Figure 3. Evaluation of subpopulations of NK cells present in the infusion product over time.

(A) Median proportion of total PBMCs that were CD56⁺CD3^– at the indicated time points. Days after infusion of the CIML NK product are indicated above the time point labels. (B) UMAP of NK cell clusters defined with single-cell RNA sequencing at day +28 compared to the infusion product for all patients whose CIML NK cells expanded. The CD56^dim NK cell subpopulations that persist include Dim1, Dim2, CD56^bright, and adaptive NK cells. (C) Identification of defined NK cell subpopulations based on transcriptional profiling reveals the expansion of several CD56^dim subpopulations and adaptive CD56^dim NK cell populations. The heatmap shows the top 5 differentially expressed genes in each NK cell cluster when compared with other clusters using Wilcoxon’s rank-sum test (log[fold change] threshold = 0.25). (D) Volcano plot showing the top differentially expressed genes in all CD56^dim clusters between infusion and day +28; fold change cutoff = 0.5, P-value cutoff = 10 × 10^–32. Differential gene expression was determined using the nonparametric Wilcoxon’s rank-sum test. (E) Violin plot to show the expression of select genes within the CD56^dim, CD56^bright, and adaptive CD56^dim NK populations at infusion (red) and day +28 (green) time points. Shown are the most differentially expressed markers as well as genes associated with NK cell activation. (F) Evaluation of NK cell subpopulations over time as a proportion of total NK cells in each of the clinical trial patients treated with CIML NK cell therapy. Key subpopulations include adaptive NK cells (CD56⁺CD3^–CD57⁺KIR⁺NKG2C⁺FcεRI^–), CD56^bright NK cells (CD56^hiCD3^–NKG2A⁺IL-7R⁺), CD56^dimNKG2A⁺ NK cells, and CD56^dimKi67⁺ NK cells, among others. The sample time points for each patient are labeled. INF, at time of infusion.