Journal of Experimental Medicine recent issues

Acetylation and activation of STAT3 mediated by nuclear translocation of CD44

Lee, J.-L., Wang, M.-J., Chen, J.-Y. — 2009-07-06

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

2009-07-06

The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy

Patel, P. H., Costa-Mattioli, M., Schulze, K. L., Bellen, H. J. — 2009-07-06

Accuracy and precision in quantitative fluorescence microscopy

Waters, J. C. — 2009-07-06

Pesticide-induced proliferation

Maxmen, A. — 2009-07-06

Spying on antigen hand-offs

Maxmen, A. — 2009-07-06

IL-17's tumor team

Maxmen, A. — 2009-07-06

Healthy hearts lay off LOX

Maxmen, A. — 2009-07-06

Mend that gut, STAT!

Maxmen, A. — 2009-07-06

HIV, prayer, and vintage cars: Danny Douek talks shop

Maxmen, A. — 2009-07-06

IL-17 can promote tumor growth through an IL-6-Stat3 signaling pathway

Wang, L., Yi, T., Kortylewski, M., Pardoll, D. M., Zeng, D., Yu, H. — 2009-07-06

Although the Th17 subset and its signature cytokine, interleukin (IL)-17A (IL-17), are implicated in certain autoimmune diseases, their role in cancer remains to be further explored. IL-17 has been shown to be elevated in several types of cancer, but how it might contribute to tumor growth is still unclear. We show that growth of B16 melanoma and MB49 bladder carcinoma is reduced in IL-17^–/– mice but drastically accelerated in IFN-^–/– mice, contributed to by elevated intratumoral IL-17, indicating a role of IL-17 in promoting tumor growth. Adoptive transfer studies and analysis of the tumor microenvironment suggest that CD4⁺ T cells are the predominant source of IL-17. Enhancement of tumor growth by IL-17 involves direct effects on tumor cells and tumor-associated stromal cells, which bear IL-17 receptors. IL-17 induces IL-6 production, which in turn activates oncogenic signal transducer and activator of transcription (Stat) 3, up-regulating prosurvival and proangiogenic genes. The Th17 response can thus promote tumor growth, in part via an IL-6–Stat3 pathway.

STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing

2009-07-06

Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c⁺ cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3^IEC-KO mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22–dependent mucosal wound healing.

Agricultural pesticide exposure and the molecular connection to lymphomagenesis

2009-07-06

The t(14;18) translocation constitutes the initiating event of a causative cascade leading to follicular lymphoma (FL). t(14;18) translocations are present in blood from healthy individuals, but there is a trend of increased prevalence in farmers exposed to pesticides, a group recently associated with higher risk of t(14;18)⁺ non-Hodgkin's lymphoma development. A direct connection between agricultural pesticide use, t(14;18) in blood, and malignant progression, however, has not yet been demonstrated. We followed t(14;18) clonal evolution over 9 yr in a cohort of farmers exposed to pesticides. We show that exposed individuals bear particularly high t(14;18) frequencies in blood because of a dramatic clonal expansion of activated t(14;18)⁺ B cells. We further demonstrate that such t(14;18)⁺ clones recapitulate the hallmark features of developmentally blocked FL cells, with some displaying aberrant activation-induced cytidine deaminase activity linked to malignant progression. Collectively, our data establish that expanded t(14;18)⁺ clones constitute bona fide precursors at various stages of FL development, and provide a molecular connection between agricultural pesticide exposure, t(14;18) frequency in blood, and clonal progression.

Visualizing B cell capture of cognate antigen from follicular dendritic cells

Suzuki, K., Grigorova, I., Phan, T. G., Kelly, L. M., Cyster, J. G. — 2009-07-06

The prominent display of opsonized antigen by follicular dendritic cells (FDCs) has long favored the view that they serve as antigen-presenting cells for B cells. Surprisingly, however, although B cell capture of antigen from macrophages and dendritic cells has been visualized, acquisition from FDCs has not been directly observed. Using two-photon microscopy, we visualized B cell capture of cognate antigen from FDCs. B cell CXCR5 expression was required, and encounter with FDC-associated antigen could be detected for >1 wk after immunization. B cell–FDC contact times were often brief but occasionally persisted for >30 min, and B cells sometimes acquired antigen together with FDC surface proteins. These observations establish that FDCs can serve as sites of B cell antigen capture, with their prolonged display time ensuring that even rare B cells have the chance of antigen encounter, and they suggest possible information transfer from antigen-presenting cell to B cell.

The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans

2009-07-06

Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity.

The thymic medulla: a unique microenvironment for intercellular self-antigen transfer

Koble, C., Kyewski, B. — 2009-07-06

Central tolerance is shaped by the array of self-antigens expressed and presented by various types of thymic antigen-presenting cells (APCs). Depending on the overall signal quality and/or quantity delivered in these interactions, self-reactive thymocytes either apoptose or commit to the T regulatory cell lineage. The cellular and molecular complexity underlying these events has only recently been appreciated. We analyzed the ex vivo presentation of ubiquitous or tissue-restricted self-antigens by medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs), the two major APC types present in the medulla. We found that the ubiquitously expressed nuclear neo–self-antigen ovalbumin (OVA) was efficiently presented via major histocompatibility complex class II by mTECs and thymic DCs. However, presentation by DCs was highly dependent on antigen expression by TECs, and hemopoietic cells did not substitute for this antigen source. Accordingly, efficient deletion of OVA-specific T cells correlated with OVA expression by TECs. Notably, OVA was only presented by thymic but not peripheral DCs. We further demonstrate that thymic DCs are constitutively provided in situ with cytosolic as well as membrane-bound mTEC-derived proteins. The subset of DCs displaying transferred proteins was enriched in activated DCs, with these cells being most efficient in presenting TEC-derived antigens. These data provide evidence for a unique, constitutive, and unidirectional transfer of self-antigens within the thymic microenvironment, thus broadening the cellular base for tolerance induction toward promiscuously expressed tissue antigens.

Cytokine-dependent regulation of NADPH oxidase activity and the consequences for activated T cell homeostasis

Purushothaman, D., Sarin, A. — 2009-07-06

Cellular dependence on growth factors for survival is developmentally programmed and continues in adult metazoans. Antigen-activated T cell apoptosis in the waning phase of the immune response is thought to be triggered by depletion of cytokines from the microenvironment. T cell apoptosis resulting from cytokine deprivation is mediated by reactive oxygen species (ROS), but their source and position in the apoptotic cascade is poorly understood. RNA interference approaches implicated the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neglect-induced apoptosis in T cells. Using mice deficient for the catalytic subunit gp91^phox to characterize the molecular link to activated T cell apoptosis, we show that gp91^phox-deficient T (T^–/–) cells generated mitochondrial superoxide but had diminished hydrogen peroxide production in response to neglect, which, in turn, regulated Jun N-terminal kinase–dependent Bax activation and apoptosis. Activated T^–/– cells were distinguished by improved survival after activation by superantigens in vivo, adoptive transfers into congenic hosts, and higher recall responses after immunization. Thus, the NADPH oxidase may regulate adaptive immunity in addition to its previously well-characterized role in the innate response.

Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells

Geiger, R., Duhen, T., Lanzavecchia, A., Sallusto, F. — 2009-07-06

The enormous diversity of the naive T cell repertoire is instrumental in generating an immune response to virtually any foreign antigen that can be processed into peptides that bind to MHC molecules. The low frequency of antigen-specific naive T cells, their high activation threshold, and the constrains of antigen-processing and presentation have hampered analysis of naive repertoires to complex protein antigens. In this study, libraries of polyclonally expanded naive T cells were used to determine frequency and antigen dose–response of human naive CD4⁺ T cells specific for a variety of antigens and to isolate antigen-specific T cell clones. In the naive repertoire, T cells specific for primary antigens, such as KLH and Bacillus anthracis protective antigen, and for recall antigens, such as tetanus toxoid, cytomegalovirus, and Mycobacterium tuberculosis purified protein derivative, were detected at frequencies ranging from 5 to 170 cells per 10⁶ naive T cells. Antigen concentrations required for half-maximal response (EC50) varied over several orders of magnitude for different naive T cells. In contrast, in the memory repertoire, T cells specific for primary antigens were not detected, whereas T cells specific for recall antigens were detected at high frequencies and displayed EC50 values in the low range of antigen concentrations. The method described may find applications for evaluation of vaccine candidates, for testing antigenicity of therapeutic proteins, drugs, and chemicals, and for generation of antigen-specific T cell clones for adoptive cellular immunotherapy.

Peptide immunotherapy in allergic asthma generates IL-10-dependent immunological tolerance associated with linked epitope suppression

2009-07-06

Treatment of patients with allergic asthma using low doses of peptides containing T cell epitopes from Fel d 1, the major cat allergen, reduces allergic sensitization and improves surrogate markers of disease. Here, we demonstrate a key immunological mechanism, linked epitope suppression, associated with this therapeutic effect. Treatment with selected epitopes from a single allergen resulted in suppression of responses to other ("linked") epitopes within the same molecule. This phenomenon was induced after peptide immunotherapy in human asthmatic subjects and in a novel HLA-DR1 transgenic mouse model of asthma. Tracking of allergen-specific T cells using DR1 tetramers determined that suppression was associated with the induction of interleukin (IL)-10⁺ T cells that were more abundant than T cells specific for the single-treatment peptide and was reversed by anti–IL-10 receptor administration. Resolution of airway pathophysiology in this model was associated with reduced recruitment, proliferation, and effector function of allergen-specific Th2 cells. Our results provide, for the first time, in vivo evidence of linked epitope suppression and IL-10 induction in both human allergic disease and a mouse model designed to closely mimic peptide therapy in humans.

T-bet is essential for encephalitogenicity of both Th1 and Th17 cells

2009-07-06

The extent to which myelin-specific Th1 and Th17 cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) is controversial. Combinations of interleukin (IL)-1β, IL-6, and IL-23 with transforming growth factor β were used to differentiate myelin-specific T cell receptor transgenic T cells into Th17 cells, none of which could induce EAE, whereas Th1 cells consistently transferred disease. However, IL-6 was found to promote the differentiation of encephalitogenic Th17 cells. Further analysis of myelin-specific T cells that were encephalitogenic in spontaneous EAE and actively induced EAE demonstrated that T-bet expression was critical for pathogenicity, regardless of cytokine expression by the encephalitogenic T cells. These data suggest that encephalitogenicity of myelin-specific T cells appears to be mediated by a pathway dependent on T-bet and not necessarily pathway-specific end products, such as interferon and IL-17.

Cardiac 12/15 lipoxygenase-induced inflammation is involved in heart failure

2009-07-06

To identify a novel target for the treatment of heart failure, we examined gene expression in the failing heart. Among the genes analyzed, Alox15 encoding the protein 12/15 lipoxygenase (LOX) was markedly up-regulated in heart failure. To determine whether increased expression of 12/15-LOX causes heart failure, we established transgenic mice that overexpressed 12/15-LOX in cardiomyocytes. Echocardiography showed that Alox15 transgenic mice developed systolic dysfunction. Cardiac fibrosis increased in Alox15 transgenic mice with advancing age and was associated with the infiltration of macrophages. Consistent with these observations, cardiac expression of monocyte chemoattractant protein 1 (MCP-1) was up-regulated in Alox15 transgenic mice compared with wild-type mice. Treatment with 12-hydroxy-eicosatetraenoic acid, a major metabolite of 12/15-LOX, increased MCP-1 expression in cardiac fibroblasts and endothelial cells but not in cardiomyocytes. Inhibition of MCP-1 reduced the infiltration of macrophages into the myocardium and prevented both systolic dysfunction and cardiac fibrosis in Alox15 transgenic mice. Likewise, disruption of 12/15-LOX significantly reduced cardiac MCP-1 expression and macrophage infiltration, thereby improving systolic dysfunction induced by chronic pressure overload. Our results suggest that cardiac 12/15-LOX is involved in the development of heart failure and that inhibition of 12/15-LOX could be a novel treatment for this condition.

Profound CD4+/CCR5+ T cell expansion is induced by CD8+ lymphocyte depletion but does not account for accelerated SIV pathogenesis

2009-07-06

Depletion of CD8⁺ lymphocytes during acute simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) results in irreversible prolongation of peak-level viral replication and rapid disease progression, consistent with a major role for CD8⁺ lymphocytes in determining postacute-phase viral replication set points. However, we report that CD8⁺ lymphocyte depletion is also associated with a dramatic induction of proliferation among CD4⁺ effector memory T (T_EM) cells and, to a lesser extent, transitional memory T (T_TrM) cells, raising the question of whether an increased availability of optimal (activated/proliferating), CD4⁺/CCR5⁺ SIV "target" cells contributes to this accelerated pathogenesis. In keeping with this, depletion of CD8⁺ lymphocytes in SIV^– RMs led to a sustained increase in the number of potential CD4⁺ SIV targets, whereas such depletion in acute SIV infection led to increased target cell consumption. However, we found that the excess CD4⁺ T_EM cell proliferation of CD8⁺ lymphocyte–depleted, acutely SIV-infected RMs was completely inhibited by interleukin (IL)-15 neutralization, and that this inhibition did not abrogate the rapidly progressive infection in these RMs. Moreover, although administration of IL-15 during acute infection induced robust CD4⁺ T_EM and T_TrM cell proliferation, it did not recapitulate the viral dynamics of CD8⁺ lymphocyte depletion. These data suggest that CD8⁺ lymphocyte function has a larger impact on the outcome of acute SIV infection than the number and/or activation status of target cells available for infection and viral production.

Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant

2009-07-06

Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4⁺ T cell responses to a dendritic cell (DC)–targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205⁺ DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4⁺ immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow–derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity.

Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction

2009-07-06

Plasmacytoid dendritic cells (pDCs) produce copious type I interferon (IFN) upon sensing nucleic acids through Toll-like receptor (TLR) 7 and TLR9. Uncontrolled pDC activation and IFN production are implicated in lymphopenia and autoimmune diseases; therefore, a mechanism controlling pDC IFN production is essential. Human pDCs specifically express an orphan receptor, immunoglobulin-like transcript 7 (ILT7). Here, we discovered an ILT7 ligand expressed by human cell lines and identified it as bone marrow stromal cell antigen 2 (BST2; CD317). BST2 directly binds to purified ILT7 protein, initiates signaling via the ILT7–FcRI complex, and strongly inhibits production of IFN and proinflammatory cytokines by pDCs. Readily induced by IFN and other proinflammatory cytokines, BST2 may modulate the human pDC’s IFN responses through ILT7 in a negative feedback fashion.

A mutation in Irak2c identifies IRAK-2 as a central component of the TLR regulatory network of wild-derived mice

Conner, J. R., Smirnova, I. I., Poltorak, A. — 2009-07-06

In a phenotypic screen of the wild-derived mouse strain MOLF/Ei, we describe an earlier and more potent toll-like receptor (TLR)–mediated induction of IL-6 transcription compared with the classical inbred strain C57BL/6J. The phenotype correlated with increased activity of the IB kinase axis as well as p38, but not extracellular signal-regulated kinase or c-Jun N-terminal kinase, mitogen-activated protein kinase (MAPK) phosphorylation. The trait was mapped to the Why1 locus, which contains Irak2, a gene previously implicated as sustaining the late phase of TLR responses. In the MOLF/Ei TLR signaling network, IRAK-2 promotes early nuclear factor B (NF-B) activity and is essential for the activation of p38 MAPK. We identify a deletion in the MOLF/Ei promoter of the inhibitory Irak2c gene, leading to an increased ratio of pro- to antiinflammatory IRAK-2 isoforms. These findings demonstrate that IRAK-2 is an essential component of the early TLR response in MOLF/Ei mice and show a distinct pathway of p38 and NF-B activation in this model organism. In addition, they demonstrate that studies in evolutionarily divergent model organisms are essential to complete dissection of signal transduction pathways.

CSL-MAML-dependent Notch1 signaling controls T lineage-specific IL-7R{alpha} gene expression in early human thymopoiesis and leukemia

2009-07-06

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

2009-07-06

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.

Bax activation by the BH3-only protein Puma promotes cell dependence on antiapoptotic Bcl-2 family members

2009-05-11

The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells

Kaizuka, Y., Douglass, A. D., Vardhana, S., Dustin, M. L., Vale, R. D. — 2009-05-11

FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1

2009-05-11

JEM's 2009 Tune-up

Van Epps, H. L. — 2009-05-11

Scientific journals, like cars, require periodic tune-ups to keep them running smoothly. Effective immediately, several changes to the JEM publication policies will take effect. Our aim is to address policy issues that have arisen over the past several years and, more broadly, to maintain the quality and integrity of the research we publish. The upcoming changes to JEM policies and the impetus behind them are outlined here.

A challenge to Goliath

Rossner, M. — 2009-05-11

Megapublishers obligate librarians to buy hundreds of journals they do not need in order to access the journals their constituents actually read. The time has come to challenge this business model, which is unsustainable for the libraries.

Follicular T-to-be

Maxmen, A. — 2009-05-11

Mast cells master a rash

Maxmen, A. — 2009-05-11

HO-1's versatility

Maxmen, A. — 2009-05-11

Bulldozing angiogenesis

Maxmen, A. — 2009-05-11

Shane Crotty: Exploring immune memory

Heller, K. — 2009-05-11

A20 takes on tumors: tumor suppression by an ubiquitin-editing enzyme

Malynn, B. A., Ma, A. — 2009-05-11

Many B cell cancers are characterized in part by the dysregulation of the NF-B signaling pathway. A new study identifies somatic mutations in TNFAIP3, the gene encoding the NF-B inhibitor A20, in Hodgkin lymphomas and primary mediastinal lymphomas. These data reveal the role of A20 as a tumor suppressor protein.

TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma

2009-05-11

Proliferation and survival of Hodgkin and Reed/Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), are dependent on constitutive activation of nuclear factor B (NF-B). NF-B activation through various stimuli is negatively regulated by the zinc finger protein A20. To determine whether A20 contributes to the pathogenesis of cHL, we sequenced TNFAIP3, encoding A20, in HL cell lines and laser-microdissected HRS cells from cHL biopsies. We detected somatic mutations in 16 out of 36 cHLs (44%), including missense mutations in 2 out of 16 Epstein-Barr virus–positive (EBV⁺) cHLs and a missense mutation, nonsense mutations, and frameshift-causing insertions or deletions in 14 out of 20 EBV^– cHLs. In most mutated cases, both TNFAIP3 alleles were inactivated, including frequent chromosomal deletions of TNFAIP3. Reconstitution of wild-type TNFAIP3 in A20-deficient cHL cell lines revealed a significant decrease in transcripts of selected NF-B target genes and caused cytotoxicity. Extending the mutation analysis to primary mediastinal B cell lymphoma (PMBL), another lymphoma with constitutive NF-B activity, revealed destructive mutations in 5 out of 14 PMBLs (36%). This report identifies TNFAIP3 (A20), a key regulator of NF-B activity, as a novel tumor suppressor gene in cHL and PMBL. The significantly higher frequency of TNFAIP3 mutations in EBV^– than EBV⁺ cHL suggests complementing functions of TNFAIP3 inactivation and EBV infection in cHL pathogenesis.

T follicular helper cells differentiate from Th2 cells in response to helminth antigens

2009-05-11

The relationship of T follicular helper (TFH) cells to other T helper (Th) subsets is controversial. We find that after helminth infection, or immunization with helminth antigens, reactive lymphoid organs of 4get IL-4/GFP reporter mice contain populations of IL-4/GFP-expressing CD4⁺ T cells that display the TFH markers CXCR5, PD-1, and ICOS. These TFH cells express the canonical TFH markers BCL6 and IL-21, but also GATA3, the master regulator of Th2 cell differentiation. Consistent with a relationship between Th2 and TFH cells, IL-4 protein production, reported by expression of huCD2 in IL-4 dual reporter (4get/KN2) mice, was a robust marker of TFH cells in LNs responding to helminth antigens. Moreover, the majority of huCD2/IL-4–producing Th cells were found within B cell follicles, consistent with their definition as TFH cells. TFH cell development after immunization failed to occur in mice lacking B cells or CD154. The relationship of TFH cells to the Th2 lineage was confirmed when TFH cells were found to develop from CXCR5^– PD-1^– IL-4/GFP⁺ CD4⁺ T cells after their transfer into naive mice and antigen challenge in vivo.

IL-4-producing CD4+ T cells in reactive lymph nodes during helminth infection are T follicular helper cells

King, I. L., Mohrs, M. — 2009-05-11

Interleukin (IL)-4 is the quintessential T helper type 2 (Th2) cytokine produced by CD4⁺ T cells in response to helminth infection. IL-4 not only promotes the differentiation of Th2 cells but is also critical for immunoglobulin (Ig) G1 and IgE isotype-switched antibody responses. Despite the IL-4–mediated link between Th2 cells and B lymphocytes, the location of IL-4–producing T cells in the lymph nodes is currently unclear. Using IL-4 dual reporter mice, we examined the Th2 response and IL-4 production in the draining mesenteric lymph nodes during infection with the enteric nematode Heligmosomoides polygyrus. We show that although IL-4–competent Th2 cells are found throughout the B and T cell areas, IL-4–producing Th2 cells are restricted to the B cell follicles and associate with germinal centers. Consistent with their localization, IL-4 producers express high levels of CXCR5, ICOS, PD-1, IL-21, and BCL-6, a phenotype characteristic of T follicular helper (Tfh) cells. Although IL-4 was dispensable for the generation of Th2 and Tfh cells, its deletion resulted in defective B cell expansion and maturation. Our report reveals the compartmentalization of Th2 priming and IL-4 production in the lymph nodes during infection, and identifies Tfh cells as the dominant source of IL-4 in vivo.

Identification and characterization of IL-10/IFN-{gamma}-producing effector-like T cells with regulatory function in human blood

Haringer, B., Lozza, L., Steckel, B., Geginat, J. — 2009-05-11

Two subsets of natural and adaptive regulatory T (T reg) cells have been described, but the identity of adaptive type 1 regulatory (Tr1)–like cells in humans is unclear. We analyzed a subset of human blood CD4⁺ T cells—CD45RA^–CD25^–interleukin (IL)-7 receptor (R)^– cells—that rapidly secreted high levels of IL-10 together with interferon , but produced little IL-2. These IL-7R^– T cells were rare, anergic, and largely Foxp3^–. They expressed low levels of Bcl-2 but high levels of Ki-67 and ICOS, suggesting that they have been recently activated in vivo. Consistently, they responded selectively to persistent foreign and self-antigens under steady-state conditions. Unlike natural CD25⁺ T reg cells, IL-7R^– cells suppressed naive and memory T cell proliferation in an IL-10–dependent fashion, and they required strong T cell receptor stimulation for suppression. To our knowledge, this is the first report that identifies Tr1-like cells in human blood. These IL-10–secreting cells have characteristics of chronically activated Th1 effector cells and are distinct from CD25⁺ T reg cells.

A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation

2009-05-11

A tissue-specific transcriptional enhancer, Eµ, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Eµ results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Eµ-independent and Eµ-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression.

In vivo regulation of interleukin 1{beta} in patients with cryopyrin-associated periodic syndromes

2009-05-11

The investigation of interleukin 1β (IL-1β) in human inflammatory diseases is hampered by the fact that it is virtually undetectable in human plasma. We demonstrate that by administering the anti–human IL-1β antibody canakinumab (ACZ885) to humans, the resulting formation of IL-1β–antibody complexes allowed the detection of in vivo–produced IL-1β. A two-compartment mathematical model was generated that predicted a constitutive production rate of 6 ng/d IL-1β in healthy subjects. In contrast, patients with cryopyrin-associated periodic syndromes (CAPS), a rare monogenetic disease driven by uncontrolled caspase-1 activity and IL-1 production, produced a mean of 31 ng/d. Treatment with canakinumab not only induced long-lasting complete clinical response but also reduced the production rate of IL-1β to normal levels within 8 wk of treatment, suggesting that IL-1β production in these patients was mainly IL-1β driven. The model further indicated that IL-1β is the only cytokine driving disease severity and duration of response to canakinumab. A correction for natural IL-1 antagonists was not required to fit the data. Together, the study allowed new insights into the production and regulation of IL-1β in man. It also indicated that CAPS is entirely mediated by IL-1β and that canakinumab treatment restores physiological IL-1β production.

Mast cells mediate neutrophil recruitment and vascular leakage through the NLRP3 inflammasome in histamine-independent urticaria

2009-05-11

Urticarial rash observed in cryopyrin-associated periodic syndrome (CAPS) caused by nucleotide-binding oligomerization domain–leucine-rich repeats containing pyrin domain 3 (NLRP3) mutations is effectively suppressed by anti–interleukin (IL)-1 treatment, suggesting a pathophysiological role of IL-1β in the skin. However, the cellular mechanisms regulating IL-1β production in the skin of CAPS patients remain unclear. We identified mast cells (MCs) as the main cell population responsible for IL-1β production in the skin of CAPS patients. Unlike normal MCs that required stimulation with proinflammatory stimuli for IL-1β production, resident MCs from CAPS patients constitutively produced IL-1β. Primary MCs expressed inflammasome components and secreted IL-1β via NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain when stimulated with microbial stimuli known to activate caspase-1. Furthermore, MCs expressing disease-associated but not wild-type NLRP3 secreted IL-1β and induced neutrophil migration and vascular leakage, the histological hallmarks of urticarial rash, when transplanted into mouse skin. Our findings implicate MCs as IL-1β producers in the skin and mediators of histamine-independent urticaria through the NLRP3 inflammasome.

Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination

Robert, I., Dantzer, F., Reina-San-Martin, B. — 2009-05-11

Immunoglobulin class switch recombination (CSR) is initiated by DNA breaks triggered by activation-induced cytidine deaminase (AID). These breaks activate DNA damage response proteins to promote appropriate repair and long-range recombination. Aberrant processing of these breaks, however, results in decreased CSR and/or increased frequency of illegitimate recombination between the immunoglobulin heavy chain locus and oncogenes like c-myc. Here, we have examined the contribution of the DNA damage sensors Parp1 and Parp2 in the resolution of AID-induced DNA breaks during CSR. We find that although Parp enzymatic activity is induced in an AID-dependent manner during CSR, neither Parp1 nor Parp2 are required for CSR. We find however, that Parp1 favors repair of switch regions through a microhomology-mediated pathway and that Parp2 actively suppresses IgH/c-myc translocations. Thus, we define Parp1 as facilitating alternative end-joining and Parp2 as a novel translocation suppressor during CSR.

The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

2009-05-11

The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes.

Ikaros controls isotype selection during immunoglobulin class switch recombination

Sellars, M., Reina-San-Martin, B., Kastner, P., Chan, S. — 2009-05-11

Class switch recombination (CSR) allows the humoral immune response to exploit different effector pathways through specific secondary antibody isotypes. However, the molecular mechanisms and factors that control immunoglobulin (Ig) isotype choice for CSR are unclear. We report that deficiency for the Ikaros transcription factor results in increased and ectopic CSR to IgG_2b and IgG_2a, and reduced CSR to all other isotypes, regardless of stimulation. Ikaros suppresses active chromatin marks, transcription, and activation-induced cytidine deaminase (AID) accessibility at the 2b and 2a genes to inhibit class switching to these isotypes. Further, Ikaros directly regulates isotype gene transcription as it directly binds the Igh 3' enhancer and interacts with isotype gene promoters. Finally, Ikaros-mediated repression of 2b and 2a transcription promotes switching to other isotype genes by allowing them to compete for AID-mediated recombination at the single-cell level. Thus, our results reveal transcriptional competition between constant region genes in individual cells to be a critical and general mechanism for isotype specification during CSR. We show that Ikaros is a master regulator of this competition.

M-CSF inhibition selectively targets pathological angiogenesis and lymphangiogenesis

2009-05-11

Antiangiogenic therapy for the treatment of cancer and other neovascular diseases is desired to be selective for pathological angiogenesis and lymphangiogenesis. Macrophage colony-stimulating factor (M-CSF), a cytokine required for the differentiation of monocyte lineage cells, promotes the formation of high-density vessel networks in tumors and therefore possesses therapeutic potential as an M-CSF inhibitor. However, the physiological role of M-CSF in vascular and lymphatic development, as well as the precise mechanisms underlying the antiangiogenic effects of M-CSF inhibition, remains unclear. Moreover, therapeutic potential of M-CSF inhibition in other neovascular diseases has not yet been evaluated. We used osteopetrotic (op/op) mice to demonstrate that M-CSF deficiency reduces the abundance of LYVE-1⁺ and LYVE1^– macrophages, resulting in defects in vascular and lymphatic development. In ischemic retinopathy, M-CSF was required for pathological neovascularization but was not required for the recovery of normal vasculature. In mouse osteosarcoma, M-CSF inhibition effectively suppressed tumor angiogenesis and lymphangiogenesis, and it disorganized extracellular matrices. In contrast to VEGF blockade, interruption of M-CSF inhibition did not promote rapid vascular regrowth. Continuous M-CSF inhibition did not affect healthy vascular and lymphatic systems outside tumors. These results suggest that M-CSF–targeted therapy is an ideal strategy for treating ocular neovascular diseases and cancer.

OX40 engagement and chemotherapy combination provides potent antitumor immunity with concomitant regulatory T cell apoptosis

2009-05-11

Expansion and recruitment of CD4⁺ Foxp3⁺ regulatory T (T reg) cells are mechanisms used by growing tumors to evade immune elimination. In addition to expansion of effector T cells, successful therapeutic interventions may require reduction of T reg cells within the tumor microenvironment. We report that the combined use of the alkylating agent cyclophosphamide (CTX) and an agonist antibody targeting the co-stimulatory receptor OX40 (OX86) provides potent antitumor immunity capable of regressing established, poorly immunogenic B16 melanoma tumors. CTX administration resulted in tumor antigen release, which after OX86 treatment significantly enhanced the antitumor T cell response. We demonstrated that T reg cells are an important cellular target of the combination therapy. Paradoxically, the combination therapy led to an expansion of T reg cells in the periphery. In the tumor, however, the combination therapy induced a profound T reg cell depletion that was accompanied by an influx of effector CD8⁺ T cells leading to a favorable T effector/T reg cell ratio. Closer examination revealed that diminished intratumoral T reg cell levels resulted from hyperactivation and T reg cell–specific apoptosis. Thus, we propose that CTX and OX40 engagement represents a novel and rational chemoimmunotherapy.

Low-dose rectal inoculation of rhesus macaques by SIVsmE660 or SIVmac251 recapitulates human mucosal infection by HIV-1

2009-05-11

We recently developed a novel strategy to identify transmitted HIV-1 genomes in acutely infected humans using single-genome amplification and a model of random virus evolution. Here, we used this approach to determine the molecular features of simian immunodeficiency virus (SIV) transmission in 18 experimentally infected Indian rhesus macaques. Animals were inoculated intrarectally (i.r.) or intravenously (i.v.) with stocks of SIVmac251 or SIVsmE660 that exhibited sequence diversity typical of early-chronic HIV-1 infection. 987 full-length SIV env sequences (median of 48 per animal) were determined from plasma virion RNA 1–5 wk after infection. i.r. inoculation was followed by productive infection by one or a few viruses (median 1; range 1–5) that diversified randomly with near starlike phylogeny and a Poisson distribution of mutations. Consensus viral sequences from ramp-up and peak viremia were identical to viruses found in the inocula or differed from them by only one or a few nucleotides, providing direct evidence that early plasma viral sequences coalesce to transmitted/founder viruses. i.v. infection was >2,000-fold more efficient than i.r. infection, and viruses transmitted by either route represented the full genetic spectra of the inocula. These findings identify key similarities in mucosal transmission and early diversification between SIV and HIV-1, and thus validate the SIV–macaque mucosal infection model for HIV-1 vaccine and microbicide research.

Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

2009-05-11

Netherton syndrome (NS) is a severe genetic skin disease with constant atopic manifestations that is caused by mutations in the serine protease inhibitor Kazal-type 5 (SPINK5) gene, which encodes the protease inhibitor lymphoepithelial Kazal-type–related inhibitor (LEKTI). Lack of LEKTI causes stratum corneum detachment secondary to epidermal proteases hyperactivity. This skin barrier defect favors allergen absorption and is generally regarded as the underlying cause for atopy in NS. We show for the first time that the pro-Th2 cytokine thymic stromal lymphopoietin (TSLP), the thymus and activation-regulated chemokine, and the macrophage-derived chemokine are overexpressed in LEKTI-deficient epidermis. This is part of an original biological cascade in which unregulated kallikrein (KLK) 5 directly activates proteinase-activated receptor 2 and induces nuclear factor B–mediated overexpression of TSLP, intercellular adhesion molecule 1, tumor necrosis factor , and IL8. This proinflammatory and proallergic pathway is independent of the primary epithelial failure and is activated under basal conditions in NS keratinocytes. This cell-autonomous process is already established in the epidermis of Spink5^–/– embryos, and the resulting proinflammatory microenvironment leads to eosinophilic and mast cell infiltration in a skin graft model in nude mice. Collectively, these data establish that uncontrolled KLK5 activity in NS epidermis can trigger atopic dermatitis (AD)–like lesions, independently of the environment and the adaptive immune system. They illustrate the crucial role of protease signaling in skin inflammation and point to new therapeutic targets for NS as well as candidate genes for AD and atopy.

Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue responses and apoptosis

2009-05-11

Mouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39^–/– mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium. Studies of these mice demonstrated that BRP-39^–/– animals have markedly diminished antigen-induced Th2 responses and that epithelial YKL-40 rescues the Th2 responses in these animals. The ability of interleukin13 to induce tissue inflammation and fibrosis was also markedly diminished in the absence of BRP-39. Mechanistic investigations demonstrated that BRP-39 and YKL-40 play an essential role in antigen sensitization and immunoglobulin E induction, stimulate dendritic cell accumulation and activation, and induce alternative macrophage activation. These proteins also inhibit inflammatory cell apoptosis/cell death while inhibiting Fas expression, activating protein kinase B/AKT, and inducing Faim 3. These studies establish novel regulatory roles for BRP-39/YKL-40 in the initiation and effector phases of Th2 inflammation and remodeling and suggest that these proteins are therapeutic targets in Th2- and macrophage-mediated disorders.

Myeloid heme oxygenase-1 regulates innate immunity and autoimmunity by modulating IFN-{beta} production

Tzima, S., Victoratos, P., Kranidioti, K., Alexiou, M., Kollias, G. — 2009-05-11

Heme oxygenase–1 (HO-1) is a key cytoprotective, antioxidant, and antiinflammatory molecule. The pathophysiological functions of HO-1 have been associated with its enzymatic activities in heme catabolism. We have examined the immune functions of HO-1 by its conditional ablation in myeloid cells (HO-1^M-KO mice). We demonstrate that myeloid HO-1 is required for the activation of interferon (IFN) regulatory factor (IRF) 3 after Toll-like receptor 3 or 4 stimulation, or viral infection. HO-1–deficient macrophages show reduced expression of IFN-β and of primary IRF3 target genes encoding RANTES, IP-10 and MCP-1. In the presence of polyI:C, myeloid HO-1 knockout mice infected with Listeria monocytogenes, a model dependent on IFN-β production, showed enhanced bacterial clearance and survival, whereas control mice succumbed to infection. Moreover, after induction of experimental autoimmune encephalomyelitis, mice with myeloid-specific HO-1 deficiency developed a higher incidence and an exacerbated, nonremitting clinical disease correlating with persistent activation of antigen-presenting cells, enhanced infiltration of Th17 cells, and a nonregressing myelin-specific T cell reactivity. Notably, these defects were rectified by exogenous administration of IFN-β, confirming that HO-1 functions directly upstream of this critical immune pathway. These results uncover a novel direct function for myeloid HO-1 in the regulation of IFN-β production, establishing HO-1 as a critical early mediator of the innate immune response.

Neutropenia with impaired host defense against microbial infection in mice lacking androgen receptor

2009-05-11

Neutrophils, the major phagocytes that form the first line of cell-mediated defense against microbial infection, are produced in the bone marrow and released into the circulation in response to granulocyte-colony stimulating factor (G-CSF). Here, we report that androgen receptor knockout (ARKO) mice are neutropenic and susceptible to acute bacterial infection, whereas castration only results in moderate neutrophil reduction in mice and humans. Androgen supplement can restore neutrophil counts via stabilizing AR in castrated mice, but not in ARKO and testicular feminization mutant (Tfm) mice. Our results show that deletion of the AR gene does not influence myeloid lineage commitment, but significantly reduces the proliferative activity of neutrophil precursors and retards neutrophil maturation. CXCR2-dependent migration is also decreased in ARKO neutrophils as compared with wild-type controls. G-CSF is unable to delay apoptosis in ARKO neutrophils, and ARKO mice show a poor granulopoietic response to exogenous G-CSF injection. In addition, AR can restore G-CSF–dependent granulocytic differentiation upon transduction into ARKO progenitors. We further found that AR augments G-CSF signaling by activating extracellular signal-regulated kinase 1/2 and also by sustaining Stat3 activity via diminishing the inhibitory binding of PIAS3 to Stat3. Collectively, our findings demonstrate an essential role for AR in granulopoiesis and host defense against microbial infection.

Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung

2009-05-11

Antibody to the dendritic cell surface activation antigen CD83 prevents acute graft-versus-host disease

2009-05-11

Mast cells mediate neutrophil recruitment and vascular leakage through the NLRP3 inflammasome in histamine-independent urticaria

2009-05-11

Role of interleukin 12 and costimulators in T cell anergy in vivo

2009-05-11

Regulation of epithelial-mesenchymal IL-1 signaling by PPAR{beta}/{delta} is essential for skin homeostasis and wound healing

2009-04-13

Canonical Wnts function as potent regulators of osteogenesis by human mesenchymal stem cells

2009-04-13

Immune understudies combat lupus

Leslie, M. — 2009-04-13

Flipping the cancer switch

Leslie, M. — 2009-04-13

Save yourself, cancer cell

Leslie, M. — 2009-04-13

Lasting T reg cell-mediated protection

Leslie, M. — 2009-04-13

HIV's cost/benefit analysis

Leslie, M. — 2009-04-13

Dana Philpott: Exploring the land of NOD

Heller, K. — 2009-04-13

Settling the thymus: immigration requirements

Cyster, J. G. — 2009-04-13

Thymus settling by precursor cells is essential for the production of T cells, yet the immigration requirements are poorly defined. P-selectin and CC chemokine receptor-9 (CCR9) are involved, and settling is favored when existing residents have moved on. A new study strengthens the correlation between niche emptying and the induction of thymic P-selectin and CCR9 ligand, and provides evidence for feedback from the periphery to thymic P-selectin expression via sphingosine-1-phosphate.

The Wiskott-Aldrich syndrome protein is required for iNKT cell maturation and function

2009-04-13

The Wiskott-Aldrich syndrome (WAS) protein (WASp) is a regulator of actin cytoskeleton in hematopoietic cells. Mutations of the WASp gene cause WAS. Although WASp is involved in various immune cell functions, its role in invariant natural killer T (iNKT) cells has never been investigated. Defects of iNKT cells could indeed contribute to several WAS features, such as recurrent infections and high tumor incidence. We found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was^–/– mice. iNKT cell numbers were significantly reduced in the thymus and periphery of was^–/– mice as compared with wild-type controls. Moreover analysis of was^–/– iNKT cell maturation revealed a complete arrest at the CD44⁺ NK1.1^– intermediate stage. Notably, generation of BM chimeras demonstrated a was^–/– iNKT cell-autonomous developmental defect. was^–/– iNKT cells were also functionally impaired, as suggested by the reduced secretion of interleukin 4 and interferon upon in vivo activation. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells and suggest that defects in these cells may play a role in WAS pathology.

A role for human skin-resident T cells in wound healing

2009-04-13

Epidermal T cells have been shown to play unique roles in tissue homeostasis and repair in mice through local secretion of distinct growth factors in the skin. Human epidermis contains both β⁺ and ⁺ T cells whose functional capabilities are not understood. We demonstrate that human epidermal T cells are able to produce insulin-like growth factor 1 (IGF-1) upon activation and promote wound healing in a skin organ culture model. Moreover, an analysis of the functional capabilities of T cells isolated from acute versus chronic wounds revealed a striking difference. Both β⁺ and V1⁺ T cells isolated from acute wounds actively produced IGF-1, demonstrating that they are activated during tissue damage to participate in wound repair. In contrast, IGF-1 production could not be detected in T cells isolated from chronic wounds. In fact, skin T cells isolated from chronic wounds were refractory to further stimulation, suggesting an unresponsive state. Collectively, these results define a novel role for human epidermis–resident T cells in wound healing and provide new insight into our understanding of chronic wound persistence.

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression

2009-04-13

Via a transcription factor, Foxp3, immunoregulatory CD4⁺CD25⁺ T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2–IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1–2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2–IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex–incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

Thymic progenitor homing and lymphocyte homeostasis are linked via S1P-controlled expression of thymic P-selectin/CCL25

2009-04-13

Thymic T cell progenitor (TCP) importation is a periodic, gated event that is dependent on the expression of functional P-selectin ligands on TCPs. Occupancy of intrathymic TCP niches is believed to negatively regulate TCP importation, but the nature of this feedback mechanism is not yet resolved. We show that P-selectin and CCL25 are periodically expressed in the thymus and are essential parts of the thymic gate-keeping mechanism. Periodicity of thymic TCP receptivity and the size of the earliest intrathymic TCP pool were dependent on the presence of functional P-selectin ligand on TCPs. Furthermore, we show that the numbers of peripheral blood lymphocytes directly affected thymic P-selectin expression and TCP receptivity. We identified sphingosine-1-phosphate (S1P) as one feedback signal that could mediate influence of the peripheral lymphocyte pool on thymic TCP receptivity. Our findings suggest a model whereby thymic TCP importation is controlled by both early thymic niche occupancy and the peripheral lymphocyte pool via S1P.

CSL-MAML-dependent Notch1 signaling controls T lineage-specific IL-7R{alpha} gene expression in early human thymopoiesis and leukemia

2009-04-13

Notch1 activation is essential for T-lineage specification of lymphomyeloid progenitors seeding the thymus. Progression along the T cell lineage further requires cooperative signaling provided by the interleukin 7 receptor (IL-7R), but the molecular mechanisms responsible for the dynamic and lineage-specific regulation of IL-7R during thymopoiesis are unknown. We show that active Notch1 binds to a conserved CSL-binding site in the human IL7R gene promoter and critically regulates IL7R transcription and IL-7R chain (IL-7R) expression via the CSL–MAML complex. Defective Notch1 signaling selectively impaired IL-7R expression in T-lineage cells, but not B-lineage cells, and resulted in a compromised expansion of early human developing thymocytes, which was rescued upon ectopic IL-7R expression. The pathological implications of these findings are demonstrated by the regulation of IL-7R expression downstream of Notch1 in T cell leukemias. Thus, Notch1 controls early T cell development, in part by regulating the stage- and lineage-specific expression of IL-7R.

Normally occurring NKG2D+CD4+ T cells are immunosuppressive and inversely correlated with disease activity in juvenile-onset lupus

2009-04-13

The NKG2D receptor stimulates natural killer cell and T cell responses upon engagement of ligands associated with malignancies and certain autoimmune diseases. However, conditions of persistent NKG2D ligand expression can lead to immunosuppression. In cancer patients, tumor expression and shedding of the MHC class I–related chain A (MICA) ligand of NKG2D drives proliferative expansions of NKG2D⁺CD4⁺ T cells that produce interleukin-10 (IL-10) and transforming growth factor-β, as well as Fas ligand, which inhibits bystander T cell proliferation in vitro. Here, we show that increased frequencies of functionally equivalent NKG2D⁺CD4⁺ T cells are inversely correlated with disease activity in juvenile-onset systemic lupus erythematosus (SLE), suggesting that these T cells may have regulatory effects. The NKG2D⁺CD4⁺ T cells correspond to a normally occurring small CD4 T cell subset that is autoreactive, primed to produce IL-10, and clearly distinct from proinflammatory and cytolytic CD4 T cells with cytokine-induced NKG2D expression that occur in rheumatoid arthritis and Crohn's disease. As classical regulatory T cell functions are typically impaired in SLE, it may be clinically significant that the immunosuppressive NKG2D⁺CD4⁺ T cells appear functionally uncompromised in this disease.

Ly49H signaling through DAP10 is essential for optimal natural killer cell responses to mouse cytomegalovirus infection

2009-04-13

The activating natural killer (NK) cell receptor Ly49H recognizes the mouse cytomegalovirus (MCMV) m157 glycoprotein expressed on the surface of infected cells and is required for protection against MCMV. Although Ly49H has previously been shown to signal via DAP12, we now show that Ly49H must also associate with and signal via DAP10 for optimal function. In the absence of DAP12, DAP10 enables Ly49H-mediated killing of m157-bearing target cells, proliferation in response to MCMV infection, and partial protection against MCMV. DAP10-deficient Ly49H⁺ NK cells, expressing only Ly49H–DAP12 receptor complexes, are partially impaired in their ability to proliferate during MCMV infection, display diminished ERK1/2 activation, produce less IFN- upon Ly49H engagement, and demonstrate reduced control of MCMV infection. Deletion of both DAP10 and DAP12 completely abrogates Ly49H surface expression and control of MCMV infection. Thus, optimal NK cell–mediated immunity to MCMV depends on Ly49H signaling through both DAP10 and DAP12.

Loss of STAT5 causes liver fibrosis and cancer development through increased TGF-{beta} and STAT3 activation

Hosui, A., Kimura, A., Yamaji, D., Zhu, B.-m., Na, R., Hennighausen, L. — 2009-04-13

The molecular mechanisms underlying the development of hepatocellular carcinoma are not fully understood. Liver-specific signal transducer and activator of transcription (STAT) 5A/B–null mice (STAT5-LKO) were treated with carbon tetrachloride (CCl₄), and histological analyses revealed liver fibrosis and tumors. Transforming growth factor (TGF)–β levels and STAT3 activity were elevated in liver tissue from STAT5-LKO mice upon CCl₄ treatment. To define the molecular link between STAT5 silencing and TGF-β up-regulation, as well as STAT3 activation, we examined STAT5-null mouse embryonic fibroblasts and primary hepatocytes. These cells displayed elevated TGF-β protein levels, whereas messenger RNA levels remained almost unchanged. Protease inhibitor studies revealed that STAT5 deficiency enhanced the stability of mature TGF-β. Immunoprecipitation and immunohistochemistry analyses demonstrated that STAT5, through its N-terminal sequences, could bind to TGF-β and that retroviral-mediated overexpression of STAT5 decreased TGF-β levels. To confirm the in vivo significance of the N-terminal domain of STAT5, we treated mice that expressed STAT5 lacking the N terminus (STAT5-N) with CCl₄. STAT5-N mice developed CCl₄-induced liver fibrosis but no tumors. In conclusion, loss of STAT5 results in elevated TGF-β levels and enhanced growth hormone–induced STAT3 activity. We propose that a deregulated STAT5–TGF-β–STAT3 network contributes to the development of chronic liver disease.

Netrin-1 acts as a survival factor for aggressive neuroblastoma

2009-04-13

Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in >60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression. We show that autocrine production of netrin-1, a multifunctional laminin-related molecule, conveys a selective advantage in tumor growth and dissemination in aggressive NB, as it blocks the proapoptotic activity of the UNC5H netrin-1 dependence receptors. We show that such netrin-1 up-regulation is a potential marker for poor prognosis in stage 4S and, more generally, in NB stage 4 diagnosed infants. Moreover, we propose that interference with the netrin-1 autocrine loop in malignant neuroblasts could represent an alternative therapeutic strategy, as disruption of this loop triggers in vitro NB cell death and inhibits NB metastasis in avian and mouse models.

Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response

2009-04-13

A primary goal of cancer immunotherapy is to improve the naturally occurring, but weak, immune response to tumors. Ineffective responses to cancer vaccines may be caused, in part, by low numbers of self-reactive lymphocytes surviving negative selection. Here, we estimated the frequency of CD8⁺ T cells recognizing a self-antigen to be <0.0001% (~1 in 1 million CD8⁺ T cells), which is so low as to preclude a strong immune response in some mice. Supplementing this repertoire with naive antigen-specific cells increased vaccine-elicited tumor immunity and autoimmunity, but a threshold was reached whereby the transfer of increased numbers of antigen-specific cells impaired functional benefit, most likely because of intraclonal competition in the irradiated host. We show that cells primed at precursor frequencies below this competitive threshold proliferate more, acquire polyfunctionality, and eradicate tumors more effectively. This work demonstrates the functional relevance of CD8⁺ T cell precursor frequency to tumor immunity and autoimmunity. Transferring optimized numbers of naive tumor-specific T cells, followed by in vivo activation, is a new approach that can be applied to human cancer immunotherapy. Further, precursor frequency as an isolated variable can be exploited to augment efficacy of clinical vaccine strategies designed to activate any antigen-specific CD8⁺ T cells.

Transcriptional complexes formed by NFAT dimers regulate the induction of T cell tolerance

2009-04-13

In T cells, anergy can be induced after T cell receptor engagement in the absence of costimulation. Under these conditions, the expression of a specific set of anergy-associated genes is activated. Several lines of evidence suggest that nuclear factor of activated T cells (NFAT) proteins may regulate the expression of many of those genes; however, the nature of the complexes responsible for the induction of this new program of gene expression is unknown. Here, we show that transcriptional complexes formed by NFAT homodimers are directly responsible for the activation of at least two anergy-inducing genes, Grail and Caspase3. Our data shows that Grail expression is activated by direct binding of NFAT dimers to the Grail promoter at two different sites. Consequently, a mutant NFAT protein with impaired ability to dimerize is not able to induce an unresponsive state in T cells. Our results not only identify a new biological function for NFAT dimers but also reveal the different nature of NFAT-containing complexes that induce anergy versus those that are activated during a productive immune response. These data also establish a basis for the design of immunomodulatory strategies that specifically target each type of complex.

Control of T helper cell differentiation through cytokine receptor inclusion in the immunological synapse

2009-04-13

The antigen recognition interface formed by T helper precursors (Thps) and antigen-presenting cells (APCs), called the immunological synapse (IS), includes receptors and signaling molecules necessary for Thp activation and differentiation. We have recently shown that recruitment of the interferon- receptor (IFNGR) into the IS correlates with the capacity of Thps to differentiate into Th1 effector cells, an event regulated by signaling through the functionally opposing receptor to interleukin-4 (IL4R). Here, we show that, similar to IFN- ligation, TCR stimuli induce the translocation of signal transducer and activator of transcription 1 (STAT1) to IFNGR1-rich regions of the membrane. Unexpectedly, STAT1 is preferentially expressed, is constitutively serine (727) phosphorylated in Thp, and is recruited to the IS and the nucleus upon TCR signaling. IL4R engagement controls this process by interfering with both STAT1 recruitment and nuclear translocation. We also show that in cells with deficient Th1 or constitutive Th2 differentiation, the IL4R is recruited to the IS. This observation suggest that the IL4R is retained outside the IS, similar to the exclusion of IFNGR from the IS during IL4R signaling. This study provides new mechanistic cues for the regulation of lineage commitment by mutual immobilization of functionally antagonistic membrane receptors.

Impact of a hypomorphic Artemis disease allele on lymphocyte development, DNA end processing, and genome stability

2009-04-13

Artemis was initially discovered as the gene inactivated in human radiosensitive T^–B^– severe combined immunodeficiency, a syndrome characterized by the absence of B and T lymphocytes and cellular hypersensitivity to ionizing radiation. Hypomorphic Artemis alleles have also been identified in patients and are associated with combined immunodeficiencies of varying severity. We examine the molecular mechanisms underlying a syndrome of partial immunodeficiency caused by a hypomorphic Artemis allele using the mouse as a model system. This mutation, P70, leads to premature translation termination that deletes a large portion of a nonconserved C terminus. We find that homozygous Artemis-P70 mice exhibit reduced numbers of B and T lymphocytes, thereby recapitulating the patient phenotypes. The hypomorphic mutation results in impaired end processing during the lymphoid-specific DNA rearrangement known as V(D)J recombination, defective double-strand break repair, and increased chromosomal instability. Biochemical analyses reveal that the Artemis-P70 mutant protein interacts with the DNA-dependent protein kinase catalytic subunit and retains significant, albeit reduced, exo- and endonuclease activities but does not undergo phosphorylation. Together, our findings indicate that the Artemis C terminus has critical in vivo functions in ensuring efficient V(D)J rearrangements and maintaining genome integrity.

Evolution of HLA-B5703 HIV-1 escape mutations in HLA-B5703-positive individuals and their transmission recipients

2009-04-13

HLA-B*57 is the class I allele most consistently associated with control of human immunodeficiency virus (HIV) replication, which may be linked to the specific HIV peptides that this allele presents to cytotoxic T lymphocytes (CTLs), and the resulting efficacy of these cellular immune responses. In two HIV C clade–infected populations in South Africa and Zambia, we sought to elucidate the role of HLA-B*5703 in HIV disease outcome. HLA-B*5703–restricted CTL responses select for escape mutations in three Gag p24 epitopes, in a predictable order. We show that the accumulation of these mutations sequentially reduces viral replicative capacity in vitro. Despite this, in vivo data demonstrate that there is ultimately an increase in viral load concomitant with evasion of all three HLA-B*5703–restricted CTL responses. In HLA-B*5703–mismatched recipients, the previously described early benefit of transmitted HLA-B*5703–associated escape mutations is abrogated by the increase in viral load coincident with reversion. Rapid disease progression is observed in HLA-matched recipients to whom mutated virus is transmitted. These data demonstrate that, although costly escape from CTL responses can progressively attenuate the virus, high viral loads develop in the absence of adequate, continued CTL responses. These data underline the need for a CTL vaccine against multiple conserved epitopes.

Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection

2009-04-13

Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8⁺ T cell populations in Mamu-A*01⁺ rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR β-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8⁺ T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181–189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8⁺ T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection.

Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung

2009-04-13

Differentiation and recruitment of alternatively activated macrophages (AAMacs) are hallmarks of several inflammatory conditions associated with infection, allergy, diabetes, and cancer. AAMacs are defined by the expression of Arginase 1, chitinase-like molecules, and resistin-like molecule (RELM) /FIZZ1; however, the influence of these molecules on the development, progression, or resolution of inflammatory diseases is unknown. We describe the generation of RELM-–deficient (Retnla^–/–) mice and use a model of T helper type 2 (Th2) cytokine-dependent lung inflammation to identify an immunoregulatory role for RELM-. After challenge with Schistosoma mansoni (Sm) eggs, Retnla^–/– mice developed exacerbated lung inflammation compared with their wild-type counterparts, characterized by excessive pulmonary vascularization, increased size of egg-induced granulomas, and elevated fibrosis. Associated with increased disease severity, Sm egg–challenged Retnla^–/– mice exhibited elevated expression of pathogen-specific CD4⁺ T cell–derived Th2 cytokines. Consistent with immunoregulatory properties, recombinant RELM- could bind to macrophages and effector CD4⁺ Th2 cells and inhibited Th2 cytokine production in a Bruton's tyrosine kinase–dependent manner. Additionally, Retnla^–/– AAMacs promoted exaggerated antigen-specific Th2 cell differentiation. Collectively, these data identify a previously unrecognized role for AAMac-derived RELM- in limiting the pathogenesis of Th2 cytokine-mediated pulmonary inflammation, in part through the regulation of CD4⁺ T cell responses.

Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites

2009-04-13

Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although little is yet known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. We investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase inhibitor (HDACi). We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi's such as trichostatin A and the clinically relevant compound pyrimethamine. We identify T. gondii HDAC3 (TgHDAC3) as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (nonreplicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of ~370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDACi of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and, likely, other Apicomplexa.