Hot off the presses! Jun 24 Immunity

The Jun 24 issue of the Immunity is now up on Pubget (About Immunity): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• NOD So Fast: NLRX1 Puts the Brake on Inflammation
  - Immunity 34(6):821-822 (2011)
  In this issue of Immunity, Allen et al. (2011) and Xia et al. (2011) provide in vivo and biochemical evidence that NLRX1, a member of the nucleotide binding domain and leucine-rich-repeat-containing protein family, functions as a negative regulator of RIG-I and Toll-like receptors.
• Intracellular Trafficking of Perforin: To Thwart a Killer
  - Immunity 34(6):823-825 (2011)
  How do killer cells restrain perforin, the most potent toxin known to biologists, at its point of synthesis in the endoplasmic reticulum, where conditions are ideal for its activation? In this issue of Immunity, Brennan et al. (2011) study its trafficking, offering insights into protective mechanisms.
• Lymphocyte Signaling Converges on Microtubules
  - Immunity 34(6):825-827 (2011)
  Movement of immunoreceptor microclusters tunes lymphocyte activation, but the underlying mechanisms are incompletely understood. In this issue of Immunity, Schnyder et al. (2011) and Hashimoto-Tane et al. (2011) show that cytoplasmic dynein drives microcluster centralization along microtubules.
• A Fine Romance: T Follicular Helper Cells and B Cells
  - Immunity 34(6):827-829 (2011)
  T follicular helper (Tfh) cells help B cells to generate affinity-matured antibodies. Three papers in this issue of Immunity ([Choi et al., 2011], [Kerfoot et al., 2011] and [Kitano et al., 2011]) provide information about the reciprocal relationship between B cells and Tfh cells.
• Application of ChIP-Seq and Related Techniques to the Study of Immune Function
  - Immunity 34(6):830-842 (2011)
  Behaviors observed at the cellular level such as development and acquisition of effector functions by immune cells result from transcriptional changes. The biochemical mediators of transcription are sequence-specific transcription factors (TFs), chromatin modifying enzymes, and chromatin, the complex of DNA and histone proteins. Covalent modification of DNA and histones, also termed epigenetic modification, influences the accessibility of target sequences for transcription factors on chromatin and the expression of linked genes required for immune functions. Genome-wide techniques such as ChIP-Seq have described the entire "cistrome" of transcription factors involved in specific developmental steps of B and T cells and started to define specific immune responses in terms of the binding profiles of critical effectors and epigenetic modification patterns. Current data suggest that both promoters and enhancers are prepared for action at different stages of activation ! by epigenetic modification through distinct transcription factors in different cells.
• NLRX1 Negatively Regulates TLR-Induced NF-κB Signaling by Targeting TRAF6 and IKK
  - Immunity 34(6):843-853 (2011)
  Tight regulation of NF-κB signaling is essential for innate and adaptive immune responses, yet the molecular mechanisms responsible for its negative regulation are not completely understood. Here, we report that NLRX1, a NOD-like receptor family member, negatively regulates Toll-like receptor-mediated NF-κB activation. NLRX1 interacts with TRAF6 or IκB kinase (IKK) in an activation signal-dependent fashion. Upon LPS stimulation, NLRX1 is rapidly ubiquitinated, disassociates from TRAF6, and then binds to the IKK complex, resulting in inhibition of IKKα and IKKβ phosphorylation and NF-κB activation. Knockdown of NLRX1 in various cell types markedly enhances IKK phosphorylation and the production of NF-κB-responsive cytokines after LPS stimulation. We further provide in vivo evidence that NLRX1 knockdown in mice markedly enhances susceptibility to LPS-induced septic shock and plasma IL-6 level. Our study identifies a previously unrecognized role for NLRX1 in the ne! gative regulation of TLR-induced NF-κB activation by dynamically interacting with TRAF6 and the IKK complex.
• NLRX1 Protein Attenuates Inflammatory Responses to Infection by Interfering with the RIG-I-MAVS and TRAF6-NF-κB Signaling Pathways
  - Immunity 34(6):854-865 (2011)
  The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1−/− mice exhibited increased expression of antiviral signaling molecules IFN-β, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1−/− mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macr! ophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.
• DDX1, DDX21, and DHX36 Helicases Form a Complex with the Adaptor Molecule TRIF to Sense dsRNA in Dendritic Cells
  - Immunity 34(6):866-878 (2011)
  The innate immune system detects viral infection predominantly by sensing viral nucleic acids. We report the identification of a viral sensor, consisting of RNA helicases DDX1, DDX21, and DHX36, and the adaptor molecule TRIF, by isolation and sequencing of poly I:C-binding proteins in myeloid dendritic cells (mDCs). Knockdown of each helicase or TRIF by shRNA blocked the ability of mDCs to mount type I interferon (IFN) and cytokine responses to poly I:C, influenza A virus, and reovirus. Although DDX1 bound poly I:C via its Helicase A domain, DHX36 and DDX21 bound the TIR domain of TRIF via their HA2-DUF and PRK domains, respectively. This sensor was localized within the cytosol, independent of the endosomes. Thus, the DDX1-DDX21-DHX36 complex represents a dsRNA sensor that uses the TRIF pathway to activate type I IFN responses in the cytosol of mDCs.
• Protection from Endogenous Perforin: Glycans and the C Terminus Regulate Exocytic Trafficking in Cytotoxic Lymphocytes
  - Immunity 34(6):879-892 (2011)
  Cytotoxic lymphocyte-mediated apoptosis is dependent on the delivery of perforin to secretory granules and its ability to form calcium-dependent pores in the target cell after granule exocytosis. It is unclear how cytotoxic lymphocytes synthesize and store perforin without incurring damage or death. We discovered that the extreme C terminus of perforin was essential for rapid trafficking from the endoplasmic reticulum to the Golgi compartment. Substitution of the C-terminal tryptophan residue resulted in retention of perforin in the ER followed by calcium-dependent toxic activity that eliminated host cells. We also found that N-linked glycosylation of perforin was critical for transport from the Golgi to secretory granules. Overall, an intact C terminus and N-linked glycosylation provide accurate and efficient export of perforin from the endoplasmic reticulum to the secretory granules and are critical for cytotoxic lymphocyte survival.
• Phospholipase C-β3 Regulates FcRI-Mediated Mast Cell Activation by Recruiting the Protein Phosphatase SHP-1
  - Immunity 34(6):893-904 (2011)
  Mast cells are major effectors in high-affinity IgE receptor (FcRI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-β3 is crucial for FcRI-mediated mast cell activation. Plcb3−/− mice showed blunted FcRI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, FcRI stimulation of Plcb3−/− mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3−/− cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-β3 constitutively interacts with FcRI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-β3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396,! FcRI-mediated phenotypes were similar in Plcb3−/− and SHP-1 mutant mast cells. Thus, we have defined a PLC-β3- and SHP-1-mediated signaling pathway for FcRI-mediated cytokine production.
• B Cell Receptor-Mediated Antigen Gathering Requires Ubiquitin Ligase Cbl and Adaptors Grb2 and Dok-3 to Recruit Dynein to the Signaling Microcluster
  - Immunity 34(6):905-918 (2011)
  The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to T cells. Although the amount of antigen presentation to T cells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein. Furthermore, we visualize the localization and movement of these microclusters on the underlying microtubule network. Importantly, disruption of this network or diminished dynein recruitment in Grb2-, Dok-3-, or Cbl-deficient B cells, does not influence microcluster formation or actin-dependent spreading, but abrogates directed movement of microclusters and antigen accumulation. Thus we identify a surprising but pivotal role for dynein and the m! icrotubule network alongside Grb2, Dok-3, and Cbl in antigen gathering during B cell activation.
• Dynein-Driven Transport of T Cell Receptor Microclusters Regulates Immune Synapse Formation and T Cell Activation
  - Immunity 34(6):919-931 (2011)
  When T cells recognize a peptide-major histocompatibility complex on antigen-presenting cells (APCs), T cell receptor microclusters (TCR-MCs) are generated and move to the center of the T cell-APC interface to form the central supramolecular activation cluster (cSMAC). cSMAC formation depends on stimulation strength and regulates T cell activation. We demonstrate that the dynein motor complex colocalized and coimmunoprecipitated with the TCR complex and that TCR-MCs moved along microtubules (MTs) toward the center of the immune synapse in a dynein-dependent manner to form cSMAC. MTs are located in close proximity to the plasma membrane at the activation site. TCR-MC velocity and cSMAC formation were impaired by dynein or MT inhibitors or by ablation of dynein expression. T cells with impaired cSMAC formation exhibited enhanced cellular activation including protein phosphorylation and interleukin-2 production. These results indicate that cSMAC formation by TCR-MC moveme! nt depends on dynein and MTs, and the movement regulates T cell activation.
• ICOS Receptor Instructs T Follicular Helper Cell versus Effector Cell Differentiation via Induction of the Transcriptional Repressor Bcl6
  - Immunity 34(6):932-946 (2011)
  The nature of follicular helper CD4+ T (Tfh) cell differentiation remains controversial, including the minimal signals required for Tfh cell differentiation and the time at which Tfh cell differentiation occurs. Here we determine that Tfh cell development initiates immediately during dendritic cell (DC) priming in vivo. We demonstrate that inducible costimulator (ICOS) provides a critical early signal to induce the transcription factor Bcl6, and Bcl6 then induces CXCR5, the canonical feature of Tfh cells. Strikingly, a bifurcation between Tfh and effector Th cells was measurable by the second cell division of CD4+ T cells, at day 2 after an acute viral infection: IL2Rαint cells expressed Bcl6 and CXCR5 (Tfh cell program), whereas IL2Rαhi cells exhibited strong Blimp1 expression that repressed Bcl6 (effector Th cell program). Virtually complete polarization between Bcl6+ Tfh cells and Blimp1+ effector Th cell populations developed by 72 hr, even without B cells. Tfh c! ells were subsequently lost in the absence of B cells, demonstrating a B cell requirement for maintenance of Bcl6 and Tfh cell commitment via sequential ICOS signals.
• Germinal Center B Cell and T Follicular Helper Cell Development Initiates in the Interfollicular Zone
  - Immunity 34(6):947-960 (2011)
  We identify the interfollicular (IF) zone as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first 2 days postimmunization, antigen-specific T and B cells remained confined within the IF zone, formed long-lived interactions, and upregulated the transcriptional repressor Bcl6. T cells also acquired the Tfh cell markers CXCR5, PD-1, and GL7. Responding B and T cells migrated to the follicle interior directly from the IF zone, T cell immigration preceding B cells by 1 day. Notably, in the absence of cognate B cells, Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS, and GL7 were no longer expressed on follicular Bcl6hi T cells that nevertheless persisted in the follicle. Thus, Ag-specific B cells are required for the maintenance of the PD-1hiICOShiGL7hi Tfh cell phenotype within the follicle, but not for their initial differentiation in the IF zone.
• Bcl6 Protein Expression Shapes Pre-Germinal Center B Cell Dynamics and Follicular Helper T Cell Heterogeneity
  - Immunity 34(6):961-972 (2011)
  The transcription factor Bcl6 is essential for the development of germinal center (GC) B cells and follicular helper T (Tfh) cells. However, little is known about in vivo dynamics of Bcl6 protein expression during and after development of these cells. By using a Bcl6 reporter mouse strain, we found that antigen-engaged B cells upregulated Bcl6 before clustering in GCs. Two-photon microscopic analysis indicated that Bcl6 upregulation in pre-GC B cells contributed to sustaining their interactions with helper T cells and was required for their entry to GC clusters. Our data also suggested that Tfh cells gradually downmodulated Bcl6 protein over weeks after development. The Bcl6-low Tfh cells rapidly terminated proliferation and upregulated IL-7 receptor. These results clarify the role of Bcl6 in pre-GC B cell dynamics and highlight the modulation of Bcl6 expression in Tfh cells that persist in the late phase of the antibody response.
• Mast Cells Are Key Promoters of Contact Allergy that Mediate the Adjuvant Effects of Haptens
  - Immunity 34(6):973-984 (2011)
  A prominent feature of sensitizing environmental compounds that cause allergic contact dermatitis is the rapid induction of an innate inflammatory response that seems to provide danger signals for efficient T cell priming. We generated mouse models of mast cell deficiency, mast cell-specific gene inactivation, and mast cell reporter mice for intravital imaging and showed that these adjuvant effects of contact allergens are mediated by mast cells and histamine. Mast cell deficiency resulted in impaired emigration of skin DCs to the lymph node and contact hypersensitivity was dramatically reduced in the absence of mast cells. In addition, mast cell-specific inactivation of the Il10 gene did not reveal any role for mast cell-derived IL-10 in the regulation of contact allergy. Collectively, we demonstrate that mast cells are essential promoters of contact hypersensitivity, thereby highlighting their potential to promote immune responses to antigens entering via the skin.