Patient Enrolment and Serum Cytokine Detection
The study population consisted of patients from a trial conducted in our clinical centre. The phase I study of a fully human anti-BCMA CAR (CT103A) was registered in the Chinese Clinical Trial Registry as ChiCTR1800018137)[21]. Within 24 hours after absolute quantification of peripheral CT103A by droplet digital polymerase chain reaction, the remaining serum was collected. Samples were frozen at −80 °C until use. The levels of serum cytokines were measured using the Bio-Plex Pro Human Cytokines, 48-Plex screening assay (Bio-Rad Life Sciences, Hercules, CA, USA). Concentrations below the limit of detection (LOD) were assigned a random value between 0 and the limit of detection to avoid an artificial reduction in the standard deviation.
Cell lines
All cell lines were purchased from ATCC or DSMZ and maintained in our lab. K562, RPMI-8266, and MM1S cells were stably transduced firefly luciferase and were cultured in complete medium (RPMI-1640 medium (Gibco) supplemented with 10% foetal bovine serum (FBS, Gibco) and 2 mM L-glutamine (Gibco)) in a humidified 5% CO2 atmosphere at 37 °C. The packaging cell line 293T was maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 2 mM glutamine (Gibco).
CAR-T cell generation and cell culture
The generation and transduction of CAR T cells were performed as reported[21]. For in vitro experiments, we isolated peripheral blood mononuclear cells (PBMCs) by leucapheresis from the peripheral blood of 3 healthy volunteer donors. T cells were positively selected from PBMCs using CD3 microbeads (Miltenyi Biotec) as directed by the manufacturer. T cells were activated with Dynabeads Human T-Activator CD3/CD28 magnetic beads (Invitrogen) at a 1:1 bead: cell ratio and then cultured for 24 hours in TexMACS GMP Medium (MACS) supplemented with 200 U/ml recombinant human IL-2 (PeproTech), 5% FBS (Gibco), and 2 mM glutamine (Gibco). T cells were then transduced with BCMA-CAR for 24 hours using lentiviral particles. After transduction, cells were grown ex vivo in the presence of IL-2 (200 U/ml, PeproTech) or IL-2 (200 U/ml, PeproTech) + IP-10 (10 ng/mL, PeproTech) to the desired cell density. BCMA-CAR-T cells were incubated at 37 °C in 5% CO2, and the culture medium and cytokines were replaced every two days.
In vitro migration assay
IP-10 (200 ng/mL, PeproTech) was added to serum-free medium in the lower chamber of Transwell plates (Corning). Then, CAR-T cells pretreated with or without anti-human CXCR3 were added to the upper chamber and incubated for 2, 4 or 6 hours at 37 °C. The number of CAR-T cells that migrated towards the chemokine IP-10 was determined by cell counting. The phenotype of CAR-T cells in the lower chambers was measured with flow cytometry.
Flow cytometry and antibodies
Cell surface marker expression was determined using a flow cytometer (NovoCyte D1040, ACEA Biosciences, Hangchow, China). CAR-T cells were stained with fluorescently labelled antibodies against cell surface markers as follows: CD3-APC, CD4-Percp/Cy5.5, CD8-APC-Cy7, CD45RA-Brilliant Violet 605, CD62L-PE, PD1-BrilliantViolet 421, CD183-APC, LAG-3-PE/Cy7, and TIM-3-APC (all from Biolegend). CAR-T cells were stained with FITC-labelled human BCMA Fc Tag protein (Acro Company) to evaluate specific binding and identify CAR-positive cells. Cells were washed with PBS and counted. A total of 106 cells were suspended in phosphate-buffered saline (PBS, Gibco) with 1% bovine serum albumin (BSA, Gibco) for 15 minutes and then incubated with the specific antibody for 30 minutes at 4 °C. Data were acquired by FACS and analysed with NovoExpress 1.5.0 and FlowJo 10.8.1 software.
Proliferation assay
CAR-T cells were isolated 14 days after the first stimulation and stained for flow cytometry with the CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher) according to the manufacturer's instructions. CAR-T cells were cocultured with tumour cells in vitro at an effector:target ratio of 1:1 in 24-well plates for three days and then analysed by flow cytometry.
CD107a Degranulation Assay
CAR-T cells were resuspended in RPMI medium and then cocultured with the target cells (RPMI-8266) at a 1:1 ratio in an incubator at 37 °C. The anti-CD107a-APC antibody (BioLegend) was added at a concentration of 20 μL/mL. Monensin (Golgi-Stop, BD Biosciences) was added at a final concentration of 6 mg/mL one hour later, and incubation was allowed to continue for another three hours. CD107a expression on CAR- and CD8-double-positive cells was identified using CAR-FITC-labelled Human BCMA Fc Tag protein (Acro) and anti-CD8-APC-Cy7 (BioLegend) antibodies.
Cytotoxicity assay.
The cytotoxicity of CT103A CAR-T cells was evaluated by a luciferase assay. Fluc-expressing target cells (K562, RPMI-8226, and MM1S) were resuspended at 1 ×105 cells per well in RPMI medium supplemented with 10% FBS in 96-well tissue culture plates. CAR-T cells were loaded at different effector: target cell ratios. Cells were lysed in lysis reagent after a 24-hour incubation period (Promega). The luminescence of the lysates was measured using an enzyme labelling instrument (SynergyTM 2, Bio–Tek, Vermont, USA).
Cytokine secretion
CAR-T cells were cultured with target tumour cells in 96-well round-bottom plates (Corning) at a 1:1 (1×105 cells each) effector:target ratio for 24 hours. The supernatant was then collected, and the production of IL-2, Granzyme B, IFN-γ, and TNF-α
by CAR-T cells was determined by an enzyme-linked immunosorbent assay (ELISA) (Neobioscience) per the manufacturer's instructions. The cytokine concentration was quantified from an eight-point standard curve. Data were acquired on a full wavelength enzyme labelling instrument (Epoch2, Bio–Tek, Vermont, USA), and concentrations were obtained in units of pg/mL.
Statistical analysis
The mean ± SD values were determined in the experiments. The nonparametric test or student’s t test was used to determine significant differences between the experimental and control groups. Comparison of survival curves was performed using the log-rank test. Statistical significance was defined as * (0.01 < P < 0.05), ** (P < 0.01), and *** (P < 0.001). All data analyses were performed using Prism software (GraphPad Prism 8) and IBM SPSS Statistics software (version 25).