Figure 5.

Activation and degranulation of primed T cells in presence of modified NK cells. (A) Representative flow plots showing the frequency of CD137⁺ cells within CD8⁺ (upper panel) and CD4⁺ (lower panel) T cell subsets after 24 h of co-incubation with the different NK cell cultures depicted above the plots. (B) Quantification of CD137⁺ frequencies among CD8⁺ (left) and CD4⁺ (right) T cells at two time points. Statistical analysis was performed by two-way ANOVA with matching by time points and Holm-Sidak testing. At both time points, the frequency of CD137⁺ cells within the CD8⁺ T cells was significantly lower when challenged with sc-HLA-E/B2M-KO NK compared to parental and sc-HLA-E NK cell containing cultures (mean ± SD; n = 7 for medium control, parental, sc-HLA-E, sc-HLA-E/B2M-KO NK cells, n = 4 for autologous and 3^rd party controls; black line and grey box indicate the mean of the baseline measurements ± 95% confidence interval). (C) Levels of CD107a normalized to the medium controls in CD8⁺ (left) and CD4⁺ (right) T cells 24 h after co-incubation with NK cells. Statistical analysis was performed using Friedman test with Dunn’s correction for multiple comparisons. Levels of CD107a were significantly lower for CD8⁺ T cells incubated with sc-HLA-E/B2M-KO NK cells compared to T cells incubated with parental or sc-HLA-E NK cells (mean ± SD; n = 7 for medium control, parental, sc-HLA-E, sc-HLA-E/B2M-KO NK cells, n = 4 for autologous and 3^rd party controls). (D) Lysis of NK cells by primed T cells at the effector targets ratios 4:1, 2:1 and 1:1 (calculated on CD8⁺ T cells) after 20 h of co-incubation. Statistical analysis was performed by two-way ANOVA with Holm-Sidak testing for multiple comparisons. The reduced lysis of sc-HLA-E/B2M-KO NK cells was statistically significant at E:T ratio 4:1 compared to parental and sc-HLA-E NK cells and at ratio 1:1 compared to sc-HLA-E NK cells but not to parental NK cells, yet the p value almost met the criterion with p = 0.0516 (mean ± SD; n = 5).