*Purpose: Injected adult allo-spleen (SC) and bone marrow cells (BMC) traffic to secondary lymphoid organs, proliferate and spread throughout neonatal mice; allo-SC consistently cause acute GVHD (aGVHD) whereas allo-BMC induce non-robust heart transplant tolerance. To understand these differences we examined CD8 T cells from both inocula before and after injection into neonatal mice, analyzing their molecular phenotypes and trafficking/fates.

*Methods: Neonatal C3H (H-2k) mice were injected iv with either total adult B6 (H-2b) GFP+ SC or BMC, or purified CD8 T cells. Cell trafficking and fates were monitored by whole body/organ imaging. GVHD was defined by reduced growth/viability and tolerance by donor-type heart transplant survival. Cell phenotypes were examined by flow cytometry.

*Results: Neonates injected with allo-SC developed lethal aGVHD whereas those injected with allo-BMC acquired a non-robust heart transplant tolerance. Both allo-SC and -BMC trafficked to secondary lymphoid organs, proliferated and then spread throughout the body by day 6, suggesting systemic inflammation. CD8 T cells purified from both inocula also proliferated and spread throughout the mice, indicating direct activation. Despite similar proliferation/ trafficking, purified SC CD8 T cells induced lethal aGVHD in 100% mice (n=4/4) whereas the same number of purified BMC CD8 T cells did so infrequently (n=1/4). To understand these differences flow cytometry was performed on SC and BMC CD8 T cells before and after injection (isolated from liver on day 6). CD8 T cells from BMC inocula expressed high levels of CD28, KLRG1, CTLA-4, PD-1 and CD44 unlike those from SC, indicating SC CD8 T cells to be naïve and BMC CD8 T cells to have a memory-like phenotype. At 6 days post-injection, CD8 T cells from both SC and BMC showed higher CTLA-4 and PD-1 expression than pre-injection, implying dampening of their immune responses. KLRG1 expression in BMC CD8 T cells was lower on day 6 than in the starting inoculum, which together with higher CTLA-4 and PD-1 expression suggests they were undergoing exhaustion. At day10, injected BMC CD8 T cells were observed in liver interacting with host CD8 T cells and undergoing fragmentation.

*Conclusions: Memory-like CD8 T cells from donor BMC start to undergo exhaustion and are attacked by the neonatal host immune system, mitigating the risk of lethal aGVHD and ultimately prolonging donor-type heart graft survival. In contrast, naïve CD8 T cells from donor SC become activated and persist to cause lethal GVHD (despite a diminished immune response over time).

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