Sophie Detienne

Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.

Phosphoinositide 3‐kinases (PI3K) are known to regulate Toll‐like receptor (TLR)‐mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll‐IL‐1 receptor domain‐containing adapter‐inducing IFN‐β (TRIF)‐dependent signaling, which induces IFN‐β gene expression downstream of TLR3 and TLR4. First, treatment of monocyte‐derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN‐β expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA‐binding activity of NF‐κB, but not interferon response factor (IRF)‐3, the key transcription factors required for TLR‐mediated IFN‐β synthesis. In parallel, wortmannin‐treated DC exhibited enhanced levels of IκB kinase (IKK)‐α/β phosphorylation and IκB‐α degradation with a concomitant increase in NF‐κB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF‐κB‐dependent promoters as well as IFN‐β promoter activities without interfering with transcription at the positive regulatory domain III‐I. Furthermore, wortmannin enhanced NF‐κB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF‐mediated NF‐κB transcriptional activity. Finally, in co‐immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF‐dependent NF‐κB activity, and thereby increases IFN‐β synthesis elicited by TLR3 or TLR4 ligands.

Critical influence of natural regulatory CD25+ T cells on the fate of allografts in the absence of immunosuppression.

Background. Allografts are occasionally accepted in the absence of immunosuppression. Because naturally occurring CD4+CD25+ regulatory T cells (natural CD25+ Treg cells) have been shown to inhibit allograft rejection, we investigated their influence on the outcome of allografts in nonimmunosuppressed mouse recipients. Methods. We compared survival times of male CBA/Ca skin grafts in female CBA/Ca recipients expressing a transgenic anti-HY T-cell receptor on a RAG-1+/+ (A1[M]RAG+) or a RAG-1−/− (A1[M]RAG−) background. Depletion of natural CD25+ Treg cells in A1[M]RAG+ mice was achieved by in vivo administration of the PC61 monoclonal antibody. The influence of natural CD25+ Treg cells on the fate of major histocompatibility complex class II-mismatched (C57BL/6× bm12)F1 skin or bm12 heart transplants in C57BL/6 recipients was also assessed. Finally, we investigated the impact of natural CD25+ Treg cells on the production of T-helper (Th)1 and Th2 cytokines in mixed lymphocyte cultures between C57BL/6 CD4+ CD25− T cells as responders and bm12 or (C57BL/6× bm12)F1 antigen-presenting cells as stimulators. Results. Male allografts were spontaneously accepted by female A1(M)RAG+ mice but readily rejected by female A1(M)RAG+ mice depleted of natural CD25+ Treg cells by pretreatment with the PC61 monoclonal antibody. Depletion of CD25+ Treg cells also enhanced eosinophil-determined rejection of (C57BL/6× bm12)F1 skin grafts or bm12 cardiac grafts in C57BL/6 recipients. Finally, natural CD25+ Treg cells inhibited the production of interleukin (IL)-2, interferon-γ, IL-5, and IL-13 in mixed lymphocyte culture in a dose-dependent manner. Conclusion. Natural CD25+ Treg cells control Th1- and Th2-type allohelper T-cell responses and thereby influence the fate of allografts in nonimmunosuppressed recipients.

Central Role of CD169 + Lymph Node Resident Macrophages in the Adjuvanticity of the QS-21 Component of AS01

Saponins represent a promising class of vaccine adjuvant. Together with the TLR4-ligand MPL, QS-21 is part of the Adjuvant System AS01, a key component of the malaria and zoster candidate vaccines that display demonstrated clinical efficacy. However, the mechanism of action of QS-21 in this liposomal formulation is poorly understood. Upon intra-muscular immunisation, we observed that QS-21 rapidly accumulated in CD169+ resident macrophages of the draining lymph node where it elicited a local innate immune response. Depletion of these cells abrogated QS-21-mediated innate cell recruitment to the lymph node, dendritic cell (DC) phenotypic maturation as well as the adjuvant effect on T-cell and antibody responses to co-administered antigens. DCs rather than lymph node-resident macrophages were directly involved in T-cell priming by QS-21, as revealed by the decrease in antigen-specific T-cell response in Batf3−/− mice. Further analysis showed that the adjuvant effect of QS-21 depended on the integration of Caspase-1 and MyD88 pathways, at least in part through the local release of HMGB1. Taken together, this work unravels the key role of lymph node sentinel macrophage in controlling the adjuvant effect of a molecule proven to improve vaccine response in humans.

Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity.

Combining immunostimulants in adjuvants can improve the quality of the immune response to vaccines. Here, we report a unique mechanism of molecular and cellular synergy between a TLR4 ligand, 3-O-desacyl-4’-monophosphoryl lipid A (MPL), and a saponin, QS-21, the constituents of the Adjuvant System AS01. AS01 is part of the malaria and herpes zoster vaccine candidates that have demonstrated efficacy in phase III studies. Hours after injection of AS01-adjuvanted vaccine, resident cells, such as NK cells and CD8+ T cells, release IFNγ in the lymph node draining the injection site. This effect results from MPL and QS-21 synergy and is controlled by macrophages, IL-12 and IL-18. Depletion strategies showed that this early IFNγ production was essential for the activation of dendritic cells and the development of Th1 immunity by AS01-adjuvanted vaccine. A similar activation was observed in the lymph node of AS01-injected macaques as well as in the blood of individuals receiving the malaria RTS,S vaccine. This mechanism, previously described for infections, illustrates how adjuvants trigger naturally occurring pathways to improve the efficacy of vaccines.Adjuvants: Vaccine components working in synergy to improve beneficial effects of vaccinationA mechanism is revealed by which vaccine components co-operate to stimulate the immune system and improve vaccine efficacy. Some vaccines are formulated with adjuvants—compounds that induce a greater immune response to the vaccine and help to elicit greater protection against future infections. Arnaud Didierlaurent and his team of researchers at GSK Vaccines, Belgium, demonstrate that the two immunostimulants in the adjuvant AS01, used in several recently developed vaccines, works in tandem to trigger the activation of important immune system moderators. The synergistic effect of the immunostimulants modulate specific immune cells at the site of the vaccination to better prepare the body against future infection. Studies such as this allow us to better understand how vaccines work and lay the foundation for more informed research into future vaccine development.

Lysosome-Dependent Activation of Human Dendritic Cells by the Vaccine Adjuvant QS-21

The adjuvant properties of the saponin QS-21 have been known for decades. It is a component of the Adjuvant System AS01 that is used in several vaccine candidates. QS-21 strongly potentiates both cellular and humoral immune responses to purified antigens, yet how it activates immune cells is largely unknown. Here, we report that QS-21 directly activated human monocyte-derived dendritic cells (moDCs) and promoted a pro-inflammatory transcriptional program. Cholesterol-dependent QS-21 endocytosis followed by lysosomal destabilization and Syk kinase activation were prerequisites for this response. Cathepsin B, a lysosomal cysteine protease, was essential for moDC activation in vitro and contributed to the adjuvant effects of QS-21 in vivo. Collectively, these findings provide new insights into the pathways involved in the direct activation of antigen-presenting cells by a clinically relevant QS-21 formulation.

Activation of the endoplasmic reticulum stress sensor IRE1α by the vaccine adjuvant AS03 contributes to its immunostimulatory properties

The oil-in-water emulsion Adjuvant System 03 (AS03) is one of the few adjuvants used in licensed vaccines. Previous work indicates that AS03 induces a local and transient inflammatory response that contributes to its adjuvant effect. However, the molecular mechanisms involved in its immunostimulatory properties are ill-defined. Upon intramuscular injection in mice, AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. In vitro, these modifications were associated with profound changes in lipid composition, alteration of endoplasmic reticulum (ER) morphology and activation of the unfolded protein response pathway. In vivo, treatment with a chemical chaperone or deletion of the ER stress sensor kinase IRE1α in myeloid cells decreased AS03-induced cytokine production and its capacity to elicit high affinity antigen-specific antibodies. In summary, our results indicate that IRE1α is a sensor for the metabolic changes induced by AS03 in monocytic cells and may constitute a canonical pathway that could be exploited for the design of novel vaccine adjuvants.Adjuvants: ER stress is critical for adjuvant efficacyAdjuvants are the ‘secret sauce’ of vaccines—by stimulating the innate arm of the immune system they potentiate activation of adaptive immunity; however, their mode of action is incompletely understood. A team led by Stanislas Goriely at the Université Libre de Bruxelles studied the functional basis of a major class of clinically-applied adjuvant—AS03—an oil-in-water emulsion adjuvant used in human influenza vaccines. Using an in vivo mouse model and cell lines they observe that AS03 triggered downregulation of genes controlling lipid metabolism. At the same time there was an increase in an ER-stress signature likely as a result of accumulation of intracellular lipid content in myeloid cells such as macrophages. ER stress was critical for the production of inflammatory cytokines by macrophages and the subsequent generation of robust and specific antibody responses following vaccination. Selective enhancement of ER stress might therefore lead to superior vaccines while minimizing side effects.

The cholinergic anti-inflammatory pathway delays TLR-induced skin allograft rejection in mice: cholinergic pathway modulates alloreactivity.

Activation of innate immunity through Toll-like receptors (TLR) can abrogate transplantation tolerance by revealing hidden T cell alloreactivity. Separately, the cholinergic anti-inflammatory pathway has the capacity to dampen macrophage activation and cytokine release during endotoxemia and ischemia reperfusion injury. However, the relevance of the α7 nicotinic acetylcholine receptor (α7nAChR)-dependent anti-inflammatory pathway in the process of allograft rejection or maintenance of tolerance remains unknown. The aim of our study is to investigate whether the cholinergic pathway could impact T cell alloreactivity and transplant outcome in mice. For this purpose, we performed minor-mismatched skin allografts using donor/recipient combinations genetically deficient for the α7nAChR. Minor-mismatched skin grafts were not rejected unless the mice were housed in an environment with endogenous pathogen exposure or the graft was treated with direct application of imiquimod (a TLR7 ligand). The α7nAChR-deficient recipient mice showed accelerated rejection compared to wild type recipient mice under these conditions of TLR activation. The accelerated rejection was associated with enhanced IL-17 and IFN-γ production by alloreactive T cells. An α7nAChR-deficiency in the donor tissue facilitated allograft rejection but not in recipient mice. In addition, adoptive T cell transfer experiments in skin-grafted lymphopenic animals revealed a direct regulatory role for the α7nAChR on T cells. Taken together, our data demonstrate that the cholinergic pathway regulates alloreactivity and transplantation tolerance at multiple levels. One implication suggested by our work is that, in an organ transplant setting, deliberate α7nAChR stimulation of brain dead donors might be a valuable approach for preventing donor tissue inflammation prior to transplant.

Author Correction: Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity

In the original version of the article, Fig. 2b was mislabelled. The second row was incorrectly labelled as “MiMPL > MiPBS and MiQS ≈ MPBS” this has now been corrected to “MiQS-21 > MiPBS and MiMPL ≈ MiPBS”. The third row was incorrectly labelled as “MiQS21 > MiPBS and MiMPL ≈ MiPBS” this has now been corrected to “MiMPL > MiPBS and MiQS-21 ≈ MiPBS”. These errors have now been corrected in the PDF and HTML versions of this article.