Purpose: Non-conditioned neonatal mice develop acute graft-versus-host disease (aGVHD) when injected with adult allogeneic spleen and bone marrow cells (allo-SC/BMC). To prevent aGVHD and induce transplant tolerance with allo-SC/BMC we reprogrammed donor/host CD4 T cells by co-stimulation blockade to preserve regulatory CD4 T cells while recapitulating depleted donor/host CD8 T cells. Methods: C3H (H-2^k) neonatal mice were injected iv with total or depleted B6 (H-2^b) GFP+ SC/BMC. CD8 T and CD49b NK cells were depleted from donor inocula using StemCell Technologies EasySep® kits. In vivo neonatal host CD8 T cells were depleted (Mab 53-6.7) and donor/host CD4 T cells reprogrammed (CD154 Mab MR1). Trafficking of injected cells was monitored by whole body/organ imaging; donor cell interactions/fates in lymphoid organs was determined by high resolution microscopy. Tolerance induction was assessed by transplanting treated mice as adults with donor-type hearts. Results: Neonatal mice injected with GFP+ allo-SC/BMC developed aGVHD, with diarrhea, reduced growth and early death. GFP+ cells proliferated and spread throughout injected mice indicating systemic inflammation. Depletion of donor/host CD8 T cells together with co-stimulation blockade of donor/host CD4 T cells (CD154) resulted in GFP signal being reduced and restricted to lymphoid organs (day 6). High resolution microscopy showed donor T and B cells positioned in PALS and follicular regions, respectively, of host spleen; lack of Ki67 immunostaining indicated many donor cells were not proliferating. Donor DC were detected in host thymus (day 6) suggesting a possible role in central tolerance. Donor-type B6 hearts transplanted into neonatally-treated adult C3H mice (n=5) (CD8 depleted/CD4 co-stimulation blockade) all continued to beat at 100 days post-transplant, 3 grafts with maximal strength and 2 grafts with diminished strength. In untreated control mice (n=3) B6 hearts stopped beating by day 10. H&E staining of a strongly beating B6 heart graft at 100 days showed undamaged cardiomyocytes with little if any cellular infiltrate and absence of macrophages and T cells by immunostaining. Conclusion: Reprograming CD4 T cells while depleting/recapitulating CD8 T cells in allo-SC/BMC treated neonates leads to 100% survival at 100 days of cardiac allograft transplanted as adults; low beat scores in 2/5 B6 grafts suggests further optimization is required. These findings provide insight into robust tolerance induction in neonates.

CITATION INFORMATION: Bascom R, Tao K, West L. CD4 and CD8 T Cells: Respectively Reprogrammed and Recapitulated in the Neonate to Induce Transplant Tolerance. Am J Transplant. 2017;17 (suppl 3).

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