Purpose: Neonatal mice are susceptible to tolerance induction with semi-allogeneic bone marrow and spleen cells (BMC/SC) while allogeneic BMC/SC cause acute lethal graft-versus-host disease (GVHD). Both injected donor types are attacked by developing neonatal effector cells. To induce transplant tolerance with allogeneic BMC/SC we depleted peripheral donor and host T cells and recapitulated their development in the immature neonatal environment in the presence of cells/antigens from both strains. Methods: C3H (H-2k) neonatal mice were injected with total or depleted B6 (H-2b) GFP+ BMC/SC. CD8a T and CD49b NK cells were depleted from donor inocula using StemCell Technologies EasySep® kits. Host CD8a and donor/host CD4 T cells were depleted in vivo by injecting monoclonal antibodies (clone 53-6.7 and GK1.5 respectively). Trafficking of injected cells was monitored by whole body/organ imaging; donor/host T cell fate in lymphoid organs was determined by high resolution microscopy. Tolerance induction was assessed by transplanting treated mice as adults with donor-type hearts. Results: Non-conditioned neonates injected with allogeneic BMC/SC developed acute GVHD, with diarrhea, weight loss and early death. At day 6 post-injection, GFP+ cells were present throughout mice indicating systemic inflammation. Depletion of donor/host effector cells resulted in a weak GFP signal in cervical lymph nodes (day6) as determined by whole body imaging. Whole organ imaging confirmed the signal and showed it restricted to lymphoid organs. In spleen and lymph node T cells were present at low numbers compared to organs from non-depleted controls; low T cell number was associated with smaller /less developed secondary lymphoid organs. Three of six donor-type B6 hearts transplanted into T cell-depleted mice (n=6) continued to beat 100 days post-transplant, two with high score (4) and one with low score (1). Cryosections from a beating heart (score 4) showed very mild infiltration by macrophages and T cells. Conclusion: Recapitulating peripheral T cell development in the immature immune environment of the neonate induced a robust transplant tolerance in 2/6 mice. Graft rejection in the remaining mice suggests incomplete depletion of host alloreactive cells and or subsequent inefficient thymic education by donor cells.

CITATION INFORMATION: Bascom R, Tao K, West L. Recapitulating Peripheral T Cell Development in Neonatal Mice to Induce Transplant Tolerance. Am J Transplant. 2016;16 (suppl 3).

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