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Supplementary Table from Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML

Supplementary Figure from Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML

Beauvericin (BEA), a mycotoxin of the enniatin family produced by various toxigenic fungi, has been attributed multiple biological activities such as anti-cancer, anti-inflammatory, and anti-microbial functions. However, effects of BEA on dendritic cells remain unknown so far. Here, we identified effects of BEA on murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow derived dendritic cells (BMDCs) and the underlying molecular mechanisms. BEA potently activates BMDCs as signified by elevated IL-12 and CD86 expression. Multiplex immunoassays performed on myeloid differentiation primary response 88 (MyD88) and toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon beta (TRIF) single or double deficient BMDCs indicate that BEA induces inflammatory cytokine and chemokine production in a MyD88/TRIF dependent manner. Furthermore, we found that BEA was not able to induce IL-12 or IFNβ production in Toll-like receptor 4 (

Background Transcription factors (TFs) control gene expression by direct binding to regulatory regions of target genes but also by impacting chromatin landscapes and modulating DNA accessibility for other TFs. In recent years several TFs have been defined that control cell fate decisions and effector functions in the immune system. Plasmacytoid dendritic cells (pDCs) are an immune cell type with the unique capacity to produce high amounts of type I interferons quickly in response to contact with viral components. Hereby, this cell type is involved in anti-infectious immune responses but also in the development of inflammatory and autoimmune diseases. To date, the global TF reservoir in pDCs early after activation remains to be fully characterized. Results To fill this gap, we have performed a comprehensive analysis in naïve versus TLR9-activated murine pDCs in a time course study covering early timepoints after stimulation (2 h, 6 h, 12 h) integrating gene expression (RNA-Seq) and chromatin landscape (ATAC-Seq) studies. To unravel the biological processes underlying the changes in TF expression on a global scale gene ontology (GO) analyses were performed. We found that 70% of all genes annotated as TFs in the mouse genome (1014 out of 1636) are expressed in pDCs for at least one stimulation time point and are covering a wide range of TF classes defined by their specific DNA binding mechanisms. GO analysis revealed involvement of TLR9-induced TFs in epigenetic modulation, NFκB and JAK-STAT signaling, and protein production in the endoplasmic reticulum. pDC activation predominantly “turned on” the chromatin regions associated with TF genes. Our in silico analyses pointed at the AP-1 family of TFs as less noticed but possibly important players in these cells after activation. AP-1 family members exhibit (1) increased gene expression, (2) enhanced chromatin accessibility in their promoter region, and (3) a TF DNA binding motif that is globally enriched in genomic regions that were found more accessible in pDCs after TLR9 activation. Conclusions In this study we define the complete set of TLR9-regulated TFs in pDCs. Further, this study identifies the AP-1 family of TFs as potentially important but so far less well characterized regulators of pDC function. Supplementary Information The online version contains supplementary material available at 10.1186/s12863-021-00991-2.

Transcription factors (TFs) control gene expression by direct binding to regulatory regions of target genes but also by impacting chromatin landscapes and thereby modulating DNA accessibility for other TFs. To date, the global TF reservoir in plasmacytoid dendritic cells (pDCs), a cell type with the unique capacity to produce unmatched amounts of type I interferons, has not been fully characterized. To fill this gap, we have performed a comprehensive analysis in naïve and TLR9-activated pDCs in a time course study covering early timepoints after stimulation (2h, 6h, 12h) integrating gene expression (RNA-Seq), chromatin landscape (ATAC-Seq) and Gene Ontology studies. We found that 70% of all described TFs are expressed in pDCs for at least one stimulation time point and that activation predominantly “turned on” the chromatin regions associated with TF genes. We hereby define the complete set of TLR9-regulated TFs in pDCs. Further, this study identifies the AP-1 family of TFs as potentially important but so far less well characterized regulators of pDC function.

Pharmacological inhibition of epigenetic enzymes can have therapeutic benefit, particularly against hematological malignancies. While these agents can affect tumor cell growth and proliferation, recent studies have demonstrated that pharmacological de-regulation of epigenetic modifiers may additionally mediate anti-tumor immune responses. Here we discovered a novel mechanism of immune regulation through the inhibition of histone deacetylases (HDACs). In a genetically engineered model of t(8;21) AML, leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor panobinostat required activation of the type I interferon (IFN) signaling pathway. Plasmacytoid dendritic cells (pDCs) were identified as the cells producing type I IFN in response to panobinostat, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated activation of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, while combined treatment of panobinostat and recombinant IFNα improved therapeutic outcomes. These discoveries offer a new therapeutic approach for t(8;21) AML and demonstrate that epigenetic rewiring of pDCs enhances anti-tumor immunity, opening the possibility of exploiting this cell type as a new target for immunotherapy.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. Significance: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397

Type I Interferons (IFNs) are hallmark cytokines produced in immune responses to all classes of pathogens. Type I IFNs can influence dendritic cell (DC) activation, maturation, migration, and survival, but also directly enhance natural killer (NK) and T/B cell activity, thus orchestrating various innate and adaptive immune effector functions. Therefore, type I IFNs have long been considered essential in the host defense against virus infections. More recently, it has become clear that depending on the type of virus and the course of infection, production of type I IFN can also lead to immunopathology or immunosuppression. Similarly, in bacterial infections type I IFN production is often associated with detrimental effects for the host. Although most cells in the body are thought to be able to produce type I IFN, plasmacytoid DCs (pDCs) have been termed the natural “IFN producing cells” due to their unique molecular adaptations to nucleic acid sensing and ability to produce high amounts of type I IFN. Findings from mouse reporter strains and depletion experiments in in vivo infection models have brought new insights and established that the role of pDCs in type I IFN production in vivo is less important than assumed. Production of type I IFN, especially the early synthesized IFNβ, is rather realized by a variety of cell types and cannot be mainly attributed to pDCs. Indeed, the cell populations responsible for type I IFN production vary with the type of pathogen, its tissue tropism, and the route of infection. In this review, we summarize recent findings from in vivo models on the cellular source of type I IFN in different infectious settings, ranging from virus, bacteria, and fungi to eukaryotic parasites. The implications from these findings for the development of new vaccination and therapeutic designs targeting the respectively defined cell types are discussed.

Type I interferons (IFNs) crucially contribute to host survival upon viral infections. Robust expression of type I IFNs (IFN-α/β) and induction of an antiviral state critically depend on amplification of the IFN signal via the type I IFN receptor (IFNAR). A small amount of type I IFN produced early upon virus infection binds the IFNAR and activates a self-enhancing positive feedback loop, resulting in induction of large, protective amounts of IFN-α. Unexpectedly, we found robust, systemic IFN-α expression upon infection of IFNAR knockout mice with the orthomyxovirus Thogoto virus (THOV). The IFNAR-independent IFN-α production required in vivo conditions and was not achieved during in vitro infection. Using replication-incompetent THOV-derived virus-like particles, we demonstrate that IFNAR-independent type I IFN induction depends on viral polymerase activity but is largely independent of viral replication. To discover the cell type responsible for this effect, we used type I IFN reporter mice and identified CD11b + F4/80 + myeloid cells within the peritoneal cavity of infected animals as the main source of IFNAR-independent type I IFN, corresponding to the particular tropism of THOV for this cell type. IMPORTANCE Type I IFNs are crucial for the survival of a host upon most viral infections, and, moreover, they shape subsequent adaptive immune responses. Production of protective amounts of type I IFN critically depends on the positive feedback amplification via the IFNAR. Unexpectedly, we observed robust IFNAR-independent type I IFN expression upon THOV infection and unraveled molecular mechanisms and determined the tissue and cell type involved. Our data indicate that the host can effectively use alternative pathways to induce type I IFN responses if the classical feedback amplification is not available. Understanding how type I IFN can be produced in large amounts independently of IFNAR-dependent enhancement will identify mechanisms which might contribute to novel therapeutic strategies to fight viral pathogens.

Major depressive disorder (MDD) is a severe mood disorder and frequently associated with alterations of the immune system characterized by enhanced levels of circulating pro-inflammatory cytokines and microglia activation in the brain. Increasing evidence suggests that dysfunction of mitochondria may play a key role in the pathogenesis of MDD. Mitochondria are regulators of numerous cellular functions including energy metabolism, maintenance of redox and calcium homeostasis, and cell death and therefore modulate many facets of the innate immune response. In depression-like behavior of rodents, mitochondrial perturbation and release of mitochondrial components have been shown to boost cytokine production and neuroinflammation. On the other hand, pro-inflammatory cytokines may influence mitochondrial functions such as oxidative phosphorylation, production of adenosine triphosphate, and reactive oxygen species, thereby aggravating inflammation. There is strong interest in a better understanding of immunometabolic pathways in MDD that may serve as diagnostic markers and therapeutic targets. Here, we review the interaction between mitochondrial metabolism and innate immunity in the pathophysiology of MDD. We specifically focus on immunometabolic processes that govern microglial and peripheral myeloid cell functions, both cellular components involved in neuroinflammation in depression-like behavior. We finally discuss microglial polarization and associated metabolic states in depression-associated behavior and in MDD.

Protective immunity against intracellular pathogens involves the induction of robust CTL responses. Vaccination with protein Ags establishes such responses only when combined with immune-stimulatory adjuvants. In this study, we compared different adjuvants and identified triphosphate RNA (3pRNA) as especially effective at inducing CTL responses. 3pRNA sensing required IPS-1/MAVS signaling and induced type I IFN in plasmacytoid dendritic cells and macrophages, with the latter being more important for the adjuvant effect. Type I IFN acted on CD11c+ cells, especially on CD8α+ Batf3-dependent dendritic cells. Vaccination with OVA in combination with 3pRNA protected mice from a subsequent OVA-encoding adenovirus infection in a CD8+ cell–dependent manner and more efficiently than other adjuvants. In summary, 3pRNA is a superior adjuvant for CTL activation and might be useful to facilitate antiviral immunization strategies.

Dysregulation of innate immune responses has frequently been reported in stress-associated psychiatric disorders such as major depression. In mice, enhanced circulating cytokine levels as well as altered innate immune cell numbers have been found after stress exposure. In addition, stress-induced recruitment of peripheral monocytes to the brain has been shown to promote anxiety-like behavior. However, it is yet unclear whether specific differences in the innate immune system are associated with stress susceptibility or resilience in mice. Utilizing chronic social defeat, a model of depression and stress vulnerability, we characterized peripheral and brain-invading myeloid cells in stress-susceptible and resilient animals. In all defeated animals, we found reduced percentages of CD11c+ dendritic cells (DCs) by flow cytometry in the spleen when compared to non-defeated controls. Exclusively in susceptible mice conventional DCs of the spleen showed up-regulated expression of MHC class II and co-stimulatory CD80 molecules pointing toward an enhanced maturation phenotype of these cells. Susceptible, but not resilient animals further exhibited an increase in inflammatory Ly6Chi monocytes and higher numbers of spleen-derived CD11b+ cells that produced the proinflammatory cytokine tumor necrosis factor (TNF) upon lipopolysaccharide (LPS) stimulation. Increased percentages of peripheral CD45hi CD11b+ cells immigrated into the brain of defeated mice, regardless of resilience or susceptibility. However, cellular infiltrates in the brain of susceptible mice contained higher percentages of CC chemokine receptor 2 (CCR2+) Ly6Chi monocytes representing an inflammatory phenotype. Thus, we defined specific stress-related immune signatures involving conventional DCs and inflammatory Ly6Chi monocytes in susceptible and resilient mice. Together, our findings suggest an impact of the innate immune system in vulnerability to stress-related disorders such as major depression.

Background The Ras-homologous GTPase Rac1 plays a key role in the regulation of gene expression, cytoskeleton-associated processes and cell death as well as carcinogenesis and inflammation. Here, we investigated the impact of Rac1 signaling on liver-mediated immune homeostasis. Methods We employed a constitutive Alb-Cre-driven rac1 knock-out and a poly I:C-inducible Mx1-Cre-based knock-out model and analyzed cytokine expression profiles in liver and other organs under basal situation and following LPS-induced endotoxemia by flow cytometry, qRT-PCR and immunocytochemistry. Results Constitutive Alb-Cre-driven rac1 knockout in hepatocytes altered the basal distribution and activation of immune cells in the liver and likewise in kidney and lung. Early systemic alterations in cytokine serum levels following LPS treatment remained unaffected by Rac1. Furthermore, lack of Rac1 in hepatocytes of untreated animals shifted the liver to a chronic inflammatory state, as depicted by an enhanced mRNA expression of marker genes related to activated macrophages. Upon acute LPS-induced endotoxemia, increased IL-10 mRNA expression in the liver of Alb-Cre Rac1-deficient mice provided an anti-inflammatory response. Employing a poly I:C-inducible Mx1-Cre-based rac1 knock-out, which allows a more widespread rac1 deletion in both hepatocytes and non-hepatocytes, we observed substantial differences regarding both basal and LPS-stimulated cytokine expression profiles as compared to the Alb-Cre system. Conclusions Rac1-dependent mechanisms in hepatocytes and non-hepatocytes contribute to the maintenance of liver immune homeostasis under basal situation and following LPS-induced endotoxemia. Disturbed Rac1-regulated hepatocyte functions may promote liver damage under pathophysiological situation involving inflammatory stress.

Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo . The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin and whether both nectin-1 and HVEM are involved are not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1-deficient epidermis, entry was strongly reduced. Almost no entry was observed, however, in nectin-1-deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1-deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis, while HVEM has a more limited role. For primary murine keratinocytes, on which nectin-1 acts as a single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we concluded that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, but the restricted presence of HVEM can potentially replace nectin-1 as a receptor, illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo . IMPORTANCE Herpes simplex virus (HSV) can cause a range of diseases in humans, from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV, and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing entry into cells grown in vitro revealed nectin-1 and HVEM as HSV receptors. To explore the contributions of nectin-1 and HVEM to entry into a natural target tissue, we established an ex vivo infection model. Using nectin-1- or HVEM-deficient mice, we demonstrated the distinct involvement of nectin-1 and HVEM for HSV-1 entry into epidermis and characterized the internalization pathways. Such advances in understanding the involvement of receptors in tissue are essential preconditions for unraveling HSV invasion of skin, which in turn will allow the development of antiviral reagents.

Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs) secrete enhanced levels of interleukin (IL) 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP), a model for human polymicrobial sepsis. Bone marrow cells (BMC) were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs) were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR) 2, the receptor for C-C chemokine ligand (CCL) 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs) that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II- to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are activated in the bone marrow and induce functional reprogramming of differentiating BMDC toward an immunosuppressive phenotype. CA Flohe

Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs) secrete enhanced levels of interleukin (IL) 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP), a model for human polymicrobial sepsis. Bone marrow cells (BMC) were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs) were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR) 2, the receptor for C-C chemokine ligand (CCL) 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs) that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II− to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are activated in the bone marrow and induce functional reprogramming of differentiating BMDC toward an immunosuppressive phenotype.

BACKGROUND: Tumor necrosis factor/tumor necrosis factor receptor superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T-cell activation and differentiation toward effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40-CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, herpesvirus entry mediator and lymphotoxin β receptor, may decrease T cell-mediated allogeneic responses. METHODS: A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT. RESULTS: We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T-cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors, herpesvirus entry mediator and lymphotoxin β receptor. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host antidonor short-term cytotoxic response in wild type B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice. CONCLUSION: The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation.

CD8(+) T cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T cell function which are still incompletely understood. Here we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B cell activating transcription factor (BATF) are necessary for sustained CD8(+) T cell effector function. Although Irf4( / ) CD8(+) T cells were initially capable of proliferation IRF4 deficiency resulted in limited CD8(+) T cell responses after infection with the lymphocytic choriomeningitis virus. Consequently Irf4( / ) mice established chronic infections but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T cell function limited immunopathology and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T cell immunity.Cell Death and Differentiation advance online publication 14 February 2014; doi:10.1038/cdd.2014.19.

Immune-mediated effector molecules can limit cancer growth, but lack of sustained immune activation in the tumour microenvironment restricts antitumour immunity. New therapeutic approaches that induce a strong and prolonged immune activation would represent a major immunotherapeutic advance. Here we show that the arenaviruses lymphocytic choriomeningitis virus (LCMV) and the clinically used Junin virus vaccine (Candid#1) preferentially replicate in tumour cells in a variety of murine and human cancer models. Viral replication leads to prolonged local immune activation, rapid regression of localized and metastatic cancers, and long-term disease control. Mechanistically, LCMV induces antitumour immunity, which depends on the recruitment of interferon-producing Ly6C+ monocytes and additionally enhances tumour-specific CD8+ T cells. In comparison with other clinically evaluated oncolytic viruses and to PD-1 blockade, LCMV treatment shows promising antitumoural benefits. In conclusion, therapeutically administered arenavirus replicates in cancer cells and induces tumour regression by enhancing local immune responses., Viruses are promising anti-tumour therapeutics due to induction of an immune response and/or oncolytic activity. Here, Kalkavan et al. show that LCMV replicates in tumour cells, without inducing cell lysis, and that its anti-tumour activity is largely mediated by recruitment of interferon-producing monocytes.

Pro-inflammatory activity and cell-mediated immune responses have been widely observed in patients with major depressive disorder (MDD). Besides their well-known function as antibody-producers, B cells play a key role in inflammatory responses by secreting pro- and anti-inflammatory factors. However, homeostasis of specific B cell subsets has not been comprehensively investigated in MDD. In this study, we characterized circulating B cells of distinct developmental steps including transitional, naive-mature, antigen-experienced switched, and non-switched memory cells, plasmablasts and regulatory B cells by multi-parameter flow cytometry. In a 6-weeks follow-up, circulating B cells were monitored in a small group of therapy responders and non-responders. Frequencies of naive lgD+CD27− B cells, but not lgD+CD27+ memory B cells, were reduced in severely depressed patients as compared to healthy donors (HD) or mildly to moderately depressed patients. Specifically, B cells with immune-regulatory capacities such as CD1d+CD5+ B cells and CD24+CD38hi transitional B cells were reduced in MDD. Also Bm1-Bm5 classification in MDD revealed reduced Bm2’ cells comprising germinal center founder cells as well as transitional B cells. We further found that reduced CD5 surface expression on transitional B cells was associated with severe depression and normalized exclusively in clinical responders. This study demonstrates a compromised peripheral B cell compartment in MDD with a reduction in B cells exhibiting a regulatory phenotype. Recovery of CD5 surface expression on transitional B cells in clinical response, a molecule involved in activation and down-regulation of B cell responses, further points towards a B cell-dependent process in the pathogenesis of MDD.

Upon infection with persistence-prone virus, type I interferon (IFN-I) mediates antiviral activity and also upregulates the expression of programmed death ligand 1 (PD-L1), and this upregulation can lead to CD8+ T-cell exhaustion. How these very diverse functions are regulated remains unknown. This study, using the lymphocytic choriomeningitis virus, showed that a subset of CD169+ macrophages in murine spleen and lymph nodes produced high amounts of IFN-I upon infection. Absence of CD169+ macrophages led to insufficient production of IFN-I, lower antiviral activity and persistence of virus. Lack of CD169+ macrophages also limited the IFN-I-dependent expression of PD-L1. Enhanced viral replication in the absence of PD-L1 led to persistence of virus and prevented CD8+ T-cell exhaustion. As a consequence, mice exhibited severe immunopathology and died quickly after infection. Therefore, CD169+ macrophages are important contributors to the IFN-I response and thereby influence antiviral activity, CD8+ T-cell exhaustion and immunopathology.

Chemokines and their receptors are key regulators of immune cell trafficking and activation. Recent findings suggest that they may also play pathophysiological roles in psychiatric diseases like depression and anxiety disorders. The CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are functionally involved in neuroinflammation as well as anti-infectious and autoimmune responses. However, their influence on behavior remains unknown. Here we characterized the functional role of the CCR4-CCL17 chemokine-receptor axis in the modulation of anxiety-related behavior, locomotor activity, and object exploration and recognition. Additionally, we investigated social exploration of CCR4 and CCL17 knockout mice and wild type (WT) controls. CCR4 knockout (CCR4(-/-)) mice exhibited fewer anxiety-related behaviors in the elevated plus-maze, diminished locomotor activity, exploratory behavior, and social exploration, while their recognition memory was not affected. In contrast, CCL17 deficient mice did not show an altered behavior compared to WT mice regarding locomotor activity, anxiety-related behavior, social exploration, and object recognition memory. In the dark-light and object recognition tests, CCL17(-/-) mice even covered longer distances than WT mice. These data demonstrate a mechanistic or developmental role of CCR4 in the regulation of locomotor and exploratory behaviors, whereas the ligand CCL17 appears not to be involved in the behaviors measured here. Thus, either CCL17 and the alternative ligand CCL22 may be redundant, or CCL22 is the main activator of CCR4 in these processes. Taken together, these findings contribute to the growing evidence regarding the involvement of chemokines and their receptors in the regulation of behavior.

Blocking HVEM–LIGHT interactions on T cells reduces the persistence of antigen-specific memory T cell populations after secondary expansion through decreased Akt activity and loss of Bcl-2 expression., Memory T helper cells (Th cells) play an important role in host defense against pathogens but also contribute to the pathogenesis of inflammatory disorders. We found that a soluble decoy lymphotoxin β receptor (LT-βR)–Fc, which can block tumor necrosis factor (TNF)–related ligands LIGHT (TNFSF14) and LT-αβ binding to the herpesvirus entry mediator (HVEM) and the LT-βR, inhibited the accumulation of memory Th2 cells after antigen encounter and correspondingly reduced inflammatory responses in vivo. Showing that this was a function of the receptor for LIGHT, antigen-specific memory CD4 T cells deficient in HVEM were also unable to persist, despite having a normal immediate response to recall antigen. HVEM−/− memory Th2 cells displayed reduced activity of PKB (protein kinase B; Akt), and constitutively active Akt rescued their survival and restored strong inflammation after antigen rechallenge. This was not restricted to Th2 memory cells as HVEM-deficient Th1 memory cells were also impaired in surviving after encounter with recall antigen. Furthermore, the absence of LIGHT on T cells recapitulated the defect seen with the absence of HVEM, suggesting that activated T cells communicate through LIGHT–HVEM interactions. Collectively, our results demonstrate a critical role of HVEM signals in the persistence of large pools of memory CD4 T cells.

Studies in experimental cerebral malaria (ECM) in mice have identified T cells and TNF family members as critical mediators of pathology. In this study we report a role for LIGHT-lymphotoxin β Receptor (LTβR) signaling in the development of ECM and control of parasite growth. Specific blockade of LIGHT-LTβR, but not LIGHT-herpesvirus entry mediator interactions, abrogated the accumulation of parasites and the recruitment of pathogenic CD8+ T cells and monocytes to the brain during infection without affecting early activation of CD4+ T cells, CD8+ T cells, or NK cells. Importantly, blockade of LIGHT-LTβR signaling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process Ag during infection, as well as reduced systemic cytokine levels when control mice displayed severe ECM symptoms. In summary, we have discovered a novel pathogenic role for LIGHT and LTβR in ECM, identifying this TNF family receptor-ligand interaction as an important immune regulator during experimental malaria.

SummaryPlasmacytoid dendritic cells (PDCs) represent a unique immune cell type specialized in type I interferon (IFN) secretion in response to viral nucleic acids. The molecular control of PDC lineage specification has been poorly understood. We report that basic helix-loop-helix transcription factor (E protein) E2-2/Tcf4 is preferentially expressed in murine and human PDCs. Constitutive or inducible deletion of murine E2-2 blocked the development of PDCs but not of other lineages and abolished IFN response to unmethylated DNA. Moreover, E2-2 haploinsufficiency in mice and in human Pitt-Hopkins syndrome patients was associated with aberrant expression profile and impaired IFN response of the PDC. E2-2 directly activated multiple PDC-enriched genes, including transcription factors involved in PDC development (SpiB, Irf8) and function (Irf7). These results identify E2-2 as a specific transcriptional regulator of the PDC lineage in mice and humans and reveal a key function of E proteins in the innate immune system.

B and T lymphocyte attenuator (BTLA) provides an inhibitory signal to B and T cells. Previously, indirect observations suggested that B7x was a ligand for BTLA. Here we show that BTLA does not bind B7x; instead, we identify herpesvirus entry mediator (HVEM) as the unique BTLA ligand. BTLA bound the most membrane-distal cysteine-rich domain of HVEM, distinct from regions where the ligands LIGHT and lymphotoxin-alpha bound HVEM. HVEM induced BTLA tyrosine phosphorylation and association of the tyrosine phosphatase SHP-2 and repressed antigen-driven T cell proliferation, providing an example of reverse signaling to a non-tumor necrosis factor family ligand. The conservation of the BTLA-HVEM interaction between mouse and human suggests that this system is an important pathway regulating lymphocyte activation and/or homeostasis in the immune response.

Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-gamma and TNF. To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LT beta R pathway. When mice deficient in LT alpha or LT beta were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LT alpha beta heterotrimers in resistance to infection. Indeed, mice deficient in the receptor for LT alpha(1)beta(2) heterotrimers (LT beta R-knockout (KO) mice) also had significantly higher numbers of M. tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection. Furthermore, LT beta R-KO mice were dramatically more susceptible than wild-type mice to i.p. infection with Listeria monocytogenes. Compared with wild-type mice, LT beta R-KO mice had similar transcript levels of TNF and IFN-gamma and recruited similar numbers of CD3(+) T cells inside granulomatous lesions in M. tuberculosis-infected lungs. Flow cytometry revealed that the LT beta R is expressed on pulmonary macrophages obtained after digestion of M. tuberculosis-infected lungs. LT beta R-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections. Since LIGHT-KO mice proved to be equally resistant to M. tuberculosis infection as wild-type mice, these data demonstrate that signaling of LT alpha(1)beta(2) heterotrimers via the LT beta R is an essential prerequisite for containment of intracellular pathogens.

Type I IFNs are critical in initiating protective antiviral immune responses, and plasmacytoid dendritic cells (pDCs) represent a major source of these cytokines. We show that only few pDCs are capable of producing IFN-β after virus infection or CpG stimulation. Using IFNβ/YFP reporter mice, we identify these IFN-β–producing cells in the spleen as a CCR9+CD9− pDC subset that is localized exclusively within the T/B cell zones. IFN-β–producing pDCs exhibit a distinct transcriptome profile, with higher expression of genes encoding cytokines and chemokines, facilitating T cell recruitment and activation. These distinctive characteristics of IFN-β–producing pDCs are independent of the type I IFNR-mediated feedback loop. Furthermore, IFN-β–producing pDCs exhibit enhanced CCR7-dependent migratory properties in vitro. Additionally, they effectively recruit T cells in vivo in a peritoneal inflammation model. We define “professional type I IFN-producing cells” as a distinct subset of pDCs specialized in coordinating cellular immune responses.

Aims Acute rejection of cardiac allografts is a major risk factor limiting survival of heart transplant recipients. Rejection is triggered by dendritic cell (DC) mediated activation of host T cells, amongst others CD4 + T helper (T H )1- and T H 17 cells. The cannabinoid receptor 2 (CB2) is an important modulator of cellular immune responses. However, its role in cardiac allograft rejection has not been studied so far. Main methods Here, we examined the effect of CB2 on cytokine release by mature DCs and its impact on CD4 + T cell differentiation by utilizing in vitro generated bone marrow-derived DCs (BM-DCs) and CD4 + T cells from CB2 knockout ( Cnr2 −/− ) mice. We further assessed the functional role of CB2 in acute allograft rejection using Cnr2 −/− mice in a fully major histocompatibility complex-mismatched mouse cardiac transplantation model. Key findings Cardiac allograft rejection was accelerated in Cnr2 −/− mice compared to wild type recipients. In vitro stimulation of BM-DCs showed enhanced secretion of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF) and the immunomodulatory cytokine TGF-β. Furthermore, secretion of the T H 1/T H 17 promoting cytokines IL-12 and IL-23 was increased in Cnr2 −/− BM-DCs. In addition, Cnr2 −/− CD4 + T cells showed an enhanced capacity to differentiate into interferon (IFN)-γ- or IL-17-producing effector cells. Significance These results demonstrate that CB2 modulates in vitro cytokine responses via DCs and directly via its influence on T H 1/T H 17 differentiation. These findings and the fact that allograft rejection is enhanced in Cnr2 −/− mice suggest that CB2 may be a promising therapeutic target in organ transplantation.

Upon treatment with vesicular stomatitis virus (VSV) particles, plasmacytoid dendritic cells (pDC) are triggered to mount substantial type I IFN responses, whereas myeloid DC (mDC) are only minor producers. Interestingly, bone marrow–derived (BM-)mDC were more vulnerable to infection with enhanced GFP (eGFP)–expressing VSV (VSVeGFP) than BM-pDC. BM-pDC stimulated with wild-type VSV mounted TLR-dependent IFN responses that were independent of RIG-I–like helicase (RLH) signaling. In contrast, in BM-pDC the VSV variant M2 induced particularly high IFN responses triggered in a TLR- and RLH-dependent manner, whereas BM-mDC stimulation was solely RLH-dependent. Importantly, VSVeGFP treatment of BM-pDC derived from IFN-β yellow fluorescent protein (YFP) reporter mice (messenger of IFN-β) resulted in YFP+ and eGFP+ single-positive cells, whereas among messenger of IFN-β–BM-mDC most YFP+ cells were also eGFP+. This observation indicated that unlike mDC, direct virus infection was not required to trigger IFN responses of pDC. VSV-infected BM-mDC triggered BM-pDC to mount significantly higher IFN responses than free virus particles. Stimulation with infected cells enhanced the percentages of pDC subsets expressing either IFN-β+ or IFN-α6+ plus IFN-β+. Irrespective of whether stimulated with free virus or infected cells, IFN induction was dependent on autophagy of pDC, whereas autophagy of the infected mDC was dispensable. Collectively, these results indicated that productive VSV infection was needed to trigger IFN responses of mDC, but not of pDC, and that IFN responses were primarily induced by virus-infected cells that stimulated pDC in a TLR-dependent manner.

Four tetrahydroxanthone dimers (1-4) and four biogenetically related monomers (5-8), including the new derivatives 4-6, were isolated from the endophyte Phomopsis longicolla. The absolute configurations of 2-4 were established for the first time by TDDFT electronic circular dichroism calculations, and that of phomoxanthone A (1) was revised by X-ray crystallography. Phomoxanthone A (1) showed the strongest pro-apoptotic activity when tested against a panel of human cancer cell lines, including cisplatin-resistant cells, whereas it was up to 100-fold less active against healthy blood cells. It was also the most potent activator of murine T lymphocytes, NK cells, and macrophages, suggesting an activation of the immune system in parallel to its pro-apoptotic activity. This dual effect in combating cancer cells could help in fighting resistance during chemotherapy. Preliminary structure-activity studies of isolated compounds and derivatives obtained by semisynthesis (9a-11) hinted at the location of the biaryl axis and the presence of acetyl groups as important structural elements for the biological activity of the studied tetrahydroxanthones.

The TNF superfamily ligand LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator [HVEM], a receptor expressed by T lymphocytes) has been shown to play a role in T cell costimulation and be involved in apoptosis of mononuclear cells. As both T cells and monocytes are key components in the development and progression of experimental autoimmune encephalomyelitis (EAE), we studied the role of LIGHT in EAE. Following immunization with myelin oligodendrocyte glycoprotein peptide (35–55), LIGHT-deficient mice developed severe EAE that resulted in an atypically high mortality rate. Histological examinations revealed intensive activation of microglia/macrophages in the CNS and higher numbers of apoptotic cells within the CNS parenchyma of LIGHT-deficient mice. However, myelin oligodendrocyte glycoprotein peptide–specific CD4+ T cells from LIGHT-deficient mice showed reduced IFN-γ and IL-17 production and migration. Serum levels of reactive nitrogen intermediates and CNS transcripts of several proinflammatory cytokines and chemokines were also substantially decreased in the absence of LIGHT. EAE adoptive transfer experiments and bone marrow chimeras indicated that expression of LIGHT on donor cells is not required for disease induction. However, its expression on CNS host cells is a decisive factor to limit disease progression and tissue damage. Together, these data show that LIGHT expression is crucially involved in controlling activated macrophages/microglia during autoimmune CNS inflammation.

Upon infection with persistence-prone virus, type I interferon (IFN-I) mediates antiviral activity and also upregulates the expression of programmed death ligand 1 (PD-L1), and this upregulation can lead to CD8+ T-cell exhaustion. How these very diverse functions are regulated remains unknown. This study, using the lymphocytic choriomeningitis virus, showed that a subset of CD169+ macrophages in murine spleen and lymph nodes produced high amounts of IFN-I upon infection. Absence of CD169+ macrophages led to insufficient production of IFN-I, lower antiviral activity and persistence of virus. Lack of CD169+ macrophages also limited the IFN-I-dependent expression of PD-L1. Enhanced viral replication in the absence of PD-L1 led to persistence of virus and prevented CD8+ T-cell exhaustion. As a consequence, mice exhibited severe immunopathology and died quickly after infection. Therefore, CD169+ macrophages are important contributors to the IFN-I response and thereby influence antiviral activity, CD8+ T-cell exhaustion and immunopathology.

Objective: To characterize changes in myeloid and lymphoid innate immune cells in patients with relapsing-remitting multiple sclerosis (MS) during a 6-month follow-up after alemtuzumab treatment. Methods: Circulating innate immune cells including myeloid cells and innate lymphoid cells (ILCs) were analyzed before and 6 and 12 months after onset of alemtuzumab treatment. Furthermore, a potential effect on granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)–23 production by myeloid cells and natural killer (NK) cell cytolytic activity was determined. Results: In comparison to CD4 + T lymphocytes, myeloid and lymphoid innate cell subsets of patients with MS expressed significantly lower amounts of CD52 on their cell surface. Six months after CD52 depletion, numbers of circulating plasmacytoid dendritic cells (DCs) and conventional DCs were reduced compared to baseline. GM-CSF and IL-23 production in DCs remained unchanged. Within the ILC compartment, the subset of CD56 bright NK cells specifically expanded under alemtuzumab treatment, but their cytolytic activity did not change. Conclusions: Our findings demonstrate that 6 months after alemtuzumab treatment, specific DC subsets are reduced, while CD56 bright NK cells expanded in patients with MS. Thus, alemtuzumab specifically restricts the DC compartment and expands the CD56 bright NK cell subset with potential immunoregulatory properties in MS. We suggest that remodeling of the innate immune compartment may promote long-term efficacy of alemtuzumab and preserve immunocompetence in patients with MS.

LIGHT (TNFSF14) is a member of the TNF superfamily involved in inflammation and defence against infection. LIGHT signals via two cell-bound receptors; herpes virus entry mediator (HVEM) and lymphotoxin-beta receptor (LTβR). We found that LIGHT is critical for control of hepatic parasite growth in mice with visceral leishmaniasis (VL) caused by infection with the protozoan parasite Leishmania donovani. LIGHT-HVEM signalling is essential for early dendritic cell IL-12/IL-23p40 production, and the generation of IFNγ- and TNF-producing T cells that control hepatic infection. However, we also discovered that LIGHT-LTβR interactions suppress anti-parasitic immunity in the liver in the first 7 days of infection by mechanisms that restrict both CD4+ T cell function and TNF-dependent microbicidal mechanisms. Thus, we have identified distinct roles for LIGHT in infection, and show that manipulation of interactions between LIGHT and its receptors may be used for therapeutic advantage., Author Summary Visceral leishmaniasis (VL) is a potentially fatal human disease caused by the intracellular protozoan parasites Leishmania donovani and L. infantum (chagasi). Parasites infect macrophages throughout the viscera, though the spleen and liver are the major sites of disease. VL is responsible for significant morbidity and mortality in the developing world, particularly in India, Sudan, Nepal, Bangladesh and Brazil. Because of the intrusive techniques required to analyse tissue in VL patients, our current understanding of the host immune response during VL largely derives from studies performed in genetically susceptible mice. We have discovered that mice which are unable to produce a cytokine called LIGHT have poor control of L. donovani infection in the liver, compared with wild-type control animals. In addition, we demonstrated that LIGHT has distinct roles during VL, depending on which of its two major cell-bound receptors it engages. Finally, we identified an antibody that stimulates the lymphotoxin β receptor (one of the LIGHT receptors), that can stimulate anti-parasitic activity during an established infection, thereby identifying this receptor as a therapeutic target during disease.

Individuals with chronic asthma show a progressive decline in lung function that is thought to be due to structural remodeling of the airways characterized by subepithelial fibrosis and smooth muscle hyperplasia. Here we show that the tumor necrosis factor (TNF) family member LIGHT is expressed on lung inflammatory cells after allergen exposure. Pharmacological inhibition of LIGHT using a fusion protein between the IgG Fc domain and lymphotoxin β receptor (LTβR) reduces lung fibrosis, smooth muscle hyperplasia and airway hyperresponsiveness in mouse models of chronic asthma, despite having little effect on airway eosinophilia. LIGHT-deficient mice also show a similar impairment in fibrosis and smooth muscle accumulation. Blockade of LIGHT suppresses expression of lung transforming growth factor-β (TGF-β) and interleukin-13 (IL-13), cytokines implicated in remodeling in humans, whereas exogenous administration of LIGHT to the airways induces fibrosis and smooth muscle hyperplasia, Thus, LIGHT may be targeted to prevent asthma-related airway remodeling.

The pleiotropic cytokine transforming growth factor-β (TGF-β) signals through different pathways among which the Smad- and the MAP-Kinase pathways are already well characterized. Both pathways utilize adaptor/chaperone molecules that facilitate or modulate the intracellular signaling events. Two of the proteins shown in vitro to play a role in Smad-dependent signaling are the TGF-β Receptor Associated Protein-1 (TRAP1, also TGFBRAP1) and its homologue VPS39, also known as Vam6 and TRAP1-Like-Protein (TLP). We generated mice deficient for TRAP1 and VPS39/TLP, respectively. Absence of TRAP1 protein results in death at either of two defined timepoints during embryogenesis, before the blastula stage or during gastrulation, whereas most of the VPS39 deficient mice die before E6.5. Heterozygous mice show no overt phenotype. In summary, our data indicate that TRAP1 and VPS39 are nonredundant and essentially required for early embryonic development.

The innate immune response plays an essential role in the prevention of early viral dissemination. We used the lymphocytic choriomeningitis virus model system to analyze the role of tissue macrophages/Kupffer cells in this process. Our findings demonstrated that Kupffer cells are essential for the efficient capture of infectious virus and for preventing viral replication. The latter process involved activation of Kupffer cells by interferon (IFN)-I and prevented viral spread to neighboring hepatocytes. In the absence of Kupffer cells, hepatocytes were not able to suppress virus replication, even in the presence of IFN-I, leading to prolonged viral replication and severe T cell-dependent immunopathology. CONCLUSION: Tissue-resident macrophages play a crucial role in early viral capture and represent the major liver cell type exhibiting responsiveness to IFN-I and providing control of viral replication.

Type I interferons, a protein family of multiple IFNalphas and a single IFNbeta, initially identified on the basis of their antiviral activities have recently been attributed important roles in bacterial and parasitic infections. To assess the cellular sources of IFNbeta, the IFN produced first in most situations, we created an IFNbeta reporter-knockin mouse, in which yellow fluorescent protein (YFP) is expressed from a bicistronic mRNA linked by an internal ribosomal entry site to the endogenous IFNbeta mRNA. This YFP expression allows spatiotemporal tracking of the initiation of the type I IFN response on a single-cell level. In vitro bone marrow-derived macrophages (BMMPhis) and bone marrow-derived dendritic cells (BMDCs) show IFNbeta production from distinct cell subpopulations in response to defined pathogen compounds. A subpopulation of GMCSF-derived BMDCs produced IFNbeta after poly(I:C), 3'5'-cytidylylguanosine (CpG), or LPS treatment, whereas Flt3-L-cultured plasmacytoid DCs (pDCs) responded mainly to CpG. After poly(I:C) injection in vivo, IFNbeta-producing cells localize to the splenic marginal zone and the lymph node subcapsular sinus. Infection with murine cytomegalovirus (MCMV) induces IFNbeta/YFP expression exclusively in few activated pDCs at the T cell/B cell interface of the splenic white pulp. This IFNbeta/YFP reporter mouse represents a reliable tool for the visualization and characterization of IFNbeta-producing cells in vitro and in vivo.

During an immune response, activated antigen (Ag)-specific T cells condition dendritic cells (DCs) to enhance DC function and survival within the inflamed draining lymph node (LN). It has been difficult to ascertain the role of the tumor necrosis factor (TNF) superfamily member lymphotoxin-alphabeta (LTalphabeta) in this process because signaling through the LTbeta-receptor (LTbetaR) controls multiple aspects of lymphoid tissue organization. To resolve this, we have used an in vivo system where the expression of TNF family ligands is manipulated only on the Ag-specific T cells that interact with and condition Ag-bearing DCs. We report that LTalphabeta is a critical participant required for optimal DC function, independent of its described role in maintaining lymphoid tissue organization. In the absence of LTalphabeta or CD40L on Ag-specific T cells, DC dysfunction could be rescued in vivo via CD40 or LTbetaR stimulation, respectively, suggesting that these two pathways cooperate for optimal DC conditioning.

Induction of type I interferons (IFNs) is one of the earliest immune responses ensuring host survival upon viral infection. It has been shown that robust type I IFN production depends on the positive feedback loop via the IFN-α/β receptor (IFNAR): Most studies show high amounts of type I IFNs in sera of virus-infected wild-type (WT) mice in contrast to IFNAR-deficient mice. Upon infection with the Orthomyxovirus SC35M , a mouse-adapted influenza H7N7 virus, WT mice showed no IFN-α response in serum. Surprisingly, large amounts of IFN-α were detected in sera of SC35M -infected IFNAR-deficient mice, despite their inability to sense IFNs and to activate the positive feedback loop. Although isolated spleen, liver, lung, and brain of SC35M -infected IFNAR-deficient mice showed high viral loads, no local type I IFN production was detectable in these organs. Nevertheless, SC35M infection of IFNAR-deficient IFN-β reporter mice, expressing luciferase under control of the endogenous IFN-β promoter, showed strong luciferase expression in the spleen of IFNAR-deficient animals. To clarify which signaling pathway was involved in IFNAR-independent type I IFN production, IFNAR-deficient mice with no functional Toll-like receptor (TLR) signaling (MyD88-/-Trif-/-IFNAR/) or no functional signaling via RIG-I-like helicases (MAVS-/-IFNAR-/-) were infected. Interestingly, in contrast to IFNAR-deficient and MAVS-/-IFNAR-/- mice, MyD88-/-Trif-/-IFNAR-/- mice showed significantly reduced IFN-α levels in serum indicating that type I IFN production is independent of signaling via RIG-I-like helicases. Collectively, upon systemic SC35M infection, we found a surprisingly strong type I IFN induction which was dependent on TLR-signaling but independent of the IFNAR feedback loop.

Adaptive immune responses require clonal expansion and differentiation of naive T cells into cytokine-secreting effector cells. After priming via signals through the T cell receptor, naive T helper cells express cytokine mRNA but do not secrete cytokine protein without additional T cell receptor stimulation. Here we show that primed T cells demonstrated phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha), a 'collapsed' polysome profile, increased expression of stress-response genes and accumulation of cytoplasmic granules associated with RNA-binding proteins, all features of the integrated stress response. Restimulation of the cells resulted in rapid eIF2alpha dephosphorylation, ribosomal mRNA loading and cytokine secretion. Interference with the function of granule-associated proteins or accumulation of phosphorylated eIF2alpha enhanced release of interleukin 4 during T helper type 2 priming. Therefore, T lymphocytes require components of the integrated stress response to uncouple differentiation from the execution of effector functions.

The importance of lymphotoxin (LT) βR (LTβR) as a regulator of lymphoid organogenesis is well established, but its role in host defense has yet to be fully defined. In this study, we report that mice deficient in LTβR signaling were highly susceptible to infection with murine CMV (MCMV) and early during infection exhibited a catastrophic loss of T and B lymphocytes, although the majority of lymphocytes were themselves not directly infected. Moreover, bone marrow chimeras revealed that lymphocyte survival required LTα expression by hemopoietic cells, independent of developmental defects in lymphoid tissue, whereas LTβR expression by both stromal and hemopoietic cells was needed to prevent apoptosis. The induction of IFN-β was also severely impaired in MCMV-infected LTα−/− mice, but immunotherapy with an agonist LTβR Ab restored IFN-β levels, prevented lymphocyte death, and enhanced the survival of these mice. IFN-αβR−/− mice were also found to exhibit profound lymphocyte death during MCMV infection, thus providing a potential mechanistic link between type 1 IFN induction and lymphocyte survival through a LTαβ-dependent pathway important for MCMV host defense.

Thymocytes depend on the interaction with thymic epithelial cells for the generation of a diverse, nonautoreactive T cell repertoire. In turn, thymic epithelial cells acquire their three-dimensional cellular organization via instructive signals from developing thymocytes. The nature of these signals has been elusive so far. We show that thymocytes and medullary epithelial cells (MECs) communicate via the lymphotoxin beta receptor (LTbetaR) signaling axis. Normal differentiation of thymic MECs requires LTbetaR ligand on thymocytes and LTbetaR together with nuclear factor-kappaB-inducing kinase (Nik) in thymic epithelial cells. Impaired lympho-epithelial cross talk in the absence of the LTbetaR causes aberrant differentiation and reduced numbers of thymic MECs, leads to the retention of mature T lymphocytes, and is associated with autoimmune phenomena, suggesting an unexpected role for LTbetaR signaling in central tolerance induction.