Recruitment of Foxp3⁺ T regulatory cells mediating allograft tolerance depends on the CCR4 chemokine receptor

Although certain chemokines and their receptors guide homeostatic recirculation of T cells and others promote recruitment of activated T cells to inflammatory sites, little is known of the mechanisms underlying a third function, migration of Foxp3⁺ regulatory T (T reg) cells to sites where they maintain unresponsiveness. We studied how T reg cells are recruited to cardiac allografts in recipients tolerized with CD154 monoclonal antibody (mAb) plus donor-specific transfusion (DST). Real-time polymerase chain reaction showed that intragraft Foxp3 levels in tolerized recipients were ∼100-fold higher than rejecting allografts or allografts associated with other therapies inducing prolonged survival but not tolerance. Foxp3⁺ cells were essential for tolerance because pretransplant thymectomy or peritransplant depletion of CD25⁺ cells prevented long-term survival, as did CD25 mAb therapy in well-functioning allografts after CD154/DST therapy. Analysis of multiple chemokine pathways showed that tolerance was accompanied by intragraft up-regulation of CCR4 and one of its ligands, macrophage-derived chemokine (CCL22), and that tolerance induction could not be achieved in CCR4^−/− recipients. We conclude that Foxp3 expression is specifically up-regulated within allografts of mice displaying donor-specific tolerance, that recruitment of Foxp3-expressing T reg cells to an allograft tissue is dependent on the chemokine receptor, CCR4, and that, in the absence of such recruitment, tolerizing strategies such as CD154 mAb therapy are ineffectual.