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 Palbociclib mw 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 U0126 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 mafosfamide 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.