A metabolic pacer ensures smooth running of the lymphocyte activation race.

Upon lymphocyte stimulation, accumulation of intracellular NAD(H) reflects the strength of antigen receptor signals and controls the rate of cell cycle entry and proliferation (see related Research Article by Turner et al.).

Apoptotic cell fragments locally activate tingible body macrophages in the germinal center.

Germinal centers (GCs) that form within lymphoid follicles during antibody responses are sites of massive cell death. Tingible body macrophages (TBMs) are tasked with apoptotic cell clearance to prevent secondary necrosis and autoimmune activation by intracellular self antigens. We show by multiple redundant and complementary methods that TBMs derive from a lymph node-resident, CD169-lineage, CSF1R-blockade-resistant precursor that is prepositioned in the follicle. Non-migratory TBMs use cytoplasmic processes to chase and capture migrating dead cell fragments using a "lazy" search strategy. Follicular macrophages activated by the presence of nearby apoptotic cells can mature into TBMs in the absence of GCs. Single-cell transcriptomics identified a TBM cell cluster in immunized lymph nodes which upregulated genes involved in apoptotic cell clearance. Thus, apoptotic B cells in early GCs trigger activation and maturation of follicular macrophages into classical TBMs to clear apoptotic debris and prevent antibody-mediated autoimmune diseases.

E3 ubiquitin ligase HECTD2 mediates melanoma progression and immune evasion.

The ubiquitin-proteasome system maintains protein homoeostasis, underpins the cell cycle, and is dysregulated in cancer. However, the role of individual E3 ubiquitin ligases, which mediate the final step in ubiquitin-mediated proteolysis, remains incompletely understood. Identified through screening for cancer-specific endogenous retroviral transcripts, we show that the little-studied E3 ubiquitin ligase HECTD2 exerts dominant control of tumour progression in melanoma. HECTD2 cell autonomously drives the proliferation of human and murine melanoma cells by accelerating the cell cycle. HECTD2 additionally regulates cancer cell production of immune mediators, initiating multiple immune suppressive pathways, which include the cyclooxygenase 2 (COX2) pathway. Accordingly, higher HECTD2 expression is associated with weaker anti-tumour immunity and unfavourable outcome of PD-1 blockade in human melanoma and counteracts immunity against a model tumour antigen in murine melanoma. This central, multifaceted role of HECTD2 in cancer cell-autonomous proliferation and in immune evasion may provide a single target for a multipronged therapy of melanoma.

Gv1, a Zinc Finger Gene Controlling Endogenous MLV Expression.

The genomes of inbred mice harbor around 50 endogenous murine leukemia virus (MLV) loci, although the specific complement varies greatly between strains. The Gv1 locus is known to control the transcription of endogenous MLVs and to be the dominant determinant of cell-surface presentation of MLV envelope, the GIX antigen. Here, we identify a single Krüppel-associated box zinc finger protein (ZFP) gene, Zfp998, as Gv1 and show it to be necessary and sufficient to determine the GIX+ phenotype. By long-read sequencing of bacterial artificial chromosome clones from 129 mice, the prototypic GIX+ strain, we reveal the source of sufficiency and deficiency as splice-acceptor variations and highlight the varying origins of the chromosomal region encompassing Gv1. Zfp998 becomes the second identified ZFP gene responsible for epigenetic suppression of endogenous MLVs in mice and further highlights the prominent role of this gene family in control of endogenous retroviruses.

Group-2 innate lymphoid cell-dependent regulation of tissue neutrophil migration by alternatively activated macrophage-secreted Ear11.

Type-2 immunity is characterised by interleukin (IL)-4, IL-5 and IL-13, eosinophilia, mucus production, IgE, and alternatively activated macrophages (AAM). However, despite the lack of neutrophil chemoattractants such as CXCL1, neutrophils, a feature of type-1 immunity, are observed in type-2 responses. Consequently, alternative mechanisms must exist to ensure that neutrophils can contribute to type-2 immune reactions without escalation of deleterious inflammation. We now demonstrate that type-2 immune-associated neutrophil infiltration is regulated by the mouse RNase A homologue, eosinophil-associated ribonuclease 11 (Ear11), which is secreted by AAM downstream of IL-25-stimulated ILC2. Transgenic overexpression of Ear11 resulted in tissue neutrophilia, whereas Ear11-deficient mice have fewer resting tissue neutrophils, whilst other type-2 immune responses are not impaired. Notably, administration of recombinant mouse Ear11 increases neutrophil motility and recruitment. Thus, Ear11 helps maintain tissue neutrophils at homoeostasis and during type-2 reactions when chemokine-producing classically activated macrophages are infrequently elicited.

Antibody-induced internalisation of retroviral envelope glycoproteins is a signal initiation event.

As obligate parasites, viruses highjack, modify and repurpose the cellular machinery for their own replication. Viral proteins have, therefore, evolved biological functions, such as signalling potential, that alter host cell physiology in ways that are still incompletely understood. Retroviral envelope glycoproteins interact with several host proteins, extracellularly with their cellular receptor and anti-envelope antibodies, and intracellularly with proteins of the cytoskeleton or sorting, endocytosis and recirculation pathways. Here, we examined the impact of endogenous retroviral envelope glycoprotein expression and interaction with host proteins, particularly antibodies, on the cell, independently of retroviral infection. We found that in the commonly used C57BL/6 substrains of mice, where murine leukaemia virus (MLV) envelope glycoproteins are expressed by several endogenous MLV proviruses, the highest expressed MLV envelope glycoprotein is under the control of an immune-responsive cellular promoter, thus linking MLV envelope glycoprotein expression with immune activation. We further showed that antibody ligation induces extensive internalisation from the plasma membrane into endocytic compartments of MLV envelope glycoproteins, which are not normally subject to constitutive endocytosis. Importantly, antibody binding and internalisation of MLV envelope glycoproteins initiates signalling cascades in envelope-expressing murine lymphocytic cell lines, leading to cellular activation. Similar effects were observed by MLV envelope glycoprotein ligation by its cellular receptor mCAT-1, and by overexpression in human lymphocytic cells, where it required an intact tyrosine-based YXXΦ motif in the envelope glycoprotein cytoplasmic tail. Together, these results suggest that signalling potential is a general property of retroviral envelope glycoproteins and, therefore, a target for intervention.

IL-33 is more potent than IL-25 in provoking IL-13-producing nuocytes (type 2 innate lymphoid cells) and airway contraction.

BACKGROUND: IL-25 and IL-33 belong to distinct cytokine families, but experimental mouse studies suggest their immunologic functions in type 2 immunity are almost entirely overlapping. However, only polymorphisms in the IL-33 pathway (IL1RL1 and IL33) have been significantly associated with asthma in large-cohort genome-wide association studies. OBJECTIVE: We sought to identify distinct pathways for IL-25 and IL-33 in the lung that might provide insight into their roles in asthma pathogenesis and potential for therapeutic intervention. METHODS: IL-25 receptor-deficient (Il17rb(-/-)), IL-33 receptor-deficient (ST2, Il1rl1(-/-)), and double-deficient (Il17rb(-/-)Il1rl1(-/-)) mice were analyzed in models of allergic asthma. Microarrays, an ex vivo lung slice airway contraction model, and Il13(+/eGFP) mice were then used to identify specific effects of IL-25 and IL-33 administration. RESULTS: Comparison of IL-25 and IL-33 pathway-deficient mice demonstrates that IL-33 signaling plays a more important in vivo role in airways hyperreactivity than IL-25. Furthermore, methacholine-induced airway contraction ex vivo increases after treatment with IL-33 but not IL-25. This is dependent on expression of the IL-33 receptor and type 2 cytokines. Confocal studies with Il13(+/eGFP) mice show that IL-33 more potently induces expansion of IL-13-producing type 2 innate lymphoid cells, correlating with airway contraction. This predominance of IL-33 activity is enforced in vivo because IL-33 is more rapidly expressed and released in comparison with IL-25. CONCLUSION: Our data demonstrate that IL-33 plays a critical role in the rapid induction of airway contraction by stimulating the prompt expansion of IL-13-producing type 2 innate lymphoid cells, whereas IL-25-induced responses are slower and less potent.

IL-33 citrine reporter mice reveal the temporal and spatial expression of IL-33 during allergic lung inflammation.

Interleukin-33 (IL-33) is an IL-1 family cytokine that signals via its receptor T1/ST2, and is a key regulator of inflammation, notably the type-2 response implicated in allergic asthma. Critical to our understanding of the role of IL-33 is the identification of the cellular sources of IL-33. Although progress has been made in this area, the development of a robust live cell reporter of expression would allow the localisation of IL-33 during ongoing immune responses. We have generated a fluorescent reporter mouse line, Il33(Cit/+), to define the expression profile of IL-33 in vivo and demonstrate its temporal and spatial expression during experimental allergic asthma responses. We found that type-2 pneumocytes constitute the major source of IL-33 upon allergic lung inflammation following exposure to OVA, fungal extract or ragweed pollen. Using Il33(Cit/Cit) mice (IL-33-deficient), we establish a role for IL-33 early in the initiation of type-2 responses and the induction of nuocytes (ILC2). We also demonstrate a potential mechanism of action by which IL-33 rapidly initiates type-2 immune responses. Il33(Cit/+) mice have enabled new insights into the initiation of type-2 responses and will provide an important tool for further dissection of this important inflammatory pathway in vivo.

Transcription factor RORα is critical for nuocyte development.

Nuocytes are essential in innate type 2 immunity and contribute to the exacerbation of asthma responses. Here we found that nuocytes arose in the bone marrow and differentiated from common lymphoid progenitors, which indicates they are distinct, previously unknown members of the lymphoid lineage. Nuocytes required interleukin 7 (IL-7), IL-33 and Notch signaling for development in vitro. Pro-T cell progenitors at double-negative stage 1 (DN1) and DN2 maintained nuocyte potential in vitro, although the thymus was not essential for nuocyte development. Notably, the transcription factor RORα was critical for the development of nuocytes and their role in the expulsion of parasitic worms.

Receptor tyrosine phosphatase PTPγ is a regulator of spinal cord neurogenesis.

During spinal cord development the proliferation, migration and survival of neural progenitors and precursors is tightly controlled, generating the fine spatial organisation of the cord. In order to understand better the control of these processes, we have examined the function of an orphan receptor protein tyrosine phosphatase (RPTP) PTPγ, in the developing chick spinal cord. Widespread expression of PTPγ occurs post-embryonic day 3 in the early cord and is consistent with a potential role in either neurogenesis or neuronal maturation. Using gain-of-function and loss-of-function approaches in ovo, we show that PTPγ perturbation significantly reduces progenitor proliferation rates and neuronal precursor numbers, resulting in hypoplasia of the neuroepithelium. PTPγ gain-of-function causes widespread suppression of Wnt/ß-catenin-driven TCF signalling. One potential target of PTPγ may therefore be ß-catenin itself, since PTPγ can dephosphorylate it in vitro, but alternative targets are also likely. PTPγ loss-of-function is not sufficient to alter TCF signalling. Instead, loss-of-function leads to increased apoptosis and defective cell-cell adhesion in progenitors and precursors. Furthermore, motor neuron precursor migration is specifically defective. PTPγ therefore regulates neurogenesis during a window of spinal cord development, with molecular targets most likely related to Wnt/ß-catenin signalling, cell survival and cell adhesion.