Introduction/Methods: Semi-allogeneic (F1; H-2k/b) splenocytes (SC) injected into newborn mice (H-2k) induce immunological tolerance whereas allogeneic SC (H-2b) lead to graft-vs-host disease (GVHD). Here we studied the effect of injected allogeneic Tregs in our model of neonatal tolerance to cardiac allografts. CD4⁺CD25⁺ Tregs were isolated using StemCell Technologies EasySep® purification kits. Injected labeled syngeneic, F1 or allogeneic SC, or purified allogeneic Tregs, were tracked in newborn mice for 6 days by OV100 small animal imaging system. Treg interactions and fates were studied by confocal microscopy, and their ability to prolong heart allograft survival in adults. Results: By whole body and organ imaging syngeneic and F1 SC tracked to lymphoid organs while allogeneic SC initially tracked there but then proliferated and distributed throughout the mice consistent with GVHD. Injected Tregs also trafficked to lymphoid organs but unlike allogeneic SC did not proliferate. Allogeneic Tregs survived post-injection in spleen suggesting they were not being attacked by the host immune system. In neonatal spleen, allogeneic Tregs interacted with host Tregs either directly or via host dendritic cells indicating a possible role in iTreg formation. Injected allogeneic Tregs prolonged survival of both donor-type and third-party heart grafts to 100 days in 50% of transplanted mice (n=6). A > 100 day functioning donor-type heart graft showed mild-moderate cellular infiltrate of macrophages and T cells including CD8+ T cells, with focal regions of myocyte damage. Conclusion: Injection of neonates with purified allogeneic Tregs prolongs survival of donor-type and third party heart grafts transplanted as adults. Prolonged third-party graft survival suggests suppression of the host immune system in a localized “non-specific” manner; alternatively given the presumed clonal diversity of allogeneic Tregs, some form of specific/cross-reactive suppression may be occurring. Allogeneic Tregs alone (at these doses) however appear insufficient to induce robust tolerance and may require additional cell types.

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