All previous studies that reported a costimulatory role of this molecule were based on the use of monoclonal antibodies to trigger the CD150 molecule on T cells (Cocks et al., 1995, Aversa et al., 1997 and Howie et al., 2002). CD150 is a self-ligating molecule and no other binding partners have been described. Thus, we wanted to analyze whether the costimulatory effect was also observed upon engagement of T cell-expressed CD150 with its natural ligand. Therefore, we generated stimulator cells expressing CD150 in conjunction with anti-CD3. When co-culturing these stimulator cells with human T cells, no significant contribution of this interaction

to T cell proliferation check details and cytokine production was observed (Fig. 5B,C). In some of our experiments reduced proliferation rates of human T cells were observed in the presence of human CD150 but additional experiments are required to

confirm that CD150 can function as a negative regulator of T cell responses. During APC–T cell interaction Ceritinib in vitro a complex interplay of numerous cell surface molecules modulates cellular immune responses by either enhancing or inhibiting T cell receptor complex signalling. Thus, assessing the function of individual costimulatory ligands using natural APC is a difficult task. With our T cell stimulator cells we have generated an experimental tool for studying individual costimulatory ligands in a cellular system, but detached from the context of numerous other molecules involved in the regulation of T cell activation that are expressed on professional APC. Whereas similar cellular systems that have been termed artificial APC (aAPC)

use cells engineered to express Fc-γ receptors (CD32 or CD64) and depend on the addition of anti-CD3 antibodies (Thomas et al., 2002, Suhoski et al., 2007 and Gong et al., 2008) we used cell lines that stably express membrane-bound anti-CD3 antibody fragments. Using different anti-CD3 expression constructs we have generated Acetophenone two cell clones that stably express different levels of anti-CD3 antibody fragments: A construct where the anti-CD3 antibody fragments are linked to the transmembrane domain of human CD28 molecules yielded Bw-aCD3low stimulator cells that give a weak signal 1 to human T cells, whereas a construct encoding anti-CD3 antibody fragments fused to the human CD14 molecule was used to generate cells expressing high levels of GPI-anchored anti-CD3 antibody fragments (Bw-aCD3high; Fig. 1). The GPI-anchored anti-CD3 antibody fragment is efficiently targeted to lipid rafts and has also successfully been used for the stimulation and manipulation of human T cells with immunosomes — virus-like particles decorated with TCR/CD3 ligands, costimulatory molecules and modified cytokines (Derdak et al., 2006, Kueng et al., 2007, Leb et al., 2009 and Kueng et al., 2010).