Immune semaphorins: a new area of semaphorin research

More than 30 members, identified from viruses to human, make up the semaphorin family (Yu and Kolodkin,1999; Pasterkamp and Kolodkin,2003) (Fig. 1). These soluble and membrane-spanning molecules fall into eight subclasses, on the basis of sequence similarity and distinctive structural features(Semaphorin Nomenclature Committee,1999). The first two semaphorin subclasses, I and II, contain semaphorins identified in invertebrate species. Subclasses III-VII contain vertebrate semaphorins and the viral semaphorins found in certain non-neurotrophic DNA viruses. Several members of the semaphorin family play roles in axonal steering, zonal segregation of axon populations, axonal fasciculation, neuronal polarity, and neuronal cell migration, acting as chemorepellents during neuronal development(Tessier-Lavigne and Goodman,1996). Recent findings, however, indicate that semaphorins play additional, diverse roles in unrelated processes to axon guidance, including organogenesis, vascularization, angiogenesis, neuronal apoptosis, and neoplastic transformation (Kitsukawa et al., 1995; Behar et al.,1996; Sekido et al.,1996; Soker et al.,1998). It is becoming increasingly clear that several semaphorins are also crucially involved in immune responses(Kikutani and Kumanogoh,2003). The physiological and pathological significance of these semaphorins in the immune system is not fully understood, but emerging work is describing the biological functions of the immune semaphorins. Here we review these current findings and address several of the controversial areas for future study throughout a broad range of research fields.

Sema4D is a 150-kDa transmembrane protein of the class IV semaphorin subfamily (Delaire et al.,1998; Kumanogoh and Kikutani,2001; Kikutani and Kumanogoh,2003). This semaphorin contains an N-terminal signal sequence followed by a sema domain, an Ig-like domain, a lysine-rich stretch, a hydrophobic transmembrane region, and a cytoplasmic tail(Furuyama et al., 1996; Hall et al., 1996). Both human and mouse Sema4D are proteolytically cleaved from their transmembrane form to generate 120-kDa soluble forms, indicating the existence of two Sema4D isoforms (Herold et al., 1995; Delaire et al., 2001; Elhabazi et al., 2001; Wang et al., 2001)(Fig. 2). Sema4D is expressed at high levels in lymphoid organs, including the spleen, thymus, and lymph nodes, and in non-lymphoid organs, such as the brain, heart, and kidney. In lymphoid organs, Sema4D is abundantly expressed on resting T cells but only weakly expressed on resting B cells and antigen-presenting cells (APCs), such as dendritic cells (DCs). Its expression, however, is upregulated in these cells following treatment with various immunological stimuli. The release of soluble Sema4D from immune cells is also dependent on cell activation.

Two receptor families, the neuropilins and plexins, mediate many semaphorin functions, such as axonal growth cone collapse(He and Tessier-Lavigne, 1997; Kolodkin et al., 1997; Comeau et al., 1998; Winberg et al., 1998; Takahashi et al., 1999; Tamagnone et al., 1999). Sema4D uses two receptors, plexin-B1 and CD72, which are expressed in non-lymphoid and lymphoid tissues(Kumanogoh and Kikutani,2001).

Plexin-B1, expressed at high levels in the fetal brain and kidney(Maestrini et al., 1996),demonstrates a high affinity(K_d=∼1×10^-9 M) for Sema4D(Tamagnone et al., 1999; Kumanogoh et al., 2000). Human Sema4D stimulation enhances the interaction of the plexin-B1 cytoplasmic region with the small GTPase Rac1 (Vikis et al., 2000). Binding of Sema4D to plexin-B1 also activates the Rho guanine-nucleotide-exchange factor PDZ-RhoGEF/LARG, leading to RhoA activation (Swiercz et al.,2002). In human epithelial cells, plexin-B1 forms a functional receptor complex with Met, the tyrosine kinase receptor for scatter factor-1/hepatocyte growth factor receptor(Giordano et al., 2002). Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of Met, leading to the phosphorylation of both the receptor and the Met substrate Gab1 (Fig. 3). Interestingly, Artigiani et al. have recently reported that the extracellular domain of plexin-B1 can be cleaved and modulates the effects of Sema4D(Artigiani et al., 2003). However, it still remains to be determined whether Sema4D can signal through plexin-B1 in the immune system.

Sema4D utilizes CD72 as its major receptor in lymphoid tissues(Kumanogoh et al., 2000; Kumanogoh and Kikutani, 2001; Kikutani and Kumanogoh, 2003; Suzuki et al., 2003). CD72 expression is detectable throughout B cell differentiation from early progenitors to mature B cells (Nakayama et al., 1989; Von Hoegen et al.,1990; Gordon,1994). Expression is downregulated, however, upon terminal differentiation into plasma cells. CD72 is expressed by professional APCs,such as macrophages and DCs (Tutt Landolfi and Parnes, 1997; Kumanogoh et al., 2002b). Several lines of evidence demonstrate that CD72 is the lymphocyte receptor for Sema4D(Kumanogoh et al., 2000):recombinant soluble Sema4D binds specifically to CD72-expressing transfectants; antibodies specific for CD72 block the binding of soluble Sema4D to B cells; and soluble mouse Sema4D fails to stimulate CD72-deficient B cells, owing to a lack of surface binding. A direct protein-protein interaction between mouse recombinant soluble Sema4D and CD72 can be detected by surface plasmon resonance. CD72, a member of the C-type lectin family, is thought to function as a negative regulator of B cell responses by recruiting SHP-1, a tyrosine phophatase, to an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic region(Adachi et al., 1998). In support of this, B cells from CD72-deficient mice are hyper-proliferative in response to various stimuli (Pan et al.,1999). Both agonistic anti-mouse CD72 monoclonal antibodies and soluble mouse Sema4D protein can induce dephosphorylation of mouse CD72,facilitating the dissociation of SHP-1 from CD72(Wu et al., 1998; Kumanogoh et al., 2000). Thus,it appears that Sema4D binding turns off inhibitory signaling by CD72(Kumanogoh et al., 2000; Kumanogoh and Kikutani, 2001)(Fig. 3).

The expression patterns of Sema4D and its high-affinity receptor,plexin-B1, imply a role in axonal guidance. In Drosophila, plexin-B controls the axonal guidance of certain motor neurons by enhancing Rho signaling (Hu et al., 2001). Sema4D binding to plexin-B1 results in RhoA activation by regulating PDZ-RhoGEF/LARG, the GEF responsible for Sema4D-induced growth cone collapse in primary hippocampal neurons (Swiercz et al., 2002). In addition, Sema4D triggers the invasive growth of epithelial cells, including cell-cell dissociation, anchorage-independent growth, and branching morphogenesis by binding to the plexin-B1-Met complex(Giordano et al., 2002). The physiological relevance of the above biological activities of Sema4D remains to be clarified, because no apparent phenotypes in nonlymphoid organs have yet been observed in Sema4D-deficient mice(Shi et al., 2000). Some of Sema4D functions might be of course compensated for by other semaphorin molecules in those mice.

A number of biological activities of Sema4D in the immune system have been reported, and some of these have been confirmed by analysis of Sema4D-deficient mice (Shi et al.,2000). Sema4D-expressing CHO cell transfectants promote the in vitro aggregation and survival of B cells(Hall et al., 1996). Human Sema4D stimulation also induces shedding of CD23 (a low affinity receptor for IgE, which is used as an activation marker for B cells) from the surface of B cells (Hall et al., 1996). Sema4D-expressing CHO cell transfectants or the addition of soluble recombinant mouse Sema4D can enhance CD40-induced proliferation and immunoglobulin production in mouse B cells(Kumanogoh et al., 2000). Similarly, B cell responses to CD40 or LPS stimulation in vitro and humoral immune responses against T-cell-dependent antigens in vivo are impaired in Sema4D-deficient mice (Shi et al.,2000). Sema4D also plays a role in the activation and maturation of DCs (Kumanogoh et al.,2002b): soluble recombinant Sema4D can enhance CD40-induced DC maturation, as measured by upregulation of CD40 and CD80, and enhanced production of interleukin 12. In Sema4D-deficeint mice, T cell priming, in which DCs play a central role, is severely impaired(Shi et al., 2000). Sema4D also seems to play a role in monocytes and macrophages: soluble human Sema4D inhibits the spontaneous and MCP-3-induced migration of freshly isolated monocytes and monocytic cell lines(Delaire et al., 2001). We have also demonstrated that both recombinant soluble human Sema4D and agonistic anti-huma-CD72 monoclonal antibodies induce the production of proinflammatory cytokines, such as IL-6 and IL-8, by human monocytes(Ishida et al., 2003). Collectively, these findings suggest that Sema4D has a crucial function in a broad range of tissues.

By contrast, some reports suggest a possible function for Sema4D as an immune receptor. Antibody crosslinking of human Sema4D enhances T cell proliferation in the presence of submitogenic doses of either monoclonal antibodies against CD3 (a component of the T cell receptor complex) or monoclonal antibodies against CD2 [a T cell surface antigen that interacts with LFA-3 (CD58)] (Bougeret et al.,1992; Herold et al.,1995). In human T cells, Sema4D is associated with an unidentified serine/threonine kinase and a protein tyrosine phosphatase (PTP), CD45(Elhabazi et al., 1997; Herold et al., 1996). A switch at the terminal stage of B cell differentiation in the type of PTP activity associated with human Sema4D has also been reported(Billard et al., 2000).

A Drosophila transmembrane semaphorin, Sema-1a, plays a role in axon guidance, acting through the plexin-A receptor(Winberg et al., 1998). However, it might also have a bi-directional role as both a ligand and a receptor in central synapse formation in Drosophila(Yu et al., 1998). This paradigm supports the plausibility of a bi-directional function for Sema4D. In what situations might Sema4D function as a receptor? Transfectants expressing human plexin-B1 sustain the proliferation of normal and leukemic CD5⁺ B cells, both of which express human Sema4D(Granziero et al., 2003). These data suggest that human Sema4D can function as a receptor for human plexin-B1. It remains to be clarified whether the cytoplasmic region of Sema4D is involved in physiological or pathological roles during the growth of normal or leukemic CD5⁺ B cells, respectively, in vivo.

Sema4A, a class IV semaphorin originally identified as semB(Puschel et al., 1995),possesses a structure similar to that of Sema4D. Its expression gradually increases during embryonic development, and Sema4A mRNA is detectable in the brain, spleen, lung, kidney, and testes of adults(Kumanogoh et al., 2002a). Sema4A is expressed by bone-marrow-derived and splenic DCs as well as by B cells, but not by resting T cells. No differences in Sema4A expression are observable between CD8α⁺ (lymphoid origin) and CD8α^- (myeloid origin) DCs. Sema4A is upregulated following B cell stimulation with anti-CD40 monoclonal antibodies. Expression of Sema4A becomes detectable upon stimulation of T cells with anti-CD3 monoclonal antibodies (Kumanogoh et al.,2002a). This expression pattern of Sema4A contrasts that of Sema4D, in which expression is preferentially seen on T cells.

Whereas Sema4D binds plexin-B1, the binding of Sema4A to plexins remains to be investigated. In the immune system, Sema4A-binding partners exist on the surface of activated T cells but not on B cells and DCs. The binding of Sema4A to the cell surface of a subset of T cell lines, such as EL-4 cells, is also observed. Expression cloning using human IgG1-Fc portion-conjugated Sema4A(Sema4A-Fc) revealed that the Sema4A receptor is a member of the T cell,immunoglobulin and mucin domain proteins (TIM) family, Tim-2(Kumanogoh et al., 2002a)(Fig. 4) - a 305-residue transmembrane protein. Sema4A-Fc binds COS7 cells transfected with Tim-2 cDNA in a specific manner (Kumanogoh et al.,2002a) and stimulation of cells with Sema4A induces Tim-2 tyrosine phosphorylation.

The Tim family of proteins is expressed on T cells and characterized by a conserved set of immunoglobulin and mucin domains. McIntire et al. originally identified a locus conferring susceptibility to mouse allergen-induced airway hypersensitivity, which they dubbed T cell and airway phenotype regulator(Tapr) (McIntire et al.,2001). Subsequent positional cloning revealed this locus to contain a new family of genes, the Tims. There are remarkable numbers of Tim-1 sequence polymorphisms within both Tapr and human Tim-1 (hHAVcr-1) (McIntire et al., 2001). The human homologue of Tim-2 has not yet been identified. Kuchroo and colleagues independently identified Tim-3 in a screen for Th1 (a functional T cell subset that preferentially produces IFN-γbut not IL-4 and plays crucial roles in cell-mediated immunity)-reactive monoclonal antibodies as a Th1 cell-specific surface protein(Monney et al., 2002). Administration of anti-Tim-3 monoclonal antibodies promotes the development of EAE, a Th1-dependent autoimmune disease. This result suggests that Tim-3 is involved in the interactions between Th1 cells and macrophages, which results in the expansion and activation of macrophages. The fact that both Tim-1 and Tim-3 play a role in T-cell-mediated immune responses underscores the functional relevance of Tim-2, a member of the Tim family, in T cell function. Although the natural ligands of Tim-1 and Tim-3 have not been identified, the known Tim protein ligands, including Sema4A, are likely to be regulatory molecules influencing the activation and differentiation of T cells.

Although recombinant soluble Sema4A does not appear to stimulate B cells and DCs, Sema4A can provide T cell costimulation(Kumanogoh et al., 2002a). The addition of recombinant soluble Sema4A enhances T cell proliferation and IL-2 production following stimulation with anti-CD3 monoclonal antibodies. Soluble Sema4A promotes the induction of either Th1-cell-mediated IFN-γproduction or Th2-cell-mediated IL-4 production, depending on the respective culture conditions. In addition, soluble Sema4A enhances mixed lymphocyte reactions (MLRs) between allogeneic T cells and DCs. Anti-Sema4A can block the MLRs, which indicates that Sema4A functions in T cell activation by influencing stimulatory interactions between T cells and DCs.

The role of Sema4A in immune responses in vivo has been clarified by use of soluble Sema4A and anti-Sema4A monoclonal antibodies(Kumanogoh et al., 2002a). Soluble Sema4A significantly enhances the generation of antigen-specific T cells. In contrast, administration of anti-Sema4A monoclonal antibodies blocks antigen-specific T cell priming. Treatment of mice with anti-Sema4A monoclonal antibodies inhibits the development of EAE induced by myelin oligodendrocyte glycoprotein (MOG)-peptide administration. Delayed administration of soluble Sema4A or anti-Sema4A antibodies does not affect the generation of antigen-specific T cells, which suggests that Sema4A acts early in T cell activation in vivo (Kumanogoh et al.,2002a; Kikutani and Kumanogoh,2003).

The functions of semaphorins as axonal chemorepellents have been well characterized in the case of the secreted class III subclass members(Tessier-Lavigne and Goodman,1996). Five of the seven semaphorin subclasses, however, are transmembrane semaphorins. Drosophila Sema-1a, a transmembrane semaphorin, functions in axonal guidance in vivo(Raper, 2000). Whether transmembrane semaphorins exert their biological activities through direct contact between ligand-expressing cells and target cells or function remotely as soluble molecules is not clear. Some transmembrane semaphorins, such as Sema4D, can be released from the cell surface by proteolytic cleavage. The generation of soluble Sema4D is highly regulated(Wang et al., 2001); release from primary T and B cells is strictly dependent on a proteolytic cascade controlled by cellular activation. A significant amount of soluble Sema4D is detectable in the sera of mice immunized with a T-cell-dependent antigen, as well as in the sera of autoimmunity-prone MRL/lpr mice. The levels of soluble Sema4D correlate well with the titers of antigen-specific antibodies or auto-antibodies; soluble Sema4D is undetectable in the sera of unimmunized normal mice (Wang et al.,2001). The profiles of soluble Sema4D expression in vivo thus suggest that soluble Sema4D has important immunological activities. Transmembrane Sema4D, however, also appears to be active. Sema4D-expressing CHO cell transfectants promote B cell proliferation and immunoglobulin production (Hall et al., 1996; Kumanogoh et al., 2000). Thus,transmembrane Sema4D might function as a transmembrane ligand and also exist as a reservoir for soluble Sema4D production. If so, proteolytic cleavage of Sema4D would be a critical step regulating its function in the immune and nervous systems.

How is proteolytic cleavage of the transmembrane form regulated? The cytoplasmic region of Sema4D appears to be necessary for this regulation. A serine kinase activity is associated with the cytoplasmic region of human Sema4D (Elhabazi et al., 1997)(Fig. 2). Staurosporine (a cell-permeable, broad specificity inhibitor of serine kinases) enhances the release of soluble Sema4D (Elhabazi et al., 2001), which suggests that serine phosphorylation is involved in the regulation of Sema4D cleavage. Despite weak cell surface expression,high levels of soluble Sema4D are detectable in the sera of transgenic mice expressing a truncated Sema4D lacking the cytoplasmic region(Watanabe et al., 2001). Thus,the cytoplasmic region of Sema4D is necessary for proper regulation of soluble form generation. Further studies will be required to determine whether these findings, particularly the regulation of soluble Sema4D production, are applicable to other transmembrane semaphorins.

Viruses encode proteins that can function as immune modulators. Such proteins modulate the host immune response to facilitate the infectious process or support viral transmission. Kolodkin et al. discovered an open reading frame, A39R, related to the semaphorin family in the genome of vaccinia virus, a member of the poxvirus family(Kolodkin et al., 1993). A39R induces robust responses in human monocytes, including cell aggregation,induction of proinflammatory cytokines (e.g. IL-6 and IL-8), and upregulation of the monocyte cell surface marker CD54 (ICAM-1)(Comeau et al., 1998), which suggests it affects the immune responses of vaccinia-infected hosts. In addition, Ensser and Fleckenstein have reported that another semaphorin,AHVsema, is encoded by the alcelaphine herpesvirus (AHV)(Ensser and Fleckenstein,1995).

Comeau et al. (Comeau et al.,1998) have found that both of these viral semaphorins, A39R and AHVsema, bind to the cellular receptor VESPR/CD232/plexin-C1. A39R-induced induction of inflammatory cytokines is abrogated by a blocking antibody against VESPR/CD232/plexin-C1 (Comeau et al., 1998). These findings suggest a pathogenic role for viral semaphorins.

Sema7A (also known as Sema-K1 or CD108) is a molecule homologus to AHVsema. It was originally discovered as the John-Milton-Hagen human blood group antigen. Sema7A is a GPI-anchored cell surface glycoprotein preferentially expressed on activated lymphocytes and thymocytes(Xu et al., 1998; Yamada et al., 1999). Sema7A specifically binds to VESPR/CD232/plexin-C1(Tamagnone et al., 1999),which confirms that this semaphorin is a cellular counterpart of AHVsema. Recombinant soluble Sema7A protein exhibits activities against monocytes similar to those of AHVsema, including induction of the production of inflammatory cytokines such as TNF-α, IL-6 and IL-8. In addition, Sema7A demonstrates chemotactic attraction for monocytes(Holmes et al., 2002), which substantiates its role in inflammatory responses. Thus, the viral semaphorins may have an immunomodulatory effect by mimicking Sema7A. It will therefore be important to determine the physiological importance of interactions between Sema7A and VESPR/CD232/plexin-C1.

Sema3A is the human homologue of collapsin-1, the first identified vertebrate semaphorin (Kolodkin et al.,1993). The function of Sema3A as an axonal guidance factor has been extensively studied in the neurobiology field. Delaire et al. reported that Sema3A also plays a role in regulating monocyte migration, Sema3A inhibiting spontaneous monocytic cell migration in a transwell assay(Delaire et al., 2001). Neuropilin-1, the known receptor of Sema3A, is not expressed on the target immune cells, however, which suggests that the inhibitory effect of Sema3A on immune-cell migration is mediated by receptors different from those previously known to bind within the nervous system(Delaire et al., 2001).

Two receptor families, plexins and neuropilins, have been implicated in mediating the functions of semaphorins in axon guidance(He and Tessier-Lavigne, 1997; Kolodkin et al., 1997; Comeau et al., 1998; Winberg et al., 1998; Takahashi et al., 1999; Tamagnone et al., 1999). Vertebrate class III secreted semaphorins use neuropilins as ligand-binding receptor components, which form the receptor complex with plexins(Takahashi et al., 1999; Pasterkamp and Kolodkin,2003). Semaphorins of invertebrates, and transmembrane and GPI-anchored semaphorins in vertebrates interact directly with plexins(Yu and Kolodkin, 1999). Viral-encoded semaphorins, A39R and AHVsema, and Sema7A, interact directly with plexin-C1, inducing activation of monocytes. Why, then, do Sema4D and Sema4A exert their functions in the immune system through CD72 and Tim-2,respectively, molecules that have no structural relationship with neuropilins and plexins. Although it is currently not known whether other semaphorins expressed in the immune system also use distinct receptor systems, the receptor systems used by Sema4D and Sema4A do not fit well into our current understanding of semaphorin receptor systems. Because Sema4D can use plexin-B1 in addition to CD72, Sema4A might also function through neuropilins or plexins during development. Sema4D and Sema4A might therefore have acquired new functions in the immune system during evolution through the use of new receptors. Such pleiotropic gene function represents an efficient way to use limited genetic resources.

The semaphorin receptor system is more complex than previously thought. Several novel components besides plexins and neuropilins are emerging, aiding our understanding of the complexity of the system. Off-track, a protein similar to receptor tyrosine kinases that contains a catalytically inactive kinase domain, associates with Drosophila melanogaster PlexinA to mediate Sema1a repulsive functions(Winberg et al., 2001). L1, a cell adhesion molecule in the immunoglobulin superfamily, plays a role in repulsive responses to the class III semaphorin Sema3A as part of a neuropilin-plexin receptor complex(Castellani et al., 2002). Moreover, as mentioned above, plexin-B1 forms a receptor complex with Met,whose intrinsic kinase activity is required for promoting epithelial cell invasive growth in response to Sema4D(Giordano et al., 2002). These recent findings suggest that many semaphorin receptors exist as large holoreceptor complexes that mediate ligand binding and signal transduction. The pairing of receptor components can also have diverse biological functions in a broad range of tissues. It will be crucial to carefully re-evaluate the composition of several semaphorin receptors formerly thought to contain one or a few different proteins, including CD72 and Tim-2. More comprehensive studies on receptor usage, including the presence or absence of additional receptor components in both lymphoid and non-lymphoid tissues, will help delineate the pleiotropic effects of the multiple semaphorin-receptor systems.

Given that semaphorins are emerging as immunological mediators, it is worth considering the clinical relevance of immune semaphorins and their ability to manipulate pathological immune responses. Novel in vivo findings suggest the potential use of Sema4D and Sema4A to enhance host immunity against pathogens. Administration of recombinant soluble Sema4D accelerates antigen-specific antibody production and antigen-specific T cell generation(Kumanogoh et al., 2000; Shi et al., 2000), as does the administration of recombinant soluble Sema4A(Kumanogoh et al., 2002a). Stimulation of antigen-pulsed DCs with recombinant mouse Sema4D in combination with anti-CD40 monoclonal antibodies significantly augments in vivo immunogenicity, resulting in enhanced generation of antigen-specific effector and memory T cells (Kumanogoh et al.,2002b). Thus, administration of Sema4D, Sema4A, or agonistic reagents that stimulate CD72 or Tim-2, in conjunction with conventional immunization may enhance immune responses, particularly those generated against infectious agents by vaccines that are weakly immunogenic. Detailed investigation of the effects of immune semaphorins in both humans and experimental animals is required, however, prior to these studies. The combination of immune semaphorin stimulation with vaccination or vigorous antibiotic therapy might be beneficial for promoting immunity against various pathogens.

In contrast, inhibition of immune semaphorins may prove to be a therapeutic approach for the control of certain immune disorders. Sema4D-deficient mice are resistant to EAE induced following administration of MOG-derived peptides,owing to the impaired generation of MOG-reactive T cells(Kumanogoh et al., 2002b). Sema4A blockade also suppresses the development of EAE(Kumanogoh et al., 2002a),which suggests that these molecules serve as potential targets in the treatment of autoimmunity. In addition, an antibody against VESPR/CD232/plexin-C1 inhibits viral semaphorin-induced induction of inflammatory cytokines by monocytes (Comeau et al., 1998). Delaire and colleagues recently reported that soluble Sema3A and Sema4D inhibits both spontaneous and MCP-3-induced monocytic cell migration (Delaire et al.,2001). They might thus prove to be useful clinical anti-inflammatory agents. The manipulation of immune semaphorin function,through either reinforcement or inhibition by recombinant proteins and specific antibodies or drugs, is a crucial area for future clinical studies.

Recent studies of Sema4D and Sema4A have uncovered crucial roles of these semaphorins in the immune system (Kikutani and Kumanogoh, 2003). However, many questions regarding interpretation of the biological significance of this protein family still remain. For instance, an intriguing issue is whether Sema4D and Sema4A can also exert their functions through CD72 and Tim-2, respectively, in non-lymphoid organs, including the nervous system. Furthermore, neuropilins or plexins might be involved in the immunological functions of these semaphorins. More comprehensive studies on their receptor usage in both lymphoid and non-lymphoid tissues will address these questions. In addition, recent studies, including work on gene-targeted mice, sometimes show results that contradict previous notions of pairing of semaphorin receptor and co-receptor systems (Cheng et al., 2001; Suto et al., 2003). Note that identification of semaphorin receptors and coreceptors has often been based only on binding analysis data or non-physiological functional assays (for instance, COS cell contraction assays). It is, therefore, crucial that we carefully reevaluate the semaphorin receptor systems.

Semaphorins other than Sema4D and Sema4A also appear to function in immunity, representing a new family of immunoregulatory molecules. Because the studies with the 'immune semaphorins' suggest that these molecules function in autoimmune diseases and viral infections, other semaphorins may prove to be viable targets for research into therapies for immune disorders and infectious diseases. Future studies clarifying the biological functions of semaphorins in the immune system should help establish a new paradigm of cell-cell communication through semaphorin networks.

We thank K. Kubota for excellent secretarial assistance. We also thank N. Takegahara for excellent artistry in the creation of figures. This study was supported by research grants from the Ministry of Education, Culture, Science,and Technology of Japan to A.K. and H.K.