Expansion and Maturation of Innate Lymphoid Cell Precursors Using Human iPSC-Derived Intestinal Organoids.

Innate lymphoid cells (ILC) are enriched at mucosal barrier sites where they play critical roles in development and disease. Mucosal organoids offer a robust platform for the simultaneous differentiation and expansion of all subsets of mature ILC from a shared peripheral blood precursor. Critically, organoid identity drives tissue-specific imprinting of the culture-derived mature innate lymphoid cells, allowing for the study of bidirectional interactions between, e.g., intestinal organoids and intestine-specific ratios and populations of ILC. This protocol reduces the need for feeder cell lines and complex cytokine cocktails used to mature and maintain ILC, instead relying on a native niche of protein signals provided by mucosal epithelial cells. This protocol details the generation of human intestinal organoids (HIO) from human-induced pluripotent stem cells (hiPSC), and the subsequent establishment of co-cultures between HIO and ILC precursors for expansion and maturation. This approach has extensive applications for mechanistic studies of fundamental biological processes and as a potential GMP-compatible source of ILC for future cell therapies.

Group 1 ILCs: Heterogeneity, plasticity, and transcriptional regulation.

Group 1 innate lymphoid cells (ILCs), comprising ILC1s and natural killer cells (NK cells), belong to a large family of developmentally related innate lymphoid cells that lack rearranged antigen-specific receptors. NK cells and ILC1s both require the transcription factor T-bet for lineage commitment but additionally rely on Eomes and Hobit, respectively, for their development and effector maturation programs. Both ILC1s and NK cells are essential for rapid responses against infections and mediate cancer immunity through production of effector cytokines and cytotoxicity mediators. ILC1s are enriched in tissues and hence generally considered tissue resident cells whereas NK cells are often considered circulatory. Despite being deemed different cell types, ILC1s and NK cells share many common features both phenotypically and functionally. Recent studies employing single cell RNA sequencing (scRNA-seq) technology have exposed previously unappreciated heterogeneity in group 1 ILCs and further broaden our understanding of these cells. Findings from these studies imply that ILC1s in different tissues and organs share a common signature but exhibit some unique characteristics, possibly stemming from tissue imprinting. Also, data from recent fate mapping studies employing Hobit, RORγt, and polychromic reporter mice have greatly advanced our understanding of the developmental and effector maturation programs of these cells. In this review, we aim to outline the fundamental traits of mouse group 1 ILCs and explore recent discoveries related to their developmental programs, phenotypic heterogeneity, plasticity, and transcriptional regulation.

Source of hematopoietic progenitor cells determines their capacity to generate innate lymphoid cells ex vivo.

BACKGROUND AIMS: The success of allogeneic hematopoietic cell transplantation (HCT) as therapy for hematologic conditions is negatively impacted by the occurrence of graft-versus-host disease (GVHD). Tissue damage, caused, for example, by chemotherapy and radiotherapy, is a key factor in GVHD pathogenesis. Innate lymphoid cells (ILCs) are important mediators of tissue repair and homeostasis. The presence of ILCs before, and enhanced ILC reconstitution after, allogeneic HCT is associated with a reduced risk to develop mucositis and GVHD. However, ILC reconstitution after allogeneic HCT is slow and often incomplete. A way to replenish the pool of ILC relies on the differentiation of hematopoietic progenitor cells (HPCs) into ILC. METHODS: We developed an ex vivo stromal cell-containing culture system to study the capacity of HPCs to differentiate into all mature helper ILC subsets. RESULTS: ILC development depended on the source of HPCs. ILCs developed at high frequencies from umbilical cord blood- and fetal liver-derived HPC and at low frequencies when HPCs were obtained from allogeneic or autologous adult HCT grafts or healthy adult bone marrow. Although all helper ILC subsets could be generated from adult HPC sources, development of tissue protective ILC2 and NKp44+ ILC3 was notoriously difficult. CONCLUSIONS: Our data suggest that slow ILC recovery after allogeneic HCT may be related to an intrinsic incapability of adult HPC to develop into ILC.

Purification of Bone Marrow Precursors to T Cells and ILCs.

T cells and innate lymphoid cells (ILCs) share expression of many key transcription factors during development and at mature stage, resulting in striking functional similarities between these lineages. Taking into account ILC contribution is thus necessary to appreciate T cell functions during immune responses. Furthermore, understanding ILC development and functions helps to understand T cells. Here we provide methods and protocols to isolate pure populations of multipotent precursors to T cells and innate lymphoid cells (ILCs) from adult mouse bone marrow, using flow cytometric sorting. These include precursors to all lymphocytes (viz., LMPPs and ALPs) and multipotent precursors to ILCs that have been recently refined (viz., specified EILPs, committed EILPs, and ILCPs).

Differential regulation of lineage-determining transcription factor expression in innate lymphoid cell and adaptive T helper cell subsets.

CD4 T helper (Th) cell subsets, including Th1, Th2 and Th17 cells, and their innate counterparts innate lymphoid cell (ILC) subsets consisting of ILC1s, ILC2s and ILC3s, display similar effector cytokine-producing capabilities during pro-inflammatory immune responses. These lymphoid cell subsets utilize the same set of lineage-determining transcription factors (LDTFs) for their differentiation, development and functions. The distinct ontogeny and developmental niches between Th cells and ILCs indicate that they may adopt different external signals for the induction of LDTF during lineage commitment. Increasing evidence demonstrates that many conserved cis-regulatory elements at the gene loci of LDTFs are often preferentially utilized for the induction of LDTF expression during Th cell differentiation and ILC development at different stages. In this review, we discuss the functions of lineage-related cis-regulatory elements in inducing T-bet, GATA3 or RORγt expression based on the genetic evidence provided in recent publications. We also review and compare the upstream signals involved in LDTF induction in Th cells and ILCs both in vitro and in vivo. Finally, we discuss the possible mechanisms and physiological importance of regulating LDTF dynamic expression during ILC development and activation.

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.

New insights into the function, development, and plasticity of type 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2s) are the most well defined group of ILCs. ILC2 development is controlled by the GATA-3 transcription factor and these cells produce archetypal type 2 cytokines, such as IL-5 and IL-13. These cytokines mediate parasite expulsion and tissue repair, but also contribute to type 2 inflammatory diseases, including allergy, asthma and chronic rhinosinusitis with nasal polyps. In response to tightly regulated local environmental cues ILCs can generate characteristics of other subtypes, a process known as plasticity. Recent advances in the ILC2 field has led to the discovery that ILC2s can promptly shift to functional IFN-γ-producing ILC1s or IL-17-producing ILC3s, depending on the cytokines and chemokines produced by antigen presenting cells or epithelial cells. Due to yet unknown triggers, this complex network of signals may become dysregulated. In this review, we will discuss general ILC characteristic, ILC2 development, plasticity, memory function, and implications in disease.

CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway.

According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.

ILC1s in Tissue Inflammation and Infection.

Innate lymphoid cells (ILCs) are innate immune cells that provide an early source of cytokines to initiate and tailor the immune response to the type of the encountered pathogen or insult. The group 1 ILCs are comprised of conventional natural killer (cNK) cells and subsets of "unconventional NK cells," termed ILC1s. Although cNK cells and ILC1s share many features, such as certain phenotypic markers and the ability to produce IFN-γ upon activation, it is now becoming apparent that these two subsets develop from different progenitors and show unique tissue distribution and functional characteristics. Recent studies have aimed at elucidating the individual contributions of cNK cells and ILC1s during protective host responses as well as during chronic inflammation. This review provides an overview of the current knowledge of the developmental origins as well as of the phenotypic and functional characteristics of ILC1s.

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G-CSF.

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high-risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft-versus-host disease and could also contribute to anti-microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34(+) cells are enriched in lymphoid-committed precursors, while PB CD34(+) cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56(+) CD161(+) ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56(+) CD161(+) ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34(+) cultures was lower for G-CSF-mobilized HSCs (good mobilizers) than for G-CSF+plerixafor-mobilized HSC (poor mobilizers). Moreover, G-CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G-CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.

Human innate lymphoid cells.

The interest in innate lymphoid cells (ILC) has rapidly grown during the last decade. ILC include distinct cell types that are collectively involved in host protection against pathogens and tumor cells and in the regulation of tissue homeostasis. Studies in mice enabled a broad characterization of ILC function and of their developmental requirements. In humans all mature ILC subsets have been characterized and their role in the pathogenesis of certain disease is emerging. Nonetheless, still limited information is available on human ILC development. Indeed, only the cell precursors committed toward NK cells or ILC3 have been described. Here, we review the most recent finding on human mature ILC, discussing their tissue localization and function. Moreover, we summarize the available data regarding human ILC development.

Development of human natural killer cells and other innate lymphoid cells.

Innate lymphoid cells (ILC) have recently gained much attention in immunology. They represent a novel developmentally related family. Three distinct subsets have been identified on the basis of phenotypic and functional criteria and termed ILC1, ILC2, and ILC3. The available data suggest that ILC play an important role in innate defenses against different pathogens, in lymphoid organogenesis, and in tissue remodeling. All these aspects are relevant in hematopoietic stem cell transplantation (HSCT), particularly in the haplo-HSCT setting, in which donor NK cells are known to play a major therapeutic role, while the involvement of other ILC is still undefined. In this context, it has been postulated that all ILC share a common precursor expressing the ID2 transcription factor. While the differentiation of human NK cells (belonging to ILC1) is now well characterized both in vitro and in vivo, limited information is available on the development of human ILC2 and ILC3 and of their relationships with NK cells. In this review, we will summarize the present knowledge on the developmental relationship among different ILC, with particular focus on early stages of NK cell differentiation, and their features shared with ILC2 and ILC3.