5). To evaluate further whether inhibition of signalling pathways modulate TG2 expression at the protein level, Caco-2 cells were incubated with TNF-α + IFN-γ in the presence of inhibitors. Western blot analysis revealed that TG2 protein induction was inhibited when treatment with TNF-α + IFN-γ was performed in the presence of sulphasalazine or wortmannin. The intensity of protein bands from TNF-α + IFN-γ-treated samples obtained in the presence of inhibitors was similar to that obtained from untreated cells. In order to evaluate further whether TG2 produced in TNF-α + IFN-γ-treated cells is correctly folded and located at the cellular membrane, flow cytometric

analysis was see more performed on THP-1 cells stimulated with TNF-α + IFN-γ for 20 h. A panel of four anti-TG2 monoclonal antibodies (named 5G7G6, 2G3H8, 4E1G9 and 1H7H9), recognizing different selleck chemical epitopes, was used to evaluate the surface expression of TG2. The four

anti-TG2 antibodies detected TG2 on the cell surface [16]. Flow cytometric analysis, using the 1H7H9 monoclonal antibody, showed that treatment of THP-1 cells with TNF-α + IFN-γ for 20 h increased TG2 protein at the cellular membrane [mean fluorescence intensity (MFI) = 30,78 in treated cells compared with MFI = 16·41 for unstimulated cells (Fig. 6). Similar results were obtained when flow cytometric analysis was performed using the anti-TG2 monoclonal antibodies 4E1G9, 5G6G7 and 2G3H8 (not shown). To evaluate whether inhibition of signalling pathways modulate the density of TG2 molecules at the cell surface, flow cytometry was performed on THP-1 cells incubated for 20h with TNF-α + IFN-γ in the presence of inhibitors. Interestingly, the induction of TG2 protein produced by the

double stimulus with TNF-α + IFN-γ was blocked completely in the presence of sulphasalazine. When the other inhibitors (Ly294002, SB203580, SP600125 and wortmannin) were tested, the expression of surface TG2 was only partially inhibited. These results are in accordance with those obtained by qRT–PCR, Western blot and luciferase activity analysis, and highlight the central role of NF-κB activity on TG2 expression. To investigate whether the synergistic induction of TG2 by TNF-α + IFN-γ Selleckchem Ponatinib in cell lines also occurred in intestinal tissue, biopsy samples from the duodenum of untreated CD patients and controls were incubated with the combination of TNF-α + IFN-γ for 24 h. Under basal conditions, intestinal mucosa of untreated CD patients had a higher TG2 mRNA content (9·8-fold increase in comparison with the housekeeping gene β-actin) than control samples (5·1-fold increase) (Fig. 7a). Intestinal tissues from untreated CD patients as well as controls showed up-regulation of TG2 mRNA (8·5- and 14·8-fold increase, respectively) when compared to unstimulated samples.

We attempted to define the exact requirements for signalling through BTLA to exert an effect on lymphocyte proliferation. We found that neither the HVEM-Fc ligand nor any of the anti-BTLA mAbs had any significant effect on B cell proliferation in selleck inhibitor vitro. We found that the ligand and some of the antibodies inhibited T cell proliferation, but only when they were cross-linked with an anti-Fc reagent; this was consistent with several different published studies. We used the beads-based system to separate the stimulus and the test agent physically and found that T

cell proliferation could be inhibited only when the test agent was juxtaposed immediately to the stimulus, and we have proposed a model for how this might occur. Other evidence in support of this hypothesis is shown by studies that demonstrate localization of BTLA to the immunological synapse during

T cell activation [32]. None of the anti-BTLA reagents tested had any significant effect on the observed in vitro T cell proliferation in other commonly used experimental systems such as the MLR or the OVA antigen-induced system. In our opinion, this observation is a reflection of our hypothesis that an anti-BTLA reagent can act to inhibit T cell proliferation only when it is juxtaposed immediately to the activating stimulus. In the MLR and DO11.10 in vitro systems, the activating stimulus this website to the T cell is either a polymorphic MHC molecule on another cell or the OVA peptide presented by an MHC molecule on another cell, respectively. Hence, the anti-BTLA test agent is physically unable to interdict the signalling complex that drives TCR signalling Interleukin-2 receptor and the subsequent T cell activation and proliferation. Indeed, as the stimulus is inherent to the cell–cell interaction, it would not be possible to mitigate the target T cell activation successfully with an exogenous anti-BTLA reagent in this experimental system. Based on our current understanding

of BTLA biology, in the frame of our current hypothesis, one would have to engineer genetically the cell that presents the stimulus to the target cell with an appropriate anti-BTLA reagent, such as the HVEM ligand, in order to interdict successfully the target T cell activation. Indeed, this was described by Sedyet al. [9], whereby the presentation was made by CHO cells transfected with the MHC IAd molecule that presented the OVA antigen to the target DO11.10 T cell, causing T cell activation. This was mitigated by transfection of the same CHO cell with the HVEM molecule, i.e. the BTLA ligand. We extended these studies to look at the effect of a BTLA-specific reagent in vivo. Of the various options for in vivo models, and bearing in mind the lack of any in vivo exposure data for any of these reagents, the most strongly indicated for T cell antagonism was judged to be the DO11.10 T cells syngeneic transfer with in vivo trapping of IL-2.