Noriko Takegahara

Class IV semaphorin Sema4A enhances T-cell activation and interacts with Tim-2

Semaphorins are a family of phylogenetically conserved soluble and transmembrane proteins. Although many soluble semaphorins deliver guidance cues to migrating axons during neuronal development, some members are involved in immune responses. For example, CD100 (also known as Sema4D), a class IV transmembrane semaphorin, signals through CD72 to effect nonredundant roles in immune responses in a ligand–receptor system that is distinct from any seen previously in the nervous system. Here we report that the class IV semaphorin Sema4A, which is expressed in dendritic cells and B cells, enhances the in vitro activation and differentiation of T cells and the in vivo generation of antigen-specific T cells. Treating mice with monoclonal antibodies against Sema4A blocks the development of an experimental autoimmune encephalomyelitis that is induced by an antigenic peptide derived from myelin oligodendrocyte glycoprotein. In addition, expression cloning shows that the Sema4A receptor is Tim-2, a member of the family of T-cell immunoglobulin domain and mucin domain (Tim) proteins that is expressed on activated T cells.

Plexin-A1 and its interaction with DAP12 in immune responses and bone homeostasis

Semaphorins and their receptors have diverse functions in axon guidance, organogenesis, vascularization and/or angiogenesis, oncogenesis and regulation of immune responses. The primary receptors for semaphorins are members of the plexin family. In particular, plexin-A1, together with ligand-binding neuropilins, transduces repulsive axon guidance signals for soluble class III semaphorins, whereas plexin-A1 has multiple functions in chick cardiogenesis as a receptor for the transmembrane semaphorin, Sema6D, independent of neuropilins. Additionally, plexin-A1 has been implicated in dendritic cell function in the immune system. However, the role of plexin-A1 in vivo, and the mechanisms underlying its pleiotropic functions, remain unclear. Here, we generated plexin-A1-deficient (plexin-A1−/−) mice and identified its important roles, not only in immune responses, but also in bone homeostasis. Furthermore, we show that plexin-A1 associates with the triggering receptor expressed on myeloid cells-2 (Trem-2), linking semaphorin-signalling to the immuno-receptor tyrosine-based activation motif (ITAM)-bearing adaptor protein, DAP12. These findings reveal an unexpected role for plexin-A1 and present a novel signalling mechanism for exerting the pleiotropic functions of semaphorins.

Semaphorin 7A initiates T-cell-mediated inflammatory responses through α1β1 integrin

Semaphorins are axon guidance factors that assist growing axons in finding appropriate targets and forming synapses. Emerging evidence suggests that semaphorins are involved not only in embryonic development but also in immune responses. Semaphorin 7A (Sema7A; also known as CD108), which is a glycosylphosphatidylinositol-anchored semaphorin, promotes axon outgrowth through β1-integrin receptors and contributes to the formation of the lateral olfactory tract. Although Sema7A has been shown to stimulate human monocytes, its function as a negative regulator of T-cell responses has also been reported. Thus, the precise function of Sema7A in the immune system remains unclear. Here we show that Sema7A, which is expressed on activated T cells, stimulates cytokine production in monocytes and macrophages through α1β1 integrin (also known as very late antigen-1) as a component of the immunological synapse, and is critical for the effector phase of the inflammatory immune response. Sema7A-deficient (Sema7a-/-) mice are defective in cell-mediated immune responses such as contact hypersensitivity and experimental autoimmune encephalomyelitis. Although antigen-specific and cytokine-producing effector T cells can develop and migrate into antigen-challenged sites in Sema7a-/- mice, Sema7a-/- T cells fail to induce contact hypersensitivity even when directly injected into the antigen-challenged sites. Thus, the interaction between Sema7A and α1β1 integrin is crucial at the site of inflammation. These findings not only identify a function of Sema7A as an effector molecule in T-cell-mediated inflammation, but also reveal a mechanism of integrin-mediated immune regulation.

Guidance of myocardial patterning in cardiac development by Sema6D reverse signalling

Cardiac chamber formation involves dynamic changes in myocardial organization, including trabeculation and expansion of the compact layer. The positional cues that regulate myocardial patterning, however, remain unclear. Through ligation of the Plexin-A1 receptor, the transmembrane-type semaphorin Sema6D regulates endocardial cell migration. Here, we demonstrate that knockdown of either Sema6D or Plexin-A1 leads to the generation of a small, thin ventricular compact layer and to defective trabeculation. In the heart, expression of the Plexin-A1 extracellular domain alone can rescue the defective trabeculation induced by suppression of Plexin-A1, but not that resulting from defective Sema6D expression. This indicates that reverse signalling by Sema6D occurs within the myocardium. In a ligand-dependent manner, Abl kinase is recruited to the cytoplasmic tail of Sema6D and activated, resulting in phosphorylation of Enabled and dissociation from Sema6D. Constitutive activation of Sema6D signalling enhances the migration of myocardial cells into the trabeculae, whereas inhibition arrests cells within the compact layer. Thus, Sema6D coordinates both compact-layer expansion and trabeculation, functioning as both a ligand and a receptor for Plexin-A1.

Requirement for the Lymphocyte Semaphorin, CD100, in the Induction of Antigen-Specific T Cells and the Maturation of Dendritic Cells

CD100 belongs to the semaphorin family, several members of which are known to act as repulsive axonal guidance factors during neuronal development. We have previously demonstrated that CD100 plays a crucial role in humoral immunity. In this study, we show that CD100 is also important for cellular immunity through the maturation of dendritic cells (DCs). CD100−/− mice fail to develop experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide, because myelin oligodendrocyte glycoprotein-specific T cells are not generated in the absence of CD100. In vitro studies with T cells from OVA-specific TCR-transgenic mice demonstrate that Ag-specific T cells lacking CD100 fail to differentiate into cells producing either IL-4 or IFN-γ in the presence of APCs and OVA peptide. In addition, DCs from CD100−/− mice display poor allostimulatory capabilities and defects in costimulatory molecule expression and IL-12 production. The addition of exogenous soluble rCD100 restores normal functions in CD100−/− DCs and further enhances functions of normal DCs. Furthermore, treatment of Ag-pulsed DCs with both soluble CD100 and anti-CD40 before immunization significantly enhances their immunogenicity. This treatment elicits improved T cell priming in vivo, enhancing both primary and memory T cell responses. Collectively, these results demonstrate that CD100, which enhances the maturation of DCs, is essential in the activation and differentiation of Ag-specific T cells.

Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II

The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.

Optic Chiasm Presentation of Semaphorin6D in the Context of Plexin-A1 and Nr-CAM Promotes Retinal Axon Midline Crossing

At the optic chiasm, retinal ganglion cells (RGCs) project ipsi- or contralaterally to establish the circuitry for binocular vision. Ipsilateral guidance programs have been characterized, but contralateral guidance programs are not well understood. Here, we identify a tripartite molecular system for contralateral RGC projections: Semaphorin6D (Sema6D) and Nr-CAM are expressed on midline radial glia and Plexin-A1 on chiasm neurons, and Plexin-A1 and Nr-CAM are also expressed on contralateral RGCs. Sema6D is repulsive to contralateral RGCs, but Sema6D in combination with Nr-CAM and Plexin-A1 converts repulsion to growth promotion. Nr-CAM functions as a receptor for Sema6D. Sema6D, Plexin-A1, and Nr-CAM are all required for efficient RGC decussation at the optic chiasm. These findings suggest a mechanism by which a complex of Sema6D, Nr-CAM, and Plexin-A1 at the chiasm midline alters the sign of Sema6D and signals Nr-CAM/Plexin-A1 receptors on RGCs to implement the contralateral RGC projection.

Roles of Sema4D-Plexin-B1 Interactions in the Central Nervous System for Pathogenesis of Experimental Autoimmune Encephalomyelitis

Although semaphorins were originally identified as axonal guidance molecules during neuronal development, it is emerging that several semaphorins play crucial roles in various phases of immune responses. Sema4D/CD100, a class IV semaphorin, has been shown to be involved in the nervous and immune systems through its receptors plexin-B1 and CD72, respectively. However, the involvement of Sema4D in neuroinflammation still remains unclear. We found that Sema4D promoted inducible NO synthase expression by primary mouse microglia, the effects of which were abolished in plexin-B1–deficient but not in CD72-deficient microglia. In addition, during the development of experimental autoimmune encephalomyelitis (EAE), which was induced by immunization with myelin oligodendrocyte glycoprotein-derived peptides, we observed that the expression of Sema4D and plexin-B1 was induced in infiltrating mononuclear cells and microglia, respectively. Consistent with these expression profiles, when myelin oligodendrocyte glycoprotein-specific T cells derived from wild-type mice were adoptively transferred into plexin-B1–deficient mice or bone marrow chimera mice with plexin-B1–deficient CNS resident cells, the development of EAE was considerably attenuated. Furthermore, blocking Abs against Sema4D significantly inhibited neuroinflammation during EAE development. Collectively, our findings demonstrate the role of Sema4D–plexin-B1 interactions in the activation of microglia and provide their pathologic significance in neuroinflammation.

Plexin-A4 negatively regulates T lymphocyte responses

Semaphorins and their receptors play crucial roles not only in axon guidance during neuronal development but also in the regulation of immune responses. Plexin-A4, a member of the plexin-A subfamily, forms a receptor complex with neuropilins and transduces signals for class III semaphorins in the nervous system. Although plexin-A4 is also expressed in the lymphoid tissues, the involvement of plexin-A4 in immune responses remains unknown. To explore the role of plexin-A4 in the immune system, we analyzed immune responses in plexin-A4-deficient (plexin-A4-/-) mice. Among immune cells, plexin-A4 mRNA was detected in T cells, dendritic cells and macrophages but not in B cells and NK cells. Plexin-A4-/- mice had normal numbers and cell surface markers for each lymphocyte subset, suggesting that plexin-A4 is not essential for lymphocyte development. However, plexin-A4-/- mice exhibited enhanced antigen-specific T cell responses and heightened sensitivity to experimental autoimmune encephalomyelitis. Plexin-A4-/- T cells exhibited hyperproliferative responses to anti-CD3 stimulation and to allogeneic dendritic cells in vitro. Furthermore, this hyperproliferation was also observed in both T cells from neuropilin-1 mutant (npn-1(Sema-)) mice, in which the binding site of class III semaphorins is disrupted, and T cells from Sema3A-deficient (Sema-3A-/-) mice. Collectively, these results suggest that plexin-A4, as a component of the receptor complex for class III semaphorins, negatively regulates T cell-mediated immune responses.

Intestinal Epithelial Cell-Derived Semaphorin 7A Negatively Regulates Development of Colitis via αvβ1 Integrin

The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Mϕs) have been implicated in repression of excessive inflammation, it remains unclear how their functions are regulated during inflammation. In this study, we report that semaphorin 7A (Sema7A), a GPI-anchored semaphorin expressed in intestinal epithelial cells (IECs), induces IL-10 production by intestinal Mφs to regulate intestinal inflammation. Sema7A-deficient mice showed severe signs of dextran sodium sulfate-induced colitis due to reduced intestinal IL-10 levels. We further identified CX3CR1+MHC class IIintF4/80hiCD11bhi Mφs as the main producers of IL-10 via αvβ1 integrin in response to Sema7A. Notably, Sema7A was predominantly expressed on the basolateral side of IECs, and its expression pattern was responsible for protective effects against dextran sodium sulfate-induced colitis and IL-10 production by Mφs during interactions between IECs and Mφs. Furthermore, we determined that the administration of recombinant Sema7A proteins ameliorated the severity of colitis, and these effects were diminished by IL-10–blocking Abs. Therefore, our findings not only indicate that Sema7A plays crucial roles in suppressing intestinal inflammation through αvβ1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and Mφs.