Non-apoptotic TRAIL function modulates NK cell activity during viral infection.

The role of death receptor signaling for pathogen control and infection-associated pathogenesis is multifaceted and controversial. Here, we show that during viral infection, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) modulates NK cell activity independently of its pro-apoptotic function. In mice infected with lymphocytic choriomeningitis virus (LCMV), Trail deficiency led to improved specific CD8+ T-cell responses, resulting in faster pathogen clearance and reduced liver pathology. Depletion experiments indicated that this effect was mediated by NK cells. Mechanistically, TRAIL expressed by immune cells positively and dose-dependently modulates IL-15 signaling-induced granzyme B production in NK cells, leading to enhanced NK cell-mediated T cell killing. TRAIL also regulates the signaling downstream of IL-15 receptor in human NK cells. In addition, TRAIL restricts NK1.1-triggered IFNγ production by NK cells. Our study reveals a hitherto unappreciated immunoregulatory role of TRAIL signaling on NK cells for the granzyme B-dependent elimination of antiviral T cells.

Cancer therapy in mice using a pure population of CD8+ T cell specific to the AH1 tumor rejection antigen.

There is a growing interest in the use of patient-derived T cells for the treatment of various types of malignancies. The expansion of a polyclonal and polyspecific population of tumor-reactive T cells, with a subsequent infusion into the same donor patient, has been implemented, sometimes with positive results. It is not known, however, whether a set of T cells with a single antigen specificity may be sufficient for an effective therapy. To gain more insights in this matter, we used naturally occurring T cells recognizing a retroviral peptide (AH1), which is endogenous in many tumor cell lines of BALB/c origin and which serves as potent tumor rejection antigen. We were able to isolate and expand this rare population of T cells to numbers suitable for therapy experiments in mice (i.e., up to 30 × 106 cells/mouse). After the expansion process, T cells efficiently killed antigen-positive tumor cells in vitro and demonstrated tumor growth inhibition in two syngeneic murine models of cancer. However, AH1-specific T cells failed to induce complete regressions of established tumors. The incomplete activity was associated with a failure of injected T cells to survive in vivo, as only a very limited amount of T cells was found in tumor or secondary lymphoid organs 72 h after injection. These data suggest that future therapeutic strategies based on autologous T cells may require the potentiation of tumor-homing and survival properties of cancer-specific T cells.

Identification of protective B cell antigens of Legionella pneumophila.

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires' disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.

Direct activation of antigen-presenting cells is required for CD8+ T-cell priming and tumor vaccination.

Successful priming of adaptive immune responses is crucially dependent on innate activation signals that convert resting antigen-presenting cells (APCs) into immunogenic ones. APCs expressing the relevant innate pattern recognition receptors can be directly activated by pathogen-associated molecular patterns (PAMPs) to become competent to prime T-cell responses. Alternatively, it has been suggested that APCs could be activated indirectly by proinflammatory mediators synthesized by PAMP-exposed cells. However, data obtained with CD4(+) T cells suggest that inflammatory signals often cannot substitute for direct pattern recognition in APC activation for the priming of T helper responses. To test whether the same is true for CD8(+) T cells, we studied cytotoxic T lymphocyte development in vitro and in mixed chimeric mice in which coexisting APCs can either present a preprocessed model antigen or directly recognize a given PAMP, but not both. We show that indirectly activated APCs promote antigen-specific proliferation of naïve CD8(+) T cells but fail to support their survival and cytotoxic T lymphocyte differentiation. Furthermore, CD8(+) T cells primed by indirectly activated APCs are unable to reject tumors. Thus, inflammation cannot substitute for direct recognition of single PAMPs in CD8(+) T-cell priming. These findings have important practical implications for vaccine design, indicating that adjuvants must be judiciously chosen to trigger the relevant pattern recognition receptors in APCs.

Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting.

The protective effect of antibodies (Abs) is generally attributed to neutralization or complement activation. Using Legionella pneumophila and Mycobacterium bovis bacillus Calmette-Guérin as a model, we discovered an additional mechanism of Ab-mediated protection effective against intracellular pathogens that normally evade lysosomal fusion. We show that Fc receptor (FcR) engagement by Abs, which can be temporally and spatially separated from bacterial infection, renders the host cell nonpermissive for bacterial replication and targets the pathogens to lysosomes. This process is strictly dependent on kinases involved in FcR signaling but not on host cell protein synthesis or protease activation. Based on these findings, we propose a mechanism whereby Abs and FcR engagement subverts the strategies by which intracellular bacterial pathogens evade lysosomal degradation.

Inflammatory signals in dendritic cell activation and the induction of adaptive immunity.

Pathogen invasion induces a rapid inflammatory response initiated through the recognition of pathogen-derived molecules by pattern recognition receptors (PRRs) expressed on both immune and non-immune cells. The initial wave of pro-inflammatory cytokines and chemokines limits pathogen spread and recruits and activates immune cells to eradicate the invaders. Dendritic cells (DCs) are responsible for initiating a subsequent phase of immunity, dominated by the action of pathogen-specific T and B cells. As for the early pro-inflammatory response, DC activation is triggered by PRR signals. These signals convert resting DCs into potent antigen-presenting cells capable of promoting the expansion and effector differentiation of naive pathogen-specific T cells. However, it has been argued that signals from PRRs are not a prerequisite for DC activation and that pro-inflammatory cytokines have the same effect. Although this may appear like an efficient way to expand the number of DCs that initiate adaptive immunity, evidence is accumulating that DCs activated indirectly by inflammatory cytokines are unable to induce functional T-cell responses. Here, we review the differences between PRR-triggered and cytokine-induced DC activation and speculate on a potential role for DCs activated by inflammatory signals in tolerance induction rather than immunity.

Th cells act via two synergistic pathways to promote antiviral CD8+ T cell responses.

The mechanisms of how Th cells promote CD8(+) T cell responses during viral infections are largely unknown. In this study, we unraveled the mechanisms of T cell help for CD8(+) T cell responses during vaccinia virus infection. Our results demonstrate that Th cells promote vaccinia virus-specific CD8(+) T cell responses via two interconnected synergistic pathways: First, CD40L expressed by activated CD4(+) T cells instructs dendritic cells to produce bioactive IL-12p70, which is directly sensed by Ag-specific CD8(+) T cells, resulting in increased IL-2Rα expression. Second, Th cells provide CD8(+) T cells with IL-2, thereby enhancing their survival. Thus, Th cells are at the center of an important communication loop with a central role for IL-2/IL-2R and bioactive IL-12.

Influenza- and MCMV-induced memory CD8 T cells control respiratory vaccinia virus infection despite residence in distinct anatomical niches.

Induction of memory CD8 T cells residing in peripheral tissues is of interest for T cell-based vaccines as these cells are located at mucosal and barrier sites and can immediately exert effector functions, thus providing protection in case of local pathogen encounter. Different memory CD8 T cell subsets patrol peripheral tissues, but it is unclear which subset is superior in providing protection upon secondary infections. We used influenza virus to induce predominantly tissue resident memory T cells or cytomegalovirus to elicit a large pool of effector-like memory cells in the lungs and determined their early protective capacity and mechanism of reactivation. Both memory CD8 T cell pools have unique characteristics with respect to their phenotype, localization, and maintenance. However, these distinct features do not translate into different capacities to control a respiratory vaccinia virus challenge in an antigen-specific manner, although differential activation mechanisms are utilized. While influenza-induced memory CD8 T cells respond to antigen by local proliferation, MCMV-induced memory CD8 T cells relocate from the vasculature into the tissue in an antigen-independent and partially chemokine-driven manner. Together these results bear relevance for the development of vaccines aimed at eliciting a protective memory CD8 T cell pool at mucosal sites.

Asymmetric cell division shapes naive and virtual memory T-cell immunity during ageing.

Efficient immune responses rely on heterogeneity, which in CD8+ T cells, amongst other mechanisms, is achieved by asymmetric cell division (ACD). Here we find that ageing, known to negatively impact immune responses, impairs ACD in murine CD8+ T cells, and that this phenotype can be rescued by transient mTOR inhibition. Increased ACD rates in mitotic cells from aged mice restore the expansion and memory potential of their cellular progenies. Further characterization of the composition of CD8+ T cells reveals that virtual memory cells (TVM cells), which accumulate during ageing, have a unique proliferation and metabolic profile, and retain their ability to divide asymmetrically, which correlates with increased memory potential. The opposite is observed for naive CD8+ T cells from aged mice. Our data provide evidence on how ACD modulation contributes to long-term survival and function of T cells during ageing, offering new insights into how the immune system adapts to ageing.

A novel role for neutrophils as critical activators of NK cells.

Neutrophils are essential players in innate immune responses to bacterial infection. Despite the striking resistance of Legionella pneumophila (Lpn) to bactericidal neutrophil function, neutrophil granulocytes are important effectors in the resolution of legionellosis. Indeed, mice depleted of neutrophils were unable to clear Lpn due to a lack of the critical cytokine IFN-gamma, which is produced by NK cells. We demonstrate that this can be ascribed to a previously unappreciated role of neutrophils as major NK cell activators. In response to Lpn infection, neutrophils activate caspase-1 and produce mature IL-18, which is indispensable for the activation of NK cells. Furthermore, we show that the IL-12p70 response in Lpn-infected neutropenic mice is also severely reduced and that the Lpn-induced IFN-gamma production by NK cells is strictly dependent on IL-12. However, since dendritic cells, and not neutrophils, are the source of Lpn-induced IL-12, its paucity is a consequence of the absence of IFN-gamma produced by NK cells rather than the absence of neutrophils per se. Therefore, neutrophil-derived IL-18, in combination with dendritic cell-produced IL-12, triggers IFN-gamma synthesis in NK cells in Lpn-infected mice. We propose a novel central role for neutrophils as essential IL-18 producers and hence NK cell "helpers" in bacterial infection.

Chronic viral infections impinge on naive bystander CD8 T cells.

INTRODUCTION: Epidemiological data suggest that persistent viral infections impair immune homeostasis and immune responsiveness. Previous studies showed that chronic virus infections negatively impact bystander T-cell differentiation and memory formation but there is limited knowledge of how chronic virus infections impinge on heterologous naive T-cell populations. METHODS: We used adoptive transfer of naive CD8 T cells with defined nonviral specificity into hosts, which were subsequently chronically infected with lymphocytic choriomeningitis virus, followed by analyses of numeric, phenotypic, and functional changes provoked in the chronically infected host. RESULTS: We demonstrate that chronic virus infections have a profound effect on the number and phenotype of naive bystander CD8 T cells. Moreover, primary expansion upon antigen encounter was severely compromised in chronically infected hosts. However, when naive bystander CD8 T cells were transferred from the chronically infected mice into naive hosts, they regained their expansion potential. Conversely, when chronically infected hosts were supplied with additional antigen-presenting cells (APCs), primary expansion of the naive CD8 T cells was restored to levels of the uninfected hosts. CONCLUSIONS: Our results document numeric, phenotypic, and functional adaptation of bystander naive CD8 T cells during nonrelated chronic viral infection. Their functional impairment was only evident in the chronically infected host, indicating that T-cell extrinsic factors, in particular the quality of priming APCs, are responsible for the impaired function of naive bystander T cells in the chronically infected hosts.

Antagonism of interferon signaling by fibroblast growth factors promotes viral replication.

Fibroblast growth factors (FGFs) play key roles in the pathogenesis of different human diseases, but the cross-talk between FGFs and other cytokines remains largely unexplored. We identified an unexpected antagonistic effect of FGFs on the interferon (IFN) signaling pathway. Genetic or pharmacological inhibition of FGF receptor signaling in keratinocytes promoted the expression of interferon-stimulated genes (ISG) and proteins in vitro and in vivo. Conversely, FGF7 or FGF10 treatment of keratinocytes suppressed ISG expression under homeostatic conditions and in response to IFN or poly(I:C) treatment. FGF-mediated ISG suppression was independent of IFN receptors, occurred at the transcriptional level, and required FGF receptor kinase and proteasomal activity. It is not restricted to keratinocytes and functionally relevant, since FGFs promoted the replication of herpes simplex virus I (HSV-1), lymphocytic choriomeningitis virus, and Zika virus. Most importantly, inhibition of FGFR signaling blocked HSV-1 replication in cultured human keratinocytes and in mice. These results suggest the use of FGFR kinase inhibitors for the treatment of viral infections.

Rapid expansion of Treg cells protects from collateral colitis following a viral trigger.

Foxp3+ regulatory T (Treg) cells are essential for maintaining peripheral tolerance and preventing autoimmunity. While genetic factors may predispose for autoimmunity, additional environmental triggers, such as viral infections, are usually required to initiate the onset of disease. Here, we show that viral infection with LCMV results in type I IFN-dependent Treg cell loss that is rapidly compensated by the conversion and expansion of Vß5+ conventional T cells into iTreg cells. Using Vß5-deficient mice, we show that these Vß5+ iTreg cells are dispensable for limiting anti-viral immunity. Rather, the delayed replenishment of Treg cells in Vß5-deficient mice compromises suppression of microbiota-dependent activation of CD8+ T cells, resulting in colitis. Importantly, recovery from clinical symptoms in IBD patients is marked by expansion of the corresponding Vß2+ Treg population in humans. Collectively, we provide a link between a viral trigger and an impaired Treg cell compartment resulting in the initiation of immune pathology.

Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner.

Chronic viral infections are widespread among humans, with ∼8-12 chronic viral infections per individual, and there is epidemiological proof that these impair heterologous immunity. We studied the impact of chronic LCMV infection on the phenotype and function of memory bystander CD8+ T cells. Active chronic LCMV infection had a profound effect on total numbers, phenotype, and function of memory bystander T cells in mice. The phenotypic changes included up-regulation of markers commonly associated with effector and exhausted cells and were induced by IL-6 in a STAT1-dependent manner in the context of chronic virus infection. Furthermore, bystander CD8 T cell functions were reduced with respect to their ability to produce inflammatory cytokines and to undergo secondary expansion upon cognate antigen challenge with major cell-extrinsic contributions responsible for the diminished memory potential of bystander CD8+ T cells. These findings open new perspectives for immunity and vaccination during chronic viral infections.

CSF1R-dependent myeloid cells are required for NK­mediated control of metastasis.

Myeloid leukocytes are essentially involved in both tumor progression and control. We show that neo-adjuvant treatment of mice with an inhibitor of CSF1 receptor (CSF1R), a drug that is used to deplete tumor-associated macrophages, unexpectedly promoted metastasis. CSF1R blockade indirectly diminished the number of NK cells due to a paucity of myeloid cells that provide the survival factor IL-15 to NK cells. Reduction of the number of NK cells resulted in increased seeding of metastatic tumor cells to the lungs but did not impact on progression of established metastases. Supplementation of mice treated with CSF1R-inhibitor with IL-15 restored numbers of NK cells and diminished metastasis. Our data suggest that CSF1R blockade should be combined with administration of IL-15 to reduce the risk of metastasis.

Post-translational modification of {alpha}-dystroglycan is not critical for lymphocytic choriomeningitis virus receptor function in vivo.

alpha-Dystroglycan (alpha-DG) is a ubiquitously expressed molecule that has been identified as a cellular receptor for lymphocytic choriomeningitis virus (LCMV) and other arenaviruses. Recently, it was demonstrated that LCMV receptor function is critically dependent on post-translational modifications, namely glycosylation. In particular, it was shown that O-mannosylation, a rare type of mammalian O-linked glycosylation, is important in determining the binding of LCMV to its cellular receptor. All studies carried out so far showed a dependence on glycosylation in LCMV receptor function in vitro. This work extended these studies to two in vivo models of alpha-DG hypoglycosylation. The results confirm earlier findings on the in vitro dependence of carbohydrate modifications in LCMV receptor function. However, experiments in animal models showed that this dependence was only very weak in vivo. It is likely that alternative receptors or alternative entry pathways may account for this attenuated in vivo phenotype.

Induction and protective role of antibodies in Legionella pneumophila infection.

Legionella pneumophila (Lpn) is a ubiquitous Gram-negative bacterium found in aquatic environments and is the causative agent of Legionnaires' disease, a severe form of pneumonia. We have used Lpn-permissive A/J mice as a model to analyze the B cell response upon intravenous (i.v.) and intranasal (i.n.) infection with Lpn. A strong antibody (Ab) response was observed upon i.v. infection with wild-type (WT) Lpn and an icmT mutant strain, which is unable to replicate within permissive host cells. In contrast to i.v. infection, only WT but not icmT mutant Lpn was able to induce specific Ab responses upon i.n. infection. After primary i.n. infection with WT Lpn, a strict compartmentalization of Lpn-specific Ab isotypes was observed, as IgG was found exclusively systemically, while IgA was detectable only locally in the lung. Regardless of the infection route, isotype switching to IgG and to IgA was strictly dependent on CD4+ T cells, whereas IgM production was completely Th-independent. Finally, we analyzed the protective capacity of the Lpn-specific Ab response. Actively or passively immunized mice or mice that were infected with opsonized Lpn had 50-100-fold reduced bacterial titers compared to naive animals, clearly demonstrating the capacity of Ab to protect against infection with Lpn.

MyD88-dependent IFN-gamma production by NK cells is key for control of Legionella pneumophila infection.

Legionella pneumophila (Lpn) is a ubiquitous Gram-negative bacterium in aquatic systems and an opportunistic intracellular pathogen in immunocompromised humans causing a severe pneumonia known as Legionnaires' disease. Using a mouse model, we investigated molecular and cellular players in the innate immune response to infection with Lpn. We observed robust levels of inflammatory cytokines in the serum upon intranasal or i.v. infection with live, virulent Lpn, but not with inactivated or avirulent bacteria lacking the Icm/Dot type IV secretion system. Interestingly, Lpn-induced serum cytokines were readily detectable regardless of the capacity of Icm/Dot-proficient Lpn to replicate in host cells and the Lpn permissiveness of the host mice. We found NK cell-derived IFN-gamma to be the key cytokine in the resolution of Lpn infection, whereas type I IFNs did not appear to play a major role in our model. Accordingly, NK cell-depleted or IFN-II-R-deficient mice carried severely increased bacterial burdens or failed to control Lpn infection, respectively. Besides the dependence of inflammatory cytokine induction on Lpn virulence, we also demonstrate a strict requirement of MyD88 for this process, suggesting the involvement of TLRs in the recognition of Lpn. However, screening of several TLR-deficient hosts did not reveal a master TLR responsible for the sensing of an Lpn infection, but provided evidence for either redundancy of individual TLRs in Lpn recognition or TLR-independent induction of inflammatory responses.

Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function.

Dendritic cells (DCs) can be activated directly by triggering of receptors for pathogens or, indirectly, by exposure to inflammatory signals. It remains unclear, however, whether the two pathways result in qualitatively similar DCs or lead to equivalent adaptive immune responses. Here we report that indirect activation by inflammatory mediators generated DCs that supported CD4(+) T cell clonal expansion but failed to direct T helper cell differentiation. In contrast, exposure to pathogen components resulted in fully activated DCs that promoted T helper responses. These results indicate that inflammation cannot substitute for contact with pathogen components in DC activation and suggest that the function of pattern recognition by DCs is to couple the quality of the adaptive immune response to the nature of the pathogen.

Newly activated T cells promote maturation of bystander dendritic cells but not IL-12 production.

The activation of dendritic cells (DC) leads to increased costimulatory activity (termed DC maturation) and, in some instances, production of immunomodulatory cytokines such as IL-12. Both innate and T cell-derived signals can promote DC activation but it is unclear to what extent the two classes of stimuli are interchangeable or regulate distinct aspects of DC function. In this study, we show that signals from newly activated CD4(+) T cells cannot initiate IL-12 synthesis although they can amplify secretion of bioactive IL-12 p70 by DC exposed to an appropriate innate stimulus. This occurs exclusively in cis and does not influence IL-12 synthesis by bystander DC that do not present Ag. In marked contrast, signals from newly activated CD4(+) T cells can induce an increase in DC costimulatory activity in the absence of any innate priming. This occurs both in cis and in trans, affecting all DC in the microenvironment, including those that do not bear specific Ag. Consistent with the latter, we show that newly activated CD4(+) T cells in vivo can deliver "help" in trans, effectively lowering the number of MHC/peptide complexes required for proliferation of third-party naive CD4(+) T cells recognizing Ag on bystander DC. These results demonstrate that DC maturation and cytokine production are regulated distinctly by innate stimuli vs signals from CD4(+) T cells and reveal a process of trans activation of DC without secretion of polarizing cytokines that takes place during T cell priming and may be involved in amplifying immune responses.