Mast Cells in Inflammation and Disease: Recent Progress and Ongoing Concerns.

Mast cells have existed long before the development of adaptive immunity, although they have been given different names. Thus, in the marine urochordate Styela plicata, they have been designated as test cells. However, based on their morphological characteristics (including prominent cytoplasmic granules) and mediator content (including heparin, histamine, and neutral proteases), test cells are thought to represent members of the lineage known in vertebrates as mast cells. So this lineage presumably had important functions that preceded the development of antibodies, including IgE. Yet mast cells are best known, in humans, as key sources of mediators responsible for acute allergic reactions, notably including anaphylaxis, a severe and potentially fatal IgE-dependent immediate hypersensitivity reaction to apparently harmless antigens, including many found in foods and medicines. In this review, we briefly describe the origins of tissue mast cells and outline evidence that these cells can have beneficial as well as detrimental functions, both innately and as participants in adaptive immune responses. We also discuss aspects of mast cell heterogeneity and comment on how the plasticity of this lineage may provide insight into its roles in health and disease. Finally, we consider some currently open questions that are yet unresolved.

Sixty Years of Discovery.

Each of us is a story. Mine is a story of doing science for 60 years, and I am honored to be asked to tell it. Even though this autobiography was written for the Annual Review of Immunology, I have chosen to describe my whole career in science because the segment that was immunology is so intertwined with all else I was doing. This article is an elongation and modification of a talk I gave at my 80th birthday celebration at Caltech on March 23, 2018.

Translating Immunology into Therapeutic Concepts for Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) defines a spectrum of complex disorders. Understanding how environmental risk factors, alterations of the intestinal microbiota, and polygenetic and epigenetic susceptibility impact on immune pathways is key for developing targeted therapies. Mechanistic understanding of polygenic IBD is complemented by Mendelian disorders that present with IBD, pharmacological interventions that cause colitis, autoimmunity, and multiple animal models. Collectively, this multifactorial pathogenesis supports a concept of immune checkpoints that control microbial-host interactions in the gut by modulating innate and adaptive immunity, as well as epithelial and mesenchymal cell responses. In addition to classical immunosuppressive strategies, we discuss how resetting the microbiota and restoring innate immune responses, in particular autophagy and epithelial barrier function, might be key for maintaining remission or preventing IBD. Targeting checkpoints in genetically stratified subgroups of patients with Mendelian disorder-associated IBD increasingly directs treatment strategies as part of personalized medicine.

Th2 Cells in Health and Disease.

Helper T (Th) cell subsets direct immune responses by producing signature cytokines. Th2 cells produce IL-4, IL-5, and IL-13, which are important in humoral immunity and protection from helminth infection and are central to the pathogenesis of many allergic inflammatory diseases. Molecular analysis of Th2 cell differentiation and maintenance of function has led to recent discoveries that have refined our understanding of Th2 cell biology. Epigenetic regulation of Gata3 expression by chromatin remodeling complexes such as Polycomb and Trithorax is crucial for maintaining Th2 cell identity. In the context of allergic diseases, memory-type pathogenic Th2 cells have been identified in both mice and humans. To better understand these disease-driving cell populations, we have developed a model called the pathogenic Th population disease induction model. The concept of defined subsets of pathogenic Th cells may spur new, effective strategies for treating intractable chronic inflammatory disorders.

APOE3ch alters microglial response and suppresses Aß-induced tau seeding and spread.

A recent case report described an individual who was a homozygous carrier of the APOE3 Christchurch (APOE3ch) mutation and resistant to autosomal dominant Alzheimer's Disease (AD) caused by a PSEN1-E280A mutation. Whether APOE3ch contributed to the protective effect remains unclear. We generated a humanized APOE3ch knock-in mouse and crossed it to an amyloid-ß (Aß) plaque-depositing model. We injected AD-tau brain extract to investigate tau seeding and spreading in the presence or absence of amyloid. Similar to the case report, APOE3ch expression resulted in peripheral dyslipidemia and a marked reduction in plaque-associated tau pathology. Additionally, we observed decreased amyloid response and enhanced microglial response around plaques. We also demonstrate increased myeloid cell phagocytosis and degradation of tau aggregates linked to weaker APOE3ch binding to heparin sulfate proteoglycans. APOE3ch influences the microglial response to Aß plaques, which suppresses Aß-induced tau seeding and spreading. The results reveal new possibilities to target Aß-induced tauopathy.

TNF-NF-κB-p53 axis restricts in vivo survival of hPSC-derived dopamine neurons.

Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.

Preclinical models for drug discovery for metastatic disease.

Despite many advances, metastatic disease remains essentially uncurable. Thus, there is an urgent need to better understand mechanisms that promote metastasis, drive tumor evolution, and underlie innate and acquired drug resistance. Sophisticated preclinical models that recapitulate the complex tumor ecosystem are key to this process. We begin with syngeneic and patient-derived mouse models that are the backbone of most preclinical studies. Second, we present some unique advantages of fish and fly models. Third, we consider the strengths of 3D culture models for resolving remaining knowledge gaps. Finally, we provide vignettes on multiplexed technologies to advance our understanding of metastatic disease.

Chimeric autoantibody receptor T cells deplete NMDA receptor-specific B cells.

Anti-NMDA receptor (NMDAR) autoantibodies cause NMDAR encephalitis, the most common autoimmune encephalitis, leading to psychosis, seizures, and autonomic dysfunction. Current treatments comprise broad immunosuppression or non-selective antibody removal. We developed NMDAR-specific chimeric autoantibody receptor (NMDAR-CAAR) T cells to selectively eliminate anti-NMDAR B cells and disease-causing autoantibodies. NMDAR-CAARs consist of an extracellular multi-subunit NMDAR autoantigen fused to intracellular 4-1BB/CD3ζ domains. NMDAR-CAAR T cells recognize a large panel of human patient-derived autoantibodies, release effector molecules, proliferate, and selectively kill antigen-specific target cell lines even in the presence of high autoantibody concentrations. In a passive transfer mouse model, NMDAR-CAAR T cells led to depletion of an anti-NMDAR B cell line and sustained reduction of autoantibody levels without notable off-target toxicity. Treatment of patients may reduce side effects, prevent relapses, and improve long-term prognosis. Our preclinical work paves the way for CAAR T cell phase I/II trials in NMDAR encephalitis and further autoantibody-mediated diseases.

A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells.

Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

PIKFYVE inhibition mitigates disease in models of diverse forms of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from many diverse genetic causes. Although therapeutics specifically targeting known causal mutations may rescue individual types of ALS, these approaches cannot treat most cases since they have unknown genetic etiology. Thus, there is a pressing need for therapeutic strategies that rescue multiple forms of ALS. Here, we show that pharmacological inhibition of PIKFYVE kinase activates an unconventional protein clearance mechanism involving exocytosis of aggregation-prone proteins. Reducing PIKFYVE activity ameliorates ALS pathology and extends survival of animal models and patient-derived motor neurons representing diverse forms of ALS including C9ORF72, TARDBP, FUS, and sporadic. These findings highlight a potential approach for mitigating ALS pathogenesis that does not require stimulating macroautophagy or the ubiquitin-proteosome system.

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

Circuit-specific gene therapy reverses core symptoms in a primate Parkinson's disease model.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Molecular mechanisms in genetically defined autoinflammatory diseases: disorders of amplified danger signaling.

Patients with autoinflammatory diseases present with noninfectious fever flares and systemic and/or disease-specific organ inflammation. Their excessive proinflammatory cytokine and chemokine responses can be life threatening and lead to organ damage over time. Studying such patients has revealed genetic defects that have helped unravel key innate immune pathways, including excessive IL-1 signaling, constitutive NF-κB activation, and, more recently, chronic type I IFN signaling. Discoveries of monogenic defects that lead to activation of proinflammatory cytokines have inspired the use of anticytokine-directed treatment approaches that have been life changing for many patients and have led to the approval of IL-1-blocking agents for a number of autoinflammatory conditions. In this review, we describe the genetically characterized autoinflammatory diseases, we summarize our understanding of the molecular pathways that drive clinical phenotypes and that continue to inspire the search for novel treatment targets, and we provide a conceptual framework for classification.

Of mice and men: From hematopoiesis in mouse models to curative gene therapy for sickle cell disease.

Through studies in mice and in humans, Stuart Orkin showed that GATA-1 is a master transcriptional regulator of hematopoiesis. He has highlighted the role of BCL11A in the fetal-adult hemoglobin switch. The Gairdner Foundation Award recognizes Orkin's contribution to the development of gene therapy of sickle cell disease.

SYK coordinates neuroprotective microglial responses in neurodegenerative disease.

Recent studies have begun to reveal critical roles for the brain's professional phagocytes, microglia, and their receptors in the control of neurotoxic amyloid beta (Aß) and myelin debris accumulation in neurodegenerative disease. However, the critical intracellular molecules that orchestrate neuroprotective functions of microglia remain poorly understood. In our studies, we find that targeted deletion of SYK in microglia leads to exacerbated Aß deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer's disease (AD). Disruption of SYK signaling in this AD model was further shown to impede the development of disease-associated microglia (DAM), alter AKT/GSK3ß-signaling, and restrict Aß phagocytosis by microglia. Conversely, receptor-mediated activation of SYK limits Aß load. We also found that SYK critically regulates microglial phagocytosis and DAM acquisition in demyelinating disease. Collectively, these results broaden our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material.

PAAN/MIF nuclease inhibition prevents neurodegeneration in Parkinson's disease.

Parthanatos-associated apoptosis-inducing factor (AIF) nuclease (PAAN), also known as macrophage migration inhibitor factor (MIF), is a member of the PD-D/E(X)K nucleases that acts as a final executioner in parthanatos. PAAN's role in Parkinson's disease (PD) and whether it is amenable to chemical inhibition is not known. Here, we show that neurodegeneration induced by pathologic α-synuclein (α-syn) occurs via PAAN/MIF nuclease activity. Genetic depletion of PAAN/MIF and a mutant lacking nuclease activity prevent the loss of dopaminergic neurons and behavioral deficits in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Compound screening led to the identification of PAANIB-1, a brain-penetrant PAAN/MIF nuclease inhibitor that prevents neurodegeneration induced by α-syn PFF, AAV-α-syn overexpression, or MPTP intoxication in vivo. Our findings could have broad relevance in human pathologies where parthanatos plays a role in the development of cell death inhibitors targeting the druggable PAAN/MIF nuclease.

Innate frequency-discrimination hyperacuity in Williams-Beuren syndrome mice.

Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of ∼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins.

Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody.

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.