New Molecular Insights into Immune Cell Development.

During development innate lymphoid cells and specialized lymphocyte subsets colonize peripheral tissues, where they contribute to organogenesis and later constitute the first line of protection while maintaining tissue homeostasis. A few of these subsets are produced only during embryonic development and remain in the tissues throughout life. They are generated through a unique developmental program initiated in lympho-myeloid-primed progenitors, which lose myeloid and B cell potential. They either differentiate into innate lymphoid cells or migrate to the thymus to give rise to embryonic T cell receptor-invariant T cells. At later developmental stages, adaptive T lymphocytes are derived from lympho-myeloid progenitors that colonize the thymus, while lymphoid progenitors become specialized in the production of B cells. This sequence of events highlights the requirement for stratification in the establishment of immune functions that determine efficient seeding of peripheral tissues by a limited number of cells.

Long noncoding RNA lncKdm2b is required for ILC3 maintenance by initiation of Zfp292 expression.

Innate lymphoid cells (ILCs) communicate with other hematopoietic and nonhematopoietic cells to regulate immunity, inflammation and tissue homeostasis. How ILC lineages develop and are maintained remains largely unknown. In this study we observed that a divergent long noncoding RNA (lncRNA), lncKdm2b, was expressed at high levels in intestinal group 3 ILCs (ILC3s). LncKdm2b deficiency in the hematopoietic system led to reductions in the number and effector functions of ILC3s. LncKdm2b expression sustained the maintenance of ILC3s by promoting their proliferation through activation of the transcription factor Zfp292. Mechanistically, lncKdm2b recruited the chromatin organizer Satb1 and the nuclear remodeling factor (NURF) complex onto the Zfp292 promoter to initiate its transcription. Deletion of Zfp292 or Bptf also abrogated the maintenance of ILC3s, leading to susceptibility to bacterial infection. Therefore, our findings reveal that lncRNAs may represent an additional layer of regulation of ILC development and function.

Arginase 1 is an innate lymphoid-cell-intrinsic metabolic checkpoint controlling type 2 inflammation.

Group 2 innate lymphoid cells (ILC2s) regulate tissue inflammation and repair after activation by cell-extrinsic factors such as host-derived cytokines. However, the cell-intrinsic metabolic pathways that control ILC2 function are undefined. Here we demonstrate that expression of the enzyme arginase-1 (Arg1) during acute or chronic lung inflammation is a conserved trait of mouse and human ILC2s. Deletion of mouse ILC-intrinsic Arg1 abrogated type 2 lung inflammation by restraining ILC2 proliferation and dampening cytokine production. Mechanistically, inhibition of Arg1 enzymatic activity disrupted multiple components of ILC2 metabolic programming by altering arginine catabolism, impairing polyamine biosynthesis and reducing aerobic glycolysis. These data identify Arg1 as a key regulator of ILC2 bioenergetics that controls proliferative capacity and proinflammatory functions promoting type 2 inflammation.

Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing.

According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)Flt3(hi)) and common myeloid progenitors (CMPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)CD41(hi)) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre-granulocyte-macrophage progenitor (pre-GM) (Lin(-)Sca-1(-)c-Kit(+)CD41(-)FcγRII/III(-)CD150(-)CD105(-)). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential.

Polychromic Reporter Mice Reveal Unappreciated Innate Lymphoid Cell Progenitor Heterogeneity and Elusive ILC3 Progenitors in Bone Marrow.

Innate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. ILCs arise from lymphoid progenitors undergoing lineage restriction and the development of specialized ILC subsets. We generated "5x polychromILC" transcription factor reporter mice to delineate ILC precursor states by revealing the multifaceted expression of key ILC-associated transcription factors (Id2, Bcl11b, Gata3, RORγt, and RORα) during ILC development in the bone marrow. This approach allowed previously unattained enrichment of rare progenitor subsets and revealed hitherto unappreciated ILC precursor heterogeneity. In vivo and in vitro assays identified precursors with potential to generate all ILC subsets and natural killer (NK) cells, and also permitted discrimination of elusive ILC3 bone marrow antecedents. Single-cell gene expression analysis identified a discrete ILC2-committed population and delineated transition states between early progenitors and a highly heterogeneous ILC1, ILC3, and NK precursor cell cluster. This diversity might facilitate greater lineage potential upon progenitor recruitment to peripheral tissues.

Characterization of Transcriptional Regulatory Networks that Promote and Restrict Identities and Functions of Intestinal Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) promote tissue homeostasis and immune defense but also contribute to inflammatory diseases. ILCs exhibit phenotypic and functional plasticity in response to environmental stimuli, yet the transcriptional regulatory networks (TRNs) that control ILC function are largely unknown. Here, we integrate gene expression and chromatin accessibility data to infer regulatory interactions between transcription factors (TFs) and genes within intestinal type 1, 2, and 3 ILC subsets. We predicted the "core" TFs driving ILC identities, organized TFs into cooperative modules controlling distinct gene programs, and validated roles for c-MAF and BCL6 as regulators affecting type 1 and type 3 ILC lineages. The ILC network revealed alternative-lineage-gene repression, a mechanism that may contribute to reported plasticity between ILC subsets. By connecting TFs to genes, the TRNs suggest means to selectively regulate ILC effector functions, while our network approach is broadly applicable to identifying regulators in other in vivo cell populations.

Calcitonin Gene-Related Peptide Negatively Regulates Alarmin-Driven Type 2 Innate Lymphoid Cell Responses.

Neuroimmune interactions have emerged as critical modulators of allergic inflammation, and type 2 innate lymphoid cells (ILC2s) are an important cell type for mediating these interactions. Here, we show that ILC2s expressed both the neuropeptide calcitonin gene-related peptide (CGRP) and its receptor. CGRP potently inhibited alarmin-driven type 2 cytokine production and proliferation by lung ILC2s both in vitro and in vivo. CGRP induced marked changes in ILC2 expression programs in vivo and in vitro, attenuating alarmin-driven proliferative and effector responses. A distinct subset of ILCs scored highly for a CGRP-specific gene signature after in vivo alarmin stimulation, suggesting CGRP regulated this response. Finally, we observed increased ILC2 proliferation and type 2 cytokine production as well as exaggerated responses to alarmins in mice lacking the CGRP receptor. Together, these data indicate that endogenous CGRP is a critical negative regulator of ILC2 responses in vivo.

Transcriptional programs define molecular characteristics of innate lymphoid cell classes and subsets.

The recognized diversity of innate lymphoid cells (ILCs) is rapidly expanding. Three ILC classes have emerged, ILC1, ILC2 and ILC3, with ILC1 and ILC3 including several subsets. The classification of some subsets is unclear, and it remains controversial whether natural killer (NK) cells and ILC1 cells are distinct cell types. To address these issues, we analyzed gene expression in ILCs and NK cells from mouse small intestine, spleen and liver, as part of the Immunological Genome Project. The results showed unique gene-expression patterns for some ILCs and overlapping patterns for ILC1 cells and NK cells, whereas other ILC subsets remained indistinguishable. We identified a transcriptional program shared by small intestine ILCs and a core ILC signature. We revealed and discuss transcripts that suggest previously unknown functions and developmental paths for ILCs.

E3 Ligase VHL Promotes Group 2 Innate Lymphoid Cell Maturation and Function via Glycolysis Inhibition and Induction of Interleukin-33 Receptor.

Group 2 innate lymphoid cells (ILC2s) are a specialized subset of lymphoid effector cells that are critically involved in allergic responses; however, the mechanisms of their regulation remain unclear. We report that conditional deletion of the E3 ubiquitin ligase VHL in innate lymphoid progenitors minimally affected early-stage bone marrow ILC2s but caused a selective and intrinsic decrease in mature ILC2 numbers in peripheral non-lymphoid tissues, resulting in reduced type 2 immune responses. VHL deficiency caused the accumulation of hypoxia-inducible factor 1α (HIF1α) and attenuated interleukin-33 (IL-33) receptor ST2 expression, which was rectified by HIF1α ablation or inhibition. HIF1α-driven expression of the glycolytic enzyme pyruvate kinase M2 downmodulated ST2 expression via epigenetic modification and inhibited IL-33-induced ILC2 development. Our study indicates that the VHL-HIF-glycolysis axis is essential for the late-stage maturation and function of ILC2s via targeting IL-33-ST2 pathway.

Interleukin-33-Activated Islet-Resident Innate Lymphoid Cells Promote Insulin Secretion through Myeloid Cell Retinoic Acid Production.

Pancreatic-islet inflammation contributes to the failure of ß cell insulin secretion during obesity and type 2 diabetes. However, little is known about the nature and function of resident immune cells in this context or in homeostasis. Here we show that interleukin (IL)-33 was produced by islet mesenchymal cells and enhanced by a diabetes milieu (glucose, IL-1ß, and palmitate). IL-33 promoted ß cell function through islet-resident group 2 innate lymphoid cells (ILC2s) that elicited retinoic acid (RA)-producing capacities in macrophages and dendritic cells via the secretion of IL-13 and colony-stimulating factor 2. In turn, local RA signaled to the ß cells to increase insulin secretion. This IL-33-ILC2 axis was activated after acute ß cell stress but was defective during chronic obesity. Accordingly, IL-33 injections rescued islet function in obese mice. Our findings provide evidence that an immunometabolic crosstalk between islet-derived IL-33, ILC2s, and myeloid cells fosters insulin secretion.

Ikaros Inhibits Group 3 Innate Lymphoid Cell Development and Function by Suppressing the Aryl Hydrocarbon Receptor Pathway.

Group 3 innate lymphoid cells (ILC3s) expressing the transcription factor (TF) RORγt are important for the defense and homeostasis of host intestinal tissues. The zinc finger TF Ikaros, encoded by Ikzf1, is essential for the development of RORγt(+) fetal lymphoid tissue inducer (LTi) cells and lymphoid organogenesis, but its role in postnatal ILC3s is unknown. Here, we show that small-intestinal ILC3s had lower Ikaros expression than ILC precursors and other ILC subsets. Ikaros inhibited ILC3s in a cell-intrinsic manner through zinc-finger-dependent inhibition of transcriptional activity of the aryl hydrocarbon receptor, a key regulator of ILC3 maintenance and function. Ablation of Ikzf1 in RORγt(+) ILC3s resulted in increased expansion and cytokine production of intestinal ILC3s and protection against infection and colitis. Therefore, in contrast to being required for LTi development, Ikaros inhibits postnatal ILC3 development and function to regulate gut immune responses at steady state and in disease.

Mass Cytometry of the Human Mucosal Immune System Identifies Tissue- and Disease-Associated Immune Subsets.

Inflammatory intestinal diseases are characterized by abnormal immune responses and affect distinct locations of the gastrointestinal tract. Although the role of several immune subsets in driving intestinal pathology has been studied, a system-wide approach that simultaneously interrogates all major lineages on a single-cell basis is lacking. We used high-dimensional mass cytometry to generate a system-wide view of the human mucosal immune system in health and disease. We distinguished 142 immune subsets and through computational applications found distinct immune subsets in peripheral blood mononuclear cells and intestinal biopsies that distinguished patients from controls. In addition, mucosal lymphoid malignancies were readily detected as well as precursors from which these likely derived. These findings indicate that an integrated high-dimensional analysis of the entire immune system can identify immune subsets associated with the pathogenesis of complex intestinal disorders. This might have implications for diagnostic procedures, immune-monitoring, and treatment of intestinal diseases and mucosal malignancies.

Transforming Growth Factor-ß Signaling Guides the Differentiation of Innate Lymphoid Cells in Salivary Glands.

The signals guiding differentiation of innate lymphoid cells (ILCs) within tissues are not well understood. Salivary gland (SG) ILCs as well as liver and intestinal intraepithelial ILC1 have markers that denote tissue residency and transforming growth factor-ß (TGF-ß) imprinting. We deleted Tgfbr2 in cells expressing the ILC and NK marker NKp46 and found that SG ILCs were reduced in number. They lost distinct tissue markers, such as CD49a, and the effector molecules TRAIL and CD73. Expression of the transcription factor Eomes, which promotes NK cell differentiation, was elevated. Conversely, Eomes deletion in NKp46(+) cells enhanced TGF-ß-imprinting of SG ILCs. Thus, TGF-ß induces SG ILC differentiation by suppressing Eomes. TGF-ß acted through a JNK-dependent, Smad4-independent pathway. Transcriptome analysis demonstrated that SG ILCs had characteristic of both NK cells and ILC1. Finally, TGF-ß imprinting of SG ILCs was synchronized with SG development, highlighting the impact of tissue microenvironment on ILC development.

White, brown, and beige; type 2 immunity gets hot.

The biogenesis of beige fat is poorly understood. In recent issues of Nature and Cell, Brestoff et al. (2014) and Lee et al. (2015) demonstrate that resident innate lymphoid cells in subcutaneous fat generate and activate beige adipocytes, producing thermogenesis.

A Gata3 enhancer necessary for ILC2 development and function.

The type 2 helper effector program is driven by the master transcription factor GATA3 and can be expressed by subsets of both innate lymphoid cells (ILCs) and adaptive CD4+ T helper (Th) cells. While ILC2s and Th2 cells acquire their type 2 differentiation program under very different contexts, the distinct regulatory mechanisms governing this common program are only partially understood. Here we show that the differentiation of ILC2s, and their concomitant high level of GATA3 expression, are controlled by a Gata3 enhancer, Gata3 +674/762, that plays only a minimal role in Th2 cell differentiation. Mice lacking this enhancer exhibited defects in several but not all type 2 inflammatory responses, depending on the respective degree of ILC2 and Th2 cell involvement. Our study provides molecular insights into the different gene regulatory pathways leading to the acquisition of the GATA3-driven type 2 helper effector program in innate and adaptive lymphocytes.

Cellular complexity in brain organoids: Current progress and unsolved issues.

Brain organoids are three-dimensional neural aggregates derived from pluripotent stem cells through self-organization and recapitulate architectural and cellular aspects of certain brain regions. Brain organoids are currently a highly exciting area of research that includes the study of human brain development, function, and dysfunction in unprecedented ways. In this Review, we discuss recent discoveries related to the generation of brain organoids that resemble diverse brain regions. We provide an overview of the strategies to complement these primarily neuroectodermal models with cell types of non-neuronal origin, such as vasculature and immune cells. Recent transplantation approaches aiming to achieve higher cellular complexity and long-term survival of these models will then be discussed. We conclude by highlighting unresolved key questions and future directions in this exciting area of human brain organogenesis.

Comparison of pan-immune-inflammation value with other inflammation markers of long-term survival after ST-segment elevation myocardial infarction.

BACKGROUND: Atherosclerosis is a process that causes coronary artery disease and is associated with the inflammatory response. In this study, we aimed to evaluate the association of Pan-Immune-Inflammation Value (PIV) with in-hospital and long-term mortality in STEMI patients. METHODS: A total of 658 patients who were admitted to the emergency department of two tertiary centers with the diagnosis of STEMI and underwent percutaneous coronary intervention (PCI) between 2018 and 2022 were retrospectively enrolled. PIV and other inflammation parameters were compared for the study population. The primary outcome was one-year all-cause of mortality. RESULTS: The mean age was 58.7 ± 17.1 years and 507 (76.9%) were male. The mean duration of the follow-up was 18.8 ± 8.5 months (median 18.9 months). PIV was superior to the neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, and systemic immune-inflammation index for the prediction of primary and secondary outcomes in STEMI. CONCLUSION: Our study reveals that PIV is a better predictor of mortality in STEMI patients. Prospective studies are needed to validate this biomarker.

Human RORγt(+)CD34(+) cells are lineage-specified progenitors of group 3 RORγt(+) innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) are defined by the expression of the transcription factor RORγt, which is selectively required for their development. The lineage-specified progenitors of ILC3s and their site of development after birth remain undefined. Here we identified a population of human CD34(+) hematopoietic progenitor cells (HPCs) that express RORγt and share a distinct transcriptional signature with ILC3s. RORγt(+)CD34(+) HPCs were located in tonsils and intestinal lamina propria (LP) and selectively differentiated toward ILC3s. In contrast, RORγt(-)CD34(+) HPCs could differentiate to become either ILC3s or natural killer (NK) cells, with differentiation toward ILC3 lineage determined by stem cell factor (SCF) and aryl hydrocarbon receptor (AhR) signaling. Thus, we demonstrate that in humans RORγt(+)CD34(+) cells are lineage-specified progenitors of IL-22(+) ILC3s and propose that tonsils and intestinal LP, which are enriched both in committed precursors and mature ILC3s, might represent preferential sites of ILC3 lineage differentiation.

Niche-Mediated Integrin Signaling Supports Steady-State Hematopoiesis in the Spleen.

Outside-in integrin signaling regulates cell fate decisions in a variety of cell types, including hematopoietic stem cells (HSCs). Our earlier published studies showed that interruption of periostin (POSTN) and integrin-αv (ITGAV) interaction induces faster proliferation in HSCs with developmental stage-dependent functional effects. In this study, we examined the role of POSTN-ITGAV axis in lymphohematopoietic activity in spleen that hosts a rare population of HSCs, the functional regulation of which is not clearly known. Vav-iCre-mediated deletion of Itgav in the hematopoietic system led to higher proliferation rates, resulting in increased frequency of primitive HSCs in the adult spleen. However, in vitro CFU-C assays demonstrated a poorer differentiation potential following Itgav deletion. This also led to a decrease in the white pulp area with a significant decline in the B cell numbers. Systemic deletion of its ligand, POSTN, phenocopied the effects noted in Vav-Itgav-/- mice. Histological examination of Postn-deficient spleen also showed an increase in the spleen trabecular areas. Importantly, these are the myofibroblasts of the trabecular and capsular areas that expressed high levels of POSTN within the spleen tissue. In addition, vascular smooth muscle cells also expressed POSTN. Through CFU-S12 assays, we showed that hematopoietic support potential of stroma in Postn-deficient splenic hematopoietic niche was defective. Overall, we demonstrate that POSTN-ITGAV interaction plays an important role in spleen lymphohematopoiesis.