Our data demonstrate a significant decrease in circulating NK cells early post-transplant in pediatric liver transplant recipients. We suggest that two plausible reasons for this decrease are the direct or indirect effects of immunosuppression or the migration of these circulating NK cells to the graft. The effects of immunosuppressive agents on NK cell numbers and function remains controversial. We have previously demonstrated both in vitro
and in vivo
that cyclosporine and tacrolimus do not effect NK cell proliferation or cytokine production although treatment with sirolimus does impair NK cell numbers and function (20
). Corticosteriods are thought to impair NK cell function and have been reported to decrease expression of the activating receptors NKp30 and NKp46 (21
). However, a recent report suggests that glucocorticoids, including methylprednisolone, in combination with IL-15 expand NK cells and retain functional capacities (22
). Our results suggest that the activation receptors, NKp46 and NKp30 are actually increased after transplant. It has been reported, in a model of allogeneic hematopoeietic stem cell transplantation, that the CD56bright
subset showed greater resistance to the effects of immunosuppressive agents as compared to the CD56dim
). Indeed, daclizumab has been reported to expand the CD56bright
NK cell subset however we did not detect any differences in the percentage of CD56bright
NK between the children who received daclizumab and those that did not. In our study NK cells were significantly decreased at one week post-transplant in all children.
NK cells may also leave the circulation and traffic to the allograft in the early weeks post-transplant. It is well established that NK cells constitute a large proportion of the lymphocytes within the liver (24
). Our results in an experimental model of liver transplant demonstrate that recipient-derived NK cells can be detected in the allograft as early as six hours post–transplant. Furthermore, there is a marked increase in NK cells in the graft and a corresponding decrease of NK cells in the circulation early post-transplant (25
). Finally, it has been shown that hepatic NK cells are enriched in the CD56bright
NK cell subset and that these cells can recirculate for two weeks after transplant thus it is possible that some of the CD56bright
NK cells in the circulation are actually of donor origin in the first week post-transplant (26
). Since the pediatric liver transplant recipients in the current study had minimal adverse events early post-transplant and our center does not perform protocol biopsies, tissue was not available to quantitate the numbers and subsets of NK cells in the liver allograft. It is important to note that the levels of NK cells stabilize and return to pre-transplant levels, by six months post-transplant, supporting a homeostatic interaction between the graft and the periphery.
The significant decrease in NK cells in early post-transplant is noteworthy since NK cells are important in the anti-viral immune response and activation receptors are essential in the recognition of virally-infected cells. Both NKp30 and NKG2D have been demonstrated to be important in the immune response to cytomegalovirus (CMV). Human NKp30 recognizes the pp65 protein of CMV and human CMV has been shown to affect expression of NKG2D ligands (27
). However, when we examined the levels of NK cell activation receptors in pediatric liver transplant recipients, we found that NKp46 and NKG2D were generally expressed at pre-transplant levels in both the CD56dim
NK cell subsets after transplant. In contrast, NKp30 was significantly increased in both NK cell subsets one week after transplant and gradually returned to pre-transplant levels over the next 12 months. Determination of NK cell function in the CD56dim
subsets is clearly of interest, however sufficient numbers of NK cells could not be isolated from the small amount of blood obtained from these children. It should be noted that the levels of NKp30, NKp46 and NKG2D were all lower at one-year post-transplant compared to pre-transplant levels. These values may represent the true “normal” levels in children and that the expression levels in the pre-transplant samples were somewhat elevated as a result of underlying disease or end-stage liver disease. Examination of samples from normal healthy, age-matched children would clarify this minor point.
In the patient experiencing acute rejection, an elevated level of NKG2D was observed in the CD56dim
subset. Numerous studies in experimental models and patient samples have detected an increase in NKG2D ligands during graft rejection. (16
). The role of NKG2D-expressing cells in mediating rejection remains unclear although Kang et al. reported that treatment with an antibody to NKG2D was effective at blocking CD28-independent rejection of cardiac allografts (16
). However this issue remains unsettled as we found that cardiac allograft survival was not significantly prolonged in the absence of NKG2D (Klrk
−/− mice) (unpublished data). Interestingly, NK cells have been shown to be essential for the induction of allograft tolerance by a mechanism involving interactions with antigen presenting cells and dendritic cells (29
Our data clearly show a transient, yet significant, decrease in both the circulating cytotoxic and cytokine producing CD56dim and CD56bright NK cell subsets in children immediately after liver transplantation. The increased expression of activation receptors on the spared NK cells may be a compensatory mechanism to help control viral infection. Further studies examining NK cell phenotypes and functions will clarify the role of NK cells in outcomes post-transplant.