Hypoxia contributes to airway inflammation and remodeling in several lung diseases; however, exactly how hypoxic pulmonary epithelium regulates allergic inflammation remains to be fully characterized. Here, we report that conditional deletion of the E3 ubiquitin ligase VHL in lung epithelial cells resulted in exacerbated type 2 responses accompanied by selective increase of group 2 innate lymphoid cells (ILC2s) at steady state and following inflammation or helminth infection. Ablation of expression of the hypoxia-inducible factor 2α (HIF2α) significantly reversed VHL-mediated ILC2 activation. VHL deficiency in lung epithelial cells caused increased expression of the peptide hormone adrenomedullin (ADM), and our data suggest that HIF2α controls Adm expression. ADM directly promoted ILC2 activation both in vitro and in vivo. Our findings indicate that the hypoxic response mediated by the VHL-HIF2α axis is critical for control of pulmonary type 2 responses by increasing ADM expression in lung epithelia, causing ILC2 activation.

CCR5 is the major HIV-1 co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Using engineered zinc-finger nucleases (ZFNs), we disrupted CCR5 in human CD34(+) hematopoietic stem/progenitor cells (HSPCs) at a mean frequency of 17% of the total alleles in a population. This procedure produces both mono- and bi-allelically disrupted cells. ZFN-treated HSPCs retained the ability to engraft NOD/SCID/IL2rgamma(null) mice and gave rise to polyclonal multi-lineage progeny in which CCR5 was permanently disrupted. Control mice receiving untreated HSPCs and challenged with CCR5-tropic HIV-1 showed profound CD4(+) T-cell loss. In contrast, mice transplanted with ZFN-modified HSPCs underwent rapid selection for CCR5(-/-) cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5(-/-) HSPCs can populate an infected animal with HIV-1-resistant, CCR5(-/-) progeny supports the use of ZFN-modified autologous hematopoietic stem cells as a clinical approach to treating HIV-1.

A system that allows manipulation of the human thymic microenvironment is needed both to elucidate the extrinsic mechanisms that control human thymopoiesis and to develop potential cell therapies for thymic insufficiency. In this report, we developed an implantable thymic microenvironment composed of two human thymic stroma populations critical for thymopoiesis; thymic epithelial cells (TECs) and thymic mesenchyme (TM). TECs and TM from postnatal human thymi were cultured in specific conditions, allowing cell expansion and manipulation of gene expression, before reaggregation into a functional thymic unit. Human CD34+ hematopoietic stem and progenitor cells (HSPC) differentiated into T cells in the aggregates in vitro and in vivo following inguinal implantation of aggregates in immune deficient mice. Cord blood HSPC previously engrafted into murine bone marrow (BM), migrated to implants, and differentiated into human T cells with a broad T cell receptor repertoire. Furthermore, lentiviral-mediated expression of vascular endothelial growth factor in TM enhanced implant size and function and significantly increased thymocyte production. These results demonstrate an in vivo system for the generation of T cells from human HSPC and represent the first model to allow manipulation of gene expression and cell composition in the microenvironment of the human thymus.

Neonatal life marks the apogee of murine thymic growth. Over the first few days after birth, growth slows and the murine thymus switches from fetal to adult morphology and function; little is known about the cues driving this dramatic transition. In this study, we show for the first time (to our knowledge) the critical role of vascular endothelial growth factor (VEGF) on thymic morphogenesis beyond its well-known role in angiogenesis. During a brief window a few days after birth, VEGF inhibition induced rapid and profound remodeling of the endothelial, mesenchymal and epithelial thymic stromal compartments, mimicking changes seen during early adult maturation. Rapid transcriptional changes were seen in each compartment after VEGF inhibition, including genes involved in migration, chemotaxis, and cell adhesion as well as induction of a proinflammatory and proadipogenic signature in endothelium, pericytes, and mesenchyme. Thymocyte numbers fell subsequent to the stromal changes. Expression patterns and functional blockade of the receptors VEGFR2 and NRP1 demonstrated that VEGF mediates its pleiotropic effects through distinct receptors on each microenvironmental compartment of the developing mouse thymus.

Innate lymphoid cells (ILCs) are important regulators of early infection at mucosal barriers. ILCs are divided into three groups based on expression profiles, and are activated by cytokines and neuropeptides. Yet, it remains unknown if ILCs integrate other signals in providing protection. We show that signaling through herpes virus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor superfamily, in ILC3 is important for host defense against oral infection with the bacterial pathogen Yersinia enterocolitica. HVEM stimulates protective interferon-γ (IFN-γ) secretion from ILCs, and mice with HVEM-deficient ILC3 exhibit reduced IFN-γ production, higher bacterial burdens and increased mortality. In addition, IFN-γ production is critical as adoptive transfer of wild-type but not IFN-γ-deficient ILC3 can restore protection to mice lacking ILCs. We identify the TNF superfamily member, LIGHT, as the ligand inducing HVEM signals in ILCs. Thus HVEM signaling mediated by LIGHT plays a critical role in regulating ILC3-derived IFN-γ production for protection following infection. VIDEO ABSTRACT.

Over the past 50 years, the use of mathematical models, derived from physical reasoning, to describe molecular and cellular systems has evolved from an art of the few to a cornerstone of biological inquiry. George Oster stood out as a pioneer of this paradigm shift from descriptive to quantitative biology not only through his numerous research accomplishments, but also through the many students and postdocs he mentored over his long career. Those of us fortunate enough to have worked with George agree that his sharp intellect, physical intuition, and passion for scientific inquiry not only inspired us as scientists but also greatly influenced the way we conduct research. We would like to share a few important lessons we learned from George in honor of his memory and with the hope that they may inspire future generations of scientists.

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3⁺TCR-αβ⁺ single-positive CD8⁺ or CD4⁺ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) initially infects the respiratory tract, it also directly or indirectly affects other organs, including the brain. However, little is known about the relative neurotropism of SARS-CoV-2 variants of concern (VOCs), including Omicron (B.1.1.529), which emerged in November 2021 and has remained the dominant pathogenic lineage since then. To address this gap, we examined the relative ability of Omicron, Beta (B.1.351), and Delta (B.1.617.2) to infect the brain in the context of a functional human immune system by using human angiotensin-converting enzyme 2 (hACE2) knock-in triple-immunodeficient NGC mice with or without reconstitution with human CD34⁺ stem cells. Intranasal inoculation of huCD34⁺-hACE2-NCG mice with Beta and Delta resulted in productive infection of the nasal cavity, lungs, and brain on day 3 post-infection, but Omicron was surprisingly unique in its failure to infect either the nasal tissue or brain. Moreover, the same infection pattern was observed in hACE2-NCG mice, indicating that antiviral immunity was not responsible for the lack of Omicron neurotropism. In independent experiments, we demonstrate that nasal inoculation with Beta or with D614G, an ancestral SARS-CoV-2 with undetectable replication in huCD34⁺-hACE2-NCG mice, resulted in a robust response by human innate immune cells, T cells, and B cells, confirming that exposure to SARS-CoV-2, even without detectable infection, is sufficient to induce an antiviral immune response. Collectively, these results suggest that modeling of the neurologic and immunologic sequelae of SARS-CoV-2 infection requires careful selection of the appropriate SARS-CoV-2 strain in the context of a specific mouse model.

Zika virus (ZIKV) and dengue virus (DENV) are arthropod-borne pathogenic flaviviruses that co-circulate in many countries. To understand some of the pressures that influence ZIKV evolution, we mimic the natural transmission cycle by repeating serial passaging of ZIKV through cultured mosquito cells and either DENV-naive or DENV-immune mice. Compared with wild-type ZIKV, the strains passaged under both conditions exhibit increased pathogenesis in DENV-immune mice. Application of reverse genetics identifies an isoleucine-to-valine mutation (I39V) in the NS2B proteins of both passaged strains that confers enhanced fitness and escape from pre-existing DENV immunity. Introduction of I39V or I39T, a naturally occurring homologous mutation detected in recent ZIKV isolates, increases the replication of wild-type ZIKV in human neuronal precursor cells and laboratory-raised mosquitoes. Our data indicate that ZIKV strains with enhanced transmissibility and pathogenicity can emerge in DENV-naive or -immune settings, and that NS2B-I39 mutants may represent ZIKV variants of interest.