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Activation and migration of donor T cells to the host target organs are critical mechanisms in the pathogenesis of graft-versus-host disease (GVHD). The role of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor CCR2 in the recruitment of T cells during immune or inflammatory response is also well known. For elucidation of the mechanism of the therapeutic effect of human bone marrow derived-mesenchymal stem cells (MSC) in GVHD, we studied the effect of these cells on migration of activated donor T cells through the CCL2-CCR2 axis in vitro. MSC were expanded from donors' bone marrow mononuclear cells. After co-culturing of IL-2-activated T cells with allogeneic MSC at different ratios, the levels of CCL2 in supernatants were measured by ELISA, and CCR2 expression in CD4+/CD8+ T cells subsets were detected by flow cytometry. The effect of MSC on the migration of activated T cells in the Transwell system was studied in the absence or presence of CCL2. Our results show that CCL2 levels in supernatants of co-cultures were significantly higher than in MSC monoculture and this increase depended on the number of MSC. MSC inhibited proliferation of T cells, but did not change the percentages of CD4+ and CD8+ T cells subsets. MSC can up-regulate the CCR2 expression in CD8+ subsets rather than in CD4+ subsets; MSC enhanced migration of IL-2-activated T cells to CCL2 by increasing the expression of CCR2. The data demonstrate that MSC can enhance chemotaxis of cytokine-activated T cells through the CCL2-CCR2 axis in vitro.

BACKGROUND The aim of this study was to evaluate the efficiency, adverse effects, and pharmacoeconomic impact of empirical and preemptive antifungal therapy for febrile neutropenic hematological malignancy patients in China. MATERIAL AND METHODS Patients with febrile neutropenia during hematological malignancy were randomly divided into an empirical group and a preemptive group. The preemptive antifungal treatment was initiated if patient status was confirmed by clinical manifestation, imaging diagnosis, 1-3-β-D glucan(G) testing, and galactomannan (GM) test. The treatment was ended 2 weeks later if the patient was recovered from neutropenia. Voriconazole was used as the first-line medicine. All patients received intravenous administration of voriconazole every 12 h, with an initiating dose of 400 mg, then the dose was reduced to 200 mg. RESULTS The overall survival rate was 97.1% and 94.6% in the empirical group and preemptive group, respectively, with no significant difference observed (χ²=1.051, P=0.305). However, the occurrence rate of invasive fungal disease (IFD) in the preemptive group was 9.2% vs. 2.2% in the empirical group. Moreover, the mortality rate due to IFD was 0.7% and 2.3% for the empirical group and preemptive group, respectively. The average duration and cost of preemptive antifungal therapy were 13.8±4.7 days and 8379.00±2253.00 RMB, respectively, which were lower than for empirical therapy. However, no significant differences were observed for incidence of adverse effects and hospital stay between the 2 groups. CONCLUSIONS Preemptive antifungal therapy for patients with febrile neutropenic hematological malignancy demonstrated a similar survival rate as with empirical therapy but is economically favorable in a Chinese population.

Myelodysplastic syndromes (MDSs) are a group of clonal disorders of hematopoietic stem cells, resulting in ineffective hematopoiesis. Previous studies have reported that decitabine (DAC) plays an essential role in cell cycle arrest and cell death induction in multiple cell types. Nevertheless, the effect of decitabine on mesenchymal stromal cells derived from bone marrow of patients with MDSs is not completely clarified. Here, we explored the apoptotic and anti-proliferative effect of DAC on MSCs isolated from patients with MDSs. Treatment with DAC inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. We found a positive relationship between cell death triggered by DAC in MSCs and the death receptor family members Fas and FasL mRNA and protein levels (***P 0.00085), cleaved caspase (-3, -8, and -9) activity, and mitochondrial membrane potential reduction. Additionally, DAC-induced apoptosis was inhibited by Kp7-6, a FasL/Fas antagonist, indicating a crucial role of FasL/Fas, a cell death receptor, in mediating the apoptotic effect of DAC. DAC also induced reactive oxygen species (ROS) generation in MSCs derived from MDSs patients (*P = 0.038). Furthermore, N-acetyl-L-cysteine (NAC), a widely accepted ROS scavenger, efficiently reversed DAC-induced apoptosis by inhibiting ROS generation (***P 0.00051) in mitochondria and restoring mitochondrial membrane potential. Furthermore, ROS production was found to be a consequence of caspase activation via caspases inhibition. Our data imply that DAC triggers ROS production in human MSCs, which serves as a crucial factor for mitochondrial membrane potential reduction, and DAC induces cell death prior to FasL/Fas stimulation.

Purpose: Our study was to detect the effect of Decitabine (DAC) combined with arsenic trioxide (AS2O3) on DAPK gene and HL-60 cell proliferation and apoptosis. Methods: DAC and AS2O3 monotherapy, combination treatment and DAC pretreatment were used in this study after incubating with HL-60 cell for 24 h, 48 h, 72 h. CCK8 was used to detect the cell proliferation of HL-60 cell. Flow cytometry was used to detect the cell apoptosis. Then, we used RT-PCR to obtain the gene expression level of DAPK. Results: HL-60 cells were treated with different concentrations of DAC (20 μmol/L, 40 μmol/L, 80 μmol/L), AS2O3 (1 μmol/L, 2.5 μmol/L, 5 μmol/L) monotherapy for 24 h, 48 h, 72 h; along with the extension of the drug concentration and time, proliferation inhibition rate had gradually increased. Monotherapy of DAC, AS2O3 could inhibit the proliferation and induce apoptosis of HL-60 cells, and was time- and dose-dependent. DAC (80 μmol/L) was firstly used for pretreatment, and then, different concentrations of AS2O3 (1 μmol/L, 2.5 μmol/L, 5 μmol/L) were used for 24 h, 48 h, 72 h. It was found that cell proliferation inhibition rate and apoptosis rate had increased significantly. When the two drugs were used together, the increasing proliferation inhibition rate, apoptosis rate and DAPK had become more obvious. Conclusion: DAC and AS2O3 had a synergetic effect for the HL-60 cell proliferation inhibition, apoptosis and expression of DAPK.

Introduction: Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has recently demonstrated high success but also shown limitations regarding their toxicity and development of CD19negative variants. Here we reported results from a phase I study designed to determine the safety of the CD19 CAR-T and CD22 CAR-T cocktail and the feasibility of making enough quantities to treat patients with CD19+CD22+ relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). Patients and Methods: From July 2017 to July 2018, a total of 15 patients with CD19+CD22+ relapsed/refractory B-ALL were treated, including 5 children and 10 adults (Table 1). All patients received fludarabine 30mg/m2/d´3d and cyclophosphamide 250mg/m2/d´2d before infusion of CAR-T cells, followed by a cocktail CAR-T cell infusion with a median number of 2 (0.9-5)´105 CD19 CAR+ T cells/kg and a median number of 0.5 (0.4-12)´105 CD22 CAR+ T cells/kg. The lentiviral backbone containing constructs of CD19 CAR and CD22 CAR are shown in Figure 1. CD19 CAR includes a truncated EGFR sequence which can be used to identify and select CAR+ cells. CD22 CAR includes a single chain variable fragment (ScFv) sequence derived from a monoclonal antibody against human PD-L1 which attempts to reduce the exhaustion of CAR-T cells by blocking the PD-1/PD-L1. Real-time quantitative PCR using primers with specificity for the ScFv of CD19 CAR and ScFv of CD22 CAR can detect the in vivo CAR-T persistence for either CAR. Sequential transduction was performed 2 days after activation of sorted T cells stimulated with CD3 and CD28 antibodies. Percentages of CD19 CAR+ and CD22 CAR+ T cells were determined by flow cytometry through staining with an antibody against EGFR and a fusion protein of CD22-Fc, respectively, and expression of anti-PDL1 ScFv in CD22 CAR-T cells were demonstrated by flow cytometry through intracellular staining with a PD-L1-Fc fusion protein. The primary end points of this study were to evaluate feasibility and toxicity, and the secondary end points included disease response and persistent CAR-T engraftment of infused CAR-T cell. Results: The median observation period was 133 days (24-392 days). The median percentage of pre-treatment bone marrow CD19+CD22+ blasts was 21.5%(0.11-74.1%). On day 20-30 after CAR-T infusion, 15/15 (100%) cases achieved complete remission (CR) or incomplete CR(CRi), 14/15 (93.3%) cases had negative minimal residual disease (MRD). Patient P098 had residual (0.58%) CD19+CD22+ BM blasts at day 30 post-infusion and thereafter achieved negative MRD after re-infusion with CD19 CAR-T cells. 11/17 patients were bridged into allo-HSCT and have remained in remission state with a median follow up of 133 (97-214) days. 2/5 patients without bridging allo-HSCT relapsed on day 240 and day 105 post-infusion, respectively. Notably, both patients (100%) relapsed with CD19+CD22+ leukemia cells. Despite achievement of a very high CR rate, a very low treatment-related toxicity was observed in this trial. Only 1 patients experienced grade 3 cytokine release syndrome (CRS) and another patient (6.7%) developed grade 3 central nervous system (CNS) toxicity; all other patients were CRS grade Conclusion: This study demonstrates technical feasibility, high efficacy and low toxicities of CD19 and CD22 CAR-T cocktail in treating patients with CD19+CD22+ relapsed/refractory B-ALL. Both patients relapsed with CD19+ leukemia suggests this cocktail treatment may reduce the risk of CD19 negative relapse. Low toxicities may relate with small number of infused CAR-T, but involvement of anti-PDL1 ScFv which is co-expressed with CD22 CAR construct cannot be excluded. Therefore, related mechanisms are currently being investigated in the lab. Disclosures No relevant conflicts of interest to declare.