Anti cd3 clone okt3

B7-H3 CAR T cells were prepared as we previously described (55 (link)). Deidentified peripheral blood mononuclear cells from normal human donor blood (1 × 10⁶ cells/well) were activated with 1 μg/mL anti-CD3 (clone OKT3; Miltenyi Biotec, Germany) and 3 μg/mL anti-CD28 antibodies (clone CD28.2; BD Biosciences, CA). T cells were expanded by addition of 10 ng/mL of IL-7 and 5 ng/mL IL-15 on day 1. Cells were transduced on day 2 with retroviral particles of the B7-H3 CAR construct using RetroNectin (Takara Bio Inc., Japan) precoated 24-well plates (39 (link)). CAR T cells were collected and stored on days 13 to 14.

Freshly isolated live cells were cultured in 24 well plates (10⁶ cells per well) in T cell culture medium supplemented with 6000 IU/ml IL-2 for ~2 weeks at 37°C 5% CO₂. Medium was refreshed approximately every 4 days. Wells were split when a monolayer of cells was observed in the entire well. After this initial culture period, cells were cultured using a standard rapid expansion protocol (REP) for another two weeks: the culture medium was replaced with T cell culture medium supplemented with 3000 IU/ml IL-2 and anti-CD3 (clone OKT3; Miltenyi Biotech), also containing 8–10 × 10⁶ irradiated (40 Gray) PBMCs pooled from 10 healthy donors. Of note, comparison of the use of allogeneic versus autologous feeder cells during the TIL expansion phase revealed that both have a similar risk of emergence of new TCR clonotypes.^{31 (link)} Cells were passaged when multiple clusters of dividing T cells were observed, which was usually once or twice during the two-week REP culture period. The expanded T cells were cryopreserved in freezing medium.

The AUTO6NG CAR T cell production method was prepared as described previously.¹¹ (link) Briefly, peripheral blood mononuclear cells from healthy donors were activated with anti-CD3 (clone OKT3, 100 ng/mL, Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-CD28 monoclonal antibodies (clone 15E8, 100 ng/mL, Miltenyi Biotec) and human recombinant human IL-7/IL-15 cytokines (10 ng/mL, PeproTech, London, UK) to induce T cell proliferation. The cells were then transduced with two retroviral vectors: Vector A encoded a GD2 CAR and SHP2/TGFβRII dominant-negative modules, and Vector B encoded a GD2 CAR and IL7R CCR module. The cells were expanded for 5 days after initial transduction and were assessed by flow cytometry for transduction efficiency and by ddPCR analysis for VCN.

Frozen PBMCs of 24 and 46 kidney transplant recipients belonging to group 3 (ABMR) and group 1 (normal/subnormal biopsies), respectively, were retrieved from Centre de Resources Biologiques of Nantes (CRB). Frozen PBMCs were thawed and rested overnight in complete RPMI medium. 10⁶ PBMCs were cultured for 4h in 24-well flat bottom plates at 37°C in 5% CO² and, when indicated, treated with plate-bound anti-CD3 (clone OKT3; 2 µg/mL), plate-bound anti-CD16 (clone 3G8; 10 µg/mL), soluble anti-CD28 (clone CD28.2; 2 µg/mL) and/or soluble IL-15 (10 ng/mL; Miltenyi Biotec). Anti-CD107a PE (clone H4A3) and Brefeldin A (5 ug/mL, Sigma) were added at culture initiation. After stimulation, cells were first stained for cell surface markers (CD3 [clone UCHT1], CD8 [clone HIT8a], CD45RA [clone HI100], CD28 [clone CD28.2] and CCR7 [clone G043H7]), followed by fixation and permeabilization (Intracellular Fixation & Permeabilization Buffer Set, Thermo Fisher) and stained for intracellular cytokines TNFα [clone Mab11] and IFNγ [Clone B27]). Cells were acquired with a Celesta flow cytometer (BD Immunocytometry Systems) and the data analyzed using FlowJo Version 10.8.0 (TreeStar). The percentage of cells expressing TNFα/IFNγ and CD107a after stimulation were compared using non-parametric tests.