CD72-semaphorin3A axis: A new regulatory pathway in systemic lupus erythematosus.

CD72 is a regulatory co-receptor on B cells, with a role in the pathogenesis of systemic lupus erythematosus (SLE) in both human and animal models. Semaphorin3A (sema3A) is a secreted member of the semaphorin family that can reconstruct B cells' regulatory functions by upregulating IL-10 expression and inhibiting the pro-inflammatory activity of B and T cells in autoimmune diseases. The aim of our present study was to identify a new ligand for CD72, namely sema3A, and exploring the signal transduction pathways following its ligation in B cells. We established that CD72 functions as sema3A binding and signal-transducing receptor. These functions of CD72 are independent of neuropilin-1 (NRP-1) (the known sema3A receptor). We discovered that sema3A induces the phosphorylation of CD72 on tyrosine residues and the association of CD72 with SHP-1 and SHP-2. In addition, the binding of sema3A to CD72 on B cells inhibits the phosphorylation of STAT-4 and HDAC-1 and induces the phosphorylation of p38-MAPK and PKC-theta in B-cells derived B-lymphoblastoid (BLCL) cells, and in primary B-cells isolated from either healthy donors or SLE patients. We concluded that sema3A is a functional regulatory ligand for CD72 on B cells. The sema3A-CD72 axis is a crucial regulatory pathway in the pathogenesis of autoimmune and inflammatory diseases namely SLE, and modulation of this pathway may have a potential therapeutic value for autoimmune diseases.

The Involvement of LAG-3positive Plasma Cells in the Development of Multiple Myeloma.

The Lymphocyte-Activation Protein 3 (LAG-3) inhibitory receptor is expressed on regulatory plasma cells (PCs). Micro-environmental cells that express LAG-3 were found to be increased during the progression of smoldering multiple myeloma (SMM). To assess the possible role of LAG-3 expression on regulatory PCs in patients with plasma cell dyscrasia. Purified Cluster of Differentiation 138 (CD138+) PCs from patients with premalignant conditions, active multiple myeloma (MM), and controls were analyzed for the expression of LAG-3 by flow cytometry. Autologous CD8+T cells were incubated with sorted LAG-3pos or LAG-3neg PCs for 24 h. The expression of granzyme (Grz) in CD8+T cells was assessed by flow cytometry. LAG-3 expression on PCs in active MM (newly diagnosed and relapse refractory MM) was significantly increased compared to monoclonal gammopathy of undetermined significance (MGUS)/ SMM. Grz expression was significantly decreased in CD8+T cells incubated with CD138+LAG-3pos PCs, compared to CD138+LAG-3neg PCs in patients with plasma cell dyscrasia, n = 31, p = 0.0041. LAG-3 expression on malignant PCs can be involved in the development of MM from MGUS by decreasing the expression of Grz in CD8+T cells.

Truncated-semaphorin3A is a potential regulatory molecule to restore immune homeostasis in immune-mediated diseases.

Regulatory molecules have recently been recognized for their beneficial effects in the treatment of immune-mediated diseases, rather than using cytotoxic immune-suppressing drugs, which are associated with many unwanted side effects. Semaphorin3A (sema3A), a unique regulatory master of the immune system, was shown to be decreased in the serum of systemic lupus erythematosus (SLE) patients, in association with disease severity. Later, we were able to show its extremely beneficial effect in treating lupus nephritis in the NZB/W mice model. The mechanisms by which sema3A maintains its regulatory effect is by binding the regulatory receptor CD72 on B cells, thereby reducing the threshold of BCR signaling on B cells and reducing the production of pro-inflammatory cytokines. The aim of this study was to generate a stable sema3A molecule, easy to produce with a higher binding capacity to CD72 receptor rather than to Neuropilin-1 (NRP-1) receptor, which is expressed in many cell types. Using the crystallographic structure of parental sema3A, we synthesized a new secreted (shorter) sema3A derivative, which we called truncated sema3A (T-sema3A). The new molecule lacked the NRP-1 binding domain (the C-terminal site) and has an artificial dimerization site at position 257 (serine residue was exchanged with a cysteine residue). To facilitate the purification of this molecule we added Histidine epitope tag in frame upstream to a stop codon. This construct was transfected using a viral vector to 293HEK cells to generate cells stably expressing T-sema3A. T-sema3A is shown to be with a higher binding ability to CD72 than to NRP-1 as demonstrated by a homemade ELISA. In addition, T-sema3A was shown to be a regulatory agent which can induce the expression of IL-10 and TGF-ß and reduce the secretion of pro-inflammatory cytokines such as IL-6, IFN-γ, and IL-17A from human T and B-lymphocytes. Keeping this in mind, T-sema3A is highly effective in maintaining immune homeostasis, therefore, becoming a potential agent in restoring the regulatory status of the immune system in immune-mediated diseases.

Semaphorin 3A Is Effective in Reducing Both Inflammation and Angiogenesis in a Mouse Model of Bronchial Asthma.

Semaphorin 3A (sema3A) belongs to the sub-family of the immune semaphorins that function as regulators of immune-mediated inflammation. Sema3A is a membrane associated molecule on T regulatory cells and on B regulatory cells. Being transiently ligated to the cell surface of these cells it is suggested to be a useful marker for evaluating their functional status. In earlier studies, we found that reduced sema3A concentration in the serum of asthma patients as well as reduced expression by Treg cells correlates with asthma disease severity. Stimulation of Treg cells with recombinant sema3A induced a significant increase in FoxP3 and IL-10 expression. To find out if sema3A can be of benefit to asthma patients, we evaluated the effect of sema3A injection in a mouse model of asthma. BALB\c-mice were sensitized using ovalbumin (OVA) + adjuvant for 15 days followed by OVA aerosol inhalation over five consecutive days. Four hours following air ways sensitization on each of the above days- 15 of these mice were injected intraperitoneally with 50 µg per mouse of recombinant human sema3A-FR and the remaining 15 mice were injected with a similarly purified vehicle. Five days later the mice were sacrificed, broncheo-alveolar lavage (BAL) was collected and formalin-fixed lung biopsies taken and analyzed. In sema3A treated mice, only 20% of the bronchioles and arterioles were infiltrated by inflammatory cells as compared to 90% in the control group (p = 0.0079). In addition, eosinophil infiltration was also significantly increased in the control group as compared with the sema3A treated mice. In sema3A treated mice we noticed only a small number of mononuclear and neutrophil cells in the BAL while in the control mice, the BAL was enriched with mononuclear and neutrophil cells. Finally, in the control mice, angiogenesis was significantly increased in comparison with sema3A treated mice as evidenced by the reduced concentration of microvessels in the lungs of sema3A treated mice. To conclude, we find that in this asthma model, sema3A functions as a potent suppressor of asthma related inflammation that has the potential to be further developed as a new therapeutic for the treatment of asthma.

The association between semaphorin 3A levels and gluten-free diet in patients with celiac disease.

Celiac disease (CD) is an inflammatory disease affecting the small intestine. We aim to assess serum level and expression of semaphorin 3A (Sema3A) on T regulatory (Treg) cells in CD patients. Twenty-six newly diagnosed celiac patients, 13 celiac patients on a gluten-free diet and 16 healthy controls included in the study. Sema3A protein level in the serum of celiac patients was significantly higher compared to healthy group (7.17±1.8ng/ml vs. 5.67±1.5ng/ml, p=0.012). Sema3A expression on Treg cells was statistically lower in celiac patients compared to healthy subjects (p=0.009) and significantly lower in celiac patients compared to celiac patients on gluten free diet (p=0.04). Negative correlation was found between Sema3A on Teg cells and the level of IgA anti-tTG antibodies (r=-0.346, p<0.01) and anti-DGP (r=-0.448, p<0.01). This study suggests involvement of the Sema3A in the pathogenesis of CD.

The Expansion of CD25 high IL-10 high FoxP3 high B Regulatory Cells Is in Association with SLE Disease Activity.

B regulatory cells (Bregs) belong to a subgroup of activated B cells tasked with maintaining self-tolerance and preventing autoimmunity. While sharing similar regulatory mechanisms such as IL-10 dependency, they also defer in exhibiting their suppressive effects by expressing Fas-Ligand, TGF-beta, and PDL-1. In this study we show, for the first time, the expansion of CD25(high)FoxP3(high) Bregs in systemic lupus erythematosus (SLE) patients compared to healthy individuals (18.5 ± 3.052% versus 11.0 ± 1.654%, p < 0.001, resp.). This expansion was also shown to correlate with SLE disease activity (r = 0.75). In addition, CD25(high)FoxP3(high) Bregs were also IL-10(high) expressing and further expanded when stimulated with semaphorin 3A. In sum we show that CD25(high)FoxP3(high) are an additional subtype of Bregs, involved in regulating SLE disease activity. Being IL-10 expressing, we may assume that they are one of the sources of increased serum IL-10 in SLE patients. Further studies are required in order to assess the relation between high serum IL-10 and CD25(high)FoxP3(high) Breg cells.