Preliminary Study On Prophylactic Infusion Of Double Negative T Cells In Allogeneic Hematopoietic Stem Cell Transplantation For Patients With Myeloid Malignancies

Objective To screen double negative T lymphocytes of third-party healthy volunteers with high cytotoxicity to primary leukemia cells.To investigate the safety and efficacy of prophylactic DNT cells infusion in allogeneic hematopoietic stem cell transplantation(allo-HSCT)for patients with myeloid malignancies.Methods The experiment was divided into two parts.The first part:Ex vivo expansion of DNTs and flow cytometry-based in vitro killing assay.Peripheral blood was extracted from healthy third-party donors and double-negative T cells were amplified and cultured in vitro.In vitro killing experiments were conducted on myeloid leukemia cell lines and primary cells of patients with recurrence after stem cell transplantation.The second part:the safety and efficacy of prophylactic DNT cells infusion in allogeneic hematopoietic stem cell transplantation(allo-HSCT)for patients with myeloid malignancies.Registered single-center clinical study to observe the safety and efficacy of DNT cells therapy and monitor the changes of DNT in vivo.Results 1.On average,0.5×10~8 DNTs were obtained from each milliliter of peripheral blood after 17-20 days ex vivo expansion with>90%purity.DNTs showed significant cytotoxicity to myeloid leukemia cell lines,and when the target ratio was 4:1,the killing efficiency of AML3,MV-411,and U937 cell lines were 88.59±3.10%,92.01±0.6%,and97.94±0.56%,respectively.The results showed that the same DNTs had different cytotoxicity for different primary cells.According to the expasion and results of killing assay,a"donor bank"is being established.2.The clinical trial registration number was Chi CTR190023499.The 3 enrolled patients all relapsed after allo-HSCT,and reached complete remission again after treatment.3patients was underwent 4 times infusion of DNTs,and no serious adverse events(SAE)occurred.The liver and kidney functions of the 3 patients were normal during treatment and after infusion,and also GVHD did not occur within 30 days after infusion.During the treatment,all patients presented with abnormal blood routine,presenting as neutrophils and thrombocytopenia,which may be related to the toxicity of chemotherapy drugs.The hematopoietic system recovered to the pre-treatment level within 2～3 weeks after the infusion.For the first patient,DNTs continue to proliferate in vivo,reaching a peak on+14d,and DNTs in vivo can maintain a certain level on 24days after infusion.One patient was considered to be chronic GVHD after UCBT.Immunosuppressive drugs were discontinued on-1d before the infusion of DNTs,and the symptoms of the c GVHD were significantly improved 28 days later.Conclusion 1.In this study,DNTs were successfully isolated and amplified in vitro using a patented technique developed by Dr.L Zhang and her team at the university of Toronto,Canada.The purity of DNTs exceeds 90%,which can achieve clinical treatment dose.DNTs have obvious killing effect on myeloid leukemia cell lines,and the killing effect on primary leukemia cells of patients was decreased with a large difference in individual killing effect.DNTs may play cytotoxic effect on leukemia cell in part through NKG2D and DNAM-1.2.Prophylactic infusion of healthy donor-derived DNT cells in patients with myeloid malignancies after allo-HSCT is safe.DNTs can further proliferate after infusion,but donor may cannot be last longer time.DNT cells may be potentially effective in reducing GVHD,but the small sample size of this study requires further recruitment of subjects to verify.