Novocyte

Peripheral blood was collected from the enrolled patient and processed with Ficoll (GE Healthcare) gradient centrifugation to isolate PBMC. T cells were purified from PBMC with the pan T isolation kit (Miltenyi). Purified T cells were activated with anti-CD3&CD28 dynabeads (Thermo Fisher) and transduced with 3rd generation lentivector encoding anti-CD19 or anti-BCMA CAR co-expressing IL6/IL1 blockers (scFv derived from Sirukumab and IL1RA), followed by electroporation with RNP complex of the sgRNA (targeting GM-CSF and TRBC) and Trucut V2 Cas9 protein (Thermo Fisher). T cells were further ex vivo expanded and analyzed for CAR expression and gene editing efficiency (Novocyte, Agilent). The CART cells were also tested for sterility and in vitro killing of leukemia cells Nalm6 (ATCC) expressing GFP or RPMI-8226 (ATCC) cells (Novocyte, Agilent).

T2 cells can be loaded with exogenous peptides and β2-microglobulin (β2m) to form specific pHLA on the cell surface. For peptide pulsing, T2 cells in the logarithmic growth phase were cultured at 5 × 10⁵ cells/mL in serum-free IMDM (Gibco, Grand Island, NY, USA) containing 10 μg/mL β2m (Sigma-Aldrich, St. Louis, MO, USA) and either 30 μg/mL irrelevant peptide RMFPNAYL or relevant peptide SLLMWITQC (synthesized by the Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) for 6–8 h at 37 °C with 5% CO₂ and 95% humidity. For the cell-binding assay, peptide-pulsed T2 cells were collected and resuspended in 100 μL ice-cold staining buffer (1% BSA in PBS) and then mixed with 1 μg NY-TCRmut (affinity-enhanced NY-TCR). After incubation on ice for 30 min, cells were washed twice with staining buffer, and FITC labelled goat anti-human IgG (H + L) (Beyotime Biotechnology, Shanghai, China) were then added and incubated on ice for 30 min. Finally, cells were examined by flow cytometry (NovoCyte^TM, ACEA Biosciences, San Diego, CA, USA) after being washed with staining buffer.

All samples were analyzed using a NovoCyteTM (ACEA Biosciences), LSR Fortessa, or C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA). The antibodies used included anti-MSLN-biotin (clone MB), Streptavidin-APC, anti-human CCR7-APC (clone 3D12), anti-human CD62L-PE (clone DREG-56), anti-human CD45RA-APC (clone HI100), anti-human CD45RO-PE (clone UCHL1), anti-human TIM3-PE (clone F38-2E2), anti-human LAG3-PerCP/Cy5.5 (clone 11C3C65), anti-human PD-1-APC (clone NAT105), anti-human CD27-PE (clone M-T271), anti-human CD28-APC (clone CD28.2), anti-human CD25-PE (clone BC96), anti-human CD69-APC (clone FN50), anti-human CD107a-APC (clone H4A3), anti-human CD3-APC (clone UCHT1), anti-human CD4-PerCP/Cy5.5 (clone OKT4), anti-human CD8-PE (clone OKT8), mouse IgG2a isotype control-APC (clone RMG2a-62), mouse IgG1kappa isotype control-PE, mouse IgG1kappa isotype control-PErCP/Cy5.5, and mouse IgG1kappa isotype control-APC (clone MOPC-21) (Biolegend, San Diego, CA, USA). All FACS-related staining procedures were performed on ice for 30 min, and cells were then washed with PBS containing 1% FBS before cytometry analysis. PB, spleen (SP), and tumor samples from mouse xenografts were treated with red blood cell lysis buffer (Biolegend), and the cells were stained with the corresponding antibodies.

Caco-2 cells were treated as described above. Following this, the cells of each group were collected and stained with DCFH-DA (15 μmol/L) for 20 min. Fluorescence intensity was recorded by flow cytometry (NovoCyte TM, ACEA Biosciences, San Diego, CA, USA) with the excitation of 485 nm and emission of 535 nm. The result was analyzed using NovoExpress software.

3 × 10⁵ DLD1 or HCT116 cells were seeded onto 6 well plates, incubated for 24 h at 37 °C, and treated as indicated. For cell cycle analysis, cells were trypsinized, resuspended and fixed with 70% ethanol at −20 °C for at least 1 h. Before analysis, the cells were resuspended in PBS containing 10 mg/mL RNaseA (Multi sciences, China) and 50 mg/mL propidium iodide (PI; Multi sciences, China) for at least 30 min. For apoptosis analysis, the supernatant was transferred to eppendorf tubes and cells were trypsinized and then collected to supernatant and washed twice with cold PBS, followed by staining with Annexin V-FITC/PI (Multi sciences, China) for 15 min. PI- and Annexin V-stained cells were analyzed immediately on a ACEA NovoCyteTM (ACEA Biosciences, USA) or Cytomic FC 500MCL (BECKMAN COULTER, USA).