Purpose: To induce robust transplant tolerance in neonates with fully allogeneic spleen and bone marrow cells (SC/BMC) it is crucial to define the complex interplay amongst the different cells of the neonatal host and the adult-derived tolerizing inoculum. Towards that end we have begun to specifically and systematically deplete donor and host effector cells in a directional manner, studying the effects of each depletion on immune outcome. Methods: C3H (H-2^k) neonates were injected iv with total or depleted GFP+ SC/BMC from B6 (H-2^b) adults. Donor effector cell types were depleted from tolerizing inocula before injection using positive selection kits. Host cell types were depleted in vivo with monoclonal antibodies. Depletion was performed in a sequential manner, with donor effector cells being depleted before equivalent neonatal ones. Trafficking, interactions and fates of injected cells were monitored by microscopy. Tolerance induction was assessed by transplanting neonatally-treated mice as adults with B6 hearts. Results: Neonates injected with GFP+ allo-SC/BMC developed acute graft-vs-host disease with diarrhea, reduced growth and early death. GFP+ cells trafficked to secondary lymphoid organs, proliferated and spread throughout injected mice indicating systemic inflammation. Donor cell proliferation in neonatal hosts was prevented when the tolerizing inoculum was depleted of CD8 T cells but not when CD4 T cells were depleted. Loss of GFP signal suggested the neonatal host immune system became dominant and cleared injected cells. Consistent with that, host CD8 T cells were abundant while donor cells fragmented. Combined depletion of CD8 T cells from both inoculum and host allowed donor cells to become dominant again with strong GFP+ signal in spleen, lymph node, lung and kidney (but not in other organs as with total allo-SC/BMC inocula). Donor CD4 T cells proliferated and were Th2 (GATA-3) skewed. B6 hearts transplanted after combined depletion (n=4) underwent delayed rejection, only one heart beating 100 days post-transplant. The beating graft showed effector cell infiltration and cardiomyocyte damage. Conclusion: Some neonatal host effector cell types may be dominant over other (injected) adult effector types (when not artificially outnumbered by the inoculum). Clinical induction of robust transplant tolerance with allo-SC/BMC will therefore likely require depletion/reprogramming of both donor and host effector cells.

CITATION INFORMATION: Bascom R., Tao K., West L. Tipping the “Immune Balance” towards Tolerance by Specific and Systematic Depletion of Donor and Host Effector Cells Am J Transplant. 2017;17 (suppl 3).

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