Anti cd8a 53 6

GD2 CARs were detected with the 14g2a anti-idiotype antibody 1A7 (National Cancer Institute BRB Repository). Additional antibodies for flow cytometry include: anti-CD4 (GK1.5, BioLegend), anti-CD8a (53–6.7, BioLegend), anti-CD62 L (IM7, BioLegend), anti-CD44 (MEL-14, BioLegend), anti-GD2 (14G2a, BioLegend). Cells were washed with PBS containing 2% Bovine Serum Albumin before the addition of the antibody at a concentration of 0.2 µg per 1 × 10⁶ cells in 100 µL volume. After 30 minutes of incubation with antibodies at 4°C in the dark, cells were washed once with PBS before being resuspended in PBS with 2% Bovine Serum Albumin (BSA). Quantitative flow cytometry was conducted using the QIFIKIT (Agilent, Santa Clara, CA). The GD2 primary antibody (14G2a, BioLegend) was added to 1 × 10⁵ cells at a saturating dose (1 µg) and incubated at 4°C for 45 minutes. This was followed by washing and the addition of 0.5 µg of the secondary stain with APC-F(ab’)2-Goat anti-Mouse IgG (H + L) secondary antibody (Thermofisher) to samples and calibration beads. After a 30 minute incubation at 4°C, samples were washed and DAPI was added. All samples were analyzed with an LSR Fortessa or FACSAria II (BD Bioscience, San Jose, CA) and data were analyzed using FlowJo.

Samples of tumor, spleen, lymph node, and blood were harvested after killing the mice. Single-cell suspensions of tumors were obtained using a Tumor Dissociation Kit (Miltenyi). The single cells were then incubated in MACS buffer (PBS supplemented with 2% FBS and 1 mM ethylenediaminetetraacetic acid [EDTA]) containing 10 µg/mL CD16/CD32 antibody (2.4G2, BD PharMingen) for 30 min at 4 °C and then stained with the antibodies. Staining reagents included anti-CD45 (30-F11, BioLegend), anti-CD45 (30-F11, BioLegend), anti-CD3 (17A2, BioLegend), anti-CD3 (17A2, BioLegend), anti-CD4 (RM4-5, BioLegend), anti-CD8a (53-6.7, BioLegend), anti-CD25 (PC61, BioLegend), anti-CD44 (IM7, BioLegend), and anti-CD62L (MEL-14, BioLegend). Data were collected on an BD FACSAria Fusion flow cytometer and analyzed with FlowJo software (TreeStar). Dead cells and cell aggregates were excluded from analyses based on a LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (eBioscience), or DAPI, with forward scatter/side scatter characteristics. The immune cell gating strategy was as follows: T cell: CD45⁺ CD3⁺, CD4⁺ T cell: CD45⁺ CD3⁺ CD4⁺, CD8⁺ T cell: CD45⁺ CD3⁺ CD8⁺, B cell: CD45⁺ CD3⁻ CD19⁺.

Anti-CD3 (17A2, cat. 561388), anti-CD4 (RM4-5, cat. 553047), anti-CD44 (IM7, cat. 561859), anti-CD62L (MEL-14, cat. 560516), anti-B220 (RA3-6B2, cat. 553091), anti-Foxp3 (MF23, cat. 562996) and anti-CD23 (B3B4, cat. 561772) were purchased from BD company (Franklin Lakes, NJ, USA). Anti-CD8a (53–6.7, cat. 100734), anti-IgM (RMM-1, cat. 406531) and anti-CD21 (7E9, cat. 123411) were obtained from BioLegend (San Diego, CA, USA). For flow cytometry experiments, total eight axillary LNs were dissected from four each of normal and tumor-bearing mice and pooled separately. Cells were harvested and washed with 1X PBS. Cells were stained with antibodies within 1:100 dilution for 40 min at 4°C with gentle mix, then washed with 1X PBS prior to Attune NxT cytometer (Thermo Fisher Scientific) analysis. For Foxp3 staining, cells were mixed with 1 ml of Fix/Perm solution and incubated at room temperature for 15 min. Cells were washed with 1X PBS and stained with anti-Foxp3 antibody for flow cytometry analysis.