CD300a TASR universally protects against NK cell alloreactivity. (A) Study design and demographic overview of the PBMC donors used. T cells are engineered to coexpress cloaking transgene and RQR8 via 2A self-cleaving peptide under control of EF1α promoter by nonviral HDR. (B) Percentage of NK cells expressing the indicated markers by flow cytometry from the 45 PBMC donors in panel A. (C) Phenotype by flow cytometry of the 3 engineered T-cell targets, gated on live single lymphocytes. Label indicates cloaking transgene. Gray dotted histograms indicate negative control T cells stained with the same markers. (D) Aggregate results as in panel C against 45 PBMC donors. Each data point represents IC50 value of the indicated cloaking ligand against PBMCs from 1 donor. Limit of quantitation in IC50 set to be twice the highest E:T ratio used. Wilcoxon matched pairs signed rank test. (E) Association of PBMC donor demographics from panel A with functional data from panel D, Kruskal-Wallis for ethnicity, Mann-Whitney for other. y-axis represents ratio of IC50 between CD300a TASR and HLA-E cloaking ligand, with 1 indicating equal protection. (F) Adaptive NK cell frequency by CMV serostatus of PBMC donors from panel A. Mann-Whitney U test. (G) Relationship between the adaptive NK cell frequency of the PBMC donor from panel F and functional potency of CD300a TASR (left) and HLA-E (right), normalized to noncloaked control from panel D. Dotted line represents linear fit of log-log transformed data. N = 45 PBMC donors.