Association Between NK Cells Reconstitution And Acute Graft-versus-host Disease

Allogeneic hematopoietic stem cell transplant(allo-HSCT)is one of effective methods in the treatment of lethal malignant hematological diseases,and has been widely used in clinical setting.Although the medical technology is developing at top speed and the cure rates of patients who receive allo-HSCT have been increasing,the acute graft-versus-host disease(aGVHD),especially the steroid-resistance aGVHD,is still a major obstacle to allo-HSCT,due to lacking of effective predictors and treatments.The development of aGVHD can be categorized as:(1)therapy-induced tissue damage initiates aGVHD;(2)T cells proliferate and activite;(3)T cells cause damage of the target organs.The treatments of aGVHD are based on inhibition of T cells,proliferation and alloreactivity,while the risk of infection and relapse increasing.Many studies show that Natural killer(NK)cells may decrease the rate of infection,induce potent anti-leukemic and even mitigate aGVHD,who bring a new hope to the aGVHD,s prevention and treatment.NK cells are the first lymphocytes to reconstitute the peripheral blood of allo-HSCT patients.The reconstructed NK cells of donors can kill the antigen presenting cells(APCs)of patients,which are the key factors of aGVHD.The surface receptors of NK cells are divided into two types: the activating receptors and inhibitory receptors.The ability of NK cells killing target cells is determined by the balance of inhibitory signals and activatory signals.The inhibitory signals are mediated by inhibitory receptors,which interact with inhibitory ligands on the target cells.While inhibitory receptors deactivate NK cells,activatory receptors activate NK cells.NK cells may be activated and kill the patients,APCs when the inhibitory signals from inhibitory receptors could be lacking because of the absence of ligands on the patients,APCs.NK cells have higher inhibitory receptor expression and lower cytotoxicity after allo-HSCT,this situation will stay until 6 months after allo-HSCT.It remained unclear whether the aGVHD was directed or mediated by the inhibited NK cells.The aims of this study were to elucidate the reconstitution of NK cells,and the association between NK cells counts/activities and aGVHD on patients receiving allo-HSCT,and explore the ability that NK cells mitigate aGVHD after reconstitution.We firstly observed the NK cells,reconstitution after allo-HSCT.From January to July 2015,a total of 26 patients were included,including 17 males and 9 females,average age of 37(12~62)years.The NK cells counts/activities in the peripheral blood of recipients on day 30,60 and 90 after allo-HSCT were monitored,which were determined using 4-color flow cytometry(FCM).The NK cells counts/activities of 26 healthy donors were monitored as the same time as the healthy control group.CD45-APC,CD56-FITC,CD16-FITC and CD3-PerCP was used to identify the frequency of CD3-CD56+CD16+ cells in peripheral blood mononuclear cells(PBMCs),and calculate the NK cells counts.Using K562 cells which were labelled by CSFE as target cells,and NK cells as effector cells to co-culture with k562 cells at a 1:1 ratio in96-well round-bottomed plates for 2.5h.After that,using PI to label all cells to identify apoptotic K652 cells.Dynamicly observe the changes of NK cells counts/activities between patients after allo-HSCT and healthy peoples,in order to evaluate the NK cells immune recovery.Our study showed: healthy peoples,NK cells counts/activities were(1198±514)cell/? and(14.0±6.8)%,patients,NK cells counts on day 30,60 and 90 after allo-HSCT were(951±403)cell/?,(1358±418)cell/? and(1255±353)cell/?.The NK cells activities were(7.3±3.7)%,(9.4±3.6)% and(9.3±2.4)%.Comparing with normal persons,patients,NK cells activities were significantly lower after allo-HSCT.NK cells counts/activities on 30 day after allo-HSCT were the lowest.Whether the low counts/activities of NK cells will affect the aGVHD remains to be further studied.In the second part,we investigated the association between NK cells counts/activities and aGVHD for patients receiving allo-HSCT.In this study,the 26 patients,clinical informations and survival prognoses of aGVHD were reviewed,the association between NK cells counts/activities and aGVHD were analyzed.Results showed that in the aGVHD group(11 cases)and the no-aGVHD group(15 cases),the NK cells counts/activities on days 30 after allo-HSCT were(655±216)cell/?l vs(1169±372)cell/?l(p=0.002)and(7.3±3.6)% vs(9.0±3.6)%(p=0.008).In the?~?grade aGVHD group(7 cases)and the 0 ~ ? grade aGVHD group(19 cases),the NK cells counts/activities were(617±220)cell/?l vs(1081±399)cell/?l(p=0.001)and(4.2±1.7)% vs(8.3±3.5)%(p=0.001).Comparing with the 0~?grade aGVHD group,patients in the ? ~ ? grade aGVHD group had higher relapse rates(57% vs 5%,p=0.010),lower 1year progression-free survival(PFS)rates(43% vs 84%,p=0.010).NK cells counts/activities on day 30 after allo-HSCT were lower in the aGVHD group,and significantly lower in the ? ~ ? grade aGVHD group.Patients with a low NK cells counts/activities would have a poorer outcome.Above the analysis,NK cells counts/activities on day 30 after allo-HSCT were the lowest,and be closely related to aGVHD,which might be a potential marker for aGVHD and provide a new target in aGVHD therapy.