Constitutive expression of CD300a TASR robustly protects B2M KO T cells against NK alloreactivity via CD300a agonism. (A) Experimental overview, NK challenge assay with T cells engineered to express cloaking transgene from the B2M locus via nonviral HDR concomitant with B2M KO. (B) TASR and HLA-I expression by flow cytometry of target T cells expressing CD300a TASR or RQR8 negative control transgene from the B2M locus, as well as HLA-I⁺ control T cells expressing GFP from the AAVS1 locus. CD300a TASR expression evaluated by antibody (left panel) and ligand-based (middle panel) staining. Dashed lines indicate negative control T cells stained with the same marker. (C) NK challenge assay with 3 NK cell donors against target T cells from panels A-B cocultured for 1 and 7 days. NK cells were cultured in 10 ng/mL IL-15, whereas resting NK cells are thawed, rested overnight, and cocultured in the absence of exogenous cytokines. N = 1 to 2 technical replicate curves per condition. (D) 1-day NK challenge assay against target T cells from panels A-B in the presence of 5 μg/mL of anti-CD300a (clone MEM-260), anti-CD300c (clone TX45), or mouse IgG1 isotype control antibody. N = average of 2 technical replicate curves per condition.