Regulation of immune cell development, differentiation and function by stromal Notch ligands

Abstract

In multicellular organisms, cell-to-cell communication is critical for the regulation of tissue organization. Notch signaling relies on direct interactions between Notch receptors on signal-receiving cells and Notch ligands on adjacent cells. Notch evolved to mediate local cellular interactions that are responsive to spatial cues via dosage-sensitive short-lived signals. Immune cells utilize these unique properties of Notch signaling to direct their development, differentiation, and function. In this review, we explore how immune cells interact through Notch receptors with stromal cells in specialized niches of lymphohematopoietic organs that express Notch-activating ligands. We emphasize factors that control these interactions and focus on how Notch signals communicate spatial, quantitative, and temporal information to program the function of signal-receiving cells in the immune system.

Introduction

Notch receptors are single-pass transmembrane receptors expressed on the surface of signal-receiving cells. They are activated when bound by Notch ligands on the surface of adjacent cells, allowing for truly local signaling interactions. Notch activation leads to conformational changes that trigger consecutive receptor cleavage by the ADAM10 metalloprotease and by the γ-secretase multiprotein complex, composed of presenilins (PSEN1/PSEN2), nicastrin (NCT), anterior pharynx-defective 1 (APH-1) and presenilin enhancer 2 (PEN-2). Notch cleavage by ADAM10 at a specific site in the extracellular domain and then by γ-secretase in the transmembrane region free the Notch intracellular domain (NICD) to translocate into the nucleus (Figure 1) [1]. NICD associates with the RBPJ transcription factor and a Mastermind-like family protein before recruiting co-activators to promote increased target gene transcription [1]. Cleaved NICD is rapidly degraded, which ensures delivery of precise time and dosage-sensitive messages to signal-receiving cells [2] (Figure 1). This mechanism of action makes Notch signaling a rational system for position sensing, patterning and cell fate decisions, as best established during embryonic development [3,4].

Figure 1. Overview of the Notch pathway and its signaling properties.

Notch signaling evolved to communicate information between adjacent cells. In the immune system, specialized stromal cells that build the structure of lymphohematopoietic organs express Notch ligands on their surface and interact with Notch receptors in hematopoietic and immune cells, passing along positional information. Ligand-receptor binding causes conformational changes in the receptor allowing for proteolytic release of the Notch intracellular domain (NICD). NICD translocates to the nucleus where it binds the RBPJ transcription factor, recruits a Mastermind-like (MAML) co-activator and induces transcriptional activation. Differential abundance of ligands or receptors and distinct ligand-receptor pairs affect the dose of Notch signal sensed by signal-receiving cells, transferring quantitative information. Once cleaved, NICD is rapidly ubiquitinated and degraded by the proteasome. Given NICD’s short half-life, persistent expression of Notch receptors and contact with ligand-expressing cells are essential for continued signaling, thus relaying time-sensitive information.

The emergence of multicellular organisms vastly increased life diversity on Earth. Ancient multicellular species developed a toolbox for intercellular communication that included the Notch pathway. Ancestral protein domains resembling those seen in Notch (e.g. epidermal growth factor-like repeats and ankyrin domains) exist across various proteins in unicellular organisms [5,6], but modern Notch receptors only emerged in the common ancestor of metazoans [7,8]. Proteins like γ-secretase that are vital to the pathway’s function may have been coopted from more ancient ancestors [7]. Notch signaling is vital throughout development and is highly conserved in the animal kingdom, even in primitive nonbilaterian species like sponges and hydra [9]. Thus, Notch evolution is intimately linked to multicellular systems.

While Drosophila, the organism in which Notch signaling was discovered, only has one Notch receptor and two Notch ligands, more complex organisms harbor additional receptors and ligands [1]. Receptors can be differentially glycosylated, increasing functional diversity [10]. In higher organisms, a mechanical force is needed to unmask the metalloprotease cleavage site during Notch activation [11]. Recent work showed that the required force threshold is mediated by a “leucine plug” found in Drosophila and vertebrates, but not in lower organisms such as C. elegans where an endocytic pulling force is not required to activate Notch [12]. Ancient ankyrin domains evolved in a species-specific manner to regulate NICD’s sensitivity and stability in response to changes in temperature [13,14]. The increased complexity of Notch signaling acquired over evolutionary time fine-tuned its versatility.

This review focuses on the role of Notch signaling in cells of the mammalian immune system with an emphasis on lymphohematopoietic organs. Notch plays critical roles at multiple stages of hematopoietic and immune cell development from the emergence of hematopoietic stem cells in the embryo to their development, differentiation and function in the fetal liver, bone marrow, thymus, spleen, and lymph nodes. We explore how immune cells interact with specialized structural cells in their microenvironment (Figure 2) and how Notch signaling facilitates communication of time-sensitive and quantitative positional information with profound effects on immune function (Figure 3).

Figure 2. Cellular specificity of Notch receptor-ligand interactions.

Notch ligands and receptors are expressed by a variety of cell types, yet only specific ligand-receptor pairs on distinct cell types effectively activate Notch signaling. We postulate that chemokines and cytokines potentiate Notch signaling in appropriate contexts by recruiting and sustaining receptor-expressing hematopoietic cells in specific ligand-expressing niches (1). Specialized stromal cells are a recurrent, established source of Notch-activating ligands that bind Notch receptors expressed on hematopoietic cells (2). Hematopoietic cells can also express Notch ligands and may play an important role in communicating signals to other hematopoietic cells in certain contexts, for example interactions between immature myeloid cells and developing hematopoietic stem cells in the fetal liver (3). Stromal elements may also use Notch signaling to communicate between themselves within lymphohematopoietic niches, for example in interactions between thymic epithelial cells in the developing thymus and between endothelial cells in the BM (4).

Figure 3. Examples of stromal niche-driven Notch signaling in the immune system.

(A) In the thymus, thymic epithelial cells express Dll4 Notch ligands to activate Notch1-mediated signaling in thymus seeding progenitors (TSPs) and downstream early T lineage progenitors, promoting T lineage development. In the absence of Notch signaling, TSPs adopt alternative cell fates. (B) In the spleen, specialized Ccl19-Cre⁺ fibroblastic reticular cells serve as a source of Dll1 ligands to activate Notch2 in transitional B cells and instruct marginal zone B cell development. Continuous exposure to Dll1 is necessary to sustain marginal zone B cell positioning and function, and access to Dll1 Notch ligands is enhanced in lymphopenia. (C) Within lymph nodes, Ccl19-Cre⁺ fibroblastic reticular cells serve as a source of Dll4 Notch ligands to promote a T follicular helper cell (Tfh) response upon antigen exposure. Tfh assist B cells in germinal center reactions to promote affinity maturation and long-lasting antibody responses, for example in the setting of vaccination.

Role of Notch signaling in the emergence of hematopoietic stem cells (HSCs)

In vertebrates, embryonic HSCs originate in the ventral aorta and selected other arteries. HSCs develop with other early hematopoietic progenitors from hemogenic endothelial cells that generate intra-aortic hematopoietic clusters. Notch signaling plays a dosage-sensitive role in directing cells towards an HSC fate [15–18]. Cells receiving high intensity Delta-like ligand 4 (Dll4)-driven Notch signals adopt an endothelial fate, while those receiving lower intensity Jagged1 (Jag1)-driven Notch signals adopt a hematopoietic fate [15] (Figure 1). Recent work showed that positioning of Notch ligands within hematopoietic clusters regulates these differentiation patterns. Dll4-expressing cells, located on the luminal layer of the aorta, restrained hematopoietic cluster growth, while Jag1 expression within clusters allowed their expansion [19]. Additionally, single-cell RNA sequencing demonstrated a transition from Notch1 to Notch2 expression as cells developed into pre-HSCs from hemogenic endothelium. In vitro, this transition was supported by immobilized agonistic anti-Notch1 and Notch2 antibodies, but not immobilized Dll4 alone [20]. Later in fetal life, hematopoietic cells themselves may also serve as an important source of Jag1 to HSCs in the fetal liver, as its absence decreased expression of HSC-specific genes, resulting in reduced engraftment after transplantation [21].

Together, these data suggest that distinct Notch ligands can impart different quantitative signal strengths and cell fate outcomes (Figure 1). Future work will help determine how signal strength affects patterns of individual Notch target gene expression. Non-hematopoietic cells serve as a vital source of Notch ligands in HSC development, but it remains less clear to what extent and in what contexts other cell types (e.g. other hematopoietic cells [21]) also contribute. Additional factors such as chemokines and cytokines might facilitate recruitment and retention of signal-receiving hematopoietic cells, thus magnifying the effects of specific microenvironmental niches expressing Notch ligands (Figure 2).

Bone marrow (BM)

In adult mammals, hematopoiesis is maintained by hematopoietic stem and progenitor cells in the BM. The BM space includes distinct niches that instruct HSCs to self-renew or differentiate into downstream progenitors with various degrees of myeloid and lymphoid potential. Delta-like and Jagged ligands expressed in BM stromal compartments are potential regulators of hematopoiesis and BM niche homeostasis [22–25]. However, only low intensity Notch activation is seen physiologically in HSCs and Notch’s role remains controversial [26]. Some studies reported that Notch signaling within the BM is important for maintenance of the HSC pool [22,23]. However, others showed that Notch signaling is non-essential for HSC maintenance [26,27]. Leukemia/lymphoma-related factor (LRF, encoded by the Zbtb7a gene), a member of the POZ domain Krüppel-like zinc finger (POK) family of transcriptional repressors, normally suppresses Dll4 expression in erythroid cells [28]. De-repression of Dll4 expression in erythroblasts of LRF-deficient mice led to Notch-driven extrathymic T cell development, highlighting how exposure to Notch ligands and Notch signaling intensity in HSCs may normally be limited through active mechanisms [28,29]. Notch signaling may also play a role extrinsic to hematopoietic cells in regulating cross-talk between stromal cells that compose the BM niche [30] (Figure 2).

HSCs give rise to downstream progenitors with distinct differentiation potential that ultimately produce mature hematopoietic cells. During regenerative hematopoiesis, Dll1 and Dll4 expression in BM endothelial cells was reported to promote lymphoid at the expense of myeloid development [25]. Dll4 expression was also described in osteocalcin⁺ osteoblast lineage cells, where its conditional deletion impaired common lymphoid progenitors (CLP) with high T cell development potential [24]. Additional work supports the idea that increased exposure to Notch signaling promotes a lymphoid and pre-T cell fate in hematopoietic progenitors even upstream of the thymus [24,31,32]. In a mouse model of hematopoietic stem cell transplantation, Notch signaling increased IL-21R expression, and transplanted IL-21-stimulated T cell progenitors proliferated more rapidly and colonized the host thymus more efficiently, leading to enhanced reconstitution of the host spleen and lymph nodes with donor-derived T cells [33]. In another system, a transplanted ectopic artificial BM scaffold containing Dll4 promoted more robust CLP and thymocyte generation, and improved systemic T cell reconstitution after BM transplantation [34]. T cell generation was much more efficient when BMP2 was also incorporated into the scaffold [34], consistent with the observation that Notch ligands cooperate with cytokines and chemokines in Notch ligand-expressing niches [35] (Figure 2).

Thymus

Thymus-seeding progenitors (TSP) migrate from the BM to the thymic corticomedullary junction, where they enter the thymus through specialized venules and encounter Notch ligands on thymic epithelial cells (TEC). TSPs enter the thymus in response to chemokine receptor CCR7/9-mediated signals from TEC-secreted chemokines [36]. TSPs require Dll4-mediated Notch1 signals to initiate and sustain the T cell transcriptional program [37–39] and shut off alternative differentiation potential [31,38,40] (Figure 3A). T cell progenitor migration through thymic niches is also influenced by Notch signals [41].

Single cell genomics data providing insights into human thymopoiesis suggest that TSPs arrive in the thymus with broad differentiation potential before high intensity Notch activation commits them to the T cell lineage [42]. Early T lineage progenitors can be divided into unique subsets that are poised for different trajectories within the thymus [42], perhaps reflecting signals (including Notch activation) received within the BM, as discussed above. Recent work in mice shows that an early thymocyte-specific enhancer controls Notch1 expression and its deletion leads to accelerated differentiation outpacing induction of lineage-restricting signals and leading to abnormal thymic production of pro-B and NK cells at the expense of T lineage cells [43]. Different Notch ligand-receptor interactions and their resulting signal strength may also direct early T lineage progenitors [42,44,45]. Interestingly, in the absence of key FoxN1-driven chemokines and cytokines including stem cell factor and CCL25, Delta-like ligands were inefficient at directing T cell fate in the thymus [35], suggesting that chemokines and cytokines were necessary to potentiate Notch signaling. These data suggest that chemokines attract and cytokines support progenitors in specialized stromal niches, where they then come into contact with rare ligand-expressing stromal cells capable of potently transducing Notch signals (Figure 2) [35,36]. Conversely, other sources of Notch ligands may be ineffective in the absence of cytokine and chemokine secretion.

Notch receptors and activating ligands also appear to play a critical role in determining cell fates of the TECs themselves [46] analogous to their effects within the BM niche described above [30,47] (Figure 2). Notch signaling was reported to regulate FoxN1 expression, which functions as a master regulator of TECs, and promote medullary TEC differentiation [48,49]. The absence of early T lineage progenitors disrupts normal thymic architecture [32], demonstrating the importance of cross-talk between stromal and hematopoietic cells in regulating immune cell development and stromal niche function.

Spleen and lymph nodes

Notch signaling plays a continued role in the function of mature lymphocytes in secondary lymphoid organs (SLOs). Specialized subsets of non-hematopoietic fibroblastic reticular cells (FRCs) support the structure of spleen and lymph nodes, but also communicate important information to immune cells. FRCs express Notch ligands that control lymphocyte responses to a variety of stimuli in SLOs [50] akin to TECs interacting with lymphoid progenitors in the thymus (Figure 3B–C). In the spleen, Dll1 expression in Ccl19-Cre⁺ FRCs, but not endothelial or hematopoietic cells, was required for the homeostasis of marginal zone B cells, a population of naïve mature B cells with innate-like characteristics [51,52]. Chemokines and cytokines secreted by FRCs may be important to sustain their capacity to deliver Notch signals to B cells and other targets (as seen in BM [34] and thymic stromal niches [35,36]) (Figure 2). After transfer of mature follicular B cells into lymphopenic recipients, Notch2-Dll1 interactions between B cells and splenic FRCs induced transdifferentiation into marginal zone B cells [53] (Figure 3B). Importantly, this process was heightened in lymphopenic as compared with lymphoid-replete mice, suggesting increased access to the Dll1⁺ niche in the setting of lymphopenia, which may serve as a regulatory mechanism to sense and regulate B cell pool size. Functionally, sustained Notch signaling in marginal zone B cells primed them for rapid plasma cell differentiation supported by Myc and mTORC signaling [54]. This regulation may extend from naïve to memory B cells, as seen in spleens from cadaveric organ donors previously immunized against smallpox where a population of long-lived antigen-specific memory B cells also had a Notch-Myc signature [55].

In lymph nodes, stroma-derived Dll4 drives Notch activation in naïve T cells to promote differentiation to T follicular helper cells after immunization [51,56] (Figure 3C). Notch signaling also acts as a key regulator of CD8⁺ T cell fate. Notch signaling promotes short-lived effector T cell differentiation in response to influenza [57], Listeria, and dendritic cell immunization [58]. In the context of graft-versus-host disease, the main immunological complication of allogeneic bone marrow or hematopoietic cell transplantation, stromal Delta-like ligands prime donor T cells for alloimmune reactivity during a short time window following transplantation by driving a pro-inflammatory T cell phenotype and inhibiting regulatory T cell expansion [59–61]. Stroma-derived Notch signals also play an important role in regulating cell surface integrins controlling homing of alloreactive T cells to the gut in mice and non-human primates [62]. A single dose of anti-Dll4 antibody at the time of allogeneic hematopoietic cell transplantation was sufficient to prevent the deleterious effects of graft-versus-host disease in the gastrointestinal tract, among the most common and most morbid sites of disease [62].

Conclusions and perspectives

Specialized stromal cells are critical as a source of Notch ligands across lymphohematopoietic organs, as best shown for epithelial cells in the thymus and FRCs in SLOs. Although Notch-activating ligands are present elsewhere (e.g. in endothelial cells), individual cell types diverge in their capacity to engage signal-receiving cells. As a common rule of engagement that defines potent Notch ligand-expressing niches, we speculate that niche-specific factors including chemokines and cytokines are necessary to prime signal-receiving cells to efficiently accept Notch signals [34,35,51] (Figure 2). Expression of Notch ligands in stromal cells embedded in a fixed anatomical space may also provide favorable biophysical properties to deliver Notch signals, while conveying positional information.

Disordered Notch signaling in lymphocytes can cause lymphomagenesis. In chronic lymphocytic leukemia [63,64], marginal zone lymphoma [65,66], and peripheral T cell lymphomas [67], cancerous cells hijack signals from ligand-expressing cells within their local niche to drive aberrant Notch signaling. Interestingly, the requirement for Notch ligands and other local signals appeared to be critical in this process, even in patients with Notch-activating mutations, as active Notch was only detected within the lymph nodes and was lost abruptly in cells extending outside these encapsulated areas [63,65,67]. Thus, local cell-to-cell contacts building on physiological signals may sustain lymphoma proliferation and be potential targets for intervention.

In summary, Notch signaling has evolved as a critical tool for cell-to-cell communication in multicellular organisms. Over evolutionary time, the pathway has been fine-tuned to allow for precise, tightly regulated communication between adjacent cells. It has been coopted by cells of the immune system to transmit spatial, quantitative, and temporal information from structural cells in lymphohematopoietic organs to hematopoietic cells (Figure 1). Notch likely acts in tandem with other signals from cytokines and chemokines to attract immune cells to appropriate niches and in certain contexts may communicate information directly between stromal cells or between hematopoietic cells (Figure 2). This signaling pathway is essential for the development, differentiation, and function of multiple immune cell subsets in the BM, thymus, spleen, and lymph nodes (Figure 3).