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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., Competing Interests: The authors declare no competing interest.

Transcription factor (TF) reporter mice have proved integral to the characterization of murine innate lymphoid cell (ILC) development and function. Here, we implemented a CRISPR/Cas9-generated combinatorial reporter approach for the simultaneous resolution of several key TFs throughout ILC development in both the fetal liver and adult bone marrow. We demonstrate that the Tcf7-expressing early innate lymphoid precursor (EILP) and the common helper ILC precursor (CHILP) both contain a heterogeneous mixture of specified ILC and lymphoid tissue inducer (LTi) precursors with restricted lineage potential rather than a shared precursor. Moreover, the earliest specified precursor to the LTi lineage was identified upstream of these populations, before Tcf7 expression. These findings match dynamic changes in chromatin accessibility associated with the expression of key TFs (i.e., GATA3 and RORγ(t)), highlighting the distinct origins of ILC and LTi lineages at the epigenetic and functional levels, and provide a revised map for ILC development., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Kasal and Bendelac.)

Zbtb16-encoded PLZF is a signature transcription factor (TF) that directs the acquisition of T-helper effector programs during the development of multiple innate lymphocyte lineages, including natural killer T cell, innate lymphoid cell, mucosal-associated invariant T cell and γδ lineages. PLZF is also essential in osteoblast and spermatogonial development. How Zbtb16 itself is regulated in different lineages is incompletely understood. Here, by systematic CRISPR/Cas9-assisted deletions of chromatin accessible regions within the Zbtb16 locus in mouse, we identify a critical enhancer controlling PLZF expression exclusively in innate lymphoid lineages. Multiple sites within this enhancer express canonical motifs for the TF Runx1, which is essential for the development of these lineages. Notably, some regulatory sites control the kinetic rather than the overall level of PLZF expression. Thus, our comprehensive, unbiased analysis of regulatory elements in vivo reveals critical mechanisms of Zbtb16 regulation shared between innate and innate-like lymphoid lineages. Zbtb16-encoded transcription factor PLZF directs the differentiation of multiple innate and innate-like cell lineages, but how Zbtb16 itself is regulated remains unclear. Here the authors show, using CRISPR gene editing, ATAC-seq and ChIP-seq, that specific Runx1-bound enhancer elements critically modulate lineage-dependent expressions of PLZF.

The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages.

The precise lineage relationship between innate lymphoid cells (ILCs) and lymphoid tissue-inducer (LTi) cells is poorly understood. Using single-cell multiplex transcriptional analysis of 100 lymphoid genes and single-cell cultures of fetal liver precursor cells, we identified the common proximal precursor to these lineages and found that its bifurcation was marked by differential induction of the transcription factors PLZF and TCF1. Acquisition of individual effector programs specific to the ILC subsets ILC1, ILC2 and ILC3 was initiated later, at the common ILC precursor stage, by transient expression of mixed ILC1, ILC2 and ILC3 transcriptional patterns, whereas, in contrast, the development of LTi cells did not go through multilineage priming. Our findings provide insight into the divergent mechanisms of the differentiation of the ILC lineage and LTi cell lineage and establish a high-resolution 'blueprint' of their development.

Among the variety of tissue-resident NK-like populations recently distinguished from recirculating classical NK (cNK) cells, liver innate lymphoid cells (ILC) type 1 (ILC1s) have been shown to represent a distinct lineage that originates from a novel promyelocytic leukaemia zinc finger (PLZF)-expressing ILC precursor (ILCP) strictly committed to the ILC1, ILC2, and ILC3 lineages. Here, using PLZF-reporter mice and cell transfer assays, we studied the developmental progression of ILC1s and demonstrated substantial overlap with stages previously ascribed to the cNK lineage, including pre-pro-NK, pre-NK precursor (pre-NKP), refined NKP (rNKP), and immature NK (iNK). Although they originated from different precursors, the ILC1 and cNK lineages followed a parallel progression at early stages and diverged later at the iNK stage, with a striking predominance of ILC1s over cNKs early in ontogeny. Although a limited set of ILC1 genes depended on PLZF for expression, characteristically including Il7r, most of these genes were also differentially expressed between ILC1s and cNKs, indicating that PLZF together with other, yet to be defined, factors contribute to the divergence between these lineages.

Innate lymphoid cells (ILCs) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers. Their diversity and similarities with previously characterized natural killer (NK) cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 solely on the basis of cytokine properties, but their developmental pathways and lineage relationships remain elusive. Here we identify and characterize a novel subset of lymphoid precursors in mouse fetal liver and adult bone marrow that transiently express high amounts of PLZF, a transcription factor previously associated with NK T cell development, by using lineage tracing and transfer studies. PLZF(high) cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1(+)DX5(-) 'NK-like' cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZF(high) precursors also expressed high amounts of ID2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages. These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILCs, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.

The transcription factor PLZF [promyelocytic leukemia zinc finger, encoded by zinc finger BTB domain containing 16 (Zbtb16)] is induced during the development of innate and innate-like lymphocytes to direct their acquisition of a T-helper effector program, but the molecular mechanisms involved are poorly understood. Using biotinylationbased ChIP-seq and microarray analysis of both natural killer T (NKT) cells and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program. First, PLZF bound and regulated genes encoding cytokine receptors as well as homing and adhesion receptors; second, PLZF bound and activated T-helper- specific transcription factor genes that in turn control T-helper-specific programs; finally, PLZF bound and suppressed the transcription of Bach2, a potent general repressor of effector differentiation in naive T cells. These findings reveal the multilayered architecture of the transcriptional program recruited by PLZF and elucidate how a single transcription factor can drive the developmental acquisition of a broad effector program. [ABSTRACT FROM AUTHOR]

Natural killer T (NKT) cells are a subset of T cells that recognize glycolipid antigens presented by the CD1d protein. The initial discovery of immunostimulatory glycolipids from a marine sponge and the T cells that respond to the compounds has led to extensive research by chemists and immunologists to understand how glycolipids are recognized, possible responses by NKT cells, and the structural features of glycolipids necessary for stimulatory activity. The presence of this cell type in humans and most mammals suggests that it plays critical roles in antigen recognition and the interface between innate and adaptive immunity. Both endogenous and exogenous natural antigens for NKT cells have been identified, and it is likely that glycolipid antigens remain to be discovered. Multiple series of structurally varied glycolipids have been synthesized and tested for stimulatory activity. The structural features of glycolipids necessary for NKT cell stimulation are moderately well understood, and designed compounds have proven to be much more potent antigens than their natural counterparts. Nevertheless, control over NKT cell responses by designed glycolipids has not been optimized, and further research will be required to fully reveal the therapeutic potential of this cell type. [ABSTRACT FROM AUTHOR]

Background: Id3 is a dominant antagonist of E protein transcription factor activity that is induced by signals emanating from the αβ and γδ T cell receptor (TCR). Mice lacking Id3 were previously shown to have subtle defects in positive and negative selection of TCRαβ+ T lymphocytes. More recently, Id3-/- mice on a C57BL/6 background were shown to have a dramatic expansion of γδ T cells. Methodology/Principal Findings: Here we report that mice lacking Id3 have reduced thymocyte numbers but increased production of γδ T cells that express a Vγ1.1+Vδ6.3+ receptor with restricted junctional diversity. These Vγ1.1+Vδ6.3+ T cells have multiple characteristics associated with ''innate'' lymphocytes such as natural killer T (NKT) cells including an activated phenotype, expression of the transcription factor PLZF, and rapid production of IFNg and interleukin-4. Moreover, like other ''innate'' lymphocyte populations, development of Id3-/- Vγ1.1+Vδ6.3+ T cells requires the signaling adapter protein SAP. Conclusions: Our data provide novel insight into the requirements for development of Vγ1.1+Vδ6.3+ T cells and indicate a role for Id3 in repressing the response of ''innate'' γδ T cells to SAP-mediated expansion or survival. [ABSTRACT FROM AUTHOR]

The S1P1 receptor, on the surface of lymphocytes and endothelial cells, regulates the unique trafficking behavior of certain lymphocyte populations. We have examined whether the S1P1 receptor also dictates the distinctive tissue distribution of Vα14-Jα18 natural killer T (NKT) cells, whose trafficking pattern is not well understood. Mice (TCS1P1KO) were established with a conditional deletion of the S1P1 receptor in thymocytes that included precursors of NKT cells. Within the thymus, NKT cells were found at normal or increased levels, indicating that S1P1 receptor expression was dispensable for NKT cell development. However, substantially reduced numbers of NKT cells were detected in the peripheral tissues of the TCS1P1KO mice. Short-term SIP1 deletion after NKT cells had established residence in the periphery did not substantially alter their distribution in tissues, except for a partial decrease in the spleen. FTY720, a S1P1 receptor ligand that has potent effects on the trafficking of conventional T cells, did not alter the preexisting distribution of NKT cells within peripheral tissues of wild-type mice. Our results indicate that the S1P1 receptor expression on NKT cells is dispensable for development within thymus but is essential for the establishment of their tissue residency in the periphery. [ABSTRACT FROM AUTHOR]

Natural killer T cells express a conserved, semi-invariant αβ T cell receptor that has specificity for self glycosphingolipids and microbial cell wall α-glycuronosylceramide antigens presented by CD1d molecules. Here we report the crystal structure of CD1d in complex with a short-chain synthetic variant of α-galactosylceramide at a resolution of 2.2 Å. This structure elucidates the basis for the high specificity of these microbial ligands and explains the restriction of the α-linkage as a unique pathogen-specific pattern-recognition motif. Comparison of the binding of altered lipid ligands to CD1d and T cell receptors suggested that the differential T helper type 1–like and T helper type 2–like properties of natural killer T cells may originate largely from differences in their 'loading' in different cell types and hence in their tissue distribution in vivo. [ABSTRACT FROM AUTHOR]

For some time, T cell receptor αβ (TCRαβ) signaling during thymocyte development was thought to induce one of three fates—negative selection to remove autoreactive cells, positive selection to enrich for host major histocompatibility complex (MHC)-restricted cells, and, in the absence of interactions with host MHC-peptides, death by neglect in experimental systems, negative selection is indeed found when thymocytes are presented with sufficient concentrations of their agonist antigen, that is, the same antigen that would activate them as mature T cells. In contrast, partial signaling mediated by variant peptides supports the positive selection of thymocytes, a step necessary for their lineage maturation into CD4 and CD8 T cells and exit from the thymus.

Summary: CD8+ T cells are selected via low-affinity interaction with MHC class I molecules on thymic epithelial cells (TECs). However, compromised T cell receptor signaling was proposed to force CD8+ T cell selection on hematopoietic cells through a SLAM-associated protein (SAP)-dependent mechanism similar to NKT cells. The outcome is an unconventional CD8+ T cell with phenotypic and functional characteristics of innate lymphocytes. Here we showed that Id3 −/− CD8+ T cells had an innate-like phenotype and required SAP for their development. However, like conventional CD8+ T cells, Id3 −/− CD8+ thymocytes were selected on TECs. The requirement for SAP and the innate-like phenotype was not intrinsic to Id3 −/− CD8+ thymocytes. Rather, an expanded population of NKT-like cells induced the innate phenotype on CD8+ T cells through production of interleukin-4. Our findings reveal that accumulation of NKT-like cells promotes conventional CD8+ thymocytes to acquire innate lymphocyte characteristics. [ABSTRACT FROM AUTHOR]

The developmental pathway of intestinal TCRαβ+CD4- CD8β- intraepithelial lymphocytes (unconventional iIELs) has remained elusive. Bendelac and colleagues demonstrate that expression of unconventional iIEL TCRs in thymocytes causes negative selection and that unconventional iIELs are derived from rare escapees of cortical negative selection. [ABSTRACT FROM AUTHOR]

Summary: The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations. [Copyright &y& Elsevier]

Rare CD1d-α-galactosylceramide-specific T cells that do not express the invariant Vα24 chain of human NKT cells were recently identified after expansion in vitro with the lipid Ag, but their phenotype and frequency in vivo and lineage relationship with NKT cells could not be elucidated. By using a CD1d tetramer-based method to enrich these cells from fresh peripheral blood, we demonstrated their naive-like CD62LhighCD45RO-CD4+ phenotype and relatively high frequency of ~10-5 in several healthy individuals. Notably, these cells expressed the NKT lineage-specific transcription promyelocytic leukemia zinc finger (PLZF), indicating a developmental relationship with NKT cells and ruling out the possibility that they were conventional MHC-restricted T cells cross-reacting against CD1d-α-galactosylceramide. Although PLZF is known to direct the effector program of NKT cells, we show in this study that the naive-like cells expressed it at a significantly lower amount than NKT cells. Further, we present mouse studies demonstrating a sharp PLZF expression threshold requirement for induction of the effector phenotype. These findings directly demonstrate in vivo the existence of naive-like CD1d-restricted human T cells marked by intermediate levels of PLZF. [ABSTRACT FROM AUTHOR]

Abstract: CD1d presentation of α-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an α-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an α-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations. [Copyright &y& Elsevier]

Summary: Invariant Vα14i NKT (iNKT) cells are a specialized subset of T lymphocytes with regulatory functions. They coexpress TCRαβ and natural killer cell markers. They differentiate through interaction of their Vα14-Jα18 invariant TCRα chains with CD1d expressed on double-positive (DP) thymocytes. Although their development has been shown to be thymus dependent, their developmental pathway has not been definitively established. By using genetic analyses, we show here that all iNKT cells are selected from a pool of DP thymocytes. Their development is absolutely dependent on Runx1 and RORγt, transcription factors that influence, but are not required for, development of conventional T cells. Our results indicate that even though CD1d binding DP thymocytes have yet to be observed, Vα14-Jα18 rearrangement in these cells is required for development of iNKT cells. [Copyright &y& Elsevier]