Comparative Behaviour of Allogeneic Spleen versus Bone Marrow Cells in Non-Conditioned Neonatal Mice: Insights into Robust Heart Transplant Tolerance

Abstract

Purpose Understanding the in vivo/in situ behaviour of fully allogeneic spleen (SC) vs bone marrow cells (BMC) in neonatal mice is important for optimizing appropriate 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 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. 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. Conclusion 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. Understanding the in vivo/in situ behaviour of fully allogeneic spleen (SC) vs bone marrow cells (BMC) in neonatal mice is important for optimizing appropriate 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. 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. 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 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. 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. 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.