Clinical Research Of Cytokine-Activated Donor Lymphocyte Infusion And Preliminary Study Of Its Mechanisms

PartⅠClinical Research of Cytokine-Activated Donor Lymphocyte InfusionObjective:To evaluate the effect and efficacy of cytokine-actived donor lymphocyte infusion (the novel DLI) in patients who relapsed after allogeneic hematopoietic stem cell transplantations (allo-HSCT).Methods:Sixteen patients with acute leukemia were retrospectively analyzed, who relapsed after allo-HSCT and were treated with the novel DLI or conventional DLI. Through multi-parameter detection of minimal residual disease, including morphology examination, flow cytometry (FCM), real time quantitive PCR (RQ-PCR), multiplex PCR amplification of short tandem repeats markers (STR-PCR), karyotype analysis with R-banding and fluorescence in situ hybridization, the therapeutic response of the novel DLI and conventional DLI were campared. Treatment regimen of two kinds of DLI were: interferon-αfollowed by DLI or infused donor lymphocyte by leukapheresis, respectively.Result:Eight patients received the novel DLI, in which 7 patients had hematological relapse and 1 patient had recurrence tendency. Other 8 patients received conventional DLI, in which 7 patients had hematological relapse and 1 patient had molecular relapse. Complete remission (CR) rate induced by the novel DLI and conventional DLI was 62.5% vs 25%, respectively. Complete remission rate for patients with hematological relapse was 57.14% vs 14.28%, respectively. The median time required for a response to CR was 7d vs 23d, respectively. In the group of receiving the novel DLI, the occurrence of GVHD and pancytopenia were 62.5% and 62.5%, whereas in the group of receiving conventional DLI, 2 of 8 patients developed GVHD and 6 patients (75%) developed pancytopenia. The overall survival at 1 year were 50% and 12.5% respectively.Conclusions:This pilot study in our institution here shows that a CR rate of 62.5% could be achieved used by the novel DLI treatment in relapsed patiens after allo-HSCT. The median remission-induction time is 7 days, and the remission state is durable. The novel DLI may be an effective therapy for patients with acute leukemia relapsed after allo-HSCT. In future, the optimal regimen of the novel DLI should be explored according to patients'disease stage, relapsing time after HSCT and transplantation modality, in order to enhance GVL and minimize GVHD as well as prolong disease-free survival.PartⅡPreliminary Study of Mechanisms for Cytokine-Activated Donor Lymphocyte InfusionObjective:To investigate the mechanism of cytokine-actived donor lymphocyte infusion (the novel DLI).Methods:1. G-CSF-primed peripheral blood stem cell (PBSC) was collected by leukapheresis. PBSC was separated by ficoll density gradient centrifugation to obtain mononuclear cells (MNC). The SHI-1 cell strains were incubated with MNC activated by various concentrations of interferon-α(500, 1000, 2000 and 4000U/ml) and different times (2, 3 and 7 days). The tumoricidal activity of interferon-αactived MNC against SHI-1 was measured by MTT; 2. The key point of time and cytokine concentration was explored while the cytotoxicity of MNC against SHI-1 was maximal, the chage of surface molecules expression of lymphocytes and dendritic cells (DC) incubated with interferon-αwas analyzed by flow cytometry (FCM); 3. Peripheral blood of 4 patients who were treated with the novel DLI was collected. The immunophenotype of lymphocytes after treatment was analyzed by FCM.Result:1. Interferon-α(IFN-α) enhanced the cytotoxicity of MNC against SHI-1 cell strains. After incubation for 2 and 3 days, high concentration of IFN-α(4000U/ml) could significantly enhance the cytotoxicity of MNC. While incubation for 7 days, conventional concentration of IFN-α(1000U/ml) could enhance the cytotoxicity of MNC against SHI-1 cell strains significantly; 2.The optimal key point of cytokine concentration and time are 4000U/ml and 3 days. The percentage of CD3~+CD4~+ cell increased significantly and the activated marker CD69 of T lymphocyte also increased markedly. The expression level of CD83, CD86 and HLA-DR of DC also increased; 3. CD3~+,CD3~+CD4~+ and CD3~+CD8~+ cell maintained at the stable level among the patients who had a response to the novel DLI, but decreased at the different level in the patients who had no response to the novel DLI. HLA-DR, the activated marker of lymphocyte, increased in the patients with response of the novel DLI. While the percentages of NK cell,CD4~+CD25~+ cell and Tγδcell did not changed during the novel DLI for all the 4 patients.Conclusions:1. In vitro, both high and low concentration of IFN-αcan enhance MNC activity to kill the target cell after incubation for 7 days; 2. IFN-αmay promote the maturation of DC and enhance the capability of presenting tumor antigen, which can stimulate T cell to active and proliferate and mediate the tumoricidal activity; 3. The GVL effect of the novel DLI may be mediated by traditional T lymphocyte.