Mef2d potentiates type-2 immune responses and allergic lung inflammation.

Innate lymphoid cells (ILCs) and adaptive T lymphocytes promote tissue homeostasis and protective immune responses. Their production depends on the transcription factor GATA3, which is further elevated specifically in ILC2s and T helper 2 cells to drive type-2 immunity during tissue repair, allergic disorders, and anti-helminth immunity. The control of this crucial up-regulation is poorly understood. Using CRISPR screens in ILCs we identified previously unappreciated myocyte-specific enhancer factor 2d (Mef2d)-mediated regulation of GATA3-dependent type-2 lymphocyte differentiation. Mef2d-deletion from ILC2s and/or T cells specifically protected against an allergen lung challenge. Mef2d repressed Regnase-1 endonuclease expression to enhance IL-33 receptor production and IL-33 signaling and acted downstream of calcium-mediated signaling to translocate NFAT1 to the nucleus to promote type-2 cytokine-mediated immunity.

Neuroprotective protein ADNP-dependent histone remodeling complex promotes T helper 2 immune cell differentiation.

Type 2 immune responses are critical in tissue homeostasis, anti-helminth immunity, and allergy. T helper 2 (Th2) cells produce interleukin-4 (IL-4), IL-5, and IL-13 from the type 2 gene cluster under regulation by transcription factors (TFs) including GATA3. To better understand transcriptional regulation of Th2 cell differentiation, we performed CRISPR-Cas9 screens targeting 1,131 TFs. We discovered that activity-dependent neuroprotector homeobox protein (ADNP) was indispensable for immune reactions to allergen. Mechanistically, ADNP performed a previously unappreciated role in gene activation, forming a critical bridge in the transition from pioneer TFs to chromatin remodeling by recruiting the helicase CHD4 and ATPase BRG1. Although GATA3 and AP-1 bound the type 2 cytokine locus in the absence of ADNP, they were unable to initiate histone acetylation or DNA accessibility, resulting in highly impaired type 2 cytokine expression. Our results demonstrate an important role for ADNP in promoting immune cell specialization.

An αvß3 integrin checkpoint is critical for efficient TH2 cell cytokine polarization and potentiation of antigen-specific immunity.

Naive CD4+ T lymphocytes initially undergo antigen-specific activation to promote a broad-spectrum response before adopting bespoke cytokine expression profiles shaped by intercellular microenvironmental cues, resulting in pathogen-focused modular cytokine responses. Interleukin (IL)-4-induced Gata3 upregulation is important for the helper type 2 T cell (TH2 cell) polarization associated with anti-helminth immunity and misdirected allergic inflammation. Whether additional microenvironmental factors participate is unclear. Using whole mouse-genome CRISPR-Cas9 screens, we discovered a previously unappreciated role for αvß3 integrin in TH2 cell differentiation. Low-level αvß3 expression by naive CD4+ T cells contributed to pan-T cell activation by promoting T-T cell clustering and IL-2/CD25/STAT5 signaling. Subsequently, IL-4/Gata3-induced selective upregulation of αvß3 licensed intercellular αvß3-Thy1 interactions among TH2 cells, enhanced mammalian target of rapamycin (mTOR) signaling, supported differentiation and promoted IL-5/IL-13 production. In mice, αvß3 was required for efficient, allergen-driven, antigen-specific lung TH2 cell responses. Thus, αvß3-expressing TH2 cells form multicellular factories to propagate and amplify TH2 cell responses.

Identification of aceNKPs, a committed common progenitor population of the ILC1 and NK cell continuum.

The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC hematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution, we have characterized bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK cell progenitors (ILC1/NKP), which we call "aceNKPs", are defined as lineage-Id2+IL-7Rα+CD25-α4ß7-NKG2A/C/E+Bcl11b-. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-γ and perforin upon IL-15 stimulation. Following reconstitution of Rag2-/-Il2rg-/- hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumor burden in a model of lung metastasis, where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing.

RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

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.