30,31 Despite their structural homology, CTLA-4 and CD28 are fundamentally different with respect to their effects

on T-cell activation. Both molecules share the same ligands [CD80 (B7-1) and CD86 (B7-2)] expressed on antigen-presenting cells or target cells, with the distinction that CTLA-4 binds to both with a higher affinity.32–34 It was demonstrated that CD80 is the preferred ligand for CTLA-4, whereas CD86 is preferred by CD28.35–37 The localization and expression patterns of these two molecules also differ. CD28 is constitutively expressed on the cell surface of naïve and activated T cells, whereas CTLA-4 is not detectable on naïve T cells and is induced only upon T-cell activation.37,38 Once Raf inhibitor expressed, CTLA-4 localizes to an endosomal compartment because it contains a tyrosine-based intracellular localization motif in its cytoplasmic tail. Our group has focused on the concept of T-cell activation by mimicking

the physiological ‘two-signal’ model39 in recent years. We developed bi-specific molecules that cross-link human T cells to antigen-positive target cells bypassing MHC restriction. Signal one is delivered by an anti-CD3 antibody of moderate activity that can be significantly enhanced by the addition of costimulatory molecules delivering signal two. To identify the most appropriate costimulatory molecule in this setting, extracellular domains of CD80 and CD86 were linked to antigen-specific single-chain fragment variable antibodies (scFv) and their potential check details to mediate T-cell proliferation and cytotoxicity were tested. Here we demonstrate that this activation method can virtually activate every single T cell and we can ‘tune’ the activation response through the costimulatory molecules used. Interestingly, we can correlate the difference in the efficiencies of T-cell activation induced by CD80 or CD86 cross-linking with Ca2+ influx. In addition, our data point to an important role of STIM2 for T-cell activation following formation of the immunological synapse after costimulation. RPMI-1640

[supplemented with 10% (volume/volume) Florfenicol heat-inactivated fetal calf serum, penicillin (100 U/ml), streptomycin (0·1 mg/ml) and glutamine (0·3 mg/ml)], all obtained from Invitrogen (Karlsruhe, Germany) was used as a standard medium (RPMI-SM). Jurkat T cells (E6-1, ATCC TIB152 and parental generated by Fanger et al.40), adherhent growing HEK-293 cells and the murine hybridoma M195 secreting an anti-human CD33 immunoglobulin G (IgG) were purchased from the American Type Culture Collection (ATCC; Manassas, VA). HEK-293 cells adapted to suspension growth were kindly provided by Professor Wurm (EPFL Lausanne, Switzerland). The Chinese hamster ovary (CHO) cell line was kindly provided by Professor Chasin (Columbia University, New York, NY).