Effect and Involved Mechanism of RSL3-induced Ferroptosis in Acute Leukemia Cells MOLM13 and Drug-resistant Cell Lines / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect and involved mechanism of RSL3 on ferroptosis action in acute leukemia cells MOLM13 and its drug-resistant cells.@*METHODS@#After MOLM13 treated with RSL3, CCK-8 assay was performed to detect cell viability, flow cytometry was used to detect the reactive oxygen species (ROS) level of the cells, RT-qPCR and Western blot were used to detect the expression of glutathione peroxidase 4 (GPX4). After MOLM13/IDA and MOLM13/Ara-C, the drug-resistant cell lines were constructed, the ferroptosis induced by RSL3 was observed. Bone marrow samples were collected from patients with acute monocytic leukemia. RT-qPCR and Western blot were performed to detect the expression of related genes and proteins involved in ferroptosis pathway.@*RESULTS@#RSL3 significantly inhibited the cell viability of MOLM13 and increased the intracellular ROS level, which were partially reversed by ferrostatin-1. The mRNA and protein expression of GPX4 decreased in MOLM13 treated with RSL3. RSL3 inhibited the viability of MOLM13/IDA and MOLM13/Ara-C cells more strongly than that of non-drug resistant cells, also increased the intracellular ROS level . The cytotoxic effects were partially reversed by ferrostatin-1. The mRNA and protein expressions of GPX4 in MOLM13/IDA and MOLM13/Ara-C cells were higher than those in non-drug resistant cells. The mRNA and protein levels of GPX4 in bone marrow of relapsed/refractory acute mononuclear leukemia patients were higher than those of ordinary acute mononuclear leukemia patients.@*CONCLUSION@#RSL3 can induce non-drug resistant cells MOLM13 ferroptosis by inhibiting GPX4 activity. MOLM13/IDA and MOLM13/Ara-C are more sensitive to RSL3 compared with non-drug resistant cells MOLM13, which may be caused by the differences in GPX4 expression. The expressions of GPX4 mRNA and protein in relapsed/refractory acute mononuclear leukemia are higher than those in ordinary acute mononuclear leukemia.

PD-1 expression, mRNA level and cytotoxicity changes in CD19CAR-T cells / 中华血液学杂志

Objective: To observe the changes of PD-1 expression, mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells. Methods: The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells. The expression of PD-1 was analyzed by flow cytometry. The mRNA level of PD-1 was analyzed by PCR. The cell proliferation was analyzed by CCK-8 assay. The cytotoxicity was analyzed by LDH assay. Results: ①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same (P>0.05) . ②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells, with or without PD-1 inhibitor were as the same (P>0.05) . ③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells (P<0.001) . There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer (P>0.05) . ④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process (P>0.05) . There was no difference of mRNA level of PD-1 in all groups during the culture process (P>0.05) . ⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells (P<0.001) . The PD-1 inhibitors could antagonize this effect. There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells (P>0.05) . Conclusion: The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells. However, the mRNA level of PD-1 of all groups did not change, even if PD-1 inhibitor was applied.

Efficacy and safety of CD19 chimeric antigen receptor T cells for the treatment of 22 patients with B-cell lymphoma / 中华血液学杂志

Objective: To investigate the efficacy and safety of CD19 chimeric antigen receptor T (CAR-T) lymphocytes for the treatment of B cell lymphoma. Methods: A total of 22 patients with B-cell lymphoma from February 1, 2017 to July 1, 2018 were reviewed to evaluate the efficacy and adverse reactions of CD19 CAR-T. Results: Of 22 patients with B-cell lymphoma received CD19 CAR-T cells, the median dose of CAR-T cells was 7.2 (2.0-12.0) ×106/kg. Nine of 12 cases of relapse refractory patients were overall response. Complete remission (CR) occurred in 2 of 12 patients, partial remission (PR) in 7 of 12 patients. The overall response in minor residual disease positive (MRD) group was 8 of 10 patients. CD19 CAR-T cells proliferated in vivo and were detectable in the blood of patients. The peak timepoints of CAR-T cells proliferated in the relapsed refractory and MRD positive groups were 12 (5-19) and 4.5 (1-12) days after treatment respectively, and among peripheral blood cells, CAR-T cells accounted for 10.10% (3.55%-24.74%) and 4.02% (2.23%-28.60%) of T lymphocytes respectively. The MRD positive patients achieved sustained remissions during a median follow-up of 8 months (rang 3-18 months) . None of all the patients relapsed during a median follow-up time of 10 months (3-18 months) . However, 7 PR responders of the relapsed refractory patients maintained a good condition for 1.5-6.0 months. One patient bridged to hematopoietic stem cell transplantation, another one sustained remission for 12 months. Cytokine-release syndrome (CRS) occurred in 14 patients with grade 1-2 CRS in MRD positive group and grade 3 CRS in relapsed refractory group. Conclusions: CAR-T cell therapy not only played a role in the rescue treatment of relapsed and refractory patients, but also produced a surprising effect in the consolidation and maintenance of B-cell lymphoma. CD19 CAR-T cells might be more effective in the treatment of MRD positive B-cell lymphoma patients than in the refractory or relapsed cases. High response rate was observed with fewer adverse reactions.

Effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells / 中华血液学杂志

Objective: To Evaluation the effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells (CD19-CAR-T) in vitro. Methods: Five patients with high PD-1 expression in peripheral blood and five healthy volunteers were selected. These peripheral blood mononuclear cells were used as the source of T cells to prepare CD19-CAR-T cells. Different doses (72, 36, 18 μg/ml) of Nivolumab was added on day 8 to the culture medium. Patient T cells incubated with 72 μg/ml Nivolumab and CD19-CAR-T cells of healthy volunteers were used as controls. CCK-8, lactate dehydrogenase (LDH) cytotoxicity assay and ELASA were used to detect the proliferation capacity, the specific cytotoxicity and the inflammatory factor secretion. Results: ①T cells from patients with high expression of PD-1 as the source of CD19-CAR-T cells did not affect transfection rate compared with that of healthy volunteers [(32.80±7.22)% vs (35.10±5.84)%, t=-0.554, P=0.593]. ②Incubation of CD19-CAR-T cells with 72 μg/ml Nivolumab did not affect CD19-CAR-T cell proliferation, but its cytotoxicity was significantly higher than that of CD19-CAR-T cells alone or patients' T cells +72 μg/ml Nivolumab (all P<0.001), there was no significant difference in the killing activity between the 72 μg/ml and 36 μg/ml Nivolumab treated CD19-CAR-T cells on Pfeiffer cells (P=0.281, 0.267, respectively), and they were all higher than those of 18 μg/ml Nivolumab treated CD19-CAR-T cells (all P<0.001). ③Different doses of PD-1 inhibitor Nivolumab combined with CD19-CAR-T cells does not affect the secretion of IFN-γ and IFN-α (all P>0.05). Conclusion: Combination of 36 μg/ml PD-1 inhibitor and CD19-CAR-T cells could reduce the drug toxicity and enhance the cytotoxicity.

Effects of Iron Overload on the Apoptosis and Function of Splenic CD8+ T Cells in Mice / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of iron overload on apoptosis and function of splenic CD8+ T cells in mice.</p><p><b>METHODS</b>Forty C57BL/6 mice were randomly divided into control groups, Iron overload (IO), IO+NAC and IO+DFX groups. The iron overload model was established by intraperitoneal injection of iron dextran, and saline was injected as the control. The levels of intracellular reactive oxygen species (ROS) and labile iron pool (LIP) were analyzed by measuring the mean fluorescence intensity (MFI) of 2-7 dichlorofluorescein (DCF) or calcein. The ratio of CD8+ T cells and the levels of IFN-γ, TNF-α, Granzyme-B, and perforin in CD8+ T cells were detected by flow cytometry. The CD8+ T cell apoptosis was determined by flow cytometry with Annexin V/PI double staining. Real-time PCR was used to detect the expression of IFN-γ, TNF-α, Granzyme-B, perforin, BCL-2, and bax at mRNA level in CD8+ T cells.</p><p><b>RESULTS</b>Iron overload was found by spleen iron staining and flow cytometry. The level of intracellular ROS in iron overload (IO) groups was higher than that of the control groups (P<0.01). The percentage of CD8+ T cells in spleen from mice with IO was lower than that in control groups (P<0.05). The expression of IFN-γ and Granzyme-B in CD8+ T cells in IO group were lower than that in control group, the expression of IFN-γ and Granzyme-B at mRNA level in CD8+ T cells was lower than that of control group (P<0.05). CD8+ T cell apoptosis in iron overload group was significantly higher than that in control groups (P<0.01); the expression of BCL-2 at mRNA level was lower than that in control group, but the expression of BAX at mRNA level was higher than that in control group (P<0.05). These effects could be reversed after treating iron-overloaded mice with DFX or NAC.</p><p><b>CONCLUSION</b>Iron overload can inhibit the ratio of CD8+ T cells of splenic cells in mice, decrease the expression of IFN-γ, Granzyme-B, increase the apoptosis of CD3+ CD8+/CD8-. These effects may be regulated through increasing the intracellular ROS level, and can be partially reversed after treating iron-overloaded mice with DFX or NAC.</p>

Establishment of an mouse model of iron-overload and its impact on bone marrow hematopoiesis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a mouse model of iron overload by intraperitoneal injection of iron dextran and investigate the impact of iron overload on bone marrow hematopoiesis.</p><p><b>METHODS</b>A total of 40 C57BL/6 mice were divided into control group, low-dose iron group (12.5 mg/ml), middle-dose iron group (25 mg/ml), and high-dose iron group (50 mg/ml). The control group received normal saline (0.2 ml), and the rest were injected with intraperitoneal iron dextran every three days for six weeks. Iron overload was confirmed by observing the bone marrow, hepatic, and splenic iron deposits and the bone marrow labile iron pool. In addition, peripheral blood and bone marrow mononuclear cells were counted and the hematopoietic function was assessed.</p><p><b>RESULTS</b>Iron deposits in bone marrow, liver, and spleen were markedly increased in the mouse models. Bone marrow iron was deposited mostly within the matrix with no significant difference in expression of labile iron pool.Compared with control group, the ability of hematopoietic colony-forming in three interventional groups were decreased significantly (P<0.05). Bone marrow mononuclear cells counts showed no significant difference. The amounts of peripheral blood cells (white blood cells, red blood cells, platelets, and hemoglobin) in different iron groups showed no significant difference among these groups;although the platelets were decreased slightly in low-dose iron group [(780.7±39.60)×10(9)/L], middle dose iron group [(676.2±21.43)×10(9)/L], and high-dose iron group [(587.3±19.67)×10(9)/L] when compared with the control group [(926.0±28.23)×10(9)/L], there was no significant difference(P>0.05).</p><p><b>CONCLUSIONS</b>The iron-overloaded mouse model was successfully established by intraperitoneal administration of iron dextran. Iron overload can damage the hepatic, splenic, and bone marrow hematopoietic function, although no significant difference was observed in peripheral blood count.</p>

Reactive oxygen species and fibrosis in tissues and organs - review / 中国实验血液学杂志

Reactive oxygen species (ROS) is a kind of molecules derived by oxygen in the metabolic process of aerobic cells, which mainly includes superoxide, hydroxyl radicals, alkoxyl, hydrogen peroxide, hypochlorous acid, ozone, etc. They can destroy the structure and function of cells through the damage of biological macromolecules such as DNA, proteins and the lipid peroxidation. ROS also can regulate the proliferation, differentiation and apoptosis of cells through several signaling pathways and participate in fibrogenesis of many organs including hepatic and pulmonary fibrosis. Recent study shows that ROS might have an important effect on the forming of myelofibrosis. Consequently, ROS plays a significant role in the fibrogenesis of tissues and organs. In this review, the relevance between ROS and common tissues and organs fibrosis is summarized.