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RATIONALE: Persistent activation of nuclear factor (NF)-kappaB has been associated with the development of asthma. Andrographolide, the principal active component of the medicinal plant Andrographis paniculata, has been shown to inhibit NF-kappaB activity. OBJECTIVES: We hypothesized that andrographolide may attenuate allergic asthma via inhibition of the NF-kappaB signaling pathway. METHODS: BALB/c mice sensitized and challenged with ovalbumin (OVA) developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Serum IgE levels were also determined. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. MEASUREMENTS AND MAIN RESULTS: Andrographolide dose-dependently inhibited OVA-induced increases in total cell count, eosinophil count, and IL-4, IL-5, and IL-13 levels recovered in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, chitinases, Muc5ac, and inducible nitric oxide synthase in lung tissues, and airway hyperresponsiveness to methacholine. In normal human bronchial epithelial cells, andrographolide blocked tumor necrosis factor-alpha-induced phosphorylation of inhibitory kappaB kinase-beta, and downstream inhibitory kappaB alpha degradation, p65 subunit of NF-kappaB phosphorylation, and p65 nuclear translocation and DNA-binding activity. Similarly, andrographolide blocked p65 nuclear translocation and DNA-binding activity in the nuclear extracts from lung tissues of OVA-challenged mice. CONCLUSIONS: Our findings implicate a potential therapeutic value of andrographolide in the treatment of asthma and it may act by inhibiting the NF-kappaB pathway at the level of inhibitory kappaB kinase-beta activation. [ABSTRACT FROM AUTHOR]

Dysfunction of adipocytes and adipose tissue is a primary defect in obesity and obesity-associated metabolic diseases. Interferon regulatory factor 3 (IRF3) has been implicated in adipogenesis. However, the role of IRF3 in obesity and obesity-associated disorders remains unclear. Here, we show that IRF3 expression in human adipose tissues is positively associated with insulin sensitivity and negatively associated with type 2 diabetes. In mouse pre-adipocytes, deficiency of IRF3 results in increased expression of PPARγ and PPARγ-mediated adipogenic genes, leading to increased adipogenesis and altered adipocyte functionality. The IRF3 knockout (KO) mice develop obesity, insulin resistance, glucose intolerance, and eventually type 2 diabetes with aging, which is associated with the development of white adipose tissue (WAT) inflammation. Increased macrophage accumulation with M1 phenotype which is due to the loss of IFNβ-mediated IL-10 expression is observed in WAT of the KO mice compared to that in wild-type mice. Bone-marrow reconstitution experiments demonstrate that the nonhematopoietic cells are the primary contributors to the development of obesity and both hematopoietic and nonhematopoietic cells contribute to the development of obesity-related complications in IRF3 KO mice. This study demonstrates that IRF3 regulates the biology of multiple cell types including adipocytes and macrophages to prevent the development of obesity and obesity-related complications and hence, could be a potential target for therapeutic interventions for the prevention and treatment of obesity-associated metabolic disorders., (© 2021. The Author(s).)

The significance of intracellular Ap 4 A levels over immune activity of dendritic cells (DCs) has been studied in Nudt2 fl/fl /CD11c-cre mice. The transgenic mice have been generated by crossing floxed NUDT2 gene mice with DC marker CD11c recombinase (cre) mice. The DCs derived from these mice have higher levels of Ap 4 A (≈30-fold) compared with those derived from Nudt2 +/+ mice. Interestingly, the elevated Ap 4 A in DCs has led them to possess higher motility and lower directional variability. In addition, the DCs are able to enhance immune protection indicated by the higher cross-presentation of antigen and priming of CD8 + OT-I T cells. Overall, the study denotes prominent impact of Ap 4 A over the functionality of DCs. The Nudt2 fl/fl /CD11c-cre mice could serve as a useful tool to study the influence of Ap 4 A in the critical immune mechanisms of DCs., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Influenza and asthma are two of the major public health concerns in the world today. During the 2009 influenza pandemic asthma was found to be the commonest comorbid illness of patients admitted to hospital. Unexpectedly, it was also observed that asthmatic patients admitted to hospital with influenza infection were less likely to die or require admission to intensive care compared with non-asthmatics. Using an in vivo model of asthma and influenza infection we demonstrate that prior exposure to Blomia tropicalis extract (BTE) leads to an altered immune response to influenza infection, comprised of less severe weight loss and faster recovery following infection. This protection was associated with significant increases in T cell numbers in the lungs of BTE sensitised and infected mice, as well as increased IFN-γ production from these cells. In addition, elevated numbers of CD11b+ dendritic cells (DCs) were found in the lung draining lymph nodes following infection of BTE sensitised mice compared to infected PBS treated mice. These CD11b+ DCs appeared to be better at priming CD8 specific T cells both in vivo and ex vivo, a function not normally attributed to CD11b+ DCs. We propose that this alteration in cross-presentation and more efficient T cell priming seen in BTE sensitised mice, led to the earlier increase in T cells in the lungs and subsequently faster clearance of the virus and reduced influenza induced pathology. We believe this data provides a novel mechanism that explains why asthmatic patients may present with less severe disease when infected with influenza.

CD8 + T cells play an important role in providing protective immunity against a wide range of pathogens, and a number of different factors control their activation. Although CD40L-mediated CD40 licensing of dendritic cells (DCs) by CD4 + T cells is known to be necessary for the generation of a robust CD8 + T cell response, the contribution of CD8 + T cell-expressed CD40L on DC licensing is less clear. We have previously shown that CD8 + T cells are able to induce the production of IL-12 p70 by DCs in a CD40L-dependent manner, providing some evidence that CD8 + T cell-mediated activation of DCs is possible. To better understand the role of CD40L on CD8 + T cell responses, we generated and characterized CD40L-expressing CD8 + T cells both in vitro and in vivo . We found that CD40L was expressed on 30-50% of effector CD8 + T cells when stimulated and that this expression was transient. The expression of CD40L on CD8 + T cells promoted the proliferation and differentiation of both the CD40L-expressing CD8 + T cells and the bystander effector CD8 + T cells. This process occurred via a cell-extrinsic manner and was mediated by DCs. These data demonstrate the existence of a mechanism where CD8 + T cells and DCs cooperate to maximize CD8 + T cell responses.

Blomia tropicalis is the major asthma allergen in the tropics comparable to Dermatophagoides pteronyssinus. However, little is known about the B. tropicalis epitopes recognized by T cells. Our aim was to identify the T-cell epitopes in the major B. tropicalis allergen, Blo t 5, and investigate the potential of the corresponding peptides to inhibit the allergic inflammatory lung response. C57BL/6 mice were immunized with plasmid DNA encoding Blo t 5 and T-cell epitopes identified using the interferon-γ ELISPOT assay with 15-mer overlapping peptides. C57BL/6 mice were sensitized with bone-marrow-derived dendritic cells (BMDC) pulsed with Blo t 5 allergen followed by intranasal Blo t 5 challenge. Two H-2 b restricted epitopes (Bt5 76-90 and Bt5 106-115 ) were recognized by CD4 T cells specific for Blo t 5, but no CD8 epitopes were identified. In mice sensitized with Blo t 5-pulsed BMDC and challenged with intranasal Blo t 5 Bt5 76-90 and Bt5 106-115 , peptide-specific CD4 T cells were found to secrete the T helper type 2 cytokines interleukin-5 and interleukin-13. Intradermal administration of synthetic peptides encoding the identified T-cell epitopes suppressed allergic airway inflammation to further allergen challenges. Hence, we have identified novel CD4 T-cell epitopes specific for Blo t 5 and demonstrated that these peptides could be employed therapeutically to suppress the T-cell response in a murine model of allergic airway inflammation., (© 2017 John Wiley & Sons Ltd.)

Sensitization of allergic patients normally takes place over several years and is the result of repeated exposure to low levels of allergen. Most mouse asthma models use a high dose of allergen administered over a short period. We have investigated the role of dose in the immune response to an inhaled respiratory allergen (Blomia tropicalis). We observed the effect of priming dose on the allergic response in mice intranasally immunized with low (0·5 μg) and high (50 μg) doses of B. tropicalis extract and killed 1 day after the last challenge. For both doses of allergen, T helper type 2 (Th2) cells and Th2 cytokines were evident as well as eosinophilic inflammation accompanied by mucus hyper-secretion. By contrast, IgE and IgG1 antibody responses were normally only detected at high-dose priming. To investigate the mechanism for these effects, we found group 2 innate lymphoid cells (ILC2s) were increased 48 hr after challenge in the low-dose-treated but not the high-dose-treated mice. Furthermore, we determined whether repeated low-dose exposure with different priming protocols could induce an antibody response. Repeated low-dose exposure to 0·5 μg three times weekly for 4 weeks (cumulative 6 μg) had the same effect as a shorter high-dose exposure (cumulative 80 μg) and increasing cumulative dose induced antibody responses. These data indicate that low doses of allergen are sufficient to prime Th2 cells and ILC2s, but insufficient to induce antibody responses. Cumulative exposure to small amounts of allergen induces both Th2 and antibody responses and may better reflect natural sensitization., (© 2017 John Wiley & Sons Ltd.)

Previous studies have highlighted the importance of lung-draining lymph nodes in the respiratory allergic immune response, whereas the lung parenchymal immune system has been largely neglected. We describe a new in vivo model of respiratory sensitization to Blomia tropicalis, the principal asthma allergen in the tropics, in which the immune response is focused on the lung parenchyma by transfer of Th2 cells from a novel TCR transgenic mouse, specific for the major B. tropicalis allergen Blo t 5, that targets the lung rather than the draining lymph nodes. Transfer of highly polarized transgenic CD4 effector Th2 cells, termed BT-II, followed by repeated inhalation of Blo t 5 expands these cells in the lung >100-fold, and subsequent Blo t 5 challenge induced decreased body temperature, reduction in movement, and a fall in specific lung compliance unseen in conventional mouse asthma models following a physiological allergen challenge. These mice exhibit lung eosinophilia; smooth muscle cell, collagen, and goblet cell hyperplasia; hyper IgE syndrome; mucus plugging; and extensive inducible BALT. In addition, there is a fall in total lung volume and forced expiratory volume at 100 ms. These pathophysiological changes were substantially reduced and, in some cases, completely abolished by administration of neutralizing mAbs specific for IL-4 and IL-13 on weeks 1, 2, and 3. This IL-4/IL-13-dependent inducible BALT model will be useful for investigating the pathophysiological mechanisms that underlie asthma and the development of more effective drugs for treating severe asthma., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Objectives: Type 1 interferons (IFN-1) are implicated in the pathogenesis of systemic lupus erythematosus (SLE), but most studies have only reported the effect of IFN-1 on mixed cell populations. We aimed to define modules of IFN-1-associated genes in purified leucocyte populations and use these as a basis for a detailed comparative analysis., Methods: CD4+ and CD8+ T cells, monocytes and neutrophils were purified from patients with SLE, other immune-mediated diseases and healthy volunteers and gene expression then determined by microarray. Modules of IFN-1-associated genes were defined using weighted gene coexpression network analysis. The composition and expression of these modules was analysed., Results: 1150 of 1288 IFN-1-associated genes were specific to myeloid subsets, compared with 11 genes unique to T cells. IFN-1 genes were more highly expressed in myeloid subsets compared with T cells. A subset of neutrophil samples from healthy volunteers (HV) and conditions not classically associated with IFN-1 signatures displayed increased IFN-1 gene expression, whereas upregulation of IFN-1-associated genes in T cells was restricted to SLE., Conclusions: Given the broad upregulation of IFN-1 genes in neutrophils including in some HV, investigators reporting IFN-1 signatures on the basis of whole blood samples should be cautious about interpreting this as evidence of bona fide IFN-1-mediated pathology. Instead, specific upregulation of IFN-1-associated genes in T cells may be a useful biomarker and a further mechanism by which elevated IFN-1 contributes to autoimmunity in SLE.

Background: Asthmatic patients are highly susceptible to air pollution and in particular to the effects of ozone (O3) inhalation, but the underlying mechanisms remain unclear., Objective: Using mouse models of O3-induced airway inflammation and airway hyperresponsiveness (AHR), we sought to investigate the role of the recently discovered group 2 innate lymphoid cells (ILC2s)., Methods: C57BL/6 and BALB/c mice were exposed to Aspergillus fumigatus, O3, or both (3 ppm for 2 hours). ILC2s were isolated by means of fluorescence-activated cell sorting and studied for Il5 and Il13 mRNA expression. ILC2s were depleted with anti-Thy1.2 mAb and replaced by means of intratracheal transfer of ex vivo expanded Thy1.1 ILC2s. Cytokine levels (ELISA and quantitative PCR), inflammatory cell profile, and AHR (flexiVent) were assessed in the mice., Results: In addition to neutrophil influx, O3 inhalation elicited the appearance of eosinophils and IL-5 in the airways of BALB/c but not C57BL/6 mice. Although O3-induced expression of IL-33, a known activator of ILC2s, in the lung was similar between these strains, isolated pulmonary ILC2s from O3-exposed BALB/c mice had significantly greater Il5 and Il13 mRNA expression than C57BL/6 mice. This suggested that an altered ILC2 function in BALB/c mice might mediate the increased O3 responsiveness. Indeed, anti-Thy1.2 treatment abolished but ILC2s added back dramatically enhanced O3-induced AHR., Conclusions: O3-induced activation of pulmonary ILC2s was necessary and sufficient to mediate asthma-like changes in BALB/c mice. This previously unrecognized role of ILC2s might help explain the heightened susceptibility of human asthmatic airways to O3 exposure., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Dengue, which is the most prevalent mosquito-borne viral disease afflicting human populations, causes a spectrum of clinical symptoms that include fever, muscle and joint pain, maculopapular skin rash, and hemorrhagic manifestations. Patients infected with dengue develop a broad antigen-specific T lymphocyte response, but the phenotype and functional properties of these cells are only partially understood. We show that natural infection induces dengue-specific CD8(+) T lymphocytes that are highly activated and proliferating, exhibit antiviral effector functions, and express CXCR3, CCR5, and the skin-homing marker cutaneous lymphocyte-associated antigen (CLA). In the same patients, bystander human cytomegalovirus -specific CD8(+) T cells are also activated during acute dengue infection but do not express the same tissue-homing phenotype. We show that CLA expression by circulating dengue-specific CD4(+) and CD8(+) T cells correlates with their in vivo ability to traffic to the skin during dengue infection. The juxtaposition of dengue-specific T cells with virus-permissive cell types at sites of possible dengue exposure represents a previously uncharacterized form of immune surveillance for this virus. These findings suggest that vaccination strategies may need to induce dengue-specific T cells with similar homing properties to provide durable protection against dengue viruses., (Copyright © 2015, American Association for the Advancement of Science.)

The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154(+) TNF-α(+) cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154(+) TNF-α(+) IFN-γ(+) IL-2(+) and CD154(+) TNF-α(+) CXCR3(+). Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

T helper (TH)-cell subsets, such as TH1 and TH17, mediate inflammation in both peripheral tissues and central nervous system. Here we show that STAT5 is required for T helper-cell pathogenicity in autoimmune neuroinflammation but not in experimental colitis. Although STAT5 promotes regulatory T cell generation and immune suppression, loss of STAT5 in CD4+ T cells resulted in diminished development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Our results showed that loss of encephalitogenic activity of STAT5-deficient autoreactive CD4+ T cells was independent of IFN-γ or interleukin 17 (IL-17) production, but was due to the impaired expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), a crucial mediator of T-cell pathogenicity. We further showed that IL-7-activated STAT5 promotes the generation of GM-CSF-producing CD4+ T cells, which were preferentially able to induce more severe EAE than TH17 or TH1 cells. Consistent with GM-CSF-producing cells being a distinct subset of TH cells, the differentiation program of these cells was distinct from that of TH17 or TH1 cells. We further found that IL-3 was secreted in a similar pattern as GM-CSF in this subset of TH cells. In conclusion, the IL-7-STAT5 axis promotes the generation of GM-CSF/IL-3-producing TH cells. These cells display a distinct transcriptional profile and may represent a novel subset of T helper cells which we designate as TH-GM.

The Blomia tropicalis dust mite is prevalent in tropical and subtropical regions of the world. Although it is a leading cause of asthma, little is known how it induces allergy. Using a novel murine asthma model induced by intranasal exposure to B. tropicalis, we observed that a single intranasal sensitization to B. tropicalis extract induces strong Th2 priming in the lung draining lymph node. Resident CD11b(+) dendritic cells (DCs) preferentially transport Ag from the lung to the draining lymph node and are crucial for the initiation of Th2 CD4(+) T cell responses. As a consequence, mice selectively deficient in CD11b(+) DCs exhibited attenuated Th2 responses and more importantly did not develop any allergic inflammation. Conversely, mice deficient in CD103(+) DCs and CCR2-dependent monocyte-derived DCs exhibited similar allergic inflammation compared with their wild-type counterparts. We also show that CD11b(+) DCs constitutively express higher levels of GM-CSF receptor compared with CD103(+) DCs and are thus selectively licensed by lung epithelial-derived GM-CSF to induce Th2 immunity. Taken together, our study identifies GM-CSF-licensed CD11b(+) lung DCs as a key component for induction of Th2 responses and represents a potential target for therapeutic intervention in allergy., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Monocytes and their derived cells have critical roles in inflammation and immune defense. However, their function in skin diseases such as allergic contact dermatitis remains poorly defined. Using a model of contact hypersensitivity (CHS) toward 2,4-dinitrochlorobenzene, we show that Ly6C+ CD11b+ monocytic cells participate in the pathophysiology of CHS and their accumulation is regulated by effector CD8 T cells. These Ly6C+ CD11b+ monocytic cells are the primary contributors of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) and derive from Ly6C(hi)CCR2+ monocytes, as they were absent in non-inflamed skin and accumulate as a consequence of inflammation in a C-C chemokine receptor type 2 (CCR2)-dependent manner. Importantly, CCR2(-/-) mice, or wild-type mice depleted of monocytes via clodronate liposomes, display a marked decrease in TNF-α and iNOS expression accompanied by attenuated skin inflammation. Using transgenic mice and antibody depletion, we show that effector CD8 T cells regulate the accumulation of Ly6C+ CD11b+ monocytic cells through IL-17 and activate them for TNF-α and iNOS through IFN-γ. CD8 T cell-derived IFN-γ was also critical for the accumulation of the major histocompatibility complex II-expressing Ly6C+ CD11b+ subset, which expressed intermediate levels of CD11c and costimulatory molecules. Taken together, our findings provide further insight into the pathophysiology of allergic contact dermatitis by showing that CD8 T cells regulate the inflammatory cascade through TNF/iNOS-expressing Ly6C+ CD11b+ monocytic cells.

The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ(-/-) and IFN-γR(-/-) compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ(-/-) mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ(-/-) and IFN-γR(-/-) mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.

Lymphangiogenesis is an important physiological response to inflammatory insult, acting to limit inflammation. Macrophages, dendritic cells, and lymphocytes are known to drive lymphangiogenesis. In this study, we show that neutrophils recruited to sites of inflammation can also coordinate lymphangiogenesis. In the absence of B cells, intranodal lymphangiogenesis induced during prolonged inflammation as a consequence of immunization is dependent on the accumulation of neutrophils. When neutrophils are depleted in wild-type mice developing skin inflammation in response to immunization or contact hypersensitization, lymphangiogenesis is decreased and local inflammation is increased. We demonstrate that neutrophils contribute to lymphangiogenesis primarily by modulating vascular endothelial growth factor (VEGF)-A bioavailability and bioactivity and, to a lesser extent, secreting VEGF-D. We further show that neutrophils increased VEGF-A bioavailability and bioactivity via the secretion of matrix metalloproteinases 9 and heparanase. Together, these findings uncover a novel function for neutrophils as organizers of lymphangiogenesis during inflammation.

CD137 and its ligand are expressed in the BM, and conflicting data exist on the regulation of myelopoiesis by the CD137 receptor-ligand system. CD137(-/-) mice have increased numbers of myeloid cells in the BM, indicating an inhibitory influence of CD137 on myelopoiesis. However, CD137 also induces proliferation of hematopoietic progenitor cells and their myeloid differentiation, arguing for an enhancing effect. Here we hypothesized that this latter case represents the situation during infections since expression of CD137 is activation dependent and strongly enhanced during inflammation. Indeed, infections with Influenza, Bordetella pertussis, Mycobacterium bovis, Bacille Calmette-Guérin or Escherichia coli or i.p. injection of LPS led to increased numbers of CD137-expressing cells, especially of CD4(+) T cells in the BM of mice. Coculture experiments confirmed that CD137 expression enables CD4(+) T cells to induce proliferation and myeloid differentiation of BM and hematopoietic progenitor cells. CD137 also enhances myelopoiesis in vivo since the infection-induced increase in myeloid cell proliferation and total myeloid cell numbers in the BM were significantly lower in CD137(-/-) mice. This study reconciles earlier conflicting data by demonstrating that while CD137-CD137L interactions inhibit myelopoiesis during steady-state conditions they increase myelopoiesis during infection., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The mechanisms regulating memory CD8(+) T cell function and homeostasis during aging are unclear. CD8(+) effector memory T cells that re-express CD45RA increase considerably in older humans and both aging and persistent CMV infection are independent factors in this process. We used MHC class I tetrameric complexes that were mutated in the CD8 binding domain to identify CMV-specific CD8(+) T cells with high Ag-binding avidity. In individuals who were HLA-A*0201, CD8(+) T cells that expressed CD45RA and were specific for the pp65 protein (NLVPMVATV epitope) had lower avidity than those that expressed CD45RO and demonstrated decreased cytokine secretion and cytolytic potential after specific activation. Furthermore, low avidity NLVPMVATV-specific CD8(+) T cells were significantly increased in older individuals. The stimulation of blood leukocytes with CMV lysate induced high levels of IFN-α that in turn induced IL-15 production. Moreover, the addition of IL-15 to CD45RA(-)CD45RO(+) CMV-specific CD8(+) T cells induced CD45RA expression while Ag activated cells remained CD45RO(+). This raises the possibility that non-specific cytokine-driven accumulation of CMV-specific CD8(+)CD45RA(+) T cells with lower Ag-binding avidity may exacerbate the effects of viral reactivation on skewing the T cell repertoire in CMV-infected individuals during aging.

Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4(+) and CD8(+) T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4(+) and 21 are CD8(+) T cell epitopes. We observe that whereas CD8(+) T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4(+) epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4(+) T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4(+) and CD8(+) T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.

Trafficking of lung dendritic cells (DCs) to the draining lymph node (dLN) is a crucial step for the initiation of T cell responses upon pathogen challenge. However, little is known about the factors that regulate lung DC migration to the dLN. In this study, using a model of influenza infection, we demonstrate that complement component C3 is critically required for efficient emigration of DCs from the lung to the dLN. C3 deficiency affect lung DC-mediated viral antigen transport to the dLN, resulting in severely compromised priming of virus-specific T cell responses. Consequently, C3-deficient mice lack effector T cell response in the lungs that affected viral clearance and survival. We further show that direct signaling by C3a and C5a through C3aR and C5aR respectively expressed on lung DCs is required for their efficient trafficking. However, among lung DCs, only CD103(+) DCs make a significant contribution to lung C5a levels and exclusively produce high levels of C3 and C5 during influenza infection. Collectively, our findings show that complement has a profound impact on immune regulation by controlling tissue DC trafficking and highlights a potential utility for complement as an adjuvant in novel vaccine strategies.

Background: We previously reported that prior nasal administration of highly attenuated Bordetella pertussis BPZE1 provides effective and sustained protection against lethal challenge with influenza A viruses. The protective effect was mediated by suppressing the production of major pro-inflammatory mediators. To further explore the anti-inflammatory properties of BPZE1, we investigated the effect of BPZE1 nasal pretreatment on two mouse models of allergic disease, allergic airway inflammation, and contact hypersensitivity (CHS)., Methods: Allergic reactions were induced in mice nasally pretreated with live attenuated BPZE1 bacteria using the ovalbumin (OVA)-induced allergic airway inflammation and dinitrochlorobenzene (DNCB)-induced CHS models., Results: Prior BPZE1 nasal treatment suppressed OVA-induced lung inflammation and inflammatory cell recruitment and significantly reduced IgE levels and cytokine production. Similarly, BPZE1 nasal pretreatment markedly inhibited ear swelling, skin inflammation, and production of pro-inflammatory cytokines in the DNCB-induced CHS model. For both models, we showed that BPZE1 pretreatment does not affect the sensitization phase. Upon challenge, BPZE1 pretreatment selectively reduced the level of cytokines whose production is increased and did not affect the basal level of other cytokines. Together, our observations suggest that BPZE1 pretreatment specifically targets those cytokine-producing effector cells that are recruited and involved in the inflammatory reaction., Conclusion: Our study demonstrates the broad anti-inflammatory properties of the attenuated B. pertussis BPZE1 vaccine candidate and supports its development as a promising agent to prevent and/or treat allergic diseases., (© 2012 John Wiley & Sons A/S.)

An effective immune response against influenza A infection depends on the generation of virus-specific T cells. NK cells are one of the first-line defenses against influenza A infection. We set out to delineate the role of NK cells in T cell immunity using a murine model of influenza A infection with A/PR/8/34. We show that early T cell recruitment mainly occurs in the posterior mediastinal lymph node (pMLN). Depletion of NK cells significantly impaired both dendritic cell (DC) and T cell recruitment into the pMLN. A similar reduction of T cell recruitment was observed when migration was blocked by pertussis toxin, suggesting that migration of pulmonary NK cells and DCs regulates cell recruitment to the pMLN. T cell recruitment was dependent on IFN-γ, and transfer of IFN-γ-competent naive NK cells into IFN-γ-/- mice restored T cell recruitment, whereas IFN-γ-deficient NK cells failed to do so. In addition, NK cell depletion reduced the uptake and transport of influenza A virus by DCs, and significantly impaired the virus-specific T cell response. Both IFN-γ-/- and perforin-/- mice showed reduced viral Ag transport by DCs, suggesting that the ability of NK cells to influence virus transport depends on IFN-γ and perforin. In summary, our data suggest that NK cells play a critical role in the initiation and shaping of the T cell response after influenza A infection.

Dengue virus (DENV) is a mosquito-borne flavivirus that affects 2.5 billion people worldwide. There are four dengue serotypes (DENV1 to DENV4), and infection with one elicits lifelong immunity to that serotype but offers only transient protection against the other serotypes. Identification of the protective determinants of the human antibody response to DENV is a vital requirement for the design and evaluation of future preventative therapies and treatments. Here, we describe the isolation of a neutralizing antibody from a DENV1-infected patient. The human antibody 14c10 (HM14c10) binds specifically to DENV1. HM14c10 neutralizes the virus principally by blocking virus attachment; at higher concentrations, a post-attachment step can also be inhibited. In vivo studies show that the HM14c10 antibody has antiviral activity at picomolar concentrations. A 7 Å resolution cryoelectron microscopy map of Fab fragments of HM14c10 in a complex with DENV1 shows targeting of a discontinuous epitope that spans the adjacent surface of envelope protein dimers. As found previously, a human antibody specific for the related West Nile virus binds to a similar quaternary structure, suggesting that this could be an immunodominant epitope. These findings provide a structural and molecular context for durable, serotype-specific immunity to DENV infection.

Background: Previous studies have shown that CD8 T cells can both prevent and cause allergic responses. However, the underlying mechanisms remain to be elucidated., Objective: We aim to investigate the potential of CD8 T cells with different IFN-γ expressions to modulate the elicitation of allergic inflammation following ovalbumin (OVA) challenge and investigate the underlying mechanisms., Methods: To study the role of IFN-γ in the effect of CD8 T cells, effector CD8 T cells from CD8 OVA transgenic (OT-I) mice and IFN-γ(-/-)OT-I mice were transferred to OVA-sensitized mice the day before 3 challenges with OVA. The effect on lung dendritic cells (DCs) exerted by CD8 T cells was studied with ex vivo culture of sorted DCs from treatment mice with CD4 T cells., Results: Effector OT-I, but not IFN-γ(-/-)OT-I CD8 T cells, attenuated eosinophilia and mucus secretion in the lungs of sensitized mice in an antigen-specific manner. Effector IFN-γ(-/-)OT-I CD8 T cells displayed a Tc2-/Tc17-biased phenotype with weaker cytotoxicity and were able to both induce and exacerbate eosinophilia as well as neutrophilia. OT-I CD8 T cells increased the ability of lung CD11b(+)CD103(-) DCs to both prime the differentiation of naive OVA-specific CD4 T cells toward a T(H)1 phenotype and enhance IFN-γ production by antigen-experienced lung CD4 T cells., Conclusion: Effector CD8 T cells attenuate pulmonary inflammation and alter the ability of DCs within the allergic lung to polarize T cells to a T(H)1 phenotype during a T(H)2 response. In the absence of IFN-γ, CD8 T cells assume a Tc2-/Tc17-biased phenotype and potentiate inflammation., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Antibody-mediated pure red cell aplasia is a very rare but devastating condition affecting patients receiving treatment with erythropoiesis-stimulating agents. New cases continue to emerge, generally in clusters, consistent with an 'environmental' trigger to its pathogenesis. Defining the causes of antibody-mediated pure red cell aplasia is clearly of importance for patients with chronic kidney disease, but any developments in this area may also have relevance to other disease areas as therapeutic delivery of endogenous proteins rapidly increases. This review focuses on the current knowledge regarding the etiology of antibody-mediated pure red cell aplasia and the current approach to therapy.

Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.

Mesenchymal stromal cells (MSCs) possess both immuno-privileged and immuno-inhibitory properties that contribute to their therapeutic effects. Ex vivo expansion is required to obtain sufficient cells for therapy, but might also alter their immunological properties. To date there has been no systematic study of MSC immunobiology during extended culture. Here, we demonstrate that both immuno-privilege and immunosuppressive properties of MSCs change with increasing passage. We demonstrate that although MSCs exhibit powerful immunosuppressive effects through secretion of transforming growth factor-β (TGF-β) and induction of interleukin-10, these effects are diminished by a concomitant increase in MSC immunogenicity. Interferon-γ treatment for 3 days induced extendedly cultured MSCs to express significantly higher levels of major histocompatibility complex class I. In vivo, this results in cells that induce significant delayed-type hypersensitivity reactions in allogeneic recipients. Importantly, these effects are alleviated by isolation of the transplanted MSCs using a semi-permeable barrier. Under these conditions, even MSCs cultured through as many as 14 passages still exhibit immuno-inhibitory effects in vivo. Furthermore, the levels of anti-inflammatory molecule TGF-β secreted by MSCs were maintained in the extended culture. These data shed light on the variable results of allogeneic MSCs in transplantation and suggest alternative strategies for prolonging the effect of allogeneic MSCs in cell-based therapy.

The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11b(low/neg)CD103(+) DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11b(low/neg)CD103(+) DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11b(high)CD103(neg) DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11b(low/neg)CD103(+) DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11b(low/neg)CD103(+) DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11b(low/neg)CD103(+) DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.

TNF/iNOS-producing dendritic cells (Tip-DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8(+) T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs that express high levels of TNF-α and iNOS. Tip-DCs exhibited T-cell priming ability, expressed high levels of MHC class II, upregulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4(+) T cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC production of TNF-α and NO. Our study hence identifies a role for CD8(+) T cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We recently reported that diadenosine tetraphosphate hydrolase (Ap(4)A hydrolase) plays a critical role in gene expression via regulation of intracellular Ap(4)A levels. This enzyme serves as a component of our newly described lysyl tRNA synthetase (LysRS)-Ap(4)A biochemical pathway that is triggered upon immunological challenge. Here we explored the mechanism of this enzyme's translocation into the nucleus and found its immunologically dependent association with importin beta. Silencing of importin beta prevented Ap(4)A hydrolase nuclear translocation and affected the local concentration of Ap(4)A, which led to an increase in microphthalmia transcription factor (MITF) transcriptional activity. Furthermore, immunological activation of mast cells resulted in dephosphorylation of Ap(4)A hydrolase, which changed the hydrolytic activity of the enzyme.

Airway epithelium functions not only as a physical barrier, but also a regulator of lung inflammation. IFN-γ plays a critical role in airway inflammation associated with respiratory viral infection. We investigated differential protein profiling in IFN-γ-stimulated normal human bronchial epithelial cells (HBEC) using a 2-dimensional gel electrophoresis followed by MALDI-TOF-MS/MS. IFN-γ markedly stimulated apolipoprotein L2 (ApoL2) protein expression in normal HBEC. ApoL2 mRNA expression was also elevated in normal human lung fibroblasts and smooth muscle cells stimulated with IFN-γ, in lung tissues from an IFN-γ-predominant influenza A virus-infected mouse lung injury model, and in cancer lung tissues from human patients. Normal HBEC showed strong resistance to IFN-γ-induced cytotoxicity. ApoL2 knockdown by siRNA promoted IFN-γ-induced cytotoxicity as revealed by a significant drop in cell viability using MTT and CyQUANT NF cell proliferation assays, and a marked increase in hypodiploid sub-G1 cell population in cell cycle analysis. Furthermore, depletion of ApoL2 facilitated IFN-γ-induced membrane damage and chromatin condensation as observed in Hoechst and propidium iodide-double staining and in transmission electron microscopy, and DNA fragmentation using a DNA laddering assay, in a caspase-dependent manner. Our results reveal a novel function for ApoL2 in conferring anti-apoptotic ability of human bronchial epithelium to the cytotoxic effects of IFN-γ, in maintaining airway epithelial layer integrity., (© 2010 Wiley-Liss, Inc.)

Foxp3⁺CD4⁺ regulatory T cells represent a T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, and there is emerging evidence to suggest these cells temper pulmonary inflammation in response to acute viral infection. Recent studies have demonstrated treatment with PC61 CD25-depleting antibody potentiates inflammation in a murine model of RSV infection, while paradoxically delaying recruitment of CD8⁺ T cells to the site of inflammation. The present study therefore sought to examine the role of these cells in a murine model of acute influenza A virus infection through the administration of PC61 CD25-depleting antibody. PC61 antibody is able to partially deplete CD25⁺Foxp3⁺ regulatory T cells to a comparable degree as seen within previous work examining RSV, however this does not alter influenza A-virus induced mortality, weight loss, viral clearance and cellularity within the lung. Collectively, these data demonstrate that partial depletion of CD4⁺CD25⁺ regulatory T cells with PC61 antibody does not alter the course of influenza A virus infection.

Bronchial mucosal CD8(+) cells are implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but there are few data on their functional properties. We have developed a novel technique to outgrow these cells from COPD patients in sufficient numbers to examine effector functions. Endobronchial biopsies from 15 COPD smokers and 12 ex-smokers, 11 control smokers and 10 non-smokers were cultured with anti-CD3/interleukin (IL)-2 ± IL-15. Outgrown CD3(+) T cells were characterized in terms of phenotype (expression of CD4, 8, 25, 28, 69 and 56), cytotoxicity and expression of COPD-related cytokines. Compared with IL-2 alone, additional IL-15 increased the yield and viability of biopsy-derived CD3(+) T cells (12-16-day culture without restimulation) without alteration of CD4(+) /CD8(+) ratios or expression of accessory/activation molecules. Biopsy-derived T cells, principally CD8(+)/CD56(+) cells, exhibited statistically significantly greater cytotoxic activity in current or ex-smokers with COPD compared with controls (P < 0·01). Elevated percentages of CD8(+) T cells expressed interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-13 (P < 0·01) in current COPD smokers compared with all comparison groups. It is possible to perform functional studies on bronchial mucosal T cells in COPD. We demonstrate increased CD8(+)CD56(+) T cell cytotoxic activity and expression of remodelling cytokines in smokers who develop COPD., (© 2010 British Society for Immunology.)

Protein inhibitor of activated STAT3 (PIAS3), the main cellular inhibitor of signal transducers and activator of transcription 3 (STAT3), has been described as a modulator of DNA binding transcription factors. The exploration of the emerging roles of PIAS3 in immune regulation is a growing and fascinating field. Recent discoveries have shed new light on the key role of PIAS3 in the regulation of transcriptional activity, and on the molecular mechanism involved. These findings suggest that the known functions of this signalling molecule are merely the "tip of the iceberg". This article reviews the challenging questions regarding the link between PIAS3 and the intracellular signalling in immune cells. Some of the known functions of PIAS3 that potentially modulate key proteins in the immune system will also be discussed., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Autoimmune diseases are common and debilitating, but their severe manifestations could be reduced if biomarkers were available to allow individual tailoring of potentially toxic immunosuppressive therapy. Gene expression-based biomarkers facilitating such tailoring of chemotherapy in cancer, but not autoimmunity, have been identified and translated into clinical practice. We show that transcriptional profiling of purified CD8(+) T cells, which avoids the confounding influences of unseparated cells, identifies two distinct subject subgroups predicting long-term prognosis in two autoimmune diseases, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), a chronic, severe disease characterized by inflammation of medium-sized and small blood vessels, and systemic lupus erythematosus (SLE), characterized by autoantibodies, immune complex deposition and diverse clinical manifestations ranging from glomerulonephritis to neurological dysfunction. We show that the subset of genes defining the poor prognostic group is enriched for genes involved in the interleukin-7 receptor (IL-7R) pathway and T cell receptor (TCR) signaling and those expressed by memory T cells. Furthermore, the poor prognostic group is associated with an expanded CD8(+) T cell memory population. These subgroups, which are also found in the normal population and can be identified by measuring expression of only three genes, raise the prospect of individualized therapy and suggest new potential therapeutic targets in autoimmunity.

The maintenance of immune homeostasis and the regulation of pro-inflammatory responses that underlie autoimmune pathology require a coordinated interplay between cytokines, cellular receptors and downstream signaling pathways. The family of fragment crystallizable receptors for immunoglobulin G (IgG) (Fc gammaR) represents a good example of how controlling the simultaneous triggering of activatory and/or inhibitory receptors results in a balanced immune response. Fc gammaRs play a crucial role in linking the antibody-mediated recognition of pathogens, allergens or auto-antigens to the cellular arm of the immune response. In this review, we detail how dysfunction in Fc gammaR pathways is linked to the development of the autoimmune disease, systemic lupus erythematosus.

Current bone disease therapy with bone marrow-derived mesenchymal stromal cells (MSC) is hampered by low efficiency. Advanced allogeneic studies on well-established mouse genetic and disease models are hindered by difficulties in isolating murine MSC (mMSC). And mMSC prepared from different laboratories exhibit significant heterogeneity. Hence, this study aimed to identify and isolate a sub-population of mMSC at an early passage number with high osteogenic potential. Enrichment of mMSC was achieved by 1-hr silica incubation and negative selection. Approximately 96% of these cells synthesized osteocalcin after 28 days of osteogenic induction in vitro, and displayed a complete dynamic alteration of alkaline phosphatase (ALP) activity with increasing osteogenic maturation and strong mineralization. Moreover, the cells displayed uniform and stable surface molecular profile, long-term survival, fast proliferation in vitro with maintenance of normal karyotype and distinct immunological properties. CD73 was found to be expressed exclusively in osteogenesis but not in adipogenesis. These cells also retained high osteogenic potential upon allogeneic transplantation in an ectopic site by the detection of bone-specific ALP, osteopontin, osteocalcin and local mineralization as early as 12 days after implantation. Hence, these cells may provide a useful source for improving current strategies in bone regenerative therapy, and for characterizing markers defining the putative MSC population.

Receptors for the fragment crystallizable region of immunoglobulin-G (FcgammaRs) play an important role in linking the humoral and cellular arms of the immune response. In this study, we present a comprehensive functional comparison of 2 human Fc-receptors, FcgammaRI and FcgammaRIIa. Activation of FcgammaRI results in a novel signaling cascade that links phospholipase D1 to sphingosine kinase-1 in U937 cells and primary human monocytes. This induces the expression of proinflammatory mediators and is associated with trafficking of immune complexes into human leukocyte antigen-DM positive antigen-processing compartments coupled with improved MHC class II-mediated antigen presentation to T lymphocytes. In contrast, activation of FcgammaRIIa elicits signaling through phospholipase Cgamma1, resulting in increases in intracellular calcium, activation of nicotinamide adenine dinucleotide phosphate-oxidative burst, and differential membrane trafficking combined with impaired antigen presentation and proinflammatory cytokine expression. These data provide a mechanistic insight into the disparate activities associated with Fc receptors in immunity, namely, reinforcement of immune responses through stimulation of proinflammatory signaling and antigen presentation, versus the maintenance of immunologic homeostasis through the noninflammatory clearance of immune complexes.

Protective memory CD8 T cell responses are generally associated with the rapid and efficient acquisition of CTL function. However, the ability of memory CD8 T cells to modulate immune responses through interactions with dendritic cells (DCs) during the early states of secondary Ag exposure is poorly understood. In this study, we show that murine Ag-specific CD44(high) CD8 T cells, representing CD8 T cells of the memory phenotype, potently activate DCs to produce high levels of IL-12p70 in conjunction with stimulation of DCs with the TLR 9 ligand, unmethylated CpG DNA. IL-12p70 production was produced predominantly by CD8alpha(+) DCs and plasmacytoid DCs, and mediated by CD8 T cell-derived cytokines IFN-gamma, GM-CSF, TNF-alpha, and surface CD40L. We also find that CD44(high) memory phenotype CD8 T cells were better DC IL-12p70 stimulators than CD44(low) naive phenotype CD8 T cells, and this was attributed to higher levels of IFN-gamma and GM-CSF produced by CD44(high) memory phenotype CD8 T cells during their Ag specific interaction with DCs. Our study identifies CpG DNA as the most effective TLR ligand that cooperates with CD8 T cells for DC IL-12p70 production, and suggests that effectiveness of memory CD8 T cells could be attributed to their ability to rapidly and effectively induce protective Th1 immunity during early stages of pathogen reinfection.

Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.