Poor NKG2C+ Adaptive NK Cell Recovery at Early Stage after Allo-HSCT Increased the Occurrence of Refractory Cytomegalovirus Infection

Backgroud: Refractory cytomegalovirus (CMV) infection remains important causes of morbidity and mortality after allogeneic hematopoietic stem cells transplantation (allo-HSCT). Previous researches reported that adaptive immunity, such as CD8⁺ CMV-CTL, plays an important role in the in the control of refractory CMV infection. In mouse, Lanier et al. found there existed subsets of adaptive NK cells with the features of expanding, contracting after control of mouse CMV, and generating long-lived "memory" NK cells. In human, these adaptive NK cells were initially identified based on the high expression of the NKG2C which against HCMV through their cytotoxic potential and the production of TNF-α and IFN-γ upon Ab-mediated stimuli in vitro. Meanwhile, the expression levels of the NKG2C⁺ adaptive NK cells has been positively correlated with the NKG2C copy number. Several researchers had found that NKG2C⁺ adaptive NK cells persistent expanded and were potent producers of IFN-γ during CMV reactivation after solid-organ transplant or allo-HSCT. However, the role of NKG2C⁺ adaptive NK cells on refractory CMV reactivation were still unknown. Whether the rapid reconstitution of NKG2C⁺ adaptive NK cells can reduce the refractory CMV reactivation merit to be investigated.

Aims:In this research, we had investigated the impacts of the quantity and quality recovery of NKG2C⁺ adaptive NK cells on the occurrence of refractory CMV infection.

Method: At first, continuous 364 patients underwent allo-HSCT since June 2012 to February 2016 were prospectively enrolled and we retrospectively analyzed the correlationship between their donor NKG2C genotype and refractory CMV infection occurrence post transplantation. Secondly, the second cohort comprising continuous 125 patients underwent allo-HSCT since May 2016 to April 2017 were prospectively enrolled to analyzed the effect of donor NKG2C genotype on NKG2C⁺ adaptive NK cell recovery as well as the effect of NKG2C⁺ adaptive NK cell recovery on refractory CMV infection. The cytotoxicity of reconstituting NKG2C⁺ adaptive NK cells were evaluated against K562 cells, AD169 CMV stain infected MRC-5 cells, and UL40 peptide pulsed 721.221 cells to detect the anti-tumor or anti-CMV function of NKG2C⁺ adaptive NK cells.

Results: Firstly, from the first cohort, we found that donor NKG2C gene deletion was an independent prognostic factor for refractory CMV reactivation (P=0.010) through the multivariate analysis. Then, through in-depth investigation from the second cohort, we found that the absolute cell counts recovery and anti-tumor function of NKG2C⁺ adaptive NK cells were both significantly lower in patients accepting NKG2C^+/del donor than those patients accepting NKG2C^+/+ donors at day 30, 90, and180, respectively after transplantation. There was no NKG2C⁺ adaptive NK cell recovery post transplantation in the patients who accepted NKG2C^del/del donors. Meanwhile, anti-CMV function recovery of NKG2C⁺ adaptive NK cells in patients with NKG2C^+/del donors were significantly lower than those patients with NKG2C^+/+ donors at day 30 post transplantation. Furthermore, we further analyzed the relationship between the early reconstitution of NKG2C⁺ adaptive NK cells and refractory CMV infection occurrence. The patients were divided into three groups: no CMV, CMV reactivation (persistent time of CMV infection <2 weeks), and refractory CMV infection (persistent time of CMV infection>2 weeks). We found that the absolute cell counts of NKG2C⁺ adaptive NK cells in refractory CMV group was significantly lower than that of other two groups at day 30 transplantation. When the patients were devided into high and low level groups based on the ROC cut-off percentage of NKG2C⁺ adaptive NK cells (1.42%), the result revealed that the patients with lower level of NKG2C⁺ adaptive NK cells at day 30 post-HSCT had an higher cumulative incidence rate of refractory CMV infection (81.1%) comparing with the higher one (40.5%) (P=0.0014). Moreover, Cox regression model further demonstrated that the lower level of NKG2C⁺ adaptive NK cells at day 30 post-HSCT was significantly associated with refractory CMV infection (HR=2.578, 95% CI 1.379-4.21, P=0.003).

Summary/Conclusion: Our results indicated that donor NKG2C deletion damaged the reconstitution of NKG2C⁺ adaptive NK cells after allo-HSCT, therefore increased the occurrence of refractory CMV infection post transplantation.