Cd19 microbeads

Primary mouse B cells isolated from the peritoneal cavity or spleen of C57BL/6J mice were isolated by use of kits purchased from Miltenyi Biotec including B cell Isolation kit (130–090–862), CD19 MicroBeads (130–052–201) and B-1a Cell Isolation Kit (130–097–413). Sorted CD19⁺ or B-1a cells were stimulated with LPS (5µg per ml, Sigma), anti-CD40 Abs (10µg per ml; BioXcell) and anti-IgM Abs (5µg per ml; Jackson ImmonoResearch) for 72 hours as described (15 (link)).

Twenty million (2 × 10⁷) ε-TRuC-T, CD28ζ-T, and BBζ CAR-T cells were co-cultured with 2 × 10⁷ Raji cells (effector-to-target ratio of 1:1) for 4 h. Raji cells were depleted using CD19 microbeads on a magnetic column according to manufacturer’s instructions (Miltenyi, Bergisch Gladbach, Germany). mRNA was extracted from purified T cells and subjected to gene expression analysis using pre-designed Human Immunology Codeset version 2 on nString nCounter (Nanostring Technologies, Seattle, WA). The raw counts were generated using the Human Immunology panel-2 and normalized using endogenous control genes. The change in T cell gene expression levels upon co-culture with Raji cells was calculated by dividing the normalized counts after co-culture with the normalized counts before co-culture to generate after-to-before ratio. A venn diagram of differentially regulated genes in TRuC and CAR-T cells was generated using R-program. Genes normalized to endogenous control were selected based on fold change (greater than two-fold or lesser than 0.5-fold) or p-value < 0.05. The heat map was generated for genes with fold change greater than 1.5 or lesser than 0.5 or p < 0.05 (Student’s t-test). The data analysis was carried out with R-program based nSolver (Nanostring Technologies, Version 3).

Tumor-derived single cell suspensions were split into halves. One half was depleted of B cells using CD19 MicroBeads (Miltenyi Biotech) according to the manufacturer’s instructions. The second half was subjected to the same procedures without addition of CD19 MicroBeads. After magnetic separation, cell suspensions (6 × 10⁵ cells/ml) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, L-glutamine and penicillin-streptomycin (Invitrogen) in 48 well plates for 6 days without any additional stimuli. The viability of CD4⁺ and CD8⁺ T cells and their capacity to produce cytokines was assessed at day 1 and 6 using LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Invitrogen) and intracellular cytokine staining as described above.

Blood was obtained in accordance with the policies established by the National Yang-Ming University Institutional Review Board. Blood was withdrawn from healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS gradient centrifugation (GE Healthcare, Chicago, IL). B cells were positively isolated from PBMCs using CD19 microbeads (Miltenyi Biotec, Auburn, CA). Cell purity was determined using anti-human CD20-APC (BioLegend, San Diego, CA) staining. B cell purity using CD19 microbeads purification was > 95% in all experiments. Cells were cultured in RPMI 1640 medium containing 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 10% fetal calf serum (Life Technologies, Grand Island, NY). CTLA-4-Ig and L6-Ig (control-Ig) for use in the in vitro assays were provided by Bristol-Myers Squibb. L6-Ig is a chimeric fusion protein consisting of the V region of the murine L6 antigen and the human IgG1 Fc portion, and it was used as a control in our studies.

Human primary B cells were prepared from adenoidal mononuclear cells by Ficoll Hypaque (PAN Biotech) gradient centrifugation as described in [66 (link)]. B cells were isolated using CD19 MicroBeads and MACS separation columns (Miltenyi Biotec), according to the manufacturer’s instruction.