Escape of HLA–reduced primary human T cells from allogeneic recognition in vitro. (A) Percentage of CD137⁺ CD8⁺ effector cells after 48 hours of coculture with indicated target cells. Effector cells were peripheral mononuclear blood cells from indicated donors, cocultured for 7 days together with PBMCs from donor A (allo priming); donors only share an allele for HLA A2 or HLA A3 as indicated; target cells were T cells from donor A without (WT) or with HLA class I reduction, sorted for CD8⁺ and HLA BC⁻ and A2⁻ A3⁻, A2⁺ A3⁻ or A2⁻ A3⁺; HLA–reduced target cells with HLA BC⁻A2⁻A3⁺ (left) or HLA BC⁻A2⁺A3⁻ phenotype, respectively, signify a synthesized HLA-match; statistical testing was done using an ordinary 1-way ANOVA and Tukey multiple comparisons test, and results are only shown for selected comparisons; n = 2-3 technical replicates; ns, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (B) Quantification of CD107a⁺ NK cells (percentage of living CD56⁺ CD8⁻ lymphocytes) from 3 different indicated donors after 5 hours of coincubation with indicated target cells; statistical testing was done using an ordinary 2-way ANOVA and Tukey multiple comparisons test and results are only shown for selected comparisons; n = 2-3 technical replicates; ns, not significant; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (C) Expression of HLA-E for target populations as assessed by flow cytometry; statistical testing was done using an ordinary 2-way ANOVA and Tukey multiple comparisons test and results are only shown for selected comparisons; n = 2-3 technical replicates; ns, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. n/a, not performed experimental conditions.