient Tfh cells had strikingly increased expression of a number of interferoninducible genes, in addition to enhanced T-bet synthesis, thus adopting a T helper-1 (Th1) cell-like effector phenotype. Conversely, IFNαβ receptor blockade restored Tfh and GC B cell phenotypes in mice containing STAT3-deficient CD4+ T cells. These data suggest mutually repressive roles for STAT3 and type I IFN signaling pathways in the differentiation of Tfh cells following viral infection.