BACKGROUND Although our previous studies revealed the role of Tregs (regulatory T cells) and MDSCs (myeloid-derived suppressor cells) in a pre-sensitized cardiac transplant model, interplay between Tregs and NK cells, neutrophils, and macrophages remain undefined. MATERIAL AND METHODS Mice heart transplantation with skin pre-sensitization was performed, in which prolonged-cold ischemia time (PCI) was used for donor treatment. Syngeneic heterotopic heart transplant recipients with PCI were treated with PC61 (monoclonal anti-CD25 antibodies), adoptive cell transfer with Tregs, and rapamycin. RESULTS We unveiled that both rapamycin treatment and adoptive transfer of Tregs could lead to a remarkable decrease of frequency of splenic Gr1+ cells (P=0.058 and P=0.016, respectively). Although administration of PC61 did not affect frequency of splenic Gr1+ cells, it dramatically increased frequency of splenic F4/80+ macrophages (P=0.052). Intriguingly, use of both exogenous PC61 and rapamycin induced a dramatic augmentation of frequency of Gr-1+ neutrophils in the grafts (PC61: P=0.00029; rapamycin: P=0.0096). Noticeably, all different regimens including PC61, rapamycin, and adoptive transfer of Tregs, consistently resulted in a remarked augmentation of frequency of F4/80+ macrophages within grafts (PC61, P=0.0013; rapamycin, P=0.015; Tregs transfer, P=0.013). Although rapamycin and adoptive transfer of Tregs did not affect frequency of NK1.1+ cells, administration of PC61 dramatically increased frequency of NK1.1+ cells within grafts (P=0.033). CONCLUSIONS Tregs depletion or Tregs induced by rapamycin or exogenous cell transfer could affect frequencies of both splenic and intragraft neutrophils, macrophages, and NK cells, but not splenic NK cells. Our data might shed light on understanding sensitized transplant biology.