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VE-PTP controls blood vessel development by balancing Tie-2 activity

2009-06-08

TLR choreography

Maxmen, A. — 2009-06-08

Galvanizing allergies

Maxmen, A. — 2009-06-08

IL-17 propels infection

Maxmen, A. — 2009-06-08

Interleukins adapt to parasites

Maxmen, A. — 2009-06-08

Modeling MS

Maxmen, A. — 2009-06-08

Matthew Krummel: Visions enumerated

Maxmen, A. — 2009-06-08

Tracking the culprit: HIV-1 evolution and immune selection revealed by single-genome amplification

Brumme, Z. L., Walker, B. D. — 2009-06-08

Early control of HIV-1 infection is determined by a balance between the host immune response and the ability of the virus to escape this response. Studies using single-genome amplification now reveal new details about the kinetics and specificity of the CD8⁺ T cell response and the evolution of the virus during early HIV infection.

Inhibition of NK cell activity by IL-17 allows vaccinia virus to induce severe skin lesions in a mouse model of eczema vaccinatum

2009-06-08

Threats of bioterrorism have renewed efforts to better understand poxvirus pathogenesis and to develop a safer vaccine against smallpox. Individuals with atopic dermatitis are excluded from smallpox vaccination because of their propensity to develop eczema vaccinatum, a disseminated vaccinia virus (VACV) infection. To study the underlying mechanism of the vulnerability of atopic dermatitis patients to VACV infection, we developed a mouse model of eczema vaccinatum. Virus infection of eczematous skin induced severe primary erosive skin lesions, but not in the skin of healthy mice. Eczematous mice exhibited lower natural killer (NK) cell activity but similar cytotoxic T lymphocyte activity and humoral immune responses. The role of NK cells in controlling VACV-induced skin lesions was demonstrated by experiments depleting or transferring NK cells. The proinflammatory cytokine interleukin (IL)-17 reduced NK cell activity in mice with preexisting dermatitis. Given low NK cell activities and increased IL-17 expression in atopic dermatitis patients, these results can explain the increased susceptibility of atopic dermatitis patients to eczema vaccinatum.

Cholera toxin inhibits IL-12 production and CD8{alpha}+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

2009-06-08

Prior studies have demonstrated that cholera toxin (CT) and other cAMP-inducing factors inhibit interleukin (IL)-12 production from monocytes and dendritic cells (DCs). We show that CT inhibits Th1 responses in vivo in mice infected with Toxoplasma gondii. This correlated with low serum IL-12 levels and a selective reduction in the numbers of CD8⁺ conventional DCs (cDCs) in lymphoid organs. CT inhibited the function of interferon (IFN) regulatory factor (IRF) 8, a transcription factor known to positively regulate IL-12p35 and p40 gene expression, and the differentiation of CD8⁺ and plasmacytoid DCs (pDCs). Fluorescence recovery after photobleaching analysis showed that exposure to CT, forskolin, or dibutyryl (db) cAMP blocked LPS and IFN-–induced IRF8 binding to chromatin. Moreover, CT and dbcAMP inhibited the binding of IRF8 to the IFN-stimulated response element (ISRE)–like element in the mouse IL-12p40 promoter, likely by blocking the formation of ISRE-binding IRF1–IRF8 heterocomplexes. Furthermore, CT inhibited the differentiation of pDCs from fms-like tyrosine kinase 3 ligand–treated bone marrow cells in vitro. Therefore, because IRF8 is essential for IL-12 production and the differentiation of CD8⁺ cDCs and pDCs, these data suggest that CT and other Gs-protein agonists can affect IL-12 production and DC differentiation via a common mechanism involving IRF8.

Immunoglobulin switch {micro} sequence causes RNA polymerase II accumulation and reduces dA hypermutation

2009-06-08

Repetitive DNA sequences in the immunoglobulin switch µ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant µ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)–deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung^–/– and Aid^–/–Ung^–/– mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung^–/– B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase , decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase during hypermutation.

Neonatal tolerance revisited: a perinatal window for Aire control of autoimmunity

Guerau-de-Arellano, M., Martinic, M., Benoist, C., Mathis, D. — 2009-06-08

There has long been conceptual and experimental support for, but also challenges to, the notion that the initial period of the immune system's development is particularly important for the establishment of tolerance to self. The display of self-antigens by thymic epithelial cells is key to inducing tolerance in the T lymphocyte compartment, a process enhanced by the Aire transcription factor. Using a doxycycline-regulated transgene to target Aire expression to the thymic epithelium, complementing the Aire knockout in a temporally controlled manner, we find that Aire is essential in the perinatal period to prevent the multiorgan autoimmunity that is typical of Aire deficiency. Surprisingly, Aire could be shut down soon thereafter and remain off for long periods, with few deleterious consequences. The lymphopenic state present in neonates was a factor in this dichotomy because inducing lymphopenia during Aire turnoff in adults recreated the disease, which, conversely, could be ameliorated by supplementing neonates with adult lymphocytes. In short, Aire expression during the perinatal period is both necessary and sufficient to induce long-lasting tolerance and avoid autoimmunity. Aire-controlled mechanisms of central tolerance are largely dispensable in the adult, as a previously tolerized T cell pool can buffer newly generated autoreactive T cells that might emerge.

The first T cell response to transmitted/founder virus contributes to the control of acute viremia in HIV-1 infection

2009-06-08

Identification of the transmitted/founder virus makes possible, for the first time, a genome-wide analysis of host immune responses against the infecting HIV-1 proteome. A complete dissection was made of the primary HIV-1–specific T cell response induced in three acutely infected patients. Cellular assays, together with new algorithms which identify sites of positive selection in the virus genome, showed that primary HIV-1–specific T cells rapidly select escape mutations concurrent with falling virus load in acute infection. Kinetic analysis and mathematical modeling of virus immune escape showed that the contribution of CD8 T cell–mediated killing of productively infected cells was earlier and much greater than previously recognized and that it contributed to the initial decline of plasma virus in acute infection. After virus escape, these first T cell responses often rapidly waned, leaving or being succeeded by T cell responses to epitopes which escaped more slowly or were invariant. These latter responses are likely to be important in maintaining the already established virus set point. In addition to mutations selected by T cells, there were other selected regions that accrued mutations more gradually but were not associated with a T cell response. These included clusters of mutations in envelope that were targeted by NAbs, a few isolated sites that reverted to the consensus sequence, and bystander mutations in linkage with T cell–driven escape.

Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection

2009-06-08

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4⁺ T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12–20 mo, viruses exhibited concentrated mutations at 17–34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

Molecular explanation for the contradiction between systemic Th17 defect and localized bacterial infection in hyper-IgE syndrome

2009-06-08

Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by atopic manifestations and susceptibility to infections with extracellular pathogens, typically Staphylococcus aureus, which preferentially affect the skin and lung. Previous studies reported the defective differentiation of T helper 17 (Th17) cells in HIES patients caused by hypomorphic STAT3 mutations. However, the apparent contradiction between the systemic Th17 deficiency and the skin/lung-restricted susceptibility to staphylococcal infections remains puzzling. We present a possible molecular explanation for this enigmatic contradiction. HIES T cells showed impaired production of Th17 cytokines but normal production of classical proinflammatory cytokines including interleukin 1β. Normal human keratinocytes and bronchial epithelial cells were deeply dependent on the synergistic action of Th17 cytokines and classical proinflammatory cytokines for their production of antistaphylococcal factors, including neutrophil-recruiting chemokines and antimicrobial peptides. In contrast, other cell types were efficiently stimulated with the classical proinflammatory cytokines alone to produce such factors. Accordingly, keratinocytes and bronchial epithelial cells, unlike other cell types, failed to produce antistaphylococcal factors in response to HIES T cell–derived cytokines. These results appear to explain, at least in part, why HIES patients suffer from recurrent staphylococcal infections confined to the skin and lung in contrast to more systemic infections in neutrophil-deficient patients.

Spontaneous relapsing-remitting EAE in the SJL/J mouse: MOG-reactive transgenic T cells recruit endogenous MOG-specific B cells

2009-06-08

We describe new T cell receptor (TCR) transgenic mice (relapsing-remitting [RR] mice) carrying a TCR specific for myelin oligodendrocyte glycoprotein (MOG) peptide 92–106 in the context of I-A^s. Backcrossed to the SJL/J background, most RR mice spontaneously develop RR experimental autoimmune encephalomyelitis (EAE) with episodes often altering between different central nervous system tissues like the cerebellum, optic nerve, and spinal cord. Development of spontaneous EAE depends on the presence of an intact B cell compartment and on the expression of MOG autoantigen. There is no spontaneous EAE development in B cell–depleted mice or in transgenic mice lacking MOG. Transgenic T cells seem to expand MOG autoreactive B cells from the endogenous repertoire. The expanded autoreactive B cells produce autoantibodies binding to a conformational epitope on the native MOG protein while ignoring the T cell target peptide. The secreted autoantibodies are pathogenic, enhancing demyelinating EAE episodes. RR mice constitute the first spontaneous animal model for the most common form of multiple sclerosis (MS), RR MS.

Milk fat globule epidermal growth factor-8 blockade triggers tumor destruction through coordinated cell-autonomous and immune-mediated mechanisms

2009-06-08

Carcinogenesis reflects the dynamic interplay of transformed cells and normal host elements, but cancer treatments typically target each compartment separately. Within the tumor microenvironment, the secreted protein milk fat globule epidermal growth factor–8 (MFG-E8) stimulates disease progression through coordinated _vβ₃ integrin signaling in tumor and host cells. MFG-E8 enhances tumor cell survival, invasion, and angiogenesis, and contributes to local immune suppression. We show that systemic MFG-E8 blockade cooperates with cytotoxic chemotherapy, molecularly targeted therapy, and radiation therapy to induce destruction of various types of established mouse tumors. The combination treatments evoke extensive tumor cell apoptosis that is coupled to efficient dendritic cell cross-presentation of dying tumor cells. This linkage engenders potent antitumor effector T cells but inhibits FoxP3⁺ T reg cells, thereby achieving long-term protective immunity. Collectively, these findings suggest that systemic MFG-E8 blockade might intensify the antitumor activities of existing therapeutic regimens through coordinated cell-autonomous and immune-mediated mechanisms.

Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1

Kuang, D.-M., Zhao, Q., Peng, C., Xu, J., Zhang, J.-P., Wu, C., Zheng, L. — 2009-06-08

Macrophages (M) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of M to trigger transient activation of monocytes in peritumoral stroma. We showed that a fraction of monocytes/M in peritumoral stroma, but not in cancer nests, expresses surface PD-L1 (also termed B7-H1) molecules in tumors from patients with hepatocellular carcinoma (HCC). Monocytes activated by tumors strongly express PD-L1 proteins with kinetics similar to their activation status, and significant correlations were found between the levels of PD-L1⁺ and HLA-DR^high on tumor-infiltrating monocytes. Autocrine tumor necrosis factor and interleukin 10 released from activated monocytes stimulated monocyte expression of PD-L1. The PD-L1⁺ monocytes effectively suppressed tumor-specific T cell immunity and contributed to the growth of human tumors in vivo; the effect could be reversed by blocking PD-L1 on those monocytes. Moreover, we found that PD-L1 expression on tumor-infiltrating monocytes increased with disease progression, and the intensity of the protein was associated with high mortality and reduced survival in the HCC patients. Thus, expression of PD-L1 on activated monocytes/M may represent a novel mechanism that links the proinflammatory response to immune tolerance in the tumor milieu.

Unc93B1 biases Toll-like receptor responses to nucleic acid in dendritic cells toward DNA- but against RNA-sensing

2009-06-08

Toll-like receptors (TLRs) 3, 7, and 9 recognize microbial nucleic acids in endolysosomes and initiate innate and adaptive immune responses. TLR7/9 in dendritic cells (DCs) also respond to self-derived RNA/DNA, respectively, and drive autoantibody production. Remarkably, TLR7 and 9 appear to have mutually opposing, pathogenic or protective, impacts on lupus nephritis in MRL/lpr mice. Little is known, however, about the contrasting relationship between TLR7 and 9. We show that TLR7 and 9 are inversely linked by Unc93B1, a multiple membrane-spanning endoplasmic reticulum (ER) protein. Complementation cloning with a TLR7-unresponsive but TLR9-responsive cell line revealed that amino acid D34 in Unc93B1 repressed TLR7-mediated responses. D34A mutation rendered Unc93B1-deficient DCs hyperresponsive to TLR7 ligand but hyporesponsive to TLR9 ligand, with TLR3 responses unaltered. Unc93B1 associates with and delivers TLR7/9 from the ER to endolysosomes for ligand recognition. The D34A mutation up-regulates Unc93B1 association with endogenous TLR7 in DCs, whereas Unc93B1 association with TLR9 was down-regulated by the D34A mutation. Consistently, the D34A mutation up-regulated ligand-induced trafficking of TLR7 but down-regulated that of TLR9. Collectively, TLR response to nucleic acids in DCs is biased toward DNA-sensing by Unc93B1.

Zinc transporter Znt5/Slc30a5 is required for the mast cell-mediated delayed-type allergic reaction but not the immediate-type reaction

2009-06-08

Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency. However, it remains unknown how Zn homeostasis is regulated in mast cells and if Zn transporters are involved in allergic reactions. We show that Znt5/Slc30a5 is required for contact hypersensitivity and mast cell–mediated delayed-type allergic response but not for immediate passive cutaneous anaphylaxis. In mast cells from Znt5^–/– mice, Fc receptor I (FcRI)–induced cytokine production was diminished, but degranulation was intact. Znt5 was involved in FcRI-induced translocation of protein kinase C (PKC) to the plasma membrane and the nuclear translocation of nuclear factor B. In addition, the Zn finger–like motif of PKC was required for its plasma membrane translocation and binding to diacylglycerol. Thus, Znt5 is selectively required for the mast cell–mediated delayed-type allergic response, and it is a novel player in mast cell activation.

iNKT cell development is orchestrated by different branches of TGF-{beta} signaling

2009-06-08

Invariant natural killer T (iNKT) cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although various steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling carefully and specifically orchestrates several steps of iNKT cell development. In vivo, this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1 branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors, allowing TGF-β to fine tune the iNKT cell differentiation program.

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

2009-06-08

CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of -chain–associated protein of 70 kD, phospholipase-, and protein kinase C- into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca²⁺ mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-B were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigeen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion.

Parasites represent a major selective force for interleukin genes and shape the genetic predisposition to autoimmune conditions

2009-06-08

Many human genes have adapted to the constant threat of exposure to infectious agents; according to the "hygiene hypothesis," lack of exposure to parasites in modern settings results in immune imbalances, augmenting susceptibility to the development of autoimmune and allergic conditions. Here, by estimating the number of pathogen species/genera in a specific geographic location (pathogen richness) for 52 human populations and analyzing 91 interleukin (IL)/IL receptor genes (IL genes), we show that helminths have been a major selective force on a subset of these genes. A population genetics analysis revealed that five IL genes, including IL7R and IL18RAP, have been a target of balancing selection, a selection process that maintains genetic variability within a population. Previous identification of polymorphisms in some of these loci, and their association with autoimmune conditions, prompted us to investigate the relationship between adaptation and disease. By searching for variants in IL genes identified in genome-wide association studies, we verified that six risk alleles for inflammatory bowel (IBD) or celiac disease are significantly correlated with micropathogen richness. These data support the hygiene hypothesis for IBD and provide a large set of putative targets for susceptibility to helminth infections.

CD1c bypasses lysosomes to present a lipopeptide antigen with 12 amino acids

2009-06-08

The recent discovery of dideoxymycobactin (DDM) as a ligand for CD1a demonstrates how a nonribosomal lipopeptide antigen is presented to T cells. DDM contains an unusual acylation motif and a peptide sequence present only in mycobacteria, but its discovery raises the possibility that ribosomally produced viral or mammalian proteins that commonly undergo lipidation might also function as antigens. To test this, we measured T cell responses to synthetic acylpeptides that mimic lipoproteins produced by cells and viruses. CD1c presented an N-acyl glycine dodecamer peptide (lipo-12) to human T cells, and the response was specific for the acyl linkage as well as the peptide length and sequence. Thus, CD1c represents the second member of the CD1 family to present lipopeptides. lipo-12 was efficiently recognized when presented by intact cells, and unlike DDM, it was inactivated by proteases and augmented by protease inhibitors. Although lysosomes often promote antigen presentation by CD1, rerouting CD1c to lysosomes by mutating CD1 tail sequences caused reduction in lipo-12 presentation. Thus, although certain antigens require antigen processing in lysosomes, others are destroyed there, providing a hypothesis for the evolutionary conservation of large CD1 families containing isoforms that survey early endosomal pathways.

Priming of protective T cell responses against virus-induced tumors in mice with human immune system components

2009-06-08

Many pathogens that cause human disease infect only humans. To identify the mechanisms of immune protection against these pathogens and also to evaluate promising vaccine candidates, a small animal model would be desirable. We demonstrate that primary T cell responses in mice with reconstituted human immune system components control infection with the oncogenic and persistent Epstein-Barr virus (EBV). These cytotoxic and interferon-–producing T cell responses were human leukocyte antigen (HLA) restricted and specific for EBV-derived peptides. In HLA-A2 transgenic animals and similar to human EBV carriers, T cell responses against lytic EBV antigens dominated over recognition of latent EBV antigens. T cell depletion resulted in elevated viral loads and emergence of EBV-associated lymphoproliferative disease. Both loss of CD4⁺ and CD8⁺ T cells abolished immune control. Therefore, this mouse model recapitulates features of symptomatic primary EBV infection and generates T cell–mediated immune control that resists oncogenic transformation.

Deficiency of the DNA repair enzyme ATM in rheumatoid arthritis

Shao, L., Fujii, H., Colmegna, I., Oishi, H., Goronzy, J. J., Weyand, C. M. — 2009-06-08

In rheumatoid arthritis (RA), dysfunctional T cells sustain chronic inflammatory immune responses in the synovium. Even unprimed T cells are under excessive replication pressure, suggesting an intrinsic defect in T cell regeneration. In naive CD4 CD45RA⁺ T cells from RA patients, DNA damage load and apoptosis rates were markedly higher than in controls; repair of radiation-induced DNA breaks was blunted and delayed. DNA damage was highest in newly diagnosed untreated patients. RA T cells failed to produce sufficient transcripts and protein of the DNA repair kinase ataxia telangiectasia (AT) mutated (ATM). NBS1, RAD50, MRE11, and p53 were also repressed. ATM knockdown mimicked the biological effects characteristic for RA T cells. Conversely, ATM overexpression reconstituted DNA repair capabilities, response patterns to genotoxic stress, and production of MRE11 complex components and rescued RA T cells from apoptotic death. In conclusion, ATM deficiency in RA disrupts DNA repair and renders T cells sensitive to apoptosis. Apoptotic attrition of naive T cells imposes lymphopenia-induced proliferation, leading to premature immunosenescence and an autoimmune-biased T cell repertoire. Restoration of DNA repair mechanisms emerges as an important therapeutic target in RA.