*Purpose: Comparing the behaviour of fully allogeneic spleen (SC) vs bone marrow cells (BMC) in non-conditioned neonatal mice is important for maximizing synergistic interactions between the two to induce robust transplant tolerance. Allo-SC and -BMC were therefore injected either as a mixture or separately into neonates; their trafficking, fates, and effects on GVHD, HVGD and tolerance compared.

*Methods: C3H (H-2k) neonates were injected with adult B6 (H2-2b) GFP+ SC or BMC. Cell trafficking, interactions and fates were monitored by whole body/organ imaging and high-resolution microscopy. GVHD was defined by reduced mouse growth/viability, HVGD by donor cell fragmentation and tolerance by donor-type heart transplantation.

*Results: Allo-SC inoculum contained more immune effector cells than allo-BMC as determined by flow cytometry (CD8 T, NK, CD4 T cells). Neonates injected with allo-SC developed aGVHD with diarrhea, severe reduction in growth and death by 14 days post-injection (n=7/7) whereas allo-BMC-injected mice developed aGVHD infrequently (n=1/8). Mice injected with the allo-SC/BMC mixture showed intermediate disease progression (n=6/7). Whole body/organ imaging showed allo-SC and -BMC each trafficked to secondary lymphoid organs, became activated, proliferated and spread throughout the mice suggesting systemic inflammation. GFP signal was stronger in mice injected with allo-SC than -BMC suggesting more cells from allo-SC proliferated. Proliferating allo-SC and -BMC both contained T cells, including CD8 T cells. GFP signal intensities in syngeneic-SC and -BMC-injected mice were low at day 6 and confined to lymphoid organs indicating proliferation due to lymphopenia was limited. Surviving allo-BMC-injected mice transplanted as adults (n=6) with donor-type hearts showed moderate (2-3) to low (≤1) allograft beat scores at 100 days post-transplant. A 100-day allograft (score=1) showed cellular infiltration consisting of macrophages and T cells including CD8 T cells.

*Conclusions: Both injected allo-SC and -BMC trafficked to secondary lymphoid organs, proliferated and spread throughout neonatal mice; allo-SC consistently caused aGVHD whereas allo-BMC induced non-robust heart transplant tolerance. These promising results suggest that robust tolerance induction will require different optimization procedures for allo-SC and -BMC.

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