Ontogeny and Function of Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) represent a unique cell type within the innate immune system. Their defining property is the recognition of pathogen-derived nucleic acids through endosomal Toll-like receptors and the ensuing production of type I interferon and other soluble mediators, which orchestrate innate and adaptive responses. We review several aspects of pDC biology that have recently come to the fore. We discuss emerging questions regarding the lineage affiliation and origin of pDCs and argue that these cells constitute an integral part of the dendritic cell lineage. We emphasize the specific function of pDCs as innate sentinels of virus infection, particularly their recognition of and distinct response to virus-infected cells. This essential evolutionary role of pDCs has been particularly important for the control of coronaviruses, as demonstrated by the recent COVID-19 pandemic. Finally, we highlight the key contribution of pDCs to systemic lupus erythematosus, in which therapeutic targeting of pDCs is currently underway.

The Immunological Conundrum of Endogenous Retroelements.

Our defenses against infection rely on the ability of the immune system to distinguish invading pathogens from self. This task is exceptionally challenging, if not seemingly impossible, in the case of retroviruses that have integrated almost seamlessly into the host. This review examines the limits of innate and adaptive immune responses elicited by endogenous retroviruses and other retroelements, the targets of immune recognition, and the consequences for host health and disease. Contrary to theoretical expectation, endogenous retroelements retain substantial immunogenicity, which manifests most profoundly when their epigenetic repression is compromised, contributing to autoinflammatory and autoimmune disease and age-related inflammation. Nevertheless, recent evidence suggests that regulated immune reactivity to endogenous retroelements is integral to immune system development and function, underpinning cancer immunosurveillance, resistance to infection, and responses to the microbiota. Elucidation of the interaction points with endogenous retroelements will therefore deepen our understanding of immune system function and contribution to disease.

Interfering with Interferons: A Critical Mechanism for Critical COVID-19 Pneumonia.

Infection with SARS-CoV-2 results in clinical outcomes ranging from silent or benign infection in most individuals to critical pneumonia and death in a few. Genetic studies in patients have established that critical cases can result from inborn errors of TLR3- or TLR7-dependent type I interferon immunity, or from preexisting autoantibodies neutralizing primarily IFN-α and/or IFN-ω. These findings are consistent with virological studies showing that multiple SARS-CoV-2 proteins interfere with pathways of induction of, or response to, type I interferons. They are also congruent with cellular studies and mouse models that found that type I interferons can limit SARS-CoV-2 replication in vitro and in vivo, while their absence or diminution unleashes viral growth. Collectively, these findings point to insufficient type I interferon during the first days of infection as a general mechanism underlying critical COVID-19 pneumonia, with implications for treatment and directions for future research.

Functional Hallmarks of Healthy Macrophage Responses: Their Regulatory Basis and Disease Relevance.

Macrophages are first responders for the immune system. In this role, they have both effector functions for neutralizing pathogens and sentinel functions for alerting other immune cells of diverse pathologic threats, thereby initiating and coordinating a multipronged immune response. Macrophages are distributed throughout the body-they circulate in the blood, line the mucosal membranes, reside within organs, and survey the connective tissue. Several reviews have summarized their diverse roles in different physiological scenarios and in the initiation or amplification of different pathologies. In this review, we propose that both the effector and the sentinel functions of healthy macrophages rely on three hallmark properties: response specificity, context dependence, and stimulus memory. When these hallmark properties are diminished, the macrophage's biological functions are impaired, which in turn results in increased risk for immune dysregulation, manifested by immune deficiency or autoimmunity. We review the evidence and the molecular mechanisms supporting these functional hallmarks.

Self-Awareness: Nucleic Acid-Driven Inflammation and the Type I Interferonopathies.

Recognition of foreign nucleic acids is the primary mechanism by which a type I interferon-mediated antiviral response is triggered. Given that human cells are replete with DNA and RNA, this evolutionary strategy poses an inherent biological challenge, i.e., the fundamental requirement to reliably differentiate self-nucleic acids from nonself nucleic acids. We suggest that the group of Mendelian inborn errors of immunity referred to as the type I interferonopathies relate to a breakdown of self/nonself discrimination, with the associated mutant genotypes involving molecules playing direct or indirect roles in nucleic acid signaling. This perspective begs the question as to the sources of self-derived nucleic acids that drive an inappropriate immune response. Resolving this question will provide fundamental insights into immune tolerance, antiviral signaling, and complex autoinflammatory disease states. Here we develop these ideas, discussing type I interferonopathies within the broader framework of nucleic acid-driven inflammation.

Type-I-interferon-responsive microglia shape cortical development and behavior.

Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional states have been defined, but their functional significance is unclear. Here, we identify a type I interferon (IFN-I)-responsive microglial state in the developing somatosensory cortex (postnatal day 5) that is actively engulfing whole neurons. This population expands during cortical remodeling induced by partial whisker deprivation. Global or microglial-specific loss of the IFN-I receptor resulted in microglia with phagolysosomal dysfunction and an accumulation of neurons with nuclear DNA damage. IFN-I gain of function increased neuronal engulfment by microglia in both mouse and zebrafish and restricted the accumulation of DNA-damaged neurons. Finally, IFN-I deficiency resulted in excess cortical excitatory neurons and tactile hypersensitivity. These data define a role for neuron-engulfing microglia during a critical window of brain development and reveal homeostatic functions of a canonical antiviral signaling pathway in the brain.

Selection of epigenetically privileged HIV-1 proviruses during treatment with panobinostat and interferon-α2a.

CD4+ T cells with latent HIV-1 infection persist despite treatment with antiretroviral agents and represent the main barrier to a cure of HIV-1 infection. Pharmacological disruption of viral latency may expose HIV-1-infected cells to host immune activity, but the clinical efficacy of latency-reversing agents for reducing HIV-1 persistence remains to be proven. Here, we show in a randomized-controlled human clinical trial that the histone deacetylase inhibitor panobinostat, when administered in combination with pegylated interferon-α2a, induces a structural transformation of the HIV-1 reservoir cell pool, characterized by a disproportionate overrepresentation of HIV-1 proviruses integrated in ZNF genes and in chromatin regions with reduced H3K27ac marks, the molecular target sites for panobinostat. By contrast, proviruses near H3K27ac marks were actively selected against, likely due to increased susceptibility to panobinostat. These data suggest that latency-reversing treatment can increase the immunological vulnerability of HIV-1 reservoir cells and accelerate the selection of epigenetically privileged HIV-1 proviruses.

Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/ß-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/ß immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/ß. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/ß-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/ß-dependent antiviral immunity.

Opioid-induced fragile-like regulatory T cells contribute to withdrawal.

Dysregulation of the immune system is a cardinal feature of opioid addiction. Here, we characterize the landscape of peripheral immune cells from patients with opioid use disorder and from healthy controls. Opioid-associated blood exhibited an abnormal distribution of immune cells characterized by a significant expansion of fragile-like regulatory T cells (Tregs), which was positively correlated with the withdrawal score. Analogously, opioid-treated mice also showed enhanced Treg-derived interferon-γ (IFN-γ) expression. IFN-γ signaling reshaped synaptic morphology in nucleus accumbens (NAc) neurons, modulating subsequent withdrawal symptoms. We demonstrate that opioids increase the expression of neuron-derived C-C motif chemokine ligand 2 (Ccl2) and disrupted blood-brain barrier (BBB) integrity through the downregulation of astrocyte-derived fatty-acid-binding protein 7 (Fabp7), which both triggered peripheral Treg infiltration into NAc. Our study demonstrates that opioids drive the expansion of fragile-like Tregs and favor peripheral Treg diapedesis across the BBB, which leads to IFN-γ-mediated synaptic instability and subsequent withdrawal symptoms.

Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity.

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.

A neutrophil response linked to tumor control in immunotherapy.

Neutrophils accumulate in solid tumors, and their abundance correlates with poor prognosis. Neutrophils are not homogeneous, however, and could play different roles in cancer therapy. Here, we investigate the role of neutrophils in immunotherapy, leading to tumor control. We show that successful therapies acutely expanded tumor neutrophil numbers. This expansion could be attributed to a Sellhi state rather than to other neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene signature, also seen in human patients, and appeared essential for successful therapy, as loss of the interferon-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil response depended on key components of anti-tumor immunity, including BATF3-dependent DCs, IL-12, and IFNγ. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in mediating effective cancer therapy.

Biochemistry, Cell Biology, and Pathophysiology of the Innate Immune cGAS-cGAMP-STING Pathway.

In the decade since the discovery of the innate immune cyclic GMP-AMP synthase (cGAS)-2'3'-cyclic GMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway, its proper activation and dysregulation have been rapidly implicated in many aspects of human disease. Understanding the biochemical, cellular, and regulatory mechanisms of this pathway is critical to developing therapeutic strategies that either harness it to boost defense or inhibit it to prevent unwanted inflammation. In this review, we first discuss how the second messenger cGAMP is synthesized by cGAS in response to double-stranded DNA and cGAMP's subsequent activation of cell-type-dependent STING signaling cascades with differential physiological consequences. We then review how cGAMP as an immunotransmitter mediates tightly controlled cell-cell communication by being exported from producing cells and imported into responding cells via cell-type-specific transporters. Finally, we review mechanisms by which thecGAS-cGAMP-STING pathway responds to different sources of mislocalized double-stranded DNA in pathogen defense, cancer, and autoimmune diseases.

Insights into cytokine-receptor interactions from cytokine engineering.

Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. However, the desired immunotherapeutic effects of native cytokines are often mitigated by toxicity or lack of efficacy, either of which results from cytokine receptor pleiotropy and/or undesired activation of off-target cells. As our understanding of the structural principles of cytokine-receptor interactions has advanced, mechanism-based manipulation of cytokine signaling through protein engineering has become an increasingly feasible and powerful approach. Modified cytokines, both agonists and antagonists, have been engineered with narrowed target cell specificities, and they have also yielded important mechanistic insights into cytokine biology and signaling. Here we review the theory and practice of cytokine engineering and rationalize the mechanisms of several engineered cytokines in the context of structure. We discuss specific examples of how structure-based cytokine engineering has opened new opportunities for cytokines as drugs, with a focus on the immunotherapeutic cytokines interferon, interleukin-2, and interleukin-4.

EMSY inhibits homologous recombination repair and the interferon response, promoting lung cancer immune evasion.

Non-small cell lung cancers (NSCLCs) harboring KEAP1 mutations are often resistant to immunotherapy. Here, we show that KEAP1 targets EMSY for ubiquitin-mediated degradation to regulate homologous recombination repair (HRR) and anti-tumor immunity. Loss of KEAP1 in NSCLC induces stabilization of EMSY, producing a BRCAness phenotype, i.e., HRR defects and sensitivity to PARP inhibitors. Defective HRR contributes to a high tumor mutational burden that, in turn, is expected to prompt an innate immune response. Notably, EMSY accumulation suppresses the type I interferon response and impairs innate immune signaling, fostering cancer immune evasion. Activation of the type I interferon response in the tumor microenvironment using a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of KEAP1-mutant tumors. Our results suggest that targeting PARP and STING pathways, individually or in combination, represents a therapeutic strategy in NSCLC patients harboring alterations in KEAP1.

Actin cytoskeleton remodeling primes RIG-I-like receptor activation.

The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design.

Facile discovery of surrogate cytokine agonists.

Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.

Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.

Spatial CRISPR genomics identifies regulators of the tumor microenvironment.

While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFß and TGFß-mediated fibroblast activation, indicating that TGFß-receptor loss on cancer cells increased TGFß bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFß responsiveness of cancer cells can affect the TME.

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.