Anti cd3 cd28 antibodies

Splenic B cells were isolated from 5 or 12 weeks old MRL/Lpr mice using B Cell Isolation Kit (Miltenyi Biotec). The purity of isolated B cells was over 97%. B cells were stained with CSFE before co-culturing with purified TCRβ+CD138+ or TCRβ+CD138- cells in the presence of anti-CD3/CD28 antibodies (BD Biosciences), phorbol 12-myristate 13-acetate (PMA)/ionomycin, or autoantigens [1 μg/ml of DNA or SM (Immunovision)]. DNA was isolated from MRL/Lpr splenocytes by hyperthemo treatment at 42°C for 4 h. After 3 to 4 days of incubation, cells were analyzed for CFSE dilution by flow cytometry. In other assays, after 10 days of culture, culture supernatants were analyzed for antibody production as well as IL-2 and IFNγ secretion by ELISA (R&D Systems). In CD4 blocking expriments, Ultra-LEAF™ purified CD4 antibody (clone GK1.5, Biolegend), or control rat IgG (Sigma-Aldrich) were added to T and B cell co-cultures. In some co-culture experiments, T and B cells were incubated either as mixed or separated with 0.4 μm pore sized polyester Corning Transwell^® membrane insert (Sigma-Aldrich).

To transduce human ATCs replication incompetent retroviral supernatant was prepared by transfecting 293T with DNA encoding our construct of interest, the Peg-Pam-e plasmid containing the sequence for MoMLV gag-pol, and the DRF plasmid containing the sequence for the RD114 envelope, as previously described.[48 (link)] Supernatant harvested at 48 or 72 hours post transfection was used to transduce human ATCs. ATCs were generated using PBMCs from healthy volunteer donors or patients with CD33⁺ AML, activated with anti-CD3/CD28 antibodies (BD Biosciences), and expanded with rh-IL2 50–100 I.U./mL twice weekly (Miltenyi Biotec; San Diego, CA). For ΔCD19 sel. iC9-CAR.CD33 CAR T-cells, we sorted ΔCD19 expressed cells with magnetic conjugated anti-CD19 antibody beads through MACS column (Miltenyi Biotec) between 7 to 9 days after ATCs expansion.

Rab37 knockout (KO) mice were generated as described in Kuo's report 23 (link). CD4⁺ and CD8⁺ T cells were isolated from splenocytes derived from wild-type (WT) and Rab37 KO mice using anti-mouse CD4 magnetic particles (BD Bioscience, #551539) and mouse CD8 T lymphocyte enrichment set (BD Bioscience, # 558471) according to the manufacturer's instructions. Cells were cultured in medium containing 10% Fetal Bovine Serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco), and 1% sodium pyruvate (Gibco) and incubated at 37 ^oC with 5% CO₂ in air. Purified naïve CD4⁺ and CD8⁺ T cells were stimulated with 2 μg/mL anti-CD3/CD28 antibodies (BD Bioscience, #553057/ #553294) for 24 h or 48 h.