IFN-γ plays a pivotal role in BN-CD38 antileukemic activity. (A-I,L-M) Total MNCs of 7 patients with AML (see supplemental Table 1) were treated with 1.0 ng/mL of BN-CD38, BN-CD38Mut, or control human IgG for 5 days. Moreover, cells from 4 of the patients were also treated with BN-CD38 (1.0 ng/mL) and 2.0 μg of αIFN-γ antibody for 5 days. The treated MNCs were subjected to CyTOF immunophenotyping comprising 36 surface markers tailored to detect AML primary cells and different immune subsets. Analysis was performed with the Cytobank platform. (A) Supervised high-fidelity FlowSOM (“self-organizing maps”) based on vi-SNE 2D analysis for 2 representative patients with AML (1 PB and 1 BM) showing that BN-CD38 reduces CD38^pos and CD38^neg AML cells and expands T-cell subsets. Equal number of events were analyzed for each treatment group for each patient, and bulk MNCs were gated. (B-C) Violin plots showing BN-CD38 but not BN-CD38Mut significantly decreases CD38^pos and CD38^neg AML cells event count. (D-F) Violin plots showing BN-CD38 and not BN-CD38Mut expands CD8^pos effector memory (EM) and terminally differentiated EM CD45RA^pos cells (TEMRA), memory T regulatory cells (Tregs), and NK T cells. (G-H) Data-driven self-stratifying CITRUS (cluster identification, characterization, and regression) analysis of 5 CD34^pos AML PB samples subjected to CyTOF immunophenotyping revealed that 7 clusters (red circles) were significantly changed (FDR < 0.01) between BN-CD38 and control groups (IgG and BN-CD38Mut). Clusters (599995, 599997, and 599998) were significantly less abundant with BN-CD38 treatment and were enriched with AML specific markers CD34, CD33, and CD117 (c-Kit), and negative for CD3 T-cell marker. Clusters (599990, 599993, 599994, and 599996) were significantly abundant with BN-CD38 treatment and are enriched with T-cell surface markers and phagocytic classical monocytes (CD14⁺CD16^negHLA-DR⁺). (I) Bar graphs of 7 clusters differentially and significantly (FDR < 0.01) changed with BN-CD38 compared with control human IgG and BN-CD38Mut. (J) Total AML cells of 4 patients with AML were treated with 1.0 ng/mL BN-CD38, BN-CD38Mut, CD38 NB, and control human IgG in combination with 2.0 μg rat control IgG or 2.0 μg rat anti-human IFN-γ antibody for 48 hours. RNA was extracted and subjected to CD38 qRT-PCR. Each sample was normalized to GAPDH followed by normalization to control human IgG for each patient and shown as F.C. over IgG. Two-way ANOVA with multiple comparisons was used to calculate statistical significance between different groups; ∗∗∗∗P < .0001. (K) Cells treated in panel J were also collected and subjected to flow cytometry surface staining of CD45, CD34, and CD38. CD38 expression was determined in CD45^DIM AML population. Two-way ANOVA with multiple comparisons was used to calculate statistical significance between different groups; ∗∗∗P < .001 and ∗∗∗∗P < .0001. (L) Four of the patient samples (4 PB samples from patients with CD34^pos) were also treated with BN-CD38 (A; 1.0 ng/mL) and 2.0 μg of αIFN-γ antibody for 5 days. The treated MNCs were subjected to CyTOF immunophenotyping. Unsupervised high-fidelity FlowSOM (self-organizing maps) based on vi-SNE 2D analysis for PB-derived MNCs of 3 representative patients with AML showing that anti-human IFN-γ antibody restores AML cells, specifically LSCs in the presence of BN-CD38. (M) Violin plot representation showing that anti-human IFN-γ antibody in the presence of BN-CD38 rescues CD34^posCD38^neg LSCs compared with BN-CD38 alone. For panels B through F and panel M, event counts of BN-CD38 (A) and BN-CD38Mut were normalized to control human IgG and normalization was converted to log scale F.C. The paired Student t test was used to calculate statistical significance; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.