Preliminary Study On Double-negative T Cells For Relapsed Myeloid Malignancies After Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation(allo HSCT)is an important medical method to cure malignant hematological diseases.In recent years,with the continuous improvement of transplantation technology,transplantation related mortality(TRM)has decreased significantly,but the recurrence rate of primary disease after transplantation has not decreased,which is still the main cause of death in patients with hematological malignancies after transplantation.According to the research data of CIBMTR from 2016 to 2017,recurrence of primary disease is still the leading cause of death in patients after unrelated donor transplantation and HLA identical sibling transplantation,accounting for 59%and 51%respectively.Patients with refractory/unresponsive status or high level of minimal residual disease(MRD)before transplantation have higher recurrence rate,which can reach 50%-80%.Donor lymphocyte infusion(DLI)is one of the most effective methods of adoptive immunotherapy for cancer.It is often used to prevent or treat relapse after allo HSCT.However,due to the lack of donors in unrelated cord blood transplantation(UCBT),the traditional DLI treatment can not be used.Once the primary disease relapses after UCBT,the treatment method is very limited.Our study suggests that patients with malignant hematologic diseases who have not been remitted before transplantation,even if they undergo unrelated umbilical cord blood transplantation,have a high risk of recurrent death.The treatment of relapse after allo HSCT mainly includes chemical drug induction therapy,secondary transplantation and immunotherapy.The re remission rate is about 30?40%in patients with chemotherapy,but the remission time is short,and the patients often die of recurrence.The second transplantation is only suitable for about 20%of patients,the OS is only 28%?29%,and the recurrence rate is as high as 60%Immunotherapy is the most common and important treatment for recurrence after transplantation.First of all,immunosuppressive therapy is the first step in the treatment of early relapsed patients without severe GVHD.It may be effective for some patients with slow disease progression,but it is not suitable for patients with complete bone marrow recurrence and rapid disease progression.DLI is an adoptive immunotherapy,which infuses peripheral blood lymphocytes from normal donors into patients to induce graft-versus-leukemia(GVL)effect,so as to eliminate leukemia cells in vivo and achieve the purpose of treating relapse.However,the main limitation of DLI is still to induce severe GVHD.Improper treatment and poor efficacy will lead to death.In addition,the biggest limitation of recurrence treatment after UCBT is the lack of traditional DLI treatment.Therefore,actively looking for new treatment strategies to make up for the defect of no traditional DLI treatment for recurrence after UCBT,and improve the survival rate of patients with recurrence after UCBT,is an urgent problem for transplant workers,which has very important clinical significance.Most T cells in human peripheral blood express CD4 or CD8 molecules,but nearly 1-5%of T cells only express CD3 but not CD4,CD8 and CD56.These cells are called DNT cells.Due to the low content of DNT cells in peripheral blood and the lack of effective cell amplification methods,the role of DNT cells in human tumor immunity is still poorly understood.Zhang L,et al.From the University of Toronto,Canada,developed a new method:DNT cells can be isolated from peripheral blood of patients with acute myeloid leukemia(AML)or healthy volunteers in complete remission(CR)stage shortly after high-dose chemotherapy(January),It can effectively kill leukemia cell lines and primary leukemia cells in vitro.In addition,DNT cells not only have selective anti leukemia effect,but also do not cause GVHD.In this study,the third-party healthy donor DNT cells were cultured in vitro to verify its anti leukemia effect to select donors,and further verified the safety and effectiveness of DNT cells in vivo through clinical research.Part one:Salvaged single-unit cord blood transplantation for 26 patients with hematologic malignancies not in remissionObjectives:To investigate the curative effect of salvage umbilical cord blood transplantation in patients with unrelieved malignant hematologic diseases.Methods:26 patients,which included acute leukemia in 19 patients,MDS(RAEB)in 5,and non-Hodgkin's lymphoma in 2 patients,received 1 CBT unit ?2 loci HLA-mismatched following myeloablative conditioning regimens from July 2005 to July 2014 were analyzed retrospectively.All of them were in non-remission(NR)before transplantation.Results:Median follow up was 27(range 5-74)months.In all,14 cases survived who were in sustained CR and 12 cases died,3 from relapse and 9 from NRM(6 from severe infection,3 from aGVHD).The estimated 2 year overall survival(OS),disease-free survival(DFS),relapse and NRM rate were 50.5%(95%CI:31.3%-69.7%),40.3%(95%CI:21.4%-59.2%),28.9%(95%CI:11.5%-46.3%)and 35.2%(95%CI:16.8%-53.6%).Conclusion:Salvaged CBT might be a promising modality for treatment of hematologic malignancies,even with high leukemia burden.However,recurrence after transplantation should not be ignored.Part two:DNT cell isolation,amplification,culture and killing experiment in vitroObjectives:1.To screen out the double negative T lymphocytes of the third party healthy volunteers who are sensitive and tolerant to the killing of primary leukemia cells.2.Search for target genes with high sensitivity/tolerance to DNT cells.Methods:1.Using the third-party healthy donors' peripheral blood culture and amplification to obtain a suspension rich in DNT cells,flow cytometry and ELISA methods were used to detect the killing activity of DNT cells in vitro to 11 AML patients' primordial cells,so as to determine the sensitivity of DNT cells in vitro to the killing of prokaryotic cells from different AML patients.In flow cytometry,DNT cells expanded in vitro for 14-17 days were selected as effector cells,and primordial cells from 11 AML patients were used as target cells.The biological efficacy of DNT cells was determined after incubation for 2 hours with the ratio of effect to target of 4:1.After CO incubation of effector cells and target cells,the supernatant was collected and the content of IFN-? in the supernatant was detected by ELISA to indirectly determine the killing activity of DNT cells.The AML tumor cell line mv-4-11 was used as the positive control.Combined with the data obtained by the two methods,the results of DNT cells killing 11 AML patients were analyzed comprehensively.2.Primary cells of AML patients sensitive to DNT or resistant to DNT were screened for high-throughput RNA SEQ and microarray analysis.Objective to search for target genes with high sensitivity/tolerance to DNT cells.Results:1.After 17-20 days of in vitro amplification,0.5×108 DNT cells per ml ofperipheral blood were obtained,and the purity was more than 90%.2.The killing activity of DNT cells in vitro to 11 AML patients' primordial cells was detected.Among the 4 AML primordial cells meeting the requirements of flow cytometry,DNT cells had killing activity on these 4 AML patients' primordial cells,which was consistent with the results obtained by ELISA,and the two methods were highly correlated(100%consistent);3 patients who failed to be detected by flow cytometry showed that the killing activity of DNT cells against the primordial cells of 11 AML patients The other 4 patients who were not detected by flow cytometry showed no killing activity by ELISA.Therefore,flow cytometry and/or ELISA were used to determine the sensitivity of DNT cells in vitro killing in 11 patients,accounting for 64%(7/11)of the total samples,which was close to the results reported in the literature.3.Combined with literature review and second-generation sequencing,SNHG5 gene,KLF6 gene,HLA-E gene,DUSP1 gene,CXCL8 gene.cd96 gene,cav1 gene,pou4f1 gene,runxlt1 gene were down regulated,and vim-2p gene and spink2 gene were up-regulated.This still needs to be verified by large sample size and functional experiments of related genes.Conclusion:1.Through the patented technology of DNT cell culture developed by Dr.I Zhang of University of Toronto and his team,DNT cells were successfully isolated and expanded in vitro with purity>90%,meeting the c linical treatment dose;2 The killing effect of DNT cells on leukemia cells was different.About 64%(7/11)patients were sensitive to DNT cells in vitro,which was close to the results reported in the literature.There may be AML genes which are highly sensitive and tolerant to DNT cells.significance:The screening of DNT sensitive/tolerant genes can be used as biological markers to better screen patients with effective treatment of DNT cells,and can be targeted to intervene patients with DNT cell tolerance in advance,better play the therapeutic role of DNT cells,and solve the problem of relapse after UCBT without traditional DLI treatment.Part three:a single center clinical study of modified DLI(third-party healthy donor DNT cells)in the treatment of relapsed myeloid malignancies after allogeneic hematopoietic stem cell transplantationObjectives1.To study the safety of modified DLI(third-party healthy donor DNT cells)in the treatment of relapsed myeloid malignant blood after allogeneic hematopoietic stem cell transplantation.2.To study the efficacy by DNT cells from third-party healthy donorsMethods:The safety and efficacy of DNT cells from third-party healthy donors in allo HSCT of myeloid malignancies were evaluated in 12 patients.The changes of DNT and inflammatory factors were monitored.Before the first DNT cell infusion,the immunosuppressive therapy of fludarabine(Flu)combined with cyclophosphamide(CTX)was given.The recommended number of DNT cells for transfusion was 5×107/kg(recipient body weight),1×108 cells/kg(recipient body weight)and 2-4×108/kg(recipient body weight).The second and third cell reinfusion was performed 7 days and 14 days after the first DNT cell infusion.Main outcome measures:complete remission rate(CR).Secondary,outcome measures:hematopoietic cell recovery time,Cr duration,early mortality,1-year overall survival rate(OS),1-year leukemia free survival rate(LFS),minimal residual(MRD),WT-1 gene,tumor gene.chromosome growth and decline rate before and after treatment.Main safety measures:treatment related mortality(TRM)secondary safety indicators treatment-related mortality(TRM)The incidence of grade ?-? and grade ?-?GVHD,adverse events,serious adverse events,the number of DNT cells in peripheral blood,C-reactive protein(CRP),serum ferritin(SF),serum cytokines:interleukin(IL)-6,interferon(IFN)-?,IL-8,IL-10.tumor necrosis factor-?,macrophage inflammatory protein(MIP)-1?,MIP-1?,monocyte chemoattractant protein(m)in peripheral blood Cp-l)and soluble receptors IL-1R ? and IL-2R ?.Results:1.The process of reinfusion was smooth in 12 cases,all of them developed chills and fever after transfusion,and improved after symptomatic treatment.There were no grade 2 or above adverse reactions in all patients.The functions of heart,lung,liver,kidney and other organs were normal.There was no cytokine release syndrome(CRS)or acute/chronic GVHD.2.As of August 1,2020,the median follow-up time of all patients was 4.3(1.7-12.2)months,and one patient was withdrawn due to personal reasons.Of the remaining 11 patients,6(54.5%)achieved complete remission,of which 5(45.4%)turned negative.I patients(9.1%)achieved partial remission.Up to the last follow-up,4 patients(36.3%)were still in complete remission3.In most patients,donor DNT cells began to rise at 6h after the first reinfusion,peaked at 24h after reinfusion,and then decreased gradually.After the second and third reinfusion,donor DNT cells only increased slightly,but lasted for about 2 weeks in vivo.Notably,the DNT cell levels at the last follow-up were still higher than the pre-infusion level in five patients who achieved CR after DNT cell treatment(patient 5,6,7,9),while patient 8 who did not respond to DNT therapy had reduced DNT cell counts at the last follow-up compared to the pre-infusion level.Further,patient 7 who achieved MRD-negative CR without the disease relapse at the last follow up had 19.8%of DNT cells in his bone marrow T cells on day 102.4.In comparison to the pre-infusion,serum levels of CRS-related cytokines,IL-6,IL-10,MIP-1?,MIP-1,and MCP-l were elevated in all nine evaluable patients shortly after each DNT cell infusion and returned to normal within 48 hours.Notable increase in serum IFN ?(2.03±0.28 folds)and TNF?(1.93±0.33 folds)levels were observed 6 hours after each infusion,supporting the induction of anti-leukemic activity by DNT cells after each DNT cell infusion.Conclusion:In this study,modified DLI(third-party healthy donor DNT cells)was used to treat relapsed myeloid malignancies after allogeneic hematopoietic stem cell transplantation.It was safe and well tolerated.Some patients were effective.After infusion,the total DNT cells in patients showed a continuous upward trend.