Efficiency of T-cell costimulation by CD80 and CD86 cross-linking correlates with calcium entry.

Costimulation is a fundamental principle of T-cell activation. In addition to T-cell receptor engagement, the interaction between CD80 and/or CD86 with CD28 and/or cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptors is required to regulate T-cell activation and tolerance. While the importance of costimulation is clearly established, the exact molecular mechanism is unknown. We demonstrate that T-cell proliferation and the ability of CD8(+) T-effector cells to kill were enhanced slightly by CD80 but dramatically by CD86 costimulation. To further analyse the cellular process of costimulation, we developed a single-cell assay to analyse Ca(2+) signals following costimulation with bi-specific antibodies. We found that this stimulation method worked in every human T-cell that was analysed, making it one of the most efficient T-cell activation methods to date for primary human T cells. The enhanced proliferation and killing by costimulation was paralleled by an increase of Ca(2+) influx following CD86 costimulation and it was dependent on CD28/CTLA-4 expression. The enhanced Ca(2+) influx following CD86 costimulation was abrogated by an antibody that interfered with CD28 function. The differences in Ca(2+) influx between CD80 and CD86 costimulation were not dependent on the depletion of Ca(2+) stores but were eliminated by the application of 10 mum 2-aminoethyldiphenyl borate which has recently been shown to enhance stromal interaction molecule 2 (STIM2)-dependent Ca(2+) entry while reducing STIM1-dependent Ca(2+) entry. Our data indicate that differences in the efficiency of costimulation are linked to differences in Ca(2+) entry.

Tumor-specific crosslinking of GITR as costimulation for immunotherapy.

Activation of murine glucocorticoid-induced tumor necrosis factor-related receptor (mGITR) by its natural ligand (GITRL) or antiGITR agonist mAb enhances T-cell responses, inhibits regulatory T-cell (Treg)-mediated suppression and induces tumor immunity in a variety of murine tumor models. However, systemic administration of these costimulatory agents can lead to global T-cell activation and autoimmunity. To specifically manipulate the T-cell compartment in the tumor microenvironment we propose to target the tumor infiltrating T cells with a bispecific mGITRL fusion protein. For that purpose, mGITRL is linked to a single-chain antibody targeting fibroblast activation protein (FAP) as FAP expression is restricted to cancer-associated fibroblasts (CAFs) found in the stroma of epithelial cancers. AntiFAP-mGITRL fusion protein forms dimers and binds to murine GITR with 1.2 µM affinity and to murine FAP with 4.5 nM. The construct is able to costimulate CD8+ and CD4+ effector T cells resulting in increased proliferation, IFN-γ and IL-2 production. This costimulatory effect is enhanced when the fusion protein is bound to a FAP-positive cell line mimicking FAP CAFs. In suppression assays, membrane-bound antiFAP-mGITRL is 100-fold more effective in overcoming Treg-mediated suppression than unbound fusion protein. These studies suggest that targeted tumor therapy with antiFAP-mGITRL fusion protein could induce tumor rejection while minimizing autoimmune side effects.