The Innate Lymphoid Cell Precursor.

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.

Eomes expression identifies the early bone marrow precursor to classical NK cells.

Natural killer (NK) cells traffic through the blood and mount cytolytic and interferon-γ (IFNγ)-focused responses to intracellular pathogens and tumors. Type 1 innate lymphoid cells (ILC1s) also produce type 1 cytokines but reside in tissues and are not cytotoxic. Whether these differences reflect discrete lineages or distinct states of a common cell type is not understood. Using single-cell RNA sequencing and flow cytometry, we focused on populations of TCF7+ cells that contained precursors for NK cells and ILC1s and identified a subset of bone marrow lineage-negative NK receptor-negative cells that expressed the transcription factor Eomes, termed EomeshiNKneg cells. Transfer of EomeshiNKneg cells into Rag2-/-Il2rg-/- recipients generated functional NK cells capable of preventing metastatic disease. By contrast, transfer of PLZF+ ILC precursors generated a mixture of ILC1s, ILC2s and ILC3s that lacked cytotoxic potential. These findings identified EomeshiNKneg cells as the bone marrow precursor to classical NK cells and demonstrated that the NK and ILC1 lineages diverged early during development.

GATA4 controls regionalization of tissue immunity and commensal-driven immunopathology.

There is growing recognition that regionalization of bacterial colonization and immunity along the intestinal tract has an important role in health and disease. Yet, the mechanisms underlying intestinal regionalization and its dysregulation in disease are not well understood. This study found that regional epithelial expression of the transcription factor GATA4 controls bacterial colonization and inflammatory tissue immunity in the proximal small intestine by regulating retinol metabolism and luminal IgA. Furthermore, in mice without jejunal GATA4 expression, the commensal segmented filamentous bacteria promoted pathogenic inflammatory immune responses that disrupted barrier function and increased mortality upon Citrobacter rodentium infection. In celiac disease patients, low GATA4 expression was associated with metabolic alterations, mucosal Actinobacillus, and increased IL-17 immunity. Taken together, these results reveal broad impacts of GATA4-regulated intestinal regionalization on bacterial colonization and tissue immunity, highlighting an elaborate interdependence of intestinal metabolism, immunity, and microbiota in homeostasis and disease.

Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissue-inducer cell lineage.

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.

IgA Responses to Microbiota.

Various immune mechanisms are deployed in the mucosa to confront the immense diversity of resident bacteria. A substantial fraction of the commensal microbiota is coated with immunoglobulin A (IgA) antibodies, and recent findings have established the identities of these bacteria under homeostatic and disease conditions. Here we review the current understanding of IgA biology, and present a framework wherein two distinct types of humoral immunity coexist in the gastrointestinal mucosa. Homeostatic IgA responses employ a polyreactive repertoire to bind a broad but taxonomically distinct subset of microbiota. In contrast, mucosal pathogens and vaccines elicit high-affinity, T cell-dependent antibody responses. This model raises fundamental questions including how polyreactive IgA specificities are generated, how these antibodies exert effector functions, and how they exist together with other immune responses during homeostasis and disease.

Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling.

Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the interleukin 2 (IL-2) receptor ß-chain, and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.

Crossreactive αß T Cell Receptors Are the Predominant Targets of Thymocyte Negative Selection.

The precise impact of thymic positive and negative selection on the T cell receptor (TCR) repertoire remains controversial. Here, we used unbiased, high-throughput cloning and retroviral expression of individual pre-selection TCRs to provide a direct assessment of these processes at the clonal level in vivo. We found that 15% of random TCRs induced signaling and directed positive (7.5%) or negative (7.5%) selection, depending on strength of signal, whereas the remaining 85% failed to induce signaling or selection. Most negatively selected TCRs exhibited promiscuous crossreactivity toward multiple other major histocompatibility complex (MHC) haplotypes. In contrast, TCRs that were positively selected or non-selected were minimally crossreactive. Negative selection of crossreactive TCRs led to clonal deletion but also recycling into intestinal CD4(-)CD8ß(-) intraepithelial lymphocytes (iIELs). Thus, broadly crossreactive TCRs arise at low frequency in the pre-selection repertoire but constitute the primary drivers of thymic negative selection and iIEL lineage differentiation.

Innate and Adaptive Humoral Responses Coat Distinct Commensal Bacteria with Immunoglobulin A.

Immunoglobulin A (IgA) is prominently secreted at mucosal surfaces and coats a fraction of the intestinal microbiota. However, the commensal bacteria bound by IgA are poorly characterized and the type of humoral immunity they elicit remains elusive. We used bacterial flow cytometry coupled with 16S rRNA gene sequencing (IgA-Seq) in murine models of immunodeficiency to identify IgA-bound bacteria and elucidate mechanisms of commensal IgA targeting. We found that residence in the small intestine, rather than bacterial identity, dictated induction of specific IgA. Most commensals elicited strong T-independent (TI) responses that originated from the orphan B1b lineage and from B2 cells, but excluded natural antibacterial B1a specificities. Atypical commensals including segmented filamentous bacteria and Mucispirillum evaded TI responses but elicited T-dependent IgA. These data demonstrate exquisite targeting of distinct commensal bacteria by multiple layers of humoral immunity and reveal a specialized function of the B1b lineage in TI mucosal IgA responses.

The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe.

Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus, are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens.

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.

Elevated T cell receptor signaling identifies a thymic precursor to the TCRαß(+)CD4(-)CD8ß(-) intraepithelial lymphocyte lineage.

The origin and developmental pathway of intestinal T cell receptor αß(+) CD4(-)CD8ß(-) intraepithelial lymphocytes (unconventional iIELs), a major population of innate-like resident cytolytic T cells, have remained elusive. By cloning and expressing several TCRs isolated from unconventional iIELs, we identified immature CD4(lo)CD8(lo)(DP(lo))CD69(hi)PD-1(hi) thymocytes as the earliest postsignaling precursors for these cells. Although these precursors displayed multiple signs of elevated TCR signaling, a sizeable fraction of them escaped deletion to selectively engage in unconventional iIEL differentiation. Conversely, TCRs cloned from DP(lo)CD69(hi)PD-1(hi) thymocytes, a population enriched in autoreactive thymocytes, selectively gave rise to unconventional iIELs upon transgenic expression. Thus, the unconventional iIEL precursor overlaps with the DP(lo) population undergoing negative selection, indicating that, concomitant with the downregulation of both CD4 and CD8 coreceptors, a balance between apoptosis and survival signals results in outcomes as divergent as clonal deletion and differentiation to the unconventional iIEL lineage.

The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian α-linked glycosylceramides.

Glycosylceramides in mammalian species are thought to be present in the form of ß-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylceramide synthase, both ß-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.

Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδ T cells.

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.

A committed precursor to innate lymphoid cells.

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.

SAP protein-dependent natural killer T-like cells regulate the development of CD8(+) T cells with innate lymphocyte characteristics.

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.

Multiple layers of transcriptional regulation by PLZF in NKT-cell development.

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 biotinylation-based 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.

BTB-ZF factors recruit the E3 ligase cullin 3 to regulate lymphoid effector programs.

The differentiation of several T- and B-cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodelling. Here we report that promyelocytic leukaemia zinc finger (PLZF), the BTB-zinc finger (BTB-ZF) transcription factor directing the innate-like effector program of natural killer T-cell thymocytes, is prominently associated with cullin 3 (CUL3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF transports CUL3 to the nucleus, where the two proteins are associated within a chromatin-modifying complex. Furthermore, PLZF expression results in selective ubiquitination changes of several components of this complex. CUL3 was also found associated with the BTB-ZF transcription factor BCL6, which directs the germinal-centre B cell and follicular T-helper cell programs. Conditional CUL3 deletion in mice demonstrated an essential role for CUL3 in the development of PLZF- and BCL6-dependent lineages. We conclude that distinct lineage-specific BTB-ZF transcription factors recruit CUL3 to alter the ubiquitination pattern of their associated chromatin-modifying complex. We propose that this new function is essential to direct the differentiation of several T- and B-cell effector programs, and may also be involved in the oncogenic role of PLZF and BCL6 in leukaemias and lymphomas.

PLZF expression maps the early stages of ILC1 lineage 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.

The transcription factor PLZF directs the effector program of the NKT cell lineage.

The transcriptional control of CD1d-restricted NKT cell development has remained elusive. We report that PLZF (promyelocytic leukemia zinc finger, Zbtb16), a member of the BTB/POZ-ZF family of transcription factors that includes the CD4-lineage-specific c-Krox (Th-POK), is exquisitely specific to CD1d-restricted NKT cells and human MR1-specific MAIT cells. PLZF was induced immediately after positive selection of NKT cell precursors, and PLZF-deficient NKT cells failed to undergo the intrathymic expansion and effector differentiation that characterize their lineage. Instead, they preserved a naive phenotype and were directed to lymph nodes. Conversely, transgenic expression of PLZF induced CD4(+) thymocytes to acquire effector differentiation and migrate to nonlymphoid tissues. We suggest that PLZF is a transcriptional signature of NKT cells that directs their innate-like effector differentiation during thymic development.