Explore The Effector Cells Of Pre-engraftment Syndrome After Unrelated Cord Blood Transplantation

Objectives Pre-engraftment syndrome(PES)is an immune response after unrelated cord blood transplantation(UCBT)with frequent occurrence,which clinical manifestation is noninfectious fever and/or unexplained rash,diarrhea before neutrophil engraftment.The incidence of transplantation-related mortality(TRM)was significantly higher in patients with severe PES.To discover the cellular origin at the time of PES,we detect the donor gene chimeric rate in UCBT patients peripheral blood cells.According to soluble ST2(sST2)increased significantly at the time of PES in the serum cytokine detection,we use IL-33 and sST2 to stimulate fresh cord blood and peripheral blood stem cells to explore the effector cells of pre-engraftment syndrome and lay a foundation for study on the pathogenesis of PES.Methods There are two parts in this study.The first part is the study of cellular origin of PES after UCBT.According to the previous research from our center,we found that the median time of PES was the seventh day after UCBT(+7d).Therefore,we choose the+7d peripheral blood samples in UCBT patients,using short tandem repeat polymerase chain reaction(STR-PCR)technique to detect PES group and without PES group of donor gene chimerism,and analysis the cellular source of PES.The second part is investigating effector cells in PES.First is about the detection of various immune cells and their subsets and related cytokines after UCBT in PES.The detection include the change of T,B,NK and monocytes subsets when PES occurs and leukocytes,plasma C-reactive protein,serum cytokines at different time which are pre-transplantation,transplantation day and +7 day after transplantation.Second is about the experimental study on the external stimulus of different sources.sST2 is significantly increased in PES,and ST2 is an orphan receptor of IL-33,which is upposed to induce PES effector cells from the IL-33/ST2 signaling pathway.Stimulating fresh cord blood and peripheral blood(a collection of sibling donors mobilized)with IL-33 and sST2,each type of cell was divided into 4 groups:experimental group: A.IL-33+sST2 group;control group: B.sST2 group;C.IL-33group;D.blank group.After 0,12,24 and 72 hours of culture in vitro,the number of cells was counted before and after stimulation,and the expression of cell subsets and ST2 receptor was detected by flow cytometry before and after stimulation.Results The first part:the study of cellular origin of PES after UCBT.STR-PCR was used to detect the chimerism of the cord blood and the recipient before and after transplantation,donor chimerism(%)in PES group and non-PES group were70.58±22.86 and 37.18±18.96 respectively.There was significant statistical difference between the groups,P<0.0001.The second part:investigating effector cells in PES.First is about the detection of various immune cells and their subsets and related cytokines after UCBT in PES.1.The counts of leukocytes in both PES group and non-PES group decreased gradually to the minimum at early stage of transplantation,and there was no statistical difference between two groups at pre-transplantation,transplantation day and +7 day after transplantation,P > 0.05.2.T,B,NK and monocytes subsets when PES occurs : CD45+CD3+T lymphocytes,CD45+CD3-CD19+B lymphocytes,CD45+CD3-CD56+NK cells,CD45+CD3-CD14+monocytes accounted for 90%,0.3%,2.1% and 0.2%,respectively.There was no significant difference in T,B,NK and monocyte subsets in PES group and non-PES group,P > 0.05.3.There was no significant difference in C-reaction protein between two groups at pre-transplantation,transplantation day and +7 day after transplantation,P > 0.05.4.The variety of serum cytokines IL-6,MCP-1 and sST2 in the PES group were significantly higher than those in the on-PES group at the time of PES(P<0.05);in PES group,these cytokines was significantly higher at the time of PES than that before transplantation and on the day of transplantation(P<0.05).In PES patients with effective treatment,IL-6 and MCP-1decreased significantly and recovered to the level before onset(P<0.01),while sST2 were decreased but there was no significant difference(P > 0.05).IFN-??IL-2?IL-1R??IL-4?IL-8?IL-17A?IL-33?MIP-1??MIP-1? and TNF-?expression levels were similar in the two groups and there was no statistically significant difference(P > 0.05).At the same time,there was no significant difference in these cytokines expression level in PES group at different time points(P > 0.05).Second is about the experimental study on the external stimulus of different sources.1.Fresh cord blood and peripheral blood stem cells by IL-33 and sST2 were cultured,the number of cells before and after stimulation were not significantly increased(P>0.05),the two sources of cells cultured after 12 h showed polymorphic change,then with prolonged culture morphology did not change significantly.2.The variety of T,B,NK and monocytes subsets:fresh cord blood cells by Ficoll extracted mononuclear cells,mononuclear cells of umbilical cord blood were T lymphocytes extraction ratio of 30-50%,B lymphocytes 1-3%,NK cells 5-8%,and monocyte 5-13%.Peripheral blood stem cells were T lymphocytes ratio of 20-35%,B lymphocytes 2-5%,NK cells 3-9%,and monocyte 18-32%%.After cultured 12,24 and 72h,four groups of T,B,NK and the proportion of monocyte subsets had no significant difference(P>0.05),with prolonged incubation time,there is a small amount of apoptosis.And there was no statistically significant difference in experimental groups and control groups(P >0.05).3.ST2 receptor expression on T,B,NK and monocytes subsets: Fresh umbilical cord blood and peripheral blood stem cells in the expression of ST2 receptor is similar.The expression of ST2 receptors on monocytes was as high as 53-90%,followed by B lymphocyte subsets of about 30-60%,T lymphocytes and NK cell subsets are lower,which were 2-5% and 3-10%,respectively.However,there was no significant change in the expression of ST2 receptor in the four subsets of cells in each group(P>0.05), nd there was no significant change in the expression of ST2 in each group of cells at different time points(P>0.05).Consequences1.PES is a kind of immune response induced by a variety of cytokines involved in the initiation of donor after UCBT,and the effector cells are presumed to be derived from donor.The early chimerism of donor is a high risk factor for PES.2.Monocyte chemotactic protein-1(MCP-1)increased significantly in PES,and lymphocyte related specific cytokines(IL-2,IL-4,IL-8,IL-17A)had no significant change in PES occurs.The main clinical manifestations of PES is in skin and gut tissues,we inferred macrophages in tissue may be the main effect cells of PES.3.Proliferation of immune cells and expression of ST2 receptor could not increase with stimulation of IL-33 and sST2 in vitro culture.But we can not determine whether cytokine level changes.To observe the IL-33/ST2 signaling pathway and sST2 on the downstream signal transduction effect and cytokine secretion,further detection of cytokines in cell culture medium are needed.At the same time,we also need to measure the indexes of immune cell activation,such as HLA-DR,CD27,CCR and so on.