Regulation of immune cell responses by semaphorins and their receptors

Abstract

Semaphorins were originally identified as axon guidance factors involved in the development of the neuronal system. However, accumulating evidence indicates that several members of semaphorins, so-called ‘immune semaphorins', are crucially involved in various phases of immune responses. These semaphorins regulate both immune cell interactions and immune cell trafficking during physiological and pathological immune responses. Here, we review the following two functional aspects of semaphorins and their receptors in immune responses: their functions in cell–cell interactions and their involvement in immune cell trafficking.

Introduction

Increasing evidence indicates that the nervous and immune systems have considerable overlap and links.¹ For example, some axon guidance molecules, such as slits^{2, 3, 4} and ephrins,^{5, 6, 7, 8} have been shown to regulate immune cell migration. In addition, T-cell-antigen-presenting cell contact sites, the so-called ‘immunological synapse', is structurally similar to the ‘neurological synapse' that connects pairs of neurons. These shared molecules and interactions play critical roles in inducing proper immune responses.

Semaphorins were named for their properties that are analogous to the system of flags and lights that is used in rail and maritime communication. They were initially identified as repulsive axon guidance molecules that were required to direct neuronal axons to their appropriate targets.⁹ More than 20 types of semaphorins have been identified,¹⁰ and they have diverse functions in many physiological process,¹¹ including cardiogenesis,^{12, 13} angiogenesis,^{14, 15} vasculogenesis,¹⁶ tumor metastasis,^{17, 18, 19} osteoclastogenesis²⁰ and immune regulation.^{21, 22} In this review, we focus on two functional aspects of semaphorins, their roles in immune cell–cell interactions and immune cell trafficking. In addition, we discuss current perspectives on ‘immune semaphorin' research, including its application for immunological disorders.

Semaphorins and their receptors

Semaphorins are secreted and membrane-associated proteins that are characterized by a conserved extracellular amino-terminal ‘Sema' domain. Based on their C-terminal structures, this diverse group of proteins has been further divided into eight subclasses. Semaphorins in classes I (invertebrate) and IV–VII are membrane-associated, whereas those in classes II (invertebrate), III and VIII (virally encoded) are secreted.^{10, 23} Two groups of proteins, plexins and neuropilins (NPs), have been identified as the primary semaphorin receptors. Most membrane-bound semaphorins directly bind plexins, whereas class III semaphorins require NPs as obligate coreceptors.^{24, 25, 26} However, recent reports have suggested that semaphorin receptor usage is more complex than previously thought. For example, Sema3E signals independently of NPs through plexin-D1,¹⁶ while Sema7A uses integrins to exert its functions in both the nervous and immune systems.^{27, 28} In addition, two molecules unrelated to plexins and NPs, CD72²⁹ and T-cell immunoglobulin and mucin domain-containing protein 2 (TIM-2),³⁰ functionally interact with Sema4D and Sema4A, respectively, in the immune system (Figure 1).

Figure 1.

Representative immune semaphorins and their receptors in lymphoid and non-lymphoid cells. Sema3A binds to neuropilin-1 with high affinity to assemble a NP-1/plexin-A1 receptor complex and involves in the axon guidance events. Sema4D binds to plexin-B1 in the brain and transduces chemorepulsive signals. In the immune system, Sema4D uses CD72 as a functional receptor in B cells and DCs and enhances the activation of B cells and DCs. Sema4A binds TIM-2 and is involved in T-cell activation and differentiation in the immune system. In the non-immune system, however, Sema4A recognizes plexin-B proteins and plexin-D1. Sema6D exerts different biological activities through plexin-A1, depending on its coreceptors. During chick embryogenesis, plexin-A1 differentially associates with off-track and VEGFR2, and these receptor complexes have distinct functions in heart development. In the immune system, plexin-A1 forms a receptor complex with TREM-2 and DAP12 and, after Sema6D binds, this complex transduces signals that stimulate DCs and osteoclasts. Sema7A uses β1 integrin as receptors in both the nervous and immune systems. In the immune system, Sema7A expressed on activated T cells stimulates macrophages through α1β1 integrin to promote inflammatory responses. DC, dendritic cell; DAP12, DNAX-activating protein 12; NP-1, neuropilin-1; OTK, off-track kinase; TIM-2, T-cell immunoglobulin and mucin domain-containing protein 2; TREM-2, triggering receptor expressed on myeloid cells 2; VEGFR2, vascular endothelial growth factor receptor 2.

Plexins are canonical semaphorin receptors with a large cytoplasmic region. In the nervous system, semaphorin–plexin signaling has been shown to mediate diverse neural functions by regulating GTPase activities and cytoplasmic/receptor-type protein kinases.^{11, 31, 32} These plexin-mediated signals are involved in integrin-mediated attachment,^{15, 33, 34} actomyosin contraction^{35, 36, 37, 38} and microtubule destabilization.^{39, 40, 41} In addition, plexins can associate with different coreceptors in distinct tissues to allow semaphorins to exert pleiotropic functions. For instance, plexin-A1 is associated with the tyrosine kinase receptors off-track and vascular endothelial growth factor receptor 2 in heart morphogenesis.⁴² On the other hand, plexin-A1 forms a receptor complex with triggering receptor expressed on myeloid cell (TREM)-2/DNAX-activating protein 12 (DAP12) in osteoclastogenesis.²⁰ Furthermore, plexin-B1 has been shown to associate with the receptor tyrosine kinases Met and ErbB2, triggering invasive growth of epithelial cells.^{19, 43}

Involvement of ‘immune semaphorins' in cell–cell interactions

Sema4D: a semaphorin involved in B-cell/dendritic cell activation

Sema4D, also known as CD100, is the first semaphorin protein that was determined to have immunoregulatory functions. In the immune system, Sema4D is expressed in T cells, activated B cells and mature dendritic cells (DCs).^{29, 44, 45} Sema4D promotes the activation of B cells and DCs to induce antibody production and antigen-specific T cells, respectively.^{29, 46, 47} Plexin-B1 and CD72 were identified as the Sema4D receptors in the nervous and immune systems.^{11, 48} CD72 negatively regulates B cells by recruiting the tyrosine phosphatase Src homology phosphatase-1 (SHP1) to its immunoreceptor tyrosine-based inhibitory motifs (ITIM).^{49, 50} Ligation of Sema4D to CD72 causes SHP1 to dissociate from CD72, resulting in B-cell and DC activation.²⁹ Consistent with this function, Sema4D-deficient mice exhibit impaired antibody production and priming of antigen-specific T cells.^{46, 47} In particular, Sema4D is crucially involved in T-cell-mediated neurological inflammatory diseases. Sema4D-deficient mice are resistant to experimental autoimmune encephalomyelitis (EAE) due to impaired antigen-specific T-cell responses in the draining lymph nodes and attenuated inflammation in the central nervous system.⁵¹ In addition, T-cell-derived Sema4D has been implicated in the collapse of process extension of immature oligodendrocytes and the death of immature neural cells in the spinal cords of patients with human T-cell lymphotropic virus type 1-associated myelopathy⁵² (Table 1).

Table 1. Immune semaphorins, their receptors and diseases.

Semaphorins/receptors	Expression	Binding partner	Activities	Related diseases
Semaphorin
Sema3A	T cells	Plexin-A proteins	Inhibition of monocyte migration	Atopic dermatitis
	Tumor cells		Inhibition of T-cell activation	Cancer
	Endothelial cells		Inhibition of tumor angiogenesis
Sema4A	Dendritic cells	Plexin-B proteins	T-cell activation	EAE
	Activated T cells	Plexin-D1	Promotion of Th1 differentiation	Atopic dermatitis
	Th1 cells	TIM-2
Sema4D	T cells	Plexin-B1	B-cell activation	EAE
	Activated B cells	CD72	DC activation	HAM
	Dendritic cells		Microglial activation
			Injury of oligodendrocytes
Sema6D	T cells	Plexin-A1	DC activation	EAE
	B cells		Production of type I interferon	Osteopetrosis
	NK cells		Differentiation of osteoclast	Nasu–Halora disease
Sema7A	Activated T cells	Plexin-C1	Monocyte/macrophage activation	Contact hypersensitivity
		Integrin α1β1		EAE
				Pulmonary fibrosis
Receptor
Neuropilin-1	T cells	Class III semaphorins	Inhibition of T-cell activation	Cancer
	Treg cells	VEGF	Tumor angiogenesis
	Tumor cells
	Endothelial cells
Plexin-A1	Dendritic cells	Class VI semaphorins	DC activation	EAE
	Plasmacytoid DCs		Production of type I interferon	Osteopetrosis
	(Osteoclasts)		Differentiation of osteoclast	Nasu–Hakola disease
Plexin-A4	T cells	Class VI semaphorins	Inhibition of T-cell activation	EAE
	Dendritic cells
	Macrophages
Plexin-B1	Microglia	Class IV semaphorins	Microglial activation	EAE
	Oligodendrocytes		Injury of oligodendrocytes	HAM
TIM-2	Activated T cells	Sema4A	T-cell activation	EAE
	Th2 cells			Airway atopy
CD72	B cells	Sema4D	B-cell activation
	(Dendritic cells)		DC activation
Integrin α1β1	Monocytes	Sema7A	Monocyte/macrophage activation	EAE
	Macrophages			Pulmonary fibrosis

Abbreviations: DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; HAM, HTLV-1-associated myelopathy; NK, natural killer; Th1, T-helper type 1; Th2, T-helper type 2; TIM-2, T-cell immunoglobulin and mucin domain-containing protein 2; Treg, regulatory T cell; VEGF, vascular endothelial growth factor.

Sema4A: a semaphorin involved in T-cell activation/differentiation

Sema4A, a class IV semaphorin, plays important roles in the immune system. Sema4A is constitutively expressed in DCs and induced in polarized T-helper type 1 (Th1) cells.^{30, 53} DC-derived Sema4A is crucial for antigen-specific T-cell priming via T cell–DC-cognate cell interactions, while T cell-derived Sema4A is involved in helper T-cell differentiation via T cell–T cell cognate cell interactions. Indeed, Sema4A-deficient mice have impaired Th1 responses to heat-killed Propionibacterium acnes, a Th1-inducing bacteria. Conversely, Sema4A-deficient mice show enhanced T-helper type 2 (Th2) responses against Nippostrongylus brasiliensis, a Th2-inducing intestinal nematode.⁵³ TIM-2, a negative regulator of Th2 cells,⁵⁴ has been suggested to serve as a functional receptor for Sema4A.³⁰ Consistent with these findings, TIM-2 is preferentially upregulated on Th2 cells.⁵⁵ Furthermore, Sema4A has been suggested to have several binding partners in addition to TIM-2, and members of plexin-B and plexin-D1 have also been shown to bind to Sema4A.¹⁴

Sema4A is also involved in T-cell-mediated autoimmune diseases through mechanisms that are distinct from Sema4D. Indeed, a Sema4A deficiency results in attenuated development of autoimmune myocarditis.⁵⁶ In addition, Sema4A-deficient mice on a Th2-prone BALB/c background spontaneously develop atopic dermatitis (unpublished data). These results provide further support that Sema4A is physiologically and pathologically involved in the differentiation of helper T cells.

Sema6D and plexin-A1: an interaction involved in the T cell/DC interface

Plexin-A1 is one of the primary semaphorin receptors whose function has been extensively investigated. Class III semaphorins bind to NP-1 and then form a receptor complex with plexin-A1.²⁵ Additionally, plexin-A1 serves as a direct binding receptor for class VI semaphorins, Sema6C and Sema6D.^{42, 57}

In the immune system, plexin-A1 is specifically expressed in DCs, where it mediates the activation of T cells and the production of type I interferon.^{20, 58, 59} The generation of antigen-specific T cells is impaired in plexin-A1^−/− mice.²⁰ Sema6D, which is expressed in T cells, B cell and natural killer cells, was identified as a putative ligand for plexin-A1.²⁰ Indeed, recombinant Sema6D protein binds to and activates DCs and increases type I interferon production. Plexin-A1 forms a receptor complex with the TREM family of proteins and the adaptor molecule DAP12.^{20, 58} Both DAP12-deficient and plexin-A1-deficient mice not only develop osteopetrosis^{20, 60, 61} but also are resistant to EAE.⁶² Interestingly, genetic mutations in human DAP12 or TREM-2 result in a bone-fracture syndrome called Nasu–Hakola disease, further suggesting that plexin-A1 physiologically associates with the TREM/DAP12 complex and that this interaction is relevant to these diseases.

Sema7A: a semaphorin involved in inflammatory responses via T cell–macrophage interactions

Sema7A, also known as CD108, is a membrane-associated glycosylphosphatidylinositol-linked protein.⁶³ In the immune system, Sema7A is induced on activated T cells.²⁷ Sema7A contains an arginine–glycine–aspartate in its Sema domain that is a well-conserved integrin-binding motif.²⁸ Recombinant Sema7A protein stimulates monocytes/macrophages through α1β1 integrin, inducing proinflammatory cytokine production.²⁷ Furthermore, Sema7A receptor usage in the immune system is consistent with that in olfactory nerve outgrowth.²⁸ Sema7A is also involved in pathogenic immune responses. Sema7A-deficient mice are resistant to inflammation, including hapten-induced contact hypersensitivity and EAE.²⁷ In addition, Sema7A plays an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis by regulating transforming growth factor-β signaling.⁶⁴

NP-1: a class III semaphorin and vascular endothelial growth factor receptor that is necessary to regulate immune responses and tumor angiogenesis

As described above, NP-1 was originally identified as a cell surface glycoprotein that functions as a class III semaphorin receptor.⁶⁵ In addition, NP-1 is also a receptor for vascular endothelial growth factor (VEGF) in both endothelial cells (ECs) and tumor cells.⁶⁶ In the immune system, NP-1 is expressed in DCs and T cells,⁶⁷ where it negatively regulates immune responses. It is also noteworthy that NP-1 plays a key role in tumor angiogenesis through interactions with VEGF.⁶⁸

NP-1 in CD4⁺CD25⁺ regulatory T cells

NP-1 has been shown to help initiate primary immune responses through homophilic interactions at the contact sites between T cells and DCs.⁶⁷ In addition, NP-1 was identified as a specific marker for CD4⁺CD25⁺ regulatory T cells (Tregs).⁶⁹ Recently, one report suggested that NP-1 in Tregs contributes to prolong contact between Tregs and DCs, resulting in the inhibition of T-cell activation at steady state.⁷⁰ These findings suggest that NP-1 in Tregs exerts suppressive functions on Tregs, presumably by mediating Treg stop signals on DCs.

NP-1 in effector T cells

Several lines of evidence suggest that Sema3A/NP-1/plexin-A4 functions in the immune system.^{71, 72} Sema3A is expressed in T cells, while plexin-A4 is expressed in various cells, including T cells, DCs and macrophages. Both NP-1-mutant T cells, in which the Sema3A binding site is specifically disrupted, and plexin-A4-deficient T cells, exhibit enhanced in vitro proliferation after anti-CD3 antibody stimulation.⁷¹ Moreover, plexin-A4-deficient mice have enhanced T-cell priming and exacerbated T cell-mediated immune responses such as EAE,⁷¹ implying that the Sema3A/NP-1/plexin-A4 interactions are pathologically relevant.

NP-1 in tumor angiogenesis

Tumor progression and dissemination depend not only on the intrinsic properties of cancer cells but also on the tumor microenvironment.⁷³ NP-1 is also expressed by various kinds of human tumor-cell lines and neoplasms.⁷⁴ Clinical studies suggest that NP-1 plays a role in tumor growth and disease progression due to mediating VEGF signals.^{75, 76} Recent studies have shown that semaphorins are secreted from tumor cells as well as macrophages and fibroblasts in the tumor microenvironment, thereby influencing cancers and their microenvironments. For instance, Sema3A is secreted from tumors or ECs and suppresses the adhesion and migration of tumor cells and ECs by modulating integrin activities. In addition, Sema3A can inhibit angiogenesis in vivo.^{77, 78} Similarly, Sema3F inhibits cell spreading and migration in breast carcinoma, melanoma and ECs, resulting in reduced metastatic dissemination.^{78, 79, 80} Furthermore, since NP-1 is a receptor for both class III semaphorins and VEGF, class III semaphorins may function as antiangiogenic factors by competitively interfering with VEGF receptors.⁶⁸

Role of semaphorins in immune cell trafficking

In the nervous and cardiovascular systems, semaphorin–plexin signaling regulates cytoskeletal dynamics by activating GTPases, resulting in the modulation of integrin-mediated cell adhesion and actomyosin contractility.³² In this context, it is possible that semaphorins also regulate immune cell trafficking using similar machinery. In addition, it has recently emerged that several semaphorins are involved in immune cell trafficking in both primary and secondary lymphoid organs, although these findings are still preliminary.

Semaphorins in the thymus

The thymus is an organ that supports T-cell differentiation and selection, where interactions with the thymic environment promote the dynamic relocalization of developing lymphocytes.⁸¹ The development of thymocytes is regulated by chemokines, sphingosine-1-phosphates, adhesion molecules and cell–cell interactions between thymocytes and thymic epithelial cells or DCs.⁸¹ In addition, it has been shown that some chemorepellent molecules, including semaphorins and ephrins, affect thymocyte differentiation during their development.^{82, 83, 84}

Sema3E, which interacts with plexin-D1 in an NP-1-independent manner, was recently reported to participate in thymocyte development.⁸² Plexin-D1 expression is high in CD4⁺CD8⁺ thymocytes (double-positive, DP) but decreased in single-positive cells. Furthermore, its ligand, Sema3E, is preferentially expressed in the medulla rather than in the cortex. Sema3E binds to positively selected CD69⁺ DP cells and inhibits their CCR9-mediated migration towards corticomedullary junctions. Indeed, fetal liver cell transfer using plexin-D1-deficient embryos showed that CD69⁺ DP thymocytes are abundantly localized in the cortex and that the boundary of DP and single-positive thymocytes at the corticomedullary junction is disrupted. A similar phenotype was observed in Sema3E-deficient mice, suggesting that the development of thymocytes within the thymus is controlled by Sema3E/plexin-D1 signaling.

Semaphorins in immune cell migration

Class III semaphorins

Sema3A is reported to inhibit immune cell migration. The responsiveness of human monocytes and T cells to chemokine gradients was inhibited by Sema3A.^{85, 86} Interestingly, it was also shown that the chemokine responsiveness of T cells was enhanced when Sema3A proteins were applied against chemokine gradients.⁸⁷ Furthermore, this effect could not be abolished by interfering with the expression of collapsing response-mediator protein 2,⁸⁷ which mediates Sema3A-induced growth-cone guidance. These observations not only indicate that neuron and leukocyte migration is controlled by different molecular mechanisms but also imply that Sema3A-mediated repulsive signals depend on both cell polarity and the site of Sema3A action during immune cell migration.

Other semaphorins

Unlike soluble secreted class III semaphorins, class IV–VII semaphorins are transmembrane proteins that regulate immune cell activities. It was previously reported that these semaphorins are also involved in immune cell migration. For instance, recombinant soluble Sema4D inhibits spontaneous and chemokine (monocyte chemotactic protein-1)-induced human monocyte migration.^{86, 88} In addition, Sema7A, which can stimulate monocytes/macrophages to produce inflammatory cytokines through α1β1 integrin,²⁷ has been suggested to function as an attractant for human monocytes.⁸⁹ Furthermore, a viral semaphorin, A39R, which is a ligand for plexin-C1, inhibits DC integrin-mediated adhesion and chemokine (CCL3)-induced migration through actin cytoskeletal rearrangement.⁹⁰

Possible mechanisms of semaphorin-guided leukocyte migration

Leukocytes must traffic in order to undergo chemokine-/integrin-mediated adhesion and transmigration across ECs through cytoskeletal rearrangement. Recently, it was reported that migrating leukocytes use both integrin-mediated signals and myosin II-mediated actomyosin contraction based on the environmental demands.^{91, 92} Although the molecular mechanisms that control semaphorin-mediated immune cell trafficking are still elusive, it is plausible that signaling events are differentially used in immune cell movement in the context of different environments and pathological situations.

In recent years, new devices such as time-lapse video imaging and multiphoton microscopy have become powerful tools that can be used to evaluate cell migration and cell–cell interactions. These new technologies will further elucidate how semaphorins and their receptors regulate immune cell trafficking.

Perspectives

Accumulating evidence indicates that semaphorins and their receptors have distinct biological activities in various phases of immune responses, from immune initiation to terminal inflammatory immune responses. These semaphorins form a family of immunoregulatory molecules that are called ‘immune semaphorins'. Consistent with their proposed roles in immunity, they are pathologically involved in several immune disorders, including autoimmune diseases, allergy and congenital bone diseases. Semaphorins and their receptors are crucially responsible for maintaining immunological homeostasis by regulating and coordinating immune cell communication systems. However, several important issues are still unresolved. First, although semaphorins regulate cell motility and morphology through plexins in the nervous system, it has not been fully elucidated how and to what extent they are involved in the dynamics of immune cell movement, particularly ‘in vivo'. Second, semaphorins have been shown to regulate immune cell responses through cell–cell interactions, but it is still unclear how semaphorin-mediated signaling regulates the interface of these cell–cell interactions. Future and ongoing studies using new technologies will not only clarify the complete picture of these unique families but also identify potential therapeutic targets that can be used to treat several immune disorders.