Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

Both authors contributed equally.Themixedchimerismapproachachievesdonor-specif-ic tolerance in organ transplantation, but clinicaluse isinhibitedbythetoxicitiesofcurrentbonemarrow(BM)transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti-CD154 monoclonal anti-bodies (mAbs) is exceptionally potent in inducingmixed chimerism, but these mAbs are clinically notavailable. Defining the roles of donor and recipientCD40in amurineallogeneicBMTmodel,weshowthatCD4orCD8activationthroughanintactdirectorCD4Tcell activation through the indirect pathway is suffi-cient to trigger BM rejection despite CTLA4Ig treat-ment. In the absence of CD4 T cells, CD8 T cellactivation via the direct pathway, in contrast, leadsto a state of split tolerance. Interruption of the CD40signals in both the direct and indirect pathway ofallorecognition or lack of recipient CD154 is requiredfor the induction of chimerism and tolerance. Wedeveloped a novel BMT protocol that induces mixedchimerism and donor-specific tolerance to fully mis-matched cardiac allografts relying on CD28 costimula-tion blockade and mTOR inhibition without targetingthe CD40 pathway. Notably, MHC-mismatched/minorantigen-matched skin grafts survive indefinitelywhereasfullymismatchedgraftsarerejected,suggest-ing that non-MHC antigens cause graft rejection andsplit tolerance.Abbreviations: BM, bone marrow; BMT, bone marrowtransplantation; FACS, fluorescence-activated cellsorter; HE mAbs, monoclo-nalantibodies;MLR,mixedlymphocytereaction;MST,median survival time; TBI, total body irradiationReceived 24 September 2014, revised 12 November2014 and accepted for publication 30 November 2014