Tolerance Induction in Neonatal Mice: Exhaustion of CD8 T Cells in Allogeneic Bone Marrow Tolerizing Inoculum Fails to Induce GVHD and Leads to Prolonged Donor Heart Graft Survival

Abstract

Adult allogeneic (allo) spleen cells (SC) induce lethal aGVHD in newborn mice whereas bone marrow cells (BMC) induce non-robust 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. 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. 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. In tolerizing inocula, BMC CD8 T cells 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.