G. O'boyle, P. Newton, Y. Jenkins, S. Ali, J.A. Kirby
Applied Immunobiology And Transplantation Research Group, Institute Of Cellular Medicine, Newcastle University, Newcastle upone Tyne/UNITED KINGDOM

Body: Introduction: Chemokines are small (8–10 kDa) proteins capable of activating and promoting vectoral leukocyte migration. Clinical trials have testedspecific antagonist compounds or blocking antibodies for therapeutic manipulation of chemokine-driven inflammation. These have proven ineffective as a consequence of the promiscuity of the chemokinesystem. The chemokine receptor CXCR3 is upregulated on activated T cells and plays a fundamental role in recruiting effector cells, including Th1 cells during allograft rejection. Studies havesuggested a possibility for cross-talk between chemokine receptors and the TCR, with activation of the TCR modulating the potential for chemokine-induced signalling. This study was designed todetermine the extent and consequences of interaction between CXCR3 and the TCR. Furthermore, we describe an entirely novel strategy to desensitise CXCR3 that results in a broad anti-chemokineeffect.

Methods: Transendothelial T cell migration towards the CXCR3 ligand CXCL10 was measured after crosslinking CD3 immediately before addition to an IFN-γ andTNF-α activated endothelium in a transwell chemotaxis assay. The molecular basis for synergy between CXCR3 and the T cell receptor complex was investigated by measurement of fluorescenceresonance energy transfer (FRET). A small molecule CXCR3 receptor agonist PS5916474 was examined for its ability to elicit chemokine receptor homologous (blocking the responses to CXCR3 ligands) andheterologous (blocking the response to other chemokine receptors) desensitisation using static Boyden chamber chemotaxis assays and a dynamic shear-flow adhesion and migration assay system.

Results: Cytokine activated endothelial cells supported a specific increase in the migration of activated T cells towards CXCL10 (see figure; red labelled Tcell that has undergone recent TCR activation and CFSE labelled unactivated cells); this was most apparent after CD3 had been activated for 90 min (p<0.0001). FRET data show that CXCL10 induced aclose (<10nm) spatial association between CXCR3 and the CD3É› subunit on the T cell surface. Treatment with the CXCR3 agonist PS5916474 rendered T cells unresponsive to stimulationwith other CXCR3 ligands and heterologous chemokines such as CCL2 and CCL5 in static and dynamic migration assays.

Conclusions: Vascular endothelium in an allograft has the potential to stimulate both CXCR3 and the T cell receptor. Our data demonstrate that these signalshave a synergistic effect which might enhance the extravasation of alloantigen-specific T cells during episodes of local inflammation. The relationship between CXCR3 and the TCR highlights theimportance of CXCR3 as a therapeutic target. A soluble CXCR3 agonist that we describe can desensitise T cells to prevent, not only CXCR3-driven inflammation, but the response to the number ofchemokines that contribute to allograft rejection.

Disclosure: All authors have declared no conflicts of interest.