RNA silencing of GM-CSF in CAR-T cells reduces the secretion of multiple inflammatory cytokines

Cells and reagents

K562 (ATCC, #CCL-243) and HEK-293T (ATCC, #ACS4500) cells were obtained from ATCC. Cryopreserved human adult purified PBMCs (MT-BIO, #PB010C) were purchased from MT- BIO. The cryopreserved PBMCs were thawed and cultivated in PRIME-XV T cell CDM (Irvine SCIENTIFIC, #91,154) at 1 × 106/ml supplemented with interleukin-2 (100 IU/ml) to prepare CAR-T cells. Anti-CD3/CD28 Dynabeads (Gibco, #1132D) were added at a bead-to-cell ratio of 1:1 for 24 h to stimulate and expand T cells. The amplified T cells were resuspended at 3 × 105/ml and transfected with lentiviruses (MOI = 3 × 105vg/cell) on RetroNectin (Takara, #T100B) pre-coated plates for 72 h. Transfection efficiency was assessed by detecting GFP expression through flow cytometry and the final transfection efficiencies were approximately at 30–50%.

Generation of GM-CSF knockdown CAR-T cells

Four sets of shRNA sequences (Supplementary Table. S1) for GM-CSF silencing were modified to be constructed on the pLVX-shRNA2 plasmid. The knockdown efficiency in K562 cells was compared through Western blot assay before application to CAR-T cells. The shRNA- expressing fraction was then amplified through polymerase chain reaction and inserted into the anti-human CD19 CAR (CAR19) vector. The CAR19 involved in this research contains an anti-human CD19 single-chain variable fragment (scFv), hinge and transmembrane (TM) regions, and intracellular signaling domains including coactivator 4-1BB as well as CD3 zeta. Lentivirus was produced in 293T cells with Lipofectamine 3000 (Thermo Fisher Scientific, # L3000015) and purified using ultrafiltration kit (Millipore, #UFC910096). Transfection was conducted as described above.

Flow cytometry analysis

For CAR19 expression assay, the transfected 293T cells were incubated with diluted biotinylated human CD19 (Acrobiosystems, #CD9-H8259) and then with BV605 Streptavidin (Biolegend, #405,229) following the manufacturer’s instruction. Flow cytometry was performed on BD LSR Fortessa X-20 Cell Analyzer. As for other cell surface markers, cells were harvested, washed in Dulbecco’s phosphate-buffered saline, and incubated with Human TruStain FcX (Biolegend, #422,302) to block the Fc receptor. Anti-CD3 (Gibco, #555,335, #555,340), anti-CD4 (Gibco, #555,349), anti-CD19 (BD, #562,440), anti-CD14 (Biolegend, #301,804), and anti-CD11b (Biolegend, #101,208) antibodies and Zombie NIR Fixable Viability Kit were added as needed for flow cytometry analyses. All flow cytometric data was analyzed on FlowJo X10.0.7r2.

Enzyme-linked immunosorbent assay

Nalm6 cells were co-cultured with CAR-T cells or mock T cells at 1:1 ratio for 16 h. Cell supernatant was collected for cytokine assays. ELISA kits of human IL6 (Biolegend, #430,504) and GM-CSF (Biolegend, 432,004) were used to detect the concentration of inflammatory cytokines according to manufacturer’s instructions.

Cell killing assay

Nalm6 cells were transduced with lentivirus for co-expression of luciferase-zsGreen (Hanbio Biotechnology) beforehand. Nalm6Luc + cells were later co-incubated with CAR-T cells or mock T cells at effector to target (E: T) ratios of 0.25:1, 0.5:1, and 1:1 for 12 h. D- Luciferin (Thermo Scientific, #8829) was added simultaneously to the cell culture. Residual live cells were measured by detecting bioluminescence on a Thermo Scientific Microplate Reader.

Monocyte cocultivation and endotheliocyte activation assay

Total monocytes were isolated from cryopreserved human PBMCs of the same donor by using the MojoSortTM Human Pan Monocyte Isolation Kit (Biolegend, #480,059) immediately after thawing. CD14 + monocytes were harvested, and about 2 × 105 was placed at the upper chamber of a 24-well transwell plate (pore size:3.0 μm), with equal numbers of CAR-T cells and Nalm6 cells cultured in the lower chamber. After 16 h, supernatant was collected for cytokine measurement.

Multi-analyte flow assay

Beads-based multi-analyte flow cytometry analysis was conducted using LEGENDplexTM Human Inflammation Panel 1 (13-plex, Biolegend, #740,809), which included IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17 A, IL-18, IL-23, and IL-33, to quantify cytokines and chemokines in the supernatant of cocultured cells. Data were analyzed via Legendplex Version 8.

Statistical analysis

Statistical significance was calculated using Student’s t test on GraphPad Prism. The results were expressed as mean (SD). Two-sided p-values < 0.05 were considered statistically significant.

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