Induction of human humoral immune responses in a novel HLA-DR-expressing transgenic NOD/Shi-scid/γcnull mouse

Mounting evidence has demonstrated that NOD-Shi/scid/γc(null) (NOG) mice are one of the most suitable mouse strains for humanized mouse technologies, in which various human cells or tissues can be engrafted without rejection and autonomously maintained. We have characterized and analyzed various features of the human immune system reconstituted in NOG mice by transplanting human hematopoietic stem cells (hu-HSC). One of the problems of the quasi-immune system in these hu-HSC NOG mice is that the quality of immune responses is not always sufficient, as demonstrated by the lack of IgG production in response to antigen challenge. In this study, we established a novel transgenic NOG sub-strain of mice bearing the HLA-DRA and HLA-DRB1:0405 genes, which specifically expresses HLA-DR4 molecules in MHC II-positive cells. This mouse strain enabled us to match the haplotype of HLA-DR between the recipient mice and human donor HSC. We demonstrated that T-cell homeostasis was differentially regulated in HLA-matched hu-HSC NOG mice compared with HLA-mismatched control mice, and antibody class switching was induced after immunization with exogenous antigens in HLA-matched mice. This novel mouse strain improves the reconstituted human immune systems that develop in humanized mice and will contribute to future studies of human humoral immune responses.     

Human B cells fail to secrete type I interferons upon cytoplasmic DNA exposure

Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion.Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence.     

Polyvalent design in the cGAS-STING pathway

Polyvalent interactions mediate the formation of higher-order macromolecular assemblies to improve the sensitivity, specificity, and temporal response of biological signals. In host defense, innate immune pathways recognize danger signals to alert host of insult or foreign invasion, while limiting aberrant activation from auto-immunity and cellular senescence. Of recent attention are the unique higher-order assemblies in the cGAS-STING pathway. Natural stimulation of cGAS enzymes by dsDNA induces phase separation and enzymatic activation for switchlike production of cGAMP. Subsequent binding of cGAMP to STING induces oligomerization of STING molecules, offering a scaffold for kinase assembly and signaling transduction. Additionally, the discovery of PC7A, a synthetic polymer which activates STING through a non-canonical biomolecular condensation, illustrates the engineering design of agonists by polyvalency principles. Herein, we discuss a mechanistic and functional comparison of natural and synthetic agonists to advance our understanding in STING signaling and highlight the principles of polyvalency in innate immune activation. The combination of exogenous cGAMP along with synthetic PC7A stimulation of STING offers a synergistic strategy in spatiotemporal orchestration of the immune milieu for a safe and effective immunotherapy against cancer.     

Emerging role of the cGAS-STING signaling pathway in autoimmune diseases: Biologic function,mechanisms and clinical prospection

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS–STING) signaling pathway, as vital component of innate immune system, acts a vital role in distinguishing invasive pathogens and cytosolic DNA. Cytosolic DNA sensor cGAS first binds to cytosolic DNA and catalyzes synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which is known as the second messenger. Next, cGAMP activates the adaptor protein STING, triggering a molecular chain reaction to stimulate cytokines including interferons (IFNs). Recently, many researches have revealed that the regulatory role of cGAS-STING signaling pathway in autoimmune diseases (AIDs) such as Rheumatoid arthritis (RA), Aicardi Goutières syndrome (AGS) and systemic lupus erythematosus (SLE). Moreover, accumulated evidence have showed inhibition of the cGAS-STING signaling pathway could remarkably suppress the joint swelling and inflammatory cell infiltration in RA mice. Therefore, in this review, we describe the molecular properties, biologic function and mechanisms of the cGAS-STING signaling pathway in AIDs. In addition, potential clinical applications especially selective small molecule inhibitors targeting the cGAS-STING signaling pathway are also discussed.     

Humanized NOG mice as a model for tuberculosis vaccine‐induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4⁺ and CD8⁺ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG‐C, a liposome‐based formulation containing the M. tuberculosis antigen ESAT‐6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T‐cell response. Humanized mice provide a crucial pre‐clinical platform for evaluating human T‐cell immune responses in vaccine development against M. tuberculosis.     

Stimulator of interferon genes agonists attenuate type I diabetes progression in NOD mice

Reagents that activate the signaling adaptor stimulator of interferon genes (STING) suppress experimentally induced autoimmunity in murine models of multiple sclerosis and arthritis. In this study, we evaluated STING agonists as potential reagents to inhibit spontaneous autoimmune type I diabetes (T1D) onset in non-obese diabetic (NOD) female mice. Treatments with DNA nanoparticles (DNPs), which activate STING when cargo DNA is sensed, delayed T1D onset and reduced T1D incidence when administered before T1D onset. DNP treatment elevated indoleamine 2,3 dioxygenase (IDO) activity, which regulates T-cell immunity, in spleen, pancreatic lymph nodes and pancreas of NOD mice. Therapeutic responses to DNPs were partially reversed by inhibiting IDO and DNP treatment synergized with insulin therapy to further delay T1D onset and reduce T1D incidence. Treating pre-diabetic NOD mice with cyclic guanyl-adenyl dinucleotide (cGAMP) to activate STING directly delayed T1D onset and stimulated interferon-αβ (IFN-αβ), while treatment with cyclic diguanyl nucleotide (cdiGMP) did not delay T1D onset or induce IFN-αβ in NOD mice. DNA sequence analyses revealed that NOD mice possess a STING polymorphism that may explain differential responses to cGAMP and cdiGMP. In summary, STING agonists attenuate T1D progression and DNPs enhance therapeutic responses to insulin therapy.     

Identification and isolation of stimulator of interferon genes (STING): an innate immune sensory and adaptor gene from camelids

The mechanism by which type I interferon–mediated antiviral response is mounted by hosts against invading pathogen is an intriguing one. Of late, an endoplasmic reticulum transmembrane protein encoded by a gene called stimulator of interferon genes (STING) is implicated in the innate signalling pathways and has been identified and cloned in few mammalian species including human, mouse and pig. In this article, we report the identification of STING from three different species of a highly conserved family of mammals – the camelids. cDNAs encoding the STING of Old World camels – dromedary camel (Camelus dromedarius) and bactrian camel (Camelus bactrianus) and a New World camel – llama (Llama glama) were amplified using conserved primers and RACE. The complete STING cDNA of dromedary camel is 2171 bp long with a 706‐bp 5′ untranslated regions (UTR), an 1137‐bp open reading frame (ORF) and a 328‐bp 3′ UTR. Sequence and phylogenetic analysis of the ORF of STING from these three camelids indicate high level of similarity among camelids and conservation of critical amino acid residues across different species. Quantitative real‐time PCR analysis revealed high levels of STING mRNA expression in blood, spleen, lymph node and lung. The identification of camelid STING will help in better understanding of the role of this molecule in the innate immunity of the camelids and other mammals.     

TLR9 and STING agonists synergistically induce innate and adaptive type‐II IFN

Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type‐1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K‐type) induces IFNs only weakly and STING ligands rather induce type‐2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type‐1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING‐ligand cyclic GMP–AMP (cGAMP), culminating in NK cell IFN‐γ (type‐II IFN) production, is due to the concurrent effects of IL‐12 and type‐I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type‐1 adjuvant, capable of inducing strong T_h1‐type responses, as demonstrated by enhanced antigen‐specific IgG2c and IFN‐γ production, as well as cytotoxic CD8⁺ T‐cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen‐free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type‐II IFN inducer.     

Rapid evolution of HIV-1 to functional CD8⁺ T cell responses in humanized BLT mice

The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1-specific immune responses and viral evolution during the acute phase of infection. HIV-1-specific CD8(+) T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8(+) T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen-specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.     

Human plasmacytoid dendritic cells elicit a Type I Interferon response by sensing DNA via the cGAS‐STING signaling pathway

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the pathogenesis of IFN‐driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self‐DNA and has been linked to TLR9‐independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP‐AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS‐STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS‐STING pathway and that targeting this pathway could be of therapeutic interest.     

UNC93B1 curbs cytosolic DNA signaling by promoting STING degradation

UNC93B1 is a trafficking chaperone of endosomal Toll-like receptors (TLRs) and plays an essential role in the TLR-mediated innate signaling. However, whether it is also involved in other innate immune sensing or cellular pathways remains largely unexplored. Here we investigated the role of UNC93B1 in cytosolic DNA-triggered cGAS-STING signaling in mouse and human cell lines. We showed that while UNC93B1 deficiency blunts the signal transduction by TLR3, it augments innate immune responses to cytosolic DNA stimulation and DNA virus infection. Mechanistic study reveals a distinct action of UNC93B1 upon STING, but not other parts along the cGAS-STING-TBK1 axis, through regulating the protein level of STING at both resting and cytosolic DNA-stimulated conditions. UNC93B1 can directly interact and traffic along with STING, and the disruption of this interaction causes accumulation of STING that subsequently leads to augmented signaling responses upon its activation. These findings reveal a new function of UNC93B1 in negatively regulating STING-mediated signaling responses.     

AMPK通过抑制STING调节干扰素免疫应答通路的研究

目的探索腺苷酸活化蛋白激酶(AMPK)与cGAS-STING通路之间的联系及其在先天免疫中扮演的角色。方法利用CRISPR/Cas9技术、蛋白质印迹、RT-qPCR等方法,探究AMPK对DNA相关免疫通路的调控机制。结果在HT-DNA和cGAMP刺激下,AMPK-/-细胞株的IFN-β的表达量明显高于野生型细胞株,但这种变化在RNA信号通路中并不明显;激活AMPK可以抑制细胞内的DNA信号通路;在DNA信号通路中,AMPK-/-细胞株相较于野生型细胞株,STING在RNA和蛋白水平上都明显升高,即AMPK对cGAS-STING通路的抑制很可能是通过抑制STING起作用。结论AMPK在调节cGAS-STING介导的干扰素免疫应答中起重要作用。     

Ataxia telangiectasia mutated kinase inhibition promotes irradiation-induced PD-L1 expression in tumour-associated macrophages through IFN-I/JAK signalling pathway

Tumour-associated macrophages (TAMs) are one of the primary sources of PD-L1 expression in the tumour microenvironment (TME). Ionizing radiation (IR) promotes PD-L1 expression in tumour cells. However, the effect of IR on macrophage PD-L1 expression and the underlying mechanisms remain unclear. ATM kinase, as the key kinase for initiating DNA damage repair (DDR) process, is associated with innate immune STING axis activation. Here, we explored the molecular mechanism implicated in macrophage PD-L1 expression regulated by IR as well as the role of ATM kinase in this process. IR-regulated PD-L1 expression in macrophages and associated signalling pathways were explored by in vitro studies using murine and human macrophage cell lines. A colorectal xenograft murine model was employed to demonstrate the impact of targeting ATM and PD-L1 expression in TAMs following IR on growth of tumour in vivo. IR up-regulated PD-L1 expression in macrophages, which was further augmented by ATM kinase inhibition. ATM inhibition increased IR-induced DNA damage, which activated STING/interferon regulatory factor 3 (IRF3) signalling pathway and up-regulated type I interferon (IFN-I) expression in macrophages. IFN-I bound to the IFN α receptor 1 on macrophages, activated the downstream JAK1 and STAT1/3 signalling and eventually led to PD-L1 up-expression. ATM inhibition augmented IR-induced PD-L1 expression in macrophages and CD8⁺ T cell infiltration, and promoted anti-tumour efficacy in vivo. These results suggested that ATM inhibition promoted IR-induced PD-L1 expression through the activation of innate immunity in TAMs, which provided a novel approach to enhance the anti-tumour efficacy of RT.     

Origin and ontogeny of lung macrophages: from mice to humans

Macrophages are tissue-resident myeloid cells with essential roles in host defense, tissue repair, and organ homeostasis. The lung harbors a large number of macrophages that reside in alveoli. As a result of their strategic location, alveolar macrophages are critical sentinels of healthy lung function and barrier immunity. They phagocytose inhaled material and initiate protective immune responses to pathogens, while preventing excessive inflammatory responses and tissue damage. Apart from alveolar macrophages, other macrophage populations are found in the lung and recent single-cell RNA-sequencing studies indicate that lung macrophage heterogeneity is greater than previously appreciated. The cellular origin and development of mouse lung macrophages has been extensively studied, but little is known about the ontogeny of their human counterparts, despite the importance of macrophages for lung health. In this context, humanized mice (mice with a human immune system) can give new insights into the biology of human lung macrophages by allowing in vivo studies that are not possible in humans. In particular, we have created humanized mouse models that support the development of human lung macrophages in vivo. In this review, we will discuss the heterogeneity, development, and homeostasis of lung macrophages. Moreover, we will highlight the impact of age, the microbiota, and pathogen exposure on lung macrophage function. Altered macrophage function has been implicated in respiratory infections as well as in common allergic and inflammatory lung diseases. Therefore, understanding the functional heterogeneity and ontogeny of lung macrophages should help to develop future macrophage-based therapies for important lung diseases in humans.     

Radioresistant cells expressing TLR5 control the respiratory epithelium's innate immune responses to flagellin

Bacterial products (such as endotoxins and flagellin) trigger innate immune responses through TLRs. Flagellin‐induced signalling involves TLR5 and MyD88 and, according to some reports, TLR4. Whereas epithelial and dendritic cells are stimulated by flagellin in vitro, the cell contribution to the in vivo response is still unclear. Here, we studied the respective roles of radioresistant and radiosensitive cells in flagellin‐induced airway inflammation in mice. We found that i.n. delivery of flagellin elicits a transient change in respiratory function and an acute, pro‐inflammatory response in the lungs, characterized by TLR5‐ and MyD88‐dependent chemokine secretion and neutrophil recruitment. In contrast, TLR4, CD14 and TRIF were not essential for flagellin‐mediated responses, indicating that TLR4 does not cooperate with TLR5 in the lungs. Respiratory function, chemokine secretion and airway infiltration by neutrophils were dependent on radioresistant, TLR5‐expressing cells. Furthermore, lung haematopoietic cells also responded to flagellin by activating TNF‐α production. We suggest that the radioresistant lung epithelial cells are essential for initiating early, TLR5‐dependent signalling in response to flagellin and thus triggering the lung's innate immune responses.     

STING pathway agonism as a cancer therapeutic

The fact that a subset of human cancers showed evidence for a spontaneous adaptive immune response as reflected by the T cell‐inflamed tumor microenvironment phenotype led to the search for candidate innate immune pathways that might be driving such endogenous responses. Preclinical studies indicated a major role for the host STING pathway, a cytosolic DNA sensing pathway, as a proximal event required for optimal type I interferon production, dendritic cell activation, and priming of CD8⁺ T cells against tumor‐associated antigens. STING agonists are therefore being developed as a novel cancer therapeutic, and a greater understanding of STING pathway regulation is leading to a broadened list of candidate immune regulatory targets. Early phase clinical trials of intratumoral STING agonists are already showing promise, alone and in combination with checkpoint blockade. Further advancement will derive from a deeper understanding of STING pathway biology as well as mechanisms of response vs resistance in individual cancer patients.     

Camouflage and interception: how pathogens evade detection by intracellular nucleic acid sensors

Intracellular DNA and RNA sensors play a vital part in the innate immune response to viruses and other intracellular pathogens, causing the secretion of type I interferons, cytokines and chemokines from infected cells. Pathogen RNA can be detected by retinoic-acid inducible gene I-like receptors in the cytosol, whereas cytosolic DNA is recognized by DNA sensors such as cyclic GMP-AMP synthase (cGAS). The resulting local immune response, which is initiated within hours of infection, is able to eliminate many pathogens before they are able to establish an infection in the host. For this reason, all viruses, and some intracellular bacteria and protozoa, need to evade detection by nucleic acid sensors. Immune evasion strategies include the sequestration and modification of nucleic acids, and the inhibition or degradation of host factors involved in innate immune signalling. Large DNA viruses, such as herpesviruses, often use multiple viral proteins to inhibit signalling cascades at several different points; for instance herpes simplex virus 1 targets both DNA sensors cGAS and interferon-γ-inducible protein 16, as well as the adaptor protein STING (stimulator of interferon genes) and other signalling factors in the pathway. Viruses with a small genome encode only a few immunomodulatory proteins, but these are often multifunctional, such as the NS1 protein from influenza A virus, which inhibits RNA sensing in multiple ways. Intracellular bacteria and protozoa can also be detected by nucleic acid sensors. However, as the type I interferon response is not always beneficial for the host under these circumstances, some bacteria subvert, rather than evade, these signalling cascades for their own gain.     

Primary Tumor Suppression and Systemic Immune Activation of Macrophages through the Sting Pathway in Metastatic Skin Tumor

PurposeAgonists of the stimulator of interferon genes (STING) play a key role in activating the STING pathway by promoting the production of cytokines. In this study, we investigated the antitumor effects and activation of the systemic immune response of treatment with DMXAA (5,6-dimethylxanthenone-4-acetic acid), a STING agonist, in EML4-ALK lung cancer and CT26 colon cancer.Materials and MethodsThe abscopal effects of DMXAA in the treatment of metastatic skin nodules were assessed. EML4-ALK lung cancer and CT26 colon cancer models were used to evaluate these effects after DMXAA treatment. To evaluate the expression of macrophages and T cells, we sacrificed the tumor-bearing mice after DMXAA treatment and obtained the formalin-fixed paraffin-embedded (FFPE) tissue and tumor cells. Immunohistochemistry and flow cytometry were performed to analyze the expression of each FFPE and tumor cell.ResultsWe observed that highly infiltrating immune cells downstream of the STING pathway had increased levels of chemokines after DMXAA treatment. In addition, the levels of CD80 and CD86 in antigen-presenting cells were significantly increased after STING activation. Furthermore, innate immune activation altered the systemic T cell-mediated immune responses, induced proliferation of macrophages, inhibited tumor growth, and increased numbers of cytotoxic memory T cells. Tumor-specific lymphocytes also increased in number after treatment with DMXAA.ConclusionThe abscopal effect of DMXAA treatment on the skin strongly reduced the spread of EML4-ALK lung cancer and CT26 colon cancer through the STING pathway and induced the presentation of antigens.     

MHC class II-deficient mice allow functional human CD4+ T-cell development

Humanized mouse models have been developed to study cell-mediated immune responses to human pathogens in vivo. How immunocompetent human T cells are selected in a murine thymus in such humanized mice remains poorly explored. To gain insights into this mechanism, we investigated the differentiation of human immune compartments in mouse MHC class II-deficient immune-compromised mice (humanized Ab0 mice). We observed a strong reduction in human CD4⁺ T-cell development but despite this reduction Ab0 mice had no disadvantage during Epstein–Barr virus (EBV) infection. Viral loads were equally well controlled in humanized Ab0 mice compared to humanized NSG mice, and improved T-cell recognition of autologous EBV-transformed B cells was observed, especially with respect to cytotoxicity. MHC class II blocking experiments with CD4⁺ T cells from humanized Ab0 mice demonstrated MHC class II restriction of lymphoblastoid cell line recognition. These findings suggest that a small number of CD4⁺ T cells in humanized mice can be solely selected on human MHC class II molecules, presumably expressed by reconstituted human immune cells, leading to improved effector functions.