Biological activity of soluble CD100. I. The extracellular region of CD100 is released from the surface of T lymphocytes by regulated proteolysis.

CD100 is the first semaphorin described in lymphoid tissues, where it has been shown to be associated with a serine kinase activity. Semaphorins are molecules involved in axon pathfinding during nerve development and act as repellent guidance cues. In the nervous system semaphorins exist as either membrane-bound or secreted forms. We report here a spontaneous processing of membrane CD100, suggesting that it is also produced as a diffusable semaphorin from lymphoid cells. Monomeric and homodimeric forms of CD100 are expressed by T lymphocytes and CD100-transfected fibroblasts. We demonstrate that CD100 is released through a proteolytic process blocked by metalloprotease inhibitors. In T cells, only soluble CD100 dimers are produced, suggesting that CD100 dimerization is required for proteolysis. In agreement, we observe that increasing membrane dimers strongly favors shedding of the molecule. By expressing a CD100 molecule mutated at cysteine 674 into a COS cell system, we additionally demonstrate that this particular residue in the extracellular domain of the molecule is required for dimerization. Finally, we show that staurosporine, a serine kinase inhibitor, enhances the membrane cleavage of CD100. Together these results demonstrate that membrane CD100 is cleaved by a metalloprotease-dependent process, which is probably regulated by phosphorylation. Mainly, these findings shed light on a possible function for the semaphorin region of CD100 as a long range guidance cue in the immune system.

Biological activity of soluble CD100. II. Soluble CD100, similarly to H-SemaIII, inhibits immune cell migration.

CD100 is a human 150-kDa homodimer expressed at the surface of most hemopoietic cells, and its gene belongs to the Ig and semaphorin gene families. Semaphorin genes encode soluble and membrane-bound proteins, most of which have been shown to act as chemorepellents on growth cone guidance. CD100 is discrete, as it is a transmembrane leukocyte surface molecule that can also exist in a soluble form. While our previous studies using mAbs suggested that the transmembrane form of CD100 plays a role in lymphocyte activation, no function was shown for its soluble form. Here, we investigated the effect of soluble CD100 in a cell migration assay; both CD100 spontaneously shed from a stable transfectant and soluble recombinant CD100 inhibited spontaneous and chemokine-induced migration of human monocytes. Interestingly, only the dimeric form of CD100 exerted an effect. Moreover, soluble CD100 inhibited migration of cells from monocytic and B cell lineages. A similar inhibitory effect on migration was observed with H-SemaIII, but not H-SemaIV, semaphorins. In addition, both CD100 and H-SemaIII were recognized by two CD100 mAbs in an ELISA, and one of these mAb abolished the inhibitory effect of each of these semaphorins. We also provide evidence that CD100 and H-SemaIII act through the same receptor on immune cells, which is not neuropilin-1. Furthermore, we describe a function on immune cells for H-SemaIII, a semaphorin to date only studied in the nervous system.

G10.3 monoclonal antibody identifies novel functional cell surface structures expressed by normal B lymphocytes and various malignant cell lines.

G10.3, a unique monoclonal antibody (mAb), was produced to better characterize lymphocyte subsets. In the present study, we show that this mAb identifies 118, 83 and 51 kDa cell surface sialylated glycoproteins on the immunizing cell line YTindi. The reactivity of G10.3 mAb is restricted in normal cells to B lymphocytes, whereas within tumoral cell lines various lymphoid and non-lymphoid cells were found positive. Interestingly, functional studies revealed that triggering G10.3 mAb reactive molecules with soluble antibody led to an inhibition of growth and to an induction of programmed cell death in tumor cell lines expressing high levels of reactive molecules.

CD100 is a leukocyte semaphorin.

CD100 was originally described as an activation molecule on the surface of human T lymphocytes. Its triggering through distinct epitopes leads to different signals of costimulation with phorbol myristate acetate (PMA) or with CD3 and CD2. Interestingly, CD100 was shown to associate with different partner molecules in T cells. First, CD100 can associate with CD45, a key molecule with protein tyrosine phosphatase activity involved in T-cell transduction: this association is physical and has functional consequences for both partners. Second, CD100 interacts in its cytoplasmic domain with a Ser/Thr kinase for which it represents a preferential substrate. Recently, CD100 was identified as a member of the semaphorin gene family. This family comprises approximately 20 structurally related proteins. The first semaphorins were identified in the developing nervous system. Function has been shown for only some of them and involves repulsion during growth cone guidance. Since CD100 was the first semaphorin identified in the immune system, this raises the possibility of the involvement of members of the semaphorin family in other physiological phenomena outside the nervous system.

The human semaphorin-like leukocyte cell surface molecule CD100 associates with a serine kinase activity.

CD100 is a 150-kDa homodimeric glycoprotein broadly expressed on the surface of human hematopoietic cells. CD100 has been recently identified as the first lymphoid gene that belongs to the semaphorin gene family. Semaphorins function as chemorepellent molecules in the nervous system, but the function of CD100 remains poorly understood. In lymphoid cells, it has been suggested to play a role in homotypic cell adhesion and in T cell activation. We demonstrate that in T cells and natural killer cells a serine kinase activity is immunoprecipitated with CD100. Distinct epitopes of CD100 have been defined with specific monoclonal antibodies, mediating opposite effects at the functional level, especially in T cells. The kinase activity is retained only with an antibody against a particular epitope of CD100. Additionally, a fusion protein containing the cytoplasmic domain of the molecule retains the kinase activity in cellular lysates, and CD100 itself is presumably a favorite substrate of the kinase. These findings suggest that a serine kinase pathway may participate in the different functional effects triggered through the distinct epitopes of CD100 and is likely involved in the biological effects of this semaphorin-like leukocyte cell surface molecule.

CD100 is associated with CD45 at the surface of human T lymphocytes. Role in T cell homotypic adhesion.

CD100 is a 150-kDa human surface glycoprotein implicated in T cell activation. Using BB18 and BD16 mAbs to two discrete epitopes of the CD100 molecule, we have shown previously that triggering the CD100 molecule through the BB18 epitope is comitogenic with PMA, whereas it lacks a detectable effect on CD2- and CD3-induced PBMC proliferation. Conversely, triggering the CD100 molecule through the BD16-defined epitope only exerts an effect on CD2- and CD3-induced PBMC proliferation. In the present study, we investigated the molecular relationship between CD100 and another surface structure involved in human T cell proliferation, the CD45 phosphatase. We show in this study that CD100 is associated with CD45 and that this association has functional significance. The association was demonstrated using coimmunoprecipitation and detection of CD45 enzymatic PTPase activity. Furthermore, we show that the association is increased during T cell activation and that triggering CD45 molecules through discrete epitopes induces the down-modulation of CD100 molecules at the cell surface. Interestingly, we demonstrate that this modulation could be attributed to the shedding of a soluble form of CD100 in the culture supernatant. Finally, one of the functional consequences of this T cell activation-induced CD100-CD45 association is revealed by the finding that CD100 mAbs have an effect on CD45-induced T cell aggregation.

Functional role of PECAM-1/CD31 molecule expressed on human cord blood progenitors.

CD31/PECAM-1 (platelet endothelial cell adhesion molecule-1) is a 130 kDa integral membrane protein of the immunoglobulin gene superfamily with the distinctive feature of being expressed on several cell types associated with the vascular compartment. In the present study we report a novel, unique CD31 mAb termed IP28A which reacts with all CD34 molecule expressing hematopoietic progenitor cells and a subset of T, B and NK lymphocytes from human cord blood. Interestingly, we show that the number of CFU-GM and BFU-E was significantly augmented in cord blood progenitor cultures when purified IP28A mAb was added to rhSCF plus rhGM-CSF and rhEpo, respectively. Thus, these results are of relevance in the field of hematopoietic stem cell transplantation as they reveal an agonistic property of the IP28A/CD31 mAb on the differentiation of cord blood progenitor cells.

Effect of physical exhaustion and glucocorticoids (dexamethasone) on T-cells of trained rats.

Following a previous observation that moderate physical training (running) of rats did not impair T-cells, in this study moderately trained Wistar rats were run to exhaustion on 2 consecutive days: in one case (T-dex) this was preceded by an intraperitoneal injection of 0.5 mg.kg-1 of dexamethasone (dex) and in the other case there was no prior injection (T). Similarly one group of sedentary control rats, was injected with dex (C-dex) and the other group was not (C). Rats were killed 24 h after the last treatment (dex, exercise). Compared with the C rats, the T rats exhibited a decreased number of thymocytes (75%), in particular CD4+CD8+ thymocytes and splenocytes (55%), notably CD4+CD8- splenocytes (P < 0.01). Also noted in the T rats was a lower (45%) in vitro (+mitogen) percentage of IL2r+CD4- splenocytes (expressing the IL2 receptor), and reduced (40%, P < 0.01) or unchanged in vitro production of T-cell growth factor (TCGF) by splenocytes or blood mononucleated cells (BMC), respectively. The dex decreased the number of thymocytes and splenocytes in the same way in T-dex rats (compared to T rats) and in C-dex rats (compared to C rats, P < 0.01). In T-dex rats compared with C-dex rats, on the other hand, dex had little effect on in vitro TCGF production by BMC, and no effect on other in vitro parameters. These results would indicate that physical exhaustion was responsible for an alteration in T-cells in the moderately trained rat. This alteration was in part enhanced by dex.

Immunomodulations of thymocytes and splenocytes in trained rats.

The aim of this study was to describe the effects of training (running) on thymus and spleen cells in the rat. Young Wistar control rats (n = 6), rats trained for 4 wk (n = 5), and rats trained for 4 wk followed by 1 wk of intensive training (3 h/day, n = 6) were studied. Various lymphocyte surface and nuclear markers were determined by immunocytochemistry. The results show that 4 wk of training 1) decreased the percentage of bromodeoxyuridine (BrdU+) thymocytes (cell in phase S of the cycle, immature thymocytes; P less than 0.05) and the viability of thymocytes stimulated with concanavalin A (Con A; P less than 0.05) and 2) increased the absolute number of CD8+ (suppressor/cytotoxic T cells; 29%) and the percentage of CD8+ splenocytes (P less than 0.01). An additional week of intensive training in the 4-wk trained rats induced 1) a decrease in the absolute number of thymocytes (25%, P less than 0.05), TCR+ thymocytes, splenocytes (28%, P less than 0.01), T, CD4+ (helper T cells; 34%), and CD8+ (31%) splenocytes (P less than 0.01) and 2) an increase in the viability of splenocytes after stimulation with Con A for 72 h (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)