Anti ccr7 bv421

Expression of the various CD19-CARs on T cells was detected using either biotinylated anti-mouse FMC63 scFv monoclonal antibody (Biowan) followed by staining with streptavidin-phycoerythrin (PE) (BD Biosciences), or with an allophycocyanin (APC)-labeled anti-mouse FMC63 scFv monoclonal antibody (Biowan). 1 × 10⁶ cells were stained for cell surface markers to analyze T cell differentiation and exhaust status. The following antibodies were used: anti-CD3-FITC (BD Biosciences), anti-human CD3-APC (BD Biosciences), anti-CCR7-BV421 (BD Biosciences), anti-CD 45RO-APC (BD Biosciences), anti-CD4-BB515, anti-CD8-BV510 (BD Biosciences), anti-CD45RA-PE- Cy7 (BD Biosciences), anti-CD62L-PE (BD Biosciences), anti-PD-1-APC (BD Biosciences), anti-TIM-3APC (BD Biosciences), and anti-LAG-3-APC (BD Biosciences). Samples were analyzed on either FACSCanto II flow cytometers (BD Biosciences) or Novocyte3110 (AECE).

After resting 2-3h in complete medium at 37°C, thawed PBMC were stained with the following mAbs: anti-CD4-Alexa Fluor 700 (BioLegend Inc., San Diego, CA, USA), anti-CD8-PE-Texas Red (Invitrogen), anti-CD45RA-Qdot655, anti-CCR7-BV421, anti-HLA-DR-V500 (BD Biosciences), and anti-PD-1-BV711 (BioLegend). Cells were subsequently fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), stained with anti-Ki67-PerCP eFluor710 (eBioscences, San Diego, CA) and anti-perforin-FITC (Diaclone, Besançon, France), and acquired with a LSR II Fortessa flow cytometer (BD Biosciences). Analysis was performed with FlowJo software (FlowJo LLC, Ashland, OR); after gating on total memory CD4⁺ and CD8⁺ lymphocytes, the percentage of IL-7R^pos and IL-7R^neg cells was calculated, as well as the percentage of IL-7R^pos and IL-7R^neg CD4⁺ T cells expressing Ki67, HLA-DR, PD-1 and perforin.

For positive rate analysis, CAR-T was incubated with biotinylated goat antimouse F(ab)2 (Jackson ImmunoResearch, USA, 109-066-006) for 30 min, followed by streptavidin-PE (BD Biosciences, 554061). Flow cytometry was performed by CantoII (BD Biosciences) and analyzed by NovoExpress (Agilent, USA). B7H3 PE-conjugated antibody (Biotechne, FAB1027P) was used for the detection of B7H3 on the cell membrane. Anti-human CD3-APC (BD Biosciences, 555342), anti-CCR7-BV421 (BD Biosciences, 740052) and anti-CD45RO-APC (ThermoFisher, 17-0457-42) antibodies were used for evaluation of the CAR-T differentiation. APC-conjugated PD1 and LAG3 antibodies (ThermoFisher, 17-9981-82, 17-2239-42) were used to reflect the exhaustion. The apoptosis of CAR-T was detected after co-cultured with target cells at E:T = 1:1 for 3 days by Annexin V-FITC and PI (Vazyme, China, A211-01).