The Study Of Pathogenesis And Intervention Of Pre-engraftment Syndrome After Unrelated Cord Blood Transplantation

Umbilical cord blood is an ideal source of stem cells for allogeneic hematopoietic stem cell transplantation.The incidence of chronic graft versus host disease after unrelated cord blood transplantation is low,while the anti-leukemia effect of graft is still strong,and the quality of life of patients is high.Although unrelated cord blood transplantation has been widely used,the use of cord blood as a source of stem cells has declined in the past few years.Due to the delay in hematopoietic recovery and immune reconstruction,the high rate of early non-relapse mortality after transplantation has led to a slowdown in the widespread use of unrelated cord blood transplantation.Therefore,it is of great importance to reduce the risk of transplant-related death.A large number of studies have shown that unrelated cord blood transplantation can lead to pre-engraftment immune response,also known as pre-engraftment syndrome(PES),and the mortality of patients with severe PES is high,which seriously affects the efficiency of unrelated cord blood transplantation.However,little is known about the pathogenesis of PES.In order to explore the immunological mechanism of PES and to find an effective intervention strategy for severe PES,we conducted this study and obtained the following conclusions:1.Patients with severe PES have poor prognosis.We used competitive risk model analysis and found that unrelated cord blood transplantation patients were prone to PES,while peripheral blood stem cell transplantation patients almost did not develop PES.Cox regression analysis revealed three independent risk factors for PES,including occurred earlier than+7 days after unrelated cord blood transplantation,more than two clinical symptoms,and non-response to methylprednisolone.A scoring system for risk stratification of PES was established by using integral method,and we compared the clinical efficacy of patients with PES with different grades.The results of the competitive risk model and kaplan-meier survival analysis showed that in patients with severe PES,Ⅱ-Ⅳacute graft-versus-host disease,Ⅲ-Ⅳ acute graft-versus-host disease,and 3-year non-relapse mortality significantly increased,resulting in significantly reduced 3-year overall survival and 3-year disease-free survival.These results indicate that patients with severe PES have poor prognosis and high mortality,which is an important obstacle to the success of unrelated cord blood transplantation.2.The number of monocytes was significantly increased in patients with PES.We hypothesized that the occurrence of PES was related to the characteristics of umbilical cord blood grafts.First,we analyzed the number of cells that were infused to the patient,and the results showed that the occurrence of PES was independent of the number of total nuclear cells,stem cells,T cells,NK cells,B cells and monocytes that were infused to the patient.Then,flow cytometry was used to dynamically monitor the peripheral blood cells of patients in the early stage after transplantation.The results showed that in the early stage after unrelated cord blood transplantation,the frequency of monocytes,but not T cells and NK cells,was significantly higher in patients with PES than non-PES,whereas B cells were barely detectable in peripheral blood,similar results were observed in the absolute number of peripheral blood cells;PES patients expanded with different kinetics.Furthermore,following the infusion of cord blood,PES patients had higher monocyte counts in the peripheral blood than non-PES patients.We investigated hematopoietic chimerism in the donors via peripheral blood analysis and polymerase chain reaction(PCR)amplification of short tandem repeats(STR-PCR)on day 7 after unrelated cord blood transplantation,the higher the donor chimerism,the more severe symptoms of the PES.In summary,a large number of monocytes were expansion in patients with PES in early stage after unrelated cord blood transplantation,and early donor chimerism was an important risk factor for PES.3.Monocytes derived from cord blood have inflammatory characteristics.Monocytes play an important role in many inflammatory diseases.To identify whether the occurrence of PES is related to monocytes.We analyzed genomic expression profile microarray,the results showed that the monocytes from umbilical cord blood were different from those from peripheral blood,they have a large number of genes differentially expressed.Monocytes derived-from cord blood had high expression of pro-inflammatory cytokines GM-CSF,IL-6 and TNF-α,which was further verified by real-time fluorescence quantitative PCR and flow cytometry.4.GM-CSF drives the inflammatory signature of cord blood-derived monocytes.By flow cytometry,we found that there were a group of GM-CSF+ monocytes in cord blood,whereas no GM-CSF+cells were detected in the peripheral blood stem cells,and the expression level of GM-CSF receptor in cord blood monocytes was higher than that in peripheral monocytes.We wanted to know whether the increased expression level of pro-inflammatory cytokine IL-6 in cord blood monocytes was related to GM-CSF,then GM-CSF,G-CSF and LPS were used for in vitro stimulation.The results showed that,even in the absence of stimulation,the expression level of IL-6 in cord blood monocytes was relatively high,and the IL-6 expression level was further increased after GM-CSF stimulation,while G-CSF could not increase the expression of IL-6 in monocytes.These results suggest that cord blood has a group of inflammatory monocytes that produce GM-CSF,which activates more monocytes and promotes IL-6 expression by binding to the GM-CSF receptor on monocytes.5.Monocytes produced IL-6 in patients with PES.We next tried to ascertain whether GM-CSF plays an important role in the pathophysiology of PES.We therefore investigated dynamic changes of GM-CSF,IL-6 levels in peripheral blood plasma by ELISA.The results showed that IL-6 levels were consistent with those of GM-CSF;levels of both of these proteins peaked around the time that PES patients first showed symptoms but then returned to baseline levels.In contrast,GM-CSF and IL-6 were almost always barely detectable in the plasma of non-PES patients,and we found that levels of plasma IL-6 in patients on the day of PES were significantly higher than that of Non-PES patients,the correlation analysis showed that the plasma IL-6 and GM-CSF levels were positively correlated.Furthermore,the severity of PES is highly correlated with IL-6 levels.The higher the IL-6 level,the more severe symptoms were in patients with PES.To further confirm whether monocytes were the biologically source of IL-6,we performed intracellular staining by flow cytometry.We observed that monocytes derived from PES patients contained large numbers of IL-6-producing cells in peripheral blood.In contrast,there are no IL-6-producing cells in T cells and NK cells,indicating that monocytes were the source of IL-6 in patients with PES.6.Blocking IL-6 can ameliorate PES.In this study,we found that IL-6 is a signature cytokine of PES,we speculated that blocking the IL-6 signaling pathway would alleviate PES,and to test the efficacy of this finding into a clinical setting,we registered a clinical trial at www.chictr.org.cn(Reference:ChiCTR1800015472).This trial enrolled a total of 11 patients who suffered steroid-refractory severe PES.Eligible patients were treated with 4-8mg/kg of the anti-IL-6 receptor monoclonal antibody,tocilizumab,with additional corticosteroids.The end point used to evaluate efficacy was response,defined as an obvious improvement in fevers and rash.Clinical trials have shown that the responsivity of PES to tocilizumab,in terms of fever and skin rash,were prevented by blocking the IL-6 receptor with tocilizumab.Furthermore,all of the patients obtained neutrophil engraftment and successfully recovered from hematopoiesis without any adverse drug reactions.Non-relapse mortality was significantly increased in patients treated with tocilizumab.All of the patients enrolled in our trial recovered and were discharged from hospital.Collectively,these results suggest that tocilizumab can reverse PES.In summary,we demonstrated that umbilical cord blood-derived monocytes possess inflammatory characteristics and secrete pro-inflammatory cytokines,such as GM-CSF and IL-6.Meanwhile,cord blood monocytes also expressed a high level of GM-CSF receptor and responded to GM-CSF.After unrelated cord blood transplantation,these cells undergo rapid expansion in the recipient.Levels of both GM-CSF and IL-6 were increased in the sera of PES patients,leading to the occurrence of PES.It is worth noting that intervention with tocilizumab(TCZ),the monoclonal antibody that targets the IL-6 receptor,is an effective treatment for patients with steroid-refractory PES.In a word,this study revealed the pathological mechanism of PES after unrelated cord blood transplantation,and provides a new treatment strategy for patients with severe PES.These findings have important guiding significance for reducing the transplant-related mortality in early stage after unrelated cord blood transplantation,and further improving the safety and efficacy of unrelated cord blood transplantation.Accordingly,expanding the application of unrelated cord blood transplantation.