Data are representative of 5 experiments performed. Discussion CTLA-4 is an essential regulator of T cell function that in combination with the CD28 pathway represents a critical decision point in T cell activation. ligand, CTLA-4-dependent suppression was highly effective whereas at higher APC numbers or high levels of ligand, inhibition was lost. Accordingly, the degree of suppression correlated with the level of CD86 expression remaining on the antigen presenting cells. These data reveal clear rules for the inhibitory function of CTLA-4 on Treg which are predicted by its ability to remove ligands from antigen presenting cells. Introduction T cell activation takes place at the interface between T cells and antigen presenting cells (APC) in secondary lymphoid organs. Typically, APC at sites of infection, upregulate CD80 and CD86 in Rabbit Polyclonal to VAV1 response to signalling by Toll-like receptors or other microbial pattern recognition receptors and migrate to lymph nodes (1), (2) (3). As a result, APC increase both in number and level of costimulatory molecule expression, resulting in the initiation of T cell responses in a CD28-dependent manner (4), (5), (6). CD28 signalling is important in the expansion, survival and helper function of T cells (7), (8), (9) (10). Against this background, the inhibitory receptor CTLA-4 shares the same ligands with CD28 but opposes T cell responses such that the absence of CTLA-4 results autoimmune T cell activation with accompanying tissue infiltration and destruction (11), (12). The expression of CTLA-4 on both regulatory T cells (Treg) as well as activated T cells raises the issue of the mechanism by which CTLA-4 acts and the immunological context where inhibition takes place. A surprisingly large Aconine number of models of CTLA-4 function Aconine have been proposed, including both cell intrinsic and extrinsic mechanisms (13), (14), (15). However, the ability of these models to predict CTLA-4 functional behaviour is variable. For example, despite popular perceptions of CTLA-4 as an inhibitory signal for T cell activation a consistent body of literature indicates that the major function of CTLA-4 is via a cell-extrinsic pathway (13), i.e. that CTLA-4 influences the cells around it rather than the cell expressing it. Therefore, whilst the role of CTLA-4 as a negative regulator is well established, the context for its effective function is not. Ultimately, understanding how to predictably measure and understand CTLA-4 function in humans has considerable implications in autoimmune settings as well as other disorders involving immune dysregulation. We recently proposed a model for CTLA-4 function whereby the central feature was the ability of CTLA-4 to capture ligands (CD80 and CD86) from APC and degrade them inside the CTLA-4 expressing cell (16). Such a mechanism is a form of cell-extrinsic ligand competition that makes several predictions for CTLA-4 function. Most obvious is that CTLA-4 function should be evident only when it depletes ligands to below a level sufficient for CD28 costimulation. Aconine A corollary of this concept is that the amount of ligand on the APC relative to the amount of CTLA-4 on T cells should dictate whether the threshold for CD28 costimulation is achieved. Accordingly, in situations where the supply of ligand is limited then consumption by CTLA-4 should Aconine be more functionally effective and vice versa. We therefore set out to test how parameters such as the number of APC, and their relative ratio to CTLA-4+ cells affected the ability of CTLA-4 to regulate T cell activation. Using a model system, we demonstrate that the efficacy of suppression by CTLA-4 is dictated by the total amount of costimulatory molecules in the system. Under conditions favouring CTLA-4 function there was effective depletion of costimulatory ligands, sufficient to suppress T cell responses and the degree of suppression was tightly correlated with the observed downregulation of ligands on APC. In contrast, under un-favourable conditions with high levels of ligands, CTLA-4 continued to function however its impact on T cell proliferation was minimal since sufficient ligand still remained. Predicated on this model program, the power was tested by us of natural Treg to curb T cell responses. We noticed that relative to our model, CTLA-4-reliant suppression was profoundly influenced with the proportion of APC:Treg and corresponded using the known degree of Compact disc86 downregulation. On the other hand, no CTLA-4-reliant inhibition was noticed during arousal with Compact disc3/28 antibodies (Abs) displaying that CTLA-4 suppressive function was totally reliant on ligand-driven T cell activation. Jointly these data create immunological contexts that anticipate the functional capability of CTLA-4 that are in keeping with a model whereby a significant function of CTLA-4 is normally to deplete its ligands from APC within a T cell-extrinsic way. Materials and Strategies Cell lines and lifestyle CHO (Chinese language hamster ovary) cells transfected with individual Compact disc80, Compact disc86, Jurkat and CTLA-4 cells transduced with individual CTLA-4 as.