Unrevealing Hematopoietic Reconstruction Characteristics After Allo-UCBT And Allo-PBSCT In Pediatric Patients With Bone Marrow Failure Syndromes

Objective:Hematopoietic stem cell transplantation(HSCT)is the most classic and mature clinical application of stem cells,which can cure malignant hematological and non-hematological diseases.Umbilical cord blood(UCB)is rich in hematopoietic stem cells(HSC),hematopoietic progenitor cells(HPC),and a large number of naive T lymphocytes.At the same time,due to the advantages of low HLA compatibility and convenient collection,the clinical application of UCB is increasing by years.Clinical studies have shown that hematopoietic reconstitution is significantly delayed in allogeneic umbilical cord blood transplantation(allo-UCBT)compared with allogeneic peripheral blood stem cell transplantation(allo-PBSCT),suggesting that the cell composition and reconstruction kinetics of UCB-derived grafts are significantly different from that of mobilized peripheral blood grafts.In order to systematically analyze the characteristics of hematopoietic reconstitution of allo-UCBT and allo-PBSCT,our study aims to use single-cell transcriptome sequencing technology to systematically interpret the differences of components collected before transplantation and the changes of hematopoietic reconstitution dynamics after transplantation,to analyze the causes of delayed hematopoietic reconstruction after allo-UCBT,and to provide valuable reference for improving clinical prognosis and early intervention.Methods:1.To exclude the effects of malignant diseases such as leukemia cloning and disease recurrence,we collected 4 cases of pediatric patients with bone marrow failure syndrome(BMFs)who received allo-UCBT and 4 cases of allo-PBSCT.Bone marrow(BM)or peripheral blood(PB)were collected at different time points(d14,d21,d30,d60,d90,d180,d365)before and after transplantation,while UCB,mobilized peripheral blood stem cells(G-PBSC)and BM of normal children were collected as controls.2.Lin’CD34+HSPC in BM,G-PBSC and UCB were separated by FACS for single cell transcriptome deep sequencing(STRT-seq).Total nucleated cells(TNC)in BM,PB,G-PBSC and UCB were isolated and sequenced by 10X Genomics single cell transcriptome sequencing.3.After conducting quality control,filtering and mapping,we constructed the gene expression matrix.We integrated the data using R and python,and constructed transcriptome atlas of hematopoietic reconstruction after HSCT from different graft sources through dimension reduction,clustering and annotation.4.FateID and gene set scoring were used to analyze the stemness and differentiation tendency of each population of HSPC as well as the characteristics of mature cells,and to explain the influence of the differentiation fate bias of HSPC subpopulations from different transplantation types on hematopoietic reconstruction after transplantation.5.Differential gene expression analysis was conducted on various populations of hematopoietic cells to explore the differences between the two transplantations in different tissue types and different time periods after transplantation;6.Cell communication between immune subgroups MK_IFITM2 and Ery_SRGN with neutrophils and monocytes was analyzed using CellphoneDB to reveal their role in the inflammatory response after transplantation.Results:1.The single-cell transcriptomic profiles of hematopoietic reconstruction after allo-UCBT and allo-PBSCT were constructed using 276628 hematopoietic cells.A total of 44 cell types were annotated,including six subclasses of hematopoietic stem progenitor cell(HSPC),myeloid cell,NK/T cell,B cell,megakaryocyte and erythrocyte.2.Compared with G-PBSC,the proportion of HSC/MPPs in UCB-derived Lin-CD34+HSPC was increased,the proportion of EBM,MEP and CMP was decreased,and the gene expression levels of MHC-II molecules was decreased.HSC/MPPs in UCB showed a stronger differentiation bias towards the lymphoid and erythroid lineages,but a lower differentiation ability towards the myeloid and megakaryocytic lineages.In UCB-derived TNC,the proportion of adaptive immune cells increased,and the gene expression levels of MHC-II molecules decreased,especially T_CD4_CCR7 cell and NK_CD56dim_CD 16 cell,in which NK_CD56dim_CD 16 cell had stronger immune function.3.In the early stage after transplantation,there was no difference in the proportion of neutrophil progenitor between the two transplantations,while the differentiation trend of HSC/MPPs and CMP2 to neutrophil was weaker in allo-UCBT.In addition,the proportion of MEP cell in allo-UCBT patients decreased,and the differentiation ability of HSC/MPPs to MEP was lower.In allo-UCBT patients,the differentiation ability of HSC/MPPs and MEP to MK was all weaker than allo-PBSCT.4.In the early stage after transplantation,HSC/MPPs of allo-UCBT had stronger differentiation ability to NK and a higher proportion of NKP cell in PB was observed.5.In the early stage after transplantation,the proportion of MK_IFITM2 subset and Ery_SRGN subset increased,and there was a good correlation with adverse clinical events after transplantation in enrolled patients,such as PES and aGVHD.We found that MK_IFITM2 subset and Ery_SRGN subset were both enriched in pathways related to innate immunity.Cell communication analysis showed that MK_IFITM2 interaction with neutrophil and Ery_SRGN interaction with monocyte during inflammation were specifically expressed in pro-inflammatory interactions,such as CD74COPA and C5AR1-RPS19,suggesting that they participate in inflammation by inducing and activating the immune function of innate immune cells.6.Compared with G-PBSC,monocyte in UCB expressed higher MHC-Ⅱ molecule related genes,but the expression level of MHC-Ⅱ molecules in early reconstructed monocyte from allo-UCBT patients was significantly lower than that of allo-PBSCT.However,the level of MHC-Ⅱ molecule expression in B cell was always lower,and we found naiveB from allo-UCBT in late stage after transplantation enriched signaling pathway associated with negative immune regulation,suggesting that it may be related to the reduced incidence of GVHD in alloUCBT patients.Conclusion:1.Constructed transcriptome atlas of hematopoietic reconstruction of different transplantation to improve data resource information for further exploring the improvement strategy of allo-UCBT hematopoietic reconstruction delay.2.We revealed that the cell proportion of MEP and CMP in UCB was decreased and the differentiation ability of HSC/MPPs to both megakaryocytic and myeloid lineages were weakened,suggesting that the delayed reconstruction of neutrophil and platelet in allo-UCBT patients was related to the nature of UCB.3.We confirmed that the proportion of MEP cell in alloUCBT and the differentiation trend of HSC/MPPs to megakaryocytic and myeloid lineages were lower than that of allo-PBSCT in the early stage of allo-UCBT transplantation,which was one of the reasons for the delay of allo-UCBT transplantation.4.Our data explained that the enhancement of HSC/MPPs differentiation into NK in allo-UCBT patients in the early stage after transplantation leads to the increase of the proportion of NKP,which promotes the early reconstruction of NK cell.5.MK_IFITM2 cell and Ery_SRGN subset with immune function were found in early stage after transplantation,and cell interaction analysis indicated that they may be involved in the occurrence or development of inflammation.6.At the transcriptional level,differences in MHC-II molecular expression of antigen-presenting cells after transplantation were revealed,and we found that naiveB cell in allo-UCBT were enriched in negative immunoregulatory signaling pathways at a late stage after transplantation,which may be related to the low incidence and mild condition of chronic GVHD.