B2M KO + CD300a TASR outcompetes HLA-E, CD47, and SIRPa engager cloaking ligands and protects against T and NK cells. (A) Phenotype by flow cytometry of engineered T cells expressing the indicated transgenes at the target locus by nonviral HDR, gated single cell lymphocytes. Gray dotted histograms indicate negative control T cells stained with the same markers. CD47 is endogenously expressed on T cells, and so the RQR8 epitope tag was coexpressed using 2A self-cleaving peptide to facilitate purification transgene-expressing cells. (B) NK challenge assay with B2M KO T cells expressing cloaking transgene from the B2M locus from panel A, challenged with 3 NK cell donors. Inset shows the NK cell phenotype by flow cytometry, gated CD3^–CD56⁺. N = 2 technical replicate curves per condition. (C) Competition assay of pooled T-cell targets from panel A cocultured with the indicated allo-T-cell clone and/or NK cell effector donor. Allo-T cells express TCRαβ specific for either minor histocompatibility antigen 2 (HA-2) or EMC7 peptide presented on HLA-A2. Target T cells are serotyped HLA-A2⁺, and effector cells are HLA-A2⁻. y-axis shows frequency of the indicated T-cell member after challenge with indicated effector cell. N = 3 technical replicates per condition.