IntroductionAllogeneic stem-cell transplantation (SCT) is a potential treatment modality for different malignant and nonmalignant diseases. 1 However, the application of this approach is hampered by the high conditioning-related toxicity, graft-versus-host disease, graft rejection, and the limited availability of major histocompatibility (MHC)-matched siblings. 2 A reduction in morbidity and mortality would greatly facilitate a widespread application of hematopoietic SCT. To achieve successful acceptance of allogeneic stem cells in the absence of cytoreduction, the induction of donor-specific tolerance is mandatory. Because T cells play a key role in the rejection of allogeneic stem cells, most transplantation studies have focused on the induction of T-cell tolerance.Natural killer (NK) cells represent another barrier that prevents engraftment following transplantation over MHC barriers. Because of their ability to kill virus-infected or malignant host cells without prior sensitization, NK cells are part of the first line of defense against pathogens. Their activation is coordinated by different inhibitory and activating receptors. Classic and nonclassic MHC class I molecules function as ligands for these receptors and a perturbation of the class I expression, due to virus-infection or tumorigenesis, can result in NK-cell activation. 3-5 Differences in MHC class I expression on allogeneic hematopoietic cells can also result in NK-cell alloreactivity, making NK cells potential effector cells in the early rejection of hematopoietic stem cells. 6 However, data concerning NK-cell elimination of allogeneic stem cells have been inconclusive. Some studies demonstrate an important role for NK cells in the elimination of allogeneic cells, 7-9 whereas other studies indicate that allogeneic stem cells are relatively resistant toward NK-cell cytotoxicity. 10,11 In this study, we used an in vivo cytotoxicity assay based on the infusion of differentially carboxyfluorescein succinimidyl ester (CFSE)-labeled syngeneic and allogeneic donor splenocytes to analyze the elimination kinetics of MHC-mismatched hematopoietic cells in an anti-CD40 ligand (anti-CD40L)-based BALB/c into C57BL/6 SCT model in the complete absence of cytoreduction. We demonstrate the importance of NK cells in the elimination of MHC-mismatched hematopoietic cells after the induction of T-cell tolerance and further show that transplantation of a high dose of stem cells is required to induce NK-cell tolerance. Finally, we show that additional NK-cell depletion allows engraftment and the induction of long-term chimerism after transplantation of a reduced number of stem cells.
Materials and methods
MiceC57BL/6 (H-2 b ), BALB/c (H-2 d ), C3H/J (H-2 k ), and BALB/c nu/nu (H-2 d ) mice were purchased from Charles River Laboratories (Maastricht, The Netherlands), and C57BL/6 nu/nu (H-2 b ) mice were purchased from EDRIS A. Brunink (Valburg, The Netherlands). All mice were female, 8 to 12 weeks old at the start of each experiment, and maintained in the animal faciliti...