| BackgroundMyelodysplastic syndromes(MDS)are a group of clonal myeloid hematopoietic malignancies,which are characterized by abnormal cell differentiation,morphological abnormalities and cytopenia,and have a tendency to transform into acute myeloid leukemia(AML).At present,allogeneic hematopoietic stem cell transplantation(alloHSCT)is an effective way to cure MDS patients.However,disease relapse remains important cause of high mortality for high-risk MDS patients after allo-HSCT.Relapse for high-risk MDS patients after transplantation is as high as 20-40%,and the long-term survival for relapsed patients is less than 20%.Robust immune reconstitution is a vital factor to the success of transplantation.Natural killer(NK)cells are the first lymphocyte population to recover after allo-HSCT and mediate potent graft versus leukemia(GVL)effect particularly in the settings of T-cell depletion.However,the significance of NK cells after unmanipulated transplantation is less clear and factors affecting early NK reconstitution remain elusive.MethodsWe retrospectively analyzed 329 patients with high-risk MDS(IPSS-R>4.5)or MDSAML who underwent the first allo-HSCT between June 2012 and May 2021 from the Hematopoietic Stem Cell Transplantation Center of Blood Diseases Hospital,Chinese Academy of Medical Sciences.Patients were divided into two groups according to the absolute count of NK cells(NK30)in peripheral blood at+30d after allo-HSCT.The primary end points of this study were death and hematologic relapse after transplantation.We employed penalized splines(P-splines)to depict the shape of the association between NK30 and the primary outcome.The shape of the P-splines curve,the median,the receiver operating characteristic curve(ROC)and the maximum selection rank statistic method were used to determine the best critical value of NK30 for the risk prediction of the primary endpoint event.The least absolute shrinkage and selection operator(LASSO)-penalized Cox regression was used to identify the variables associated with the primary outcome.We further investigated underlying factors that affect NK recovery after transplantation.ResultsPenalized splines and receiver operator characteristic(ROC)curves demonstrate a strong association between NK30 and 3-year all-cause mortality with the cut-off value being close to the median value which divides patients dichotomously(NK30=82 cells/μl).There were 164 patients(49.8%)in the high NK30 group and 165 patients(50.2%)in the low NK30 group.We shows that rapid NK recovery is associated with reduced disease relapse(P<0.001)and better survival(P<0.001).Furthermore,robust NK recovery(NK30 and NK60)also correlates with lower incidence of cytomegalovirus(CMV)reactivation and opportunistic infection(P<0.001).Low-NK30 group,complex karyotype,lower CD34+content in the graft and bone marrow blasts>10%before transplantation are independent predictors of high relapse rate in high-risk MDS patients after transplantation(Cindex=0.806).Subsequently,we analyze underlying factors that affect NK recovery after transplantation.NK30 is associated with the numbers of CD34+cells(r=0.711,P<0.001)but not mature NK cells contained in the graft(r=0.077,P=0.161).Furthermore,neither T cell recovery after transplantation nor application of anti-thymocyte globulins(ATG)in the conditioning regimen(P=0.60)demonstrate suppressive effect on NK recovery.ConclusionRapid NK cell recovery is associated with improved prognosis of unmanipulated transplantation for myeloid malignancies.Manipulation of NK cell recovery represents a feasible approach to improve transplant outcomes for example by optimizing CD34+ cells in the graft.BackgroundAllogeneic hematopoietic stem cell transplantation(allo-HSCT)is an important way to cure myelodysplastic syndrome(MDS).but relapse is still the leading cause of death in MDS patients after transplantation.Clinical study shows that delayed NK reconstitution after transplantation is an independent risk factor for relapse after transplantation in MDS patients in the T-depleted settings.However,neither the mechanism of delayed NK recovery in relapsed patients nor the functional reconstitution early NK cells is not clear.The aim of this study is to analyze the molecular mechanism of early NK reconstitution and function regulation in relapsed patients with MDS after allo-HSCT by single-cell transcriptome sequencing.MethodsWe retrospectively collected 48 bone marrow(BM)and peripheral blood(PB)samples from 12 high-risk MDS/MDS-AML patients and their donors from 329 clinical cohorts at multiple time points(initial diagnosis,+30 days after transplantation and relapse for singlecell transcriptome sequencing and whole exome sequencing(WES)analysis.In a small subset(N=12)of the cohort,3 patients were continuous complete remission 3 years after transplantation and 9 patients with disease relapse within 1 year after transplantation.We first performed CD34+magnetic bead sorting on BM samples from patients at various stages,and CD34+cells were subjected to single-cell transcriptome sequencing using the 10×Genomics platform.Lin-(CD3/CD14/CD19/CD20)CD7+cells were sorted by flow cytometry on CD34-cells and analyzed by single-cell tagged reverse transcription RNA sequencing(STRT-seq).WES was performed on samples obtained from patients before transplantation and from their donors.CD34+cells and Lin-CD7+NK cells were clustered and annotated,the transcriptome characteristics of each subpopulation of cells were analyzed,differentially expressed genes were identified and pathway enrichment were performed NK cell immunophenotyping,NK degranulation and direct cytotoxicity assays,and proliferation/apoptosis assays were performed on BM and PB samples from patients at all stages by flow cytometry.ResultsAfter data filtering and quality control,we identified the source of donor and recipient cells at the single cell resolution.A total of 10,517 donor-derived CD34+hematopoietic stem/progenitor cells(HSPCs)and 2205 Lin-CD7+NK cells were obtained for further analysis.We found that:1.The compensatory differentiation of donor-derived HSPCs into NK cells was increased in the early stage after allo-HSCT for relapsed patients,and the proportion of lymphoid and NK progenitor cells were significantly increased,but most of them were in a quiescent state.2.Early reconstruction of NK cells after transplantation for relapsed patients had transcriptomic similarity with NK cells isolated from patients at disease stage(newly diagnose/relapse),and reconstructed NK cells were dysfunctional,mainly manifested as:(1)The cytotoxic function was significantly decreased;(2)The activation of NK cells was inhibited,the expression of inhibitory receptors was up-regulated,and the expression of activating molecules was significantly down-regulated;(3)The expression of genes involved in proliferation or cell cycle were decreased,and cell apoptosis was increased.3.There were significant differences in the transcriptome between the early reconstruction of NK cells from patients with sustained complete remission and relapse after transplantation.Early reconstructed NK cells in remission patients were active in proliferation or cell cycle,exerted enhanced effector function.4.Compared with the newly diagnosed state,the expression of immune checkpoint receptor NKG2 A was significantly up-regulated,and the expression of chemokine CXCR4 was decreased from NK cells at relapse after allo-HSCT.Bone marrow-derived NK cells were significantly decreased at relapse after transplantation.ConclusionsFor relapsed patients,the proliferation and activation of early reconstructed NK cells were decreased,and the compensatory differentiation of donor-derived HSPCs into NK cells was increased.In addition,early reconstructed NK cells for patients with sustained remission after transplantation had similar transcriptome characteristics with ML NK,and its effector function was significantly enhanced.In the future,immunotherapy targeted NK cells functional receptors may represent a new breakthrough in prevention and early intervention for relapse after transplantation. |
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