Cd28 37.51

Two MSCV-based γ retroviral vectors were used to target the Cd4 gene as described by Kotov et al. ²⁹ . One vector encoded Cas9 and the fluorescent protein mNeongreen and the other either Cd4 or control LacZ guide RNAs (gRNAs) and the fluorescent protein mAmetrine ³⁰ . Both vectors were produced by modifying the LMP-Amt vector from S. Crotty (La Jolla Institute) as described by Kotov et al. ²⁹ . Platinum-E cells (Cell Biolabs) were grown in complete DMEM (Life Technologies) and transfected with the aforementioned plasmids. Virus-containing supernatants were harvested several days later. B3K508 T cells were cultured with complete IMDM (MilliporeSigma) containing IL-7 (Tonbo Biosciences) and then activated in plates coated with CD3 (2C11; Bio X Cell) and CD28 (37.51; Bio X Cell) antibodies. The cells were transduced with retroviral supernatant and polybrene (MilliporeSigma) 24 and 40 hours after activation and transferred to uncoated plates without CD3 or CD28 Abs for five days, the last three in complete IMDM-containing IL-7 (Tonbo Biosciences). The cells were then stained with P5R:I-A^b or P5R:I-A^b-4E tetramers and then BV786-labeled CD4 (RM4–5; BD Biosciences) antibody and a fixable viability dye (Ghost Dye Red 780; Tonbo Biosciences). The stained cells were analyzed by flow cytometry as described below.