the control unstimulated cells and 0.01 vs. -PD1. Accordingly, we measured the transcriptomic levels of TSPAN32 in polarized T cells under Th1 and Th2 conditions and TSPAN32 resulted significantly reduced as compared with unstimulated cells. On the other hand, in Treg cells, TSPAN32 underwent minor changes upon activation. The in vitro data were finally translated into the context of multiple sclerosis (MS). Encephalitogenic T cells from Myelin Oligodendrocyte Glycoprotein (MOG)-Induced Experimental Autoimmune Encephalomyelitis (EAE) mice showed significantly lower levels of TSPAN32 and increased levels of CD9, CD53, CD82 and CD151. Similarly, in vitro-activated circulating CD4 T cells from MS patients showed lower levels of TSPAN32 as compared with cells from healthy donors. Overall, these data suggest an immunoregulatory role for TSPAN32 in T helper immune response and may represent a target of future immunoregulatory therapies for T cell-mediated autoimmune diseases. = 3 impartial replicates). (C) TSPAN32 levels in CD4+ effector T cells upon anti-CD3/CD28 stimulation, in the presence or absence of rapamycin 200 nM, were evaluated by real-time PCR (= 3 impartial replicates). (D) TSPAN32 expression levels were evaluated at different time points upon activation of Treg cells via real-time PCR (= 3 impartial replicates). (E) TSPAN32 protein levels were determined by western blot upon activation of effector and regulatory T cells at different time points (pooled proteins of cells from 3 healthy donors). Data are shown as normalized mean SD and statistical analysis performed using one-way ANOVA followed by Bonferroni multiple test correction. Anti-CD3 stimulation was sufficient to significantly downregulate TSPAN32 ( 0.05 vs. the control unstimulated cells), and its effect was potentiated by co-stimulation with anti-CD28 ( 0.001 vs. the control unstimulated cells and 0.01 vs. the anti-CD3 stimulated cells) (Physique 2B). No significant differences were observed in TSPAN32 levels after anti-CD3 stimulation and co-stimulation with anti-CTLA4, anti-ICOS or Harmaline anti-PD1 antibodies (Physique 2B). Since CD28-mediated signaling depends on the PI3K/Akt/mTOR pathway, we wanted to verify whether mTOR could be involved in the modulation Rabbit Polyclonal to INTS2 of TSPAN32 expression. As expected, treatment of T cells with the mTOR inhibitor rapamycin were shown to significantly increase the levels of TSPAN32 ( 0.05) (Figure 2C). In the Treg subset of CD4+ lymphocytes, a moderate decrease in TSPAN32 expression levels was also observed upon activation, which reached statistical significance only Harmaline at 5 h post stimulation ( 0.05 vs. the control unstimulated cells) (Physique 2D). Significantly lower levels of TSPAN32 were observed in T effector cells as compared with Treg cells at 5 and 6 h post stimulation ( 0.05). The modulation of TSPAN32 in both effector and regulatory cells was further confirmed at the protein level. As shown in Physique 2E, a marked reduction of TSPAN32 could be observed in effector T cells at 4 h post activation, while no modulation was observed in Treg cells. 2.3. Tetraspanins Expression in T Cell Polarization Next, we decided the transcriptomic levels Harmaline of TSPAN32 in polarized T cells under Th1 and Th2 conditions. TSPAN32 in both Th1 and Th2 cell subsets was significantly reduced in comparison with unstimulated cells ( 0.001 for both Th1 and Th2 cells as compared with the control unstimulated cells) (Determine 3A). Moreover, a significantly lower TSPAN32 expression was observed in Th1 cells ( 0.05) as compared with Th2 cells. Open in a separate window Physique 3 Expression of tetraspanins in human T cell polarization. (A) Expression of TSPAN32 was evaluated by real-time PCR in polarized Th1 and Th2 cells and unstimulated cells (= 3 impartial replicates). (B) Expression of CD9, CD37, CD53, CD63, CD81, CD82 and CD151 in polarized Th1 and Th2 cells and unstimulated cells as evaluated by real-time PCR (= 3 impartial replicates). Data are shown as normalized mean SD and statistical analysis performed using one-way ANOVA followed by Bonferroni multiple test correction. The expression levels of other members of the tetraspanin family were also evaluated for comparison and CD9, Harmaline CD37, Harmaline CD53, CD63, CD81, CD82 and CD151 were considered. Among them, a significant increase could be observed in both Th1 and Th2 cells for CD37 and CD82, while a significant increase in Th2 cells as compared with unstimulated cells was observed for CD63 and CD151 (Physique 3B). The expression of CD9, CD53 and CD81 did not show significant variation between the unstimulated and the polarized cells (Physique 3B). In Jurkat T cells, overexpression of TSPAN32 (induced by transient transfection with a TSPAN32-encoding plasmid) was associated with a significant reduction in the production of the pro-inflammatory cytokines TNF-alpha and IFN-gamma upon cell activation (Physique 4). Open in a separate window Physique 4 Effect of TSPAN32 overexpression in Jurkat cells. Following transient transfection of Jurkat cells with a DNA plasmid encoding for TSPAN32 (pTSPAN32) or the empty.