Ying yang git it1/31/2024 The average KD ratio in shGM2-CAR19 T cells and shGM4-CAR19 T cells reached 89% and 98.9%, respectively. The GM-CSF concentrations were 2,815.42 ng/ml in the shGM2-CAR19 T group and 284.22 ng/ml in the shGM4-CAR19 T group (Fig. The average GM-CSF concentration in the regular CAR19 T supernatant was 25,086.21 ng/ml. Cell supernatant was collected, and enzyme-linked immunosorbent assay (ELISA) experiments were conducted to detect the levels of GM-CSF. We used Nalm6 cells (a cell line of CD19 + B cell acute lymphoblastic leukemia) as target cells to coculture with CAR-T cells or mock T cells for 16 h. The transfected T cells were divided into five groups: mock T cells (GFP +), CAR19 T cells (CAR19 +, GFP +), shGM2-CAR19 T cells, shGM4-CAR19 T cells, and scramble-CAR19 T cells (Supplementary Fig. ![]() * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant)Ĭryopreserved human adult purified PBMCs were used to produce activated and amplified T cells. ( Each experiment was repeated for at least three times, and the results were expressed as mean (SD). The concentrations of IFN-α2, IL-12p70, IL-18, IL-23, and IL-33 were below the minimum value and not shown in this figure. (f) Cytokine profiles of co-cultured supernatants were measured by multi-analyte flow assay. (e) Concentration of GM-CSF (left) and IL-6 (right) in the supernatant collected after coculturing CAR-T/Nalm6/ CD14 + cells for 16 h assessed by ELISA. (d) Diagram of the space division of CAR-T, Nalm6 and CD14 + cells cocultured in transwell (pore size = 0.3 μm). (c) Cytotoxicity of conventional CAR19 T cells and GM-CSF KD CAR19 T cells were compared 12 h after cocultivation with Nalm6 Luc+ cells. Concentration of GM-CSF in the supernatant was analyzed by ELISA. (b) CAR-T cells (or mock T cells) were cocultured with CD19 + Nalm6 cells for 16 h. (a) Schematic presentation of the GM-CSF KD-shRNA-CAR19 construct used in this study. Regular manufacturing procedures would suffice to produce GM-CSF KD CAR-T cells and achieve comparable gene silencing efficiency to gene knockout, thereby promoting the clinical application of CAR-T cell therapy.Ĭharacterization and effects of GM-CSF KD CAR-T cells. In the present study, we inserted a short hairpin RNA (shRNA)-expression cassette in the CAR vector to reduce GM-CSF secreted by CAR-T cells. Meanwhile, the knockout efficiency of GM-CSF through double transduction could be unpredictable, and additional examinations would be needed. These additional manufacturing procedures might hamper the viability of CAR-T cells. However, these studies were conducted using CRISPR/Cas9 or TALENs, which require either a dual transduction of CAR-expressing vector and GM-CSF-gRNA-lentiCRISPRv2 lentiviruses or electroporation of mRNA encoding TRAC TALEN arms and subsequent AAV transfection to prepare GM-CSF KO CAR-T cells. Previous studies reported that gene editing of GM-CSF in CAR-T cells could prevent against adverse effects in vitro and in xenograft mouse models without damaging the cytotoxicity of CAR-T cells. Inflammatory cascade is thus initiated and leads to CRS and neurotoxicity. Stimulated monocytes–macrophages massively produce cytokines including IL-6 and IL-1. Activated CAR-T cells release various inflammatory cytokines including GM-CSF, which subsequently plays a major role in the stimulation and differentiation of innate monocytes–macrophages lineage. Various inflammatory factors were found elevated in serum samples after infusing CAR- T cells these factors include interleukin-6 (IL-6), interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble interleukin-6 receptor (sIL-6R), macrophage inflammatory protein-1 (MCP-1), interleukin-1 (IL-1), etc. ![]() Cytokine release syndrome (CRS manifested by fever, hypotension, hypoxia, and target-organ damage) and neurotoxicity (characterized by headaches, confusion, seizure, and other neurologic manifestations) are the most representative. However, immunotherapy-associated side effects strongly hampered the development of CAR-T cell therapy, as severe complications are life-threatening for patients. Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated extraordinary potentials in the treatment of acute lymphocytic leukemia and B-cell lymphoma.
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