Abstract
Relapse of malignancies remains to be one of the major problems after allogeneic stem cell transplantation (allo-SCT). It is well-recognized that natural killer cells (NK-cells) are predominated in early phase of immune reconstitution after allo-SCT, and several studies demonstrated that CD56 bright CD16 negative (CD56++CD16−) NK-cells, which account for only a few percentage of peripheral NK-cells in healthy individuals, constitute a large subset of NK-cells at this phase. Although CD56++CD16− NK-cells possess unique ability to proliferate and produce proinflammatory cytokines in response to monokines or IL-2, they have been regarded to be less cytotoxic and unfavorable for graft-versus leukemia effects. To verify this issue, we compared the frequency of peripheral CD56++ NK-cells among total NK-cells with subsequent relapse in 25 allo-SCT recipients. Although the ratio of CD56++ NK-cells was gradually decreased as the increased duration between phlebotomy and allo-SCT, we could divide these patients into two groups. Group 1 was consisted of patients who showed consistently elevated ratio of CD56++ NK-cells, and the remainder was categorized into group 2. The relapse after allo-SCT was seen in 1 out of 8 patients in group 1, whereas it was documented in 5 out of 17 patients in group 2. This finding suggested that CD56++ NK-cells might also have a role in preventing relapse. We have found that peripheral CD56++CD16− NK-cells from patients after allo-SCT consistently expressed TNF-related apoptosis-inducing ligand (TRAIL), although its expression was faintly detectable on circulating NK-cells from healthy volunteers. As reported, stimulation with IL-2 or IL-15 resulted in the increased expression of TRAIL on NK-cells from healthy volunteers as well as the recipients of allo-SCT. However, its expression was always stronger in the CD16- subset than CD16+ in both groups. Cultivation of purified NK-cells from healthy volunteers with 0.5 nM of IL-2 for more than 2 weeks resulted in the expansion of both NK-cell subsets, and after sorting into CD16− and CD16+ NK-cells, cytotoxic assays against Jurkat were performed in the presence or absence of concanamycin A, neutralizing anti-Fas antibody, and neutralizing anti-TRAIL antibody. Cytotoxicity was more prominent in the CD16− subset than CD16+, and blocking study revealed that TRAIL expressed on CD16− NK-cells was strongly involved in the killing of Jurkat. We could not detect TRAIL-mediated cytotoxicity in the CD16+ subset, because the expression of TRAIL was much lower in the CD16+ subset than CD16−. Next, NK-cells purified from allo-SCT recipients and healthy volunteers were overnight cultured with 0.5 nM of IL-2 and their cytotoxicity against Jurkat was examined. NK-cells from patients who received allo-SCT within 3 months and those from healthy volunteers showed equivalent cytotoxicity. In patients who showed increased ratio of CD56++CD16− NK-cells, TRAIL was strongly expressed on overnight cultured CD56++CD16− NK cells, and TRAIL-mediated cytotoxicity was also detected. In murine models, TRAIL has been reported to exert strong graft-versus-tumor effects without causing GVHD. As CD56++CD16− NK cells readily express functional TRAIL on cytokine stimulation, and they usually reconstituted shortly early after allo-SCT, they may become promising targets for immunological intervention.
Split anergized natural killer cells halt inflammation by inducing stem cell differentiation, resistance to NK cell cytotoxicity and prevention of cytokine and chemokine secretion