Regulatory T Cells Allosensitized Under the Blockade of CD80/CD86-CD28 Costimulation Prolonged Islet Allograft Survival

BACKGROUND: Although CD80/CD86-CD28 costimulation is essential for the development and homeostasis of regulatory T cells (Tregs), a Treg-based cell product (referred to herein as induced T cells with suppressive functions or iTS) manufactured by allogeneic mixed lymphocyte reaction under costimulation blockade has demonstrated potential for minimizing immunosuppression and achieving operational tolerance in organ transplantation. However, the precise immunological mechanisms underlying Treg-induction therapy and its durable efficacy against donor alloantigens remain to be fully elucidated. METHODS: Murine iTS was induced in the splenocytes of C57BL/6 mice. hCD2/Foxp3 reporter mice were cocultured with irradiated BALB/c splenocytes and antagonistic anti-CD80/CD86 monoclonal antibodies (mAbs). Tregs isolated from iTS (iTS-Tregs) were intravenously or locally transferred into diabetic C57BL/6 recipients that received BALB/c islets. Graft survival, chemokine profiles, and inhibitory functions were examined. RESULTS: Intravenous transfer of 1 × 106 iTS-Tregs significantly prolonged graft survival compared with Tregs isolated from allo-MLR under isotype control. The local delivery of 1 × 106 iTS-Tregs with islets under the kidney capsule induced long-term graft acceptance (>100 d). The CXCR4-expressing population was significantly increased among iTS-Tregs, which selectively persisted within islet grafts expressing CXCL12, the cognate ligand of CXCR4. Although CXCR4+ and CXCR4- iTS-Tregs showed comparable inhibitory functions in vitro, iTS-Tregs failed to induce long-term acceptance when the CXCR4-expressing population was depleted. CONCLUSIONS: Murine iTS-Tregs exerted enhanced inhibitory function, and CXCR4+ iTS-Tregs persisted within grafts to promote long-term islet allograft acceptance.