Targeting acute allograft rejection by immunotherapy with ex vivo-expanded natural CD4+CD25+ regulatory T cells

Background. Natural CD4(+)CD25(+) regulatory T (T-reg) cells have been implicated in suppressing alloreactivity in vitro and in vivo. We hypothesized that immunotherapy using ex vivo-expanded natural T-reg could prevent acute allograft rejection in mice. Methods. Natural CD4(+)CD25(+) T-reg were freshly purified from naive mice via automated magnetic cell sorter and expanded ex vivo by anti-CD3/CD28 monoclonal antibody (mAb)-coated Dynabeads. Suppression was assayed in vitro by mixed lymphocyte reaction and in vivo by targeting cardiac allograft rejection. Survival of T-reg or effector T (T-eff) cells after adoptive transfer in vivo was tracked by flow cytometry and all allografts were examined by histology and immunohistochemistry. Results. By day nine in culture, 26.6 +/- 5.3-fold of expansion was achieved by co-culture of fresh natural T-reg with anti-CD3/CD28 mAb-coated Dynabeads and interleukin-2. Ex vivo-expanded T-reg exerted stronger suppression than fresh ones towards alloantigens in vitro and prevented CD4(+) T-eff-mediated but only delayed CD4(+)/CD8(+) T-eff-mediated heart allograft rejection in Rag(-/-) mice. Long-term surviving allografts showed no signs of acute or chronic rejection with graft-infiltrating T-reg expressing CD25 and FoxP3. Infused T-reg persisted and expanded long-term in vivo and trafficked through the peripheral lymphoid tissues. CD25 expression was dynamic in vivo: maintained CD25 expression on T-reg was indicative for the preservation of allosuppression, while significantly enhanced CD25 expression on CD4(+) effector T cells was most likely associated with T-cell expansion and graft rejection. Conclusions. Therapeutic use of ex vivo-expanded natural CD4(+)CD25(+) T-reg may be a feasible and nontoxic modality for controlling allograft rejection or perhaps inducing allograft tolerance.