Fig. 3: Immunophenotyping by mass cytometry with stochastic clustering to compare immune reconstitution in patients compared to healthy controls and FC21-NK cell products.

Blood was obtained at the indicated timepoints, and mononuclear cells (MNC) were isolated, processed, and labeled with a 34-parameter panel of heavy-metal conjugated antibodies (Supplementary Information Table S1), along with healthy subject MNC and expanded FC21-NK cell products as controls. Events were collected on a CyTOF 2 mass cytometer (Fluidigm). Events were filtered by sequential gating on live, singlet (event length vs. 191Ir), non-apoptotic (PARP-negative), and hematopoietic (CD45+) cells, and then clustered by visual interactive stochastic neighbor embedding (ViSNE, CytoBank) on CD3, TIGIT, NKP30, NKP46, CD56, NKG2D, CD94, and CD57, using equal sampling to unbias differences in sample event number. ViSNE clusters corresponding to T-cells, standard NK cells, FC21-NK cells, and any remaining MNC were created using the reference samples. The percentage of cells in clusters 1 through 4 were quantified for each sample. Ki67 staining within the gated populations was determined as a surrogate for proliferation. A shows representative plots from two patients, one healthy subject, and one NK cell product, showing expression of key activating surface markers, perforin, and Ki67 across four broad phenotypic clusters identified. Cluster 1 (bottom left) consisting of CD3⁺ T cells, cluster 2 (top middle) of CD3⁻CD56^dimNKG2D^dimCD57⁺ “standard” NK cells, cluster 3 (top right) consisting of CD56^brNKG2D^brNKp46^brCD57⁻ (“superbright”) NK cells corresponding to the phenotype of the infused FC21-NK cell product, and cluster 4 (bottom middle) consisting of all remaining cells. Cluster 3 identifies a unique phenotypic signature associated with the FC21-NK cells that is not present in healthy subjects and persists in patients at day 14 (7 days after adoptive transfer) and later (Supplementary Information Fig. S2). B NK cell immune reconstitution in patients over time maintains FC21-NK “superbright” phenotype with high proliferation, expressed as percent of total cell events, of cluster 3 (superbright FC21-NK cells) in healthy donors, FC21-NK cell products, and in patients receiving FC21-NK cell products (across all timepoints). C Proportion of Cluster 1 (T cells) and total NK cells (Cluster 2 + Cluster 3) in blood of study subjects across time. D The ratio of NK cells and T cells for all patients and timepoints assessed (n = 24). E Ki67 staining in FC21-NK cells (Cluster 3), standard NK cells (Cluster 4), and T cells (Cluster 1) in four representative patient samples obtained at day 14 (7 days after the NK cell infusion at day 7). F, G The percent of Ki67+ and Ki67 mean metal intensity (MMI), respectively, in standard NK cells, FC21-NK cells, and T cells for all patients and timepoints assessed (n = 24). Bars and whiskers represent median ± interquartile range, P < 0.0001. H NK cells in clusters 2 (standard NK) and 3 (FC21-NK) as assessed for expression of NKG2C across all patients at all timepoints, with FC21-NK cell infusion products and healthy subjects shown for reference. I the percent of NKG2C + NK cells from all patients at all timepoints, with early (Days 7 and 14) and late (>day 28) timepoints pooled. J NK cells (CD3−/CD56+) gated and assessed for KIR expression and summed for total percentage of KIR + NK cells, and then K plotted across time, with early and late timepoints pooled.