F16IL2 and BI 836858 combination therapy expands and activates NK cells. (A) Lymphocyte subsets in BM and PB were analyzed using flow cytometry at different time points during study treatment. Lymphocytes were immunophenotypically defined as CD45⁺/FSC^low (for gating strategy see supplemental Figure 5). The most significant changes were observed in the NK-cell compartment. CD56 immunohistochemical stainings of pretherapeutic and posttherapeutic BM biopsies (UPN004) (B) and CD56 vs CD3 flow cytometry plots (UPN011) (C) illustrate progressive expansion of NK cells in BM during F16IL2 and BI 836858 combination therapy in 2 representative patients. (D) NK cell percentages (of lymphocytes) significantly increased over time in BM and PB. (E) Absolute number of NK cells in BM and PB during combination therapy. (F) F16IL2 and BI 836858 combination therapy resulted in a significant expansion of FcγRIIIA/CD16⁺ NK cells capable of mediating ADCC. (G) Ratios of CD56^dim vs CD56^bright NK cells indicated a shift toward CD56^bright cells during combination therapy, but also CD56^dim NK cells increased in absolute numbers (H). (I-J) CD56^dim and CD56^bright subsets expressing FcγRIIIA/CD16⁺ significantly expanded. (K-L) Number of NK cells expressing the natural cytotoxicity receptors NKp30 or NKp46 significantly increased over time. (M) Absolute numbers of Tregs peaked at the end of C1, with a subsequent decline during C2. P values indicate significant changes from values at screening as follows (mixed effects analysis of variance with Holm-Šidák corrections): *P < .05; **P < .01; ***P < .001. Data are presented as the mean ± standard error of the mean.