Rearrangement of immunoglobulin heavy chain genes in human T leukaemic cells shows preferential utilization of the D segment (DQ52) nearest to the J region.

The DNA rearrangements leading to the assembly of genes coding for the immunoglobulin heavy chain (IgH) in B cells and the T cell receptor for antigen in T cells are not completely lineage specific. This probably reflects the use of a common recombinase by IgH and the T cell receptor. This paper describes novel observations on the nature of these cross-lineage rearrangements. A high proportion (though not all) IgH rearrangements in human T leukaemic cells involve the D segment nearest to the J region (DQ52). This same D segment is not involved in B cell IgH rearrangements with one important exception, namely a proportion of B cell leukaemic clones with the most primitive B cell precursor phenotype. These observations have potentially important implications for early lymphoid cell differentiation and in particular support the idea that the 3' D plus J region might lie within a limited window of accessibility of the IgH gene in precursor lymphocytes.     

2B4 (CD244) signaling via chimeric receptors costimulates tumor-antigen specific proliferation and in vitro expansion of human T cells

Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and G_D2 to the signaling domains of human 2B4 and/or TCRζ were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRζ. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25^hiFoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells.     

Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates.

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Here we show that the human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with a tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. We conclude that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion.     

Plexin-A4 is expressed in oligodendrocyte precursor cells and acts as a mediator of semaphorin signals

Class 3 semaphorin acts as a guidance clue for both cell migration and nerve fiber projection. The signal of class 3 semaphorin travels via a receptor complex consisting of neuropilins and Plexin-A subfamily. Although it has been reported that class 3 semaphorin acts as a repellent for oligodendrocyte precursor cells (OPCs), which migrate actively during brain development, the expression of Plexin-A subfamily has not been reported in OPCs yet. Therefore, it is currently unclear how semaphorin signals can travel in OPCs. In the present study, the expression of Plexin-A4 (PlexA4) was first demonstrated in a newly established OPC line and OPCs in developing brain. In the OPC line, repulsion for process extension was caused by both Sema3A and Sema6A, and the effect of the semaphorins was diminished in cells expressing PlexA4 lacking the cytoplasmic domain. These results strongly suggest that PlexA4 expressed in OPCs acts as a mediator of semaphorin signals.     

Semaphorin CD100 from activated T lymphocytes induces process extension collapse in oligodendrocytes and death of immature neural cells

An inappropriate cross talk between activated T lymphocytes infiltrating the CNS and neural cells can sustain the onset and progression of demyelination and axonal degeneration in neuroinflammatory diseases. To mimic this deleterious cross talk, we designed an experimental paradigm consisting of transient cocultures of T lymphocytes chronically activated by retrovirus infection (not virus productive) with human multipotent neural precursors or primary oligodendrocytes from rat brain. We showed that activated T lymphocytes induced apoptotic death of multipotent neural progenitors and immature oligodendrocytes after a progressive collapse of their process extensions. These effects were reminiscent of those induced by brain semaphorin on neural cells. Blockade by specific Abs of soluble CD100 (sCD100)/semaphorin 4D released by activated T cells, or treatment with rsCD100, demonstrated that this immune semaphorin has the ability to collapse oligodendrocyte process extensions and to trigger neural cell apoptosis, most likely through receptors of the plexin family. The specific presence of sCD100 in the cerebrospinal fluid and of CD100-expressing T lymphocytes in the spinal cord of patients suffering with neuroinflammatory demyelination pointed to the potential pathological effect of sCD100 in the CNS. Thus, our results show that CD100 is a new important element in the deleterious T cell-neural cell cross talk during neuroinflammation and suggest its role in demyelination or absence of remyelination in neuroinflammatory diseases including multiple sclerosis and human T lymphotropic virus type 1-associated myelopathy.     

The semaphorin 4D receptor controls invasive growth by coupling with Met

Semaphorins are cell surface and soluble signals that control axonal guidance. Recently, semaphorin receptors (plexins) have been discovered and shown to be widely expressed. Their biological activities outside the nervous system and the signal transduction mechanism(s) they utilize are largely unknown. Here, we show that in epithelial cells, Semaphorin 4D (Sema 4D) triggers invasive growth, a complex programme that includes cell#150;cell dissociation, anchorage-independent growth and branching morphogenesis. Interestingly, the same response is also controlled by scatter factors through their tyrosine kinase receptors, which share striking structural homology with plexins in their extracellular domain. We found that in cells expressing the endogenous proteins, Plexin B1 (the Sema 4D Receptor) and Met (the Scatter Factor 1/ Hepatocyte Growth Factor Receptor) associate in a complex. In addition, binding of Sema 4D to Plexin B1 stimulates the tyrosine kinase activity of Met, resulting in tyrosine phosphorylation of both receptors. Finally, cells lacking Met expression do not respond to Sema 4D unless exogenous Met is expressed. This work identifies a novel biological function of semaphorins and suggests the involvement of an unexpected signalling mechanism, namely, the coupling of a plexin to a tyrosine kinase receptor.     

Identification and characterization of zebrafish semaphorin 6D

The semaphorin gene family contains a large number of secreted and transmembrane proteins; some function as repulsive and attractive cues of axon guidance during development. Here, we report cloning and characterization of zebrafish transmembrane semaphorin gene, semaphorin 6D (sema6D). Sema6D is expressed predominantly in the nervous system during embryogenesis, as determined by in situ hybridization. We also found that Sema6D binds Plexin-A1 in vitro, but not other Plexins. It induces the repulsion of dorsal root ganglion axons, but not sympathetic axons. Consequently, Sema6D might use Plexin-A1 as a receptor to repel specific types of axons during development.     

Enhanced immune responses in transgenic mice expressing a truncated form of the lymphocyte semaphorin CD100

CD100/Sema4D is a 150-kDa transmembrane protein that belongs to the semaphorin family. Binding of CD100 to CD72 enhances the immune response by turning off the negative signaling effects of CD72. To investigate the physiological functions of CD100 in vivo, we generated transgenic mice expressing a truncated form of CD100. A large amount of the soluble form of CD100 was detected in the sera of mice expressing a truncated form of CD100, although the amount of CD100 was only slightly elevated on the surface of B cells. In the mutant mice the development of conventional B and T cells appeared normal in terms of the surface marker phenotypes, while the number of CD5(+) B-1 cells in the peritoneal cavity increased in comparison with wild-type mice. In vitro proliferation and Ig production of B cells in response to CD40 stimulation were considerably enhanced in mice expressing a truncated form of CD100. Additionally, in vivo both Ab responses against T cell-dependent Ags and generation of Ag-specific T cells were enhanced. Furthermore, introduction of the CD100-transgene could restore in vitro B cell responses as well as in vivo Ab production against T cell-dependent Ag in CD100-deficient mice. Collectively, these results not only indicate that CD100 has an important role in the immune system, but also that the soluble form of CD100 released from the cell surface can exert functions in vivo.     

Identification of semaphorin 4B as a negative regulator of basophil-mediated immune responses

Basophils are strong mediators of Th2 responses during helminthic infections. Recently, basophils were shown to function as APCs and promote both Th2 skewing and humoral memory responses. However, the mechanisms that regulate basophils are still unclear. In this article, we show that a class IV semaphorin, Sema4B, negatively regulates basophil functions through T cell-basophil contacts. In a screen to identify semaphorins that function in the immune system, we determined that Sema4B is expressed in T and B cells. Interestingly, Sema4B(-/-) mice had considerably increased serum IgE levels despite normal lymphocyte and dendritic cell functions. Recombinant Sema4B significantly inhibited IL-4 and IL-6 production from basophils in response to various stimuli, including IL-3, papain, and FcεRI cross-linking. In addition, T cell-derived Sema4B, which accumulated at contact sites between basophils and CD4(+) T cells, suppressed basophil-mediated Th2 skewing, suggesting that Sema4B regulates basophil responses through cognate cell-cell contacts. Furthermore, Sema4B(-/-) mice had enhanced basophil-mediated memory IgE production, which was abolished by treating with an anti-FcεRIα Ab. Collectively, these results indicate that Sema4B negatively regulates basophil-mediated Th2 and humoral memory responses.     

Probing the interaction between feline immunodeficiency virus and CD134 by using the novel monoclonal antibody 7D6 and the CD134 (Ox40) ligand

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+ T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+ T cells, with weaker expression on CD8+ T cells, concordant with the expansion of FIV into CD8+ T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.     

Interactions between semaphorins and plexin–neuropilin receptor complexes in the membranes of live cells

Signaling of semaphorin ligands via their plexin–neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein–protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer–dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.     

The murine IL 2 receptor. III. Cellular requirements for the induction of IL 2 receptor expression on T cell subpopulations

The accessory cell requirements for the induction of the IL 2 receptor by the lectin Con A on murine T cell subsets were directly assayed with anti-IL 2 receptor monoclonal antibodies. Substantial levels of IL 2 receptor expression were induced on T lymphocytes of the MHC class I-restricted, suppressor/cytotoxic phenotype (L3T4-, Ly-2+) in the presence and absence of accessory cells. In contrast, high levels of IL 2 receptor expression could only be induced on T cells of the MHC class II-restricted, helper/inducer phenotype (L3T4+, LY-2-) in the presence, but not in the absence, of accessory cells. Ia- cells such as the P388D1 macrophage line or cultured fibroblasts (DAP X 3) were as efficient as the Ia+ B cell hybridoma LB in providing accessory cell function for the L3T4+, Ly-2- subset. PMA, but not purified human IL 1, could substitute for accessory cells for both IL 2 receptor expression and IL 2 secretion by the L3T4+, Ly-2- subset. These data suggest that IL 2 receptor induction on the L3T4+, Ly-2- subset is complex, possibly requiring a T cell-accessory cell interaction, whereas the lectin may directly trigger IL 2 receptor expression on L3T4-, Ly-2+ T cells.     

Signaling events involved in interleukin 27 (IL-27)-induced proliferation of human naive CD4+ T cells and B cells

IL-27 induces stronger proliferation of naive than memory human B cells and CD4(+) T cells. In B cells, this differential response is associated with similar levels of IL-27 receptor chains, IL-27Rα and gp130, in both subsets and stronger STAT1 and STAT3 activation by IL-27 in naive B cells. Here, we show that the stronger proliferative response of CD3-stimulated naive CD4(+) T cells to IL-27 is associated with lower levels of IL-27Rα but higher levels of gp130 compared with memory CD4(+) T cells. IL-27 signaling differs between naive and memory CD4(+) T cells, as shown by more sustained STAT1, -3, and -5 activation and weaker activation of SHP-2 in naive CD4(+) T cells. In the latter, IL-27 increases G0/G1 to S phase transition, cell division and, in some cases, cell survival. IL-27 proliferative effect on naive CD4(+) T cells is independent of MAPK, but is dependent on c-Myc and Pim-1 induction by IL-27 and is associated with induction of cyclin D2, cyclin D3, and CDK4 by IL-27 in a c-Myc and Pim-1-dependent manner. In BCR-stimulated naive B cells, IL-27 only increases entry in the S phase and induces the expression of Pim-1 and of cyclins A, D2, and D3. In these cells, inhibition of Pim-1 inhibits IL-27 effect on proliferation and cyclin induction. Altogether, these data indicate that IL-27 mediates proliferation of naive CD4(+) T cells and B cells through induction of both common and distinct sets of cell cycle regulators.     

Semaphorin 3B is a 1,25-Dihydroxyvitamin D3-induced gene in osteoblasts that promotes osteoclastogenesis and induces osteopenia in mice

The vitamin D endocrine system is important for skeletal homeostasis. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct actions of 1,25(OH)(2)D(3) in bone, relatively little is known of the mechanisms or target genes that are regulated by 1,25(OH)(2)D(3) in skeletal cells. Here, we identify semaphorin 3B (SEMA3B) as a 1,25(OH)(2)D(3)-stimulated gene in osteoblastic cells. Northern analysis revealed strong induction of SEMA3B mRNA by 1,25(OH)(2)D(3) in MG-63, ST-2, MC3T3, and primary osteoblastic cells. Moreover, differentiation of these osteogenic cells enhanced SEMA3B gene expression. Biological effects of SEMA3B in the skeletal system have not been reported. Here, we show that osteoblast-derived SEMA3B alters global skeletal homeostasis in intact animals and osteoblast function in cell culture. Osteoblast-targeted expression of SEMA3B in mice resulted in reduced bone mineral density and aberrant trabecular structure compared with nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study establishes that SEMA3B is a 1,25(OH)(2)D(3)-induced gene in osteoblasts and that osteoblast-derived SEMA3B impacts skeletal biology in vitro and in vivo. Collectively, these studies support a putative role for SEMA3B as an osteoblast protein that regulates bone mass and skeletal homeostasis.     

Ontogeny and function of B220+ L3T4+ T-cell subset of MRL/Mp-lpr/lpr mice

T-cell-enriched populations obtained from lymph nodes (LNs) of 4-month-old MRL/Mp-lpr/lpr (MRL-lpr), C3H/HeJ-lpr/lpr (C3H-lpr), and C3H/HeJ-gld/gld (C3H-gld) mice were studied for the expression of B220, L3T4, and Lyt 2 antigens. A new B220+ L3T4+ phenotype was demonstrated in T-cell populations of these mice by two-color flow cytometry with phycoerythrin-conjugated monoclonal antibodies (MoAb) to L3T4 and FITC-anti-B220 MoAb. The generation of the T subset was apparently under the influence of the lpr or gld gene, since it could not be demonstrated in T-cell-enriched populations of MRL/Mp- +/+ and normal C3H mice. The expression level of L3T4 antigen on the T subset was lower than that on B220- L3T4+ cells, while the level of B220 antigen was similar to that of B220+ L3T4- or B220+ Lyt 2- cells. The B220+ L3T4+ phenotype appeared in LNs and spleens, but not in thymuses, of MRL-lpr mice as early as 2 months of age. Its proportion to whole LN T cells at this age was equivalent to that observed in 4-month-old mice. Functional studies on FACS-sorted cell populations revealed that the T subset similar to B220+ L3T4- cells possessed deficiencies in the IL-2-IL-2 receptor system. The appearance of the T subset with an intermediate phenotype and its functional defects suggests that lpr and gld genes in these autoimmune mice exert their influences on the ontogeny and function of L3T4+ T cells and contribute to the induction of early lupus.     

Successful inhibition of tumor development by specific class-3 semaphorins is associated with expression of appropriate semaphorin receptors by tumor cells

The class-3 semaphorins (sema3s) include seven family members. Six of them bind to neuropilin-1 (np1) or neuropilin-2 (np2) receptors or to both, while the seventh, sema3E, binds to the plexin-D1 receptor. Sema3B and sema3F were previously characterized as tumor suppressors and as inhibitors of tumor angiogenesis. To determine if additional class-3 semaphorins such as sema3A, sema3D, sema3E and sema3G possess anti-angiogenic and anti-tumorigenic properties, we expressed the recombinant full length semaphorins in four different tumorigenic cell lines expressing different combinations of class-3 semaphorin receptors. We show for the first time that sema3A, sema3D, sema3E and sema3G can function as potent anti-tumorigenic agents. All the semaphorins we examined were also able to reduce the concentration of tumor associated blood vessels although the potencies of the anti-angiogenic effects varied depending on the tumor cell type. Surprisingly, there was little correlation between the ability to inhibit tumor angiogenesis and their anti-tumorigenic activity. None of the semaphorins inhibited the adhesion of the tumor cells to plastic or fibronectin nor did they modulate the proliferation of tumor cells cultured in cell culture dishes. However, various semaphorins were able to inhibit the formation of soft agar colonies from tumor cells expressing appropriate semaphorin receptors, although in this case too the inhibitory effect was not always correlated with the anti-tumorigenic effect. In contrast, the anti-tumorigenic effect of each of the semaphorins correlated very well with tumor cell expression of specific signal transducing receptors for particular semaphorins. This correlation was not broken even in cases in which the tumor cells expressed significant concentrations of endogenous semaphorins. Our results suggest that combinations of different class-3 semaphorins may be more effective than single semaphorins in cases in which tumor cells express more than one type of semaphorin receptors.     

Role of the interleukin 2 receptor in differentiation of a clone of Ly-1+ B cells

CH12.LX, an in vitro subclone of a murine B cell lymphoma that makes IgM reactive with sheep erythrocytes (SRBC), has cell surface receptors for the lymphokine interleukin 2 (IL 2). The binding of recombinant murine IL 2 to these receptors did not stimulate CH12.LX cells to differentiate and secrete antibody. However, the binding of either of two monoclonal antibodies (Mab) specific for the IL 2 receptor increased the proportion of CH12.LX cells that secrete hemolytic IgM. The effect did not require the presence of antigen. One of the Mab, 3C7, is known to block the binding of IL2 to its receptor on T cells, whereas the other, 7D4, which also reacts with the IL 2 receptor, does not block the binding of IL 2. The differentiation of CH12.LX induced by 3C7, but not that induced by 7D4, was inhibited by recombinant IL 2. Neither IL 2 (up to 200 U/ml) nor 3C7 (up to 10 micrograms/ml) had any significant influence on incorporation of [3H]thymidine; 7D4 at 10 micrograms/ml decreased thymidine incorporation by about 60%. Mitomycin C and hydroxyurea, which both inhibit the incorporation of [3H]thymidine into CH12.LX cells, also both induce antibody secretion. In both cases, the concentration necessary to cause differentiation is substantially lower than that needed to cause detectable inhibition of thymidine uptake. We conclude that the IL 2 receptor on CH12.LX cells is a functional signal transducing molecule, and we discuss the possible inverse relationship between proliferation and differentiation.     

A human CD4- T cell leukemia subclone with contact-dependent helper function.

Helper T lymphocytes provide a contact dependent signal to resting B cells that is required for optimal differentiation into Ig-secreting cells. The surface structure(s) on T cells that mediate helper function have not been identified but are known to be induced by T cell activation. A CD4- subclone of the Jurkat leukemic T cell line (D1.1) was isolated and found to be distinct from CD4+ Jurkat clones and a variety of other T and non-T cell leukemic lines in that coculture of D1.1 with resting B cells induced B cells to specifically express surface CD23 molecules, a marker of B cell activation. Furthermore, Jurkat D1.1 induced B cell proliferation and terminal B cell differentiation into IgG-secreting cells in the presence of T cell-dependent B cell mitogens. Similar to the helper effector function of activated T cells, the effects of Jurkat D1.1 were neither Ag nor MHC restricted. Paraformaldehyde fixed Jurkat D1.1 cells remained competent to activate B cells while D1.1 supernatants and diffusible factors were inactive. The effect of Jurkat D1.1 on B cell activation was distinct from that of rIL-4 and was not inhibited by antibodies to IL-4. Together these observations suggested that surface structures on D1.1 and not secreted factors, mediated contact-dependent helper effector function.