Background. Macrophages are important effector cells in T cell-mediated xenograft rejection. The aim of this study was to determine whether CD4+CD25+ regulatory T cells (Tregs) were capable of suppressing macrophage activation in vitro. Methods. Porcine cell or xenoantigen-primed human peripheral blood mononuclear cells, CD4+ T cell-depleted peripheral blood mononuclear cells, or CD14+ macrophages plus autologous CD4+CD25− T cells were cultured with or without expanded autologous Tregs. Transwell cultures were used to separate the various components to determine the need for cell-cell contact. Results. Pig cell primed CD14+ macrophages required the presence of CD4+CD25− T cells for activation and increased expression of CD40, interleukin-12, and tumor necrosis factor-&agr;. This up-regulated expression of macrophage activation markers was reduced substantially in the presence of autologous Tregs. Coculture with Tregs did not alter macrophage viability but reduced the capacity of macrophages to stimulate proliferation of responder T cells. Tregs required direct contact with CD4+CD25− T cells to inhibit macrophage activation but activated macrophage phenotype was not altered by separating the stimulated human peripheral blood mononuclear cells or CD14+ macrophages from Tregs in a transwell system. Macrophages did not require direct cell contact with porcine stimulator cells for full activation by CD4+CD25− T cells. Conclusions. Human Tregs were able to suppress xenoantigen-primed and CD4+ T-cell-mediated macrophage activation and antigen-presenting cell function. However, Tregs had no direct effect on macrophages in vitro.

BACKGROUND:Our previous study has shown that porcine antigen-primed and CD4+ T cell-activated macrophages are capable of recognition and rejection of porcine xenografts after adoptive transfer. However, whether this is an absolute xenograft specific rejection remains to be confirmed.METHODS:Mouse islet allografts and neonatal porcine islet cell cluster (NICC) xenografts were admixed and transplanted under the left kidney capsule, and NICC xenografts alone were transplanted under the right kidney capsule of strepotozotocin-induced diabetic NOD-SCID mice. After achievement of normoglycemia, the NOD-SCID recipients were transferred with macrophages purified from NICC transplant NOD-SCID mice reconstituted with CD4+ T cells. Five weeks after macrophage transfer the left kidney with the admixed grafts were removed. Graft survival and function following macrophage transfer was assessed by blood glucose measurement and immunohistochemistry.RESULTS AND CONCLUSIONS:Adoptive transfer with activated macrophages did not affect the normalized blood glucose levels in NOD-SCID recipients of admixed grafts until left nephrectomy 5 weeks post-macrophage transfer. Insulin-positive and porcine C-peptide-negative mouse islets were detected in the admixed grafts. The surviving mouse islets in the admixed grafts were surrounded but not infiltrated by macrophages. The nephrectomized recipients demonstrated sustained hyperglycemia and completely destroyed NICC xenografts in their remaining right kidneys 8 weeks after macrophage transfer. Taken together, these data provide direct evidence of porcine islet xenograft specific rejection by activated macrophages.