Donor-Derived Unlicensed NK Cells Promote the Hematopoietic Recovery after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Yiyang Ding , Zhao Ming , Wang Xiang , Xie Juan , Shang Qian-Nan , Cao Xun-Hong , Zhang Xiaohui , Huang Xiao-Jun , Zhao Xiangyu 
Published: December 2024

Introduction
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for hematopoietic malignancies. Successful engraftment of hematopoietic cells is crucial for the prognosis of the transplant, and delayed or failed engraftment remains a primary cause of transplant failure. Natural killer (NK) cells, as the earliest immune cells to recover post-transplantation, hold significant importance in regulating immune responses and exerting anti-tumor effects. NK cells expressing killer cell immunoglobulin-like receptor (KIR) that recognize major histocompatibility complex (MHC) class I are ‘licensed’, whereas ‘Unlicensed’ NK cells lack receptors for self MHC class I. This licensing process plays a crucial role in regulating NK cell activity and ensuring effective immune surveillance. Previous research has shown that Unlicensed NK cells can enhance the engraftment of hematopoietic stem cells. However, there is currently no evidence demonstrating the impact of donor-derived NK cells on hematopoietic engraftment. Considering that the recipient's NK cells are essentially depleted after conditioning regimen, it is crucial to investigate the influence of donor-derived NK cells on hematopoietic engraftment.
Results
In vitro experiments involving human (MHC-I mismatched NK cells co-cultured with CD34+ cells) and mice (NK cells from C57BL/6 co-cultured with bone marrow cells from B6D2F1) models demonstrated that NK cells enhanced the formation of hematopoietic stem cell colonies. Unlicensed NK cells exhibited a stronger effect in promoting the formation of hematopoietic stem cell colonies compared to licensed NK cells, particularly in colony forming unit-granulocyte and macrophage (GM-CFU). Transwell experiments indicated that this result was achieved through interaction with stromal cells in the bone marrow microenvironment.
Using a mouse model of HSCT (CD45.1 C57BL/6 mice into CD45.2 C57BL/6 mice), we investigated that mice receiving Unlicensed NK cell infusion showed significantly faster hematopoietic stem cell reconstitution, with shorter neutrophil recovery time and increased quantity and proportion of neutrophils. Furthermore, in the bone marrow colony-forming experiment, the Unlicensed NK cell group formed a greater number of colonies, primarily composed of granulocyte-macrophage colonies. Additionally, the Unlicensed NK cell group demonstrated significantly higher levels of neutrophils and platelets in peripheral blood. Further exploration of its mechanism revealed a significant increase in granulocyte-monocyte progenitor cells (GMPs) in the Unlicensed NK cell group. Examination of mice plasma proteomics after HSCT by LC-MS showed up-regulation of CD62L and platelet alpha-granules in the Unlicensed NK cell group.
To verify this result, we conducted a retrospective clinical study involving 272 patients following allo-HSCT, 108 patients were categorized into the Unlicensed NK group, while 164 were in the non-Unlicensed NK group, based on the KIR-MHC class I matching between donors and recipients. The mean days of neutrophil (13.81 versus 15.08, P=0.027) and platelet recovery (23.43 versus 34.97, P=0.0217) were exhibited faster in Unlicensed NK group compared to the non-Unlicensed NK group. Multivariate analyses revealed that the presence of Unlicensed NK cells served as an independent protective factor for both neutrophil and platelet recovery.
Conclusion
Our results demonstrate that Unlicensed NK cells derived from donors can promote hematopoietic recovery. The mechanism of action will be further verified. In the future, this study is expected to influence the selection of transplant donors and improve transplantation prognosis.