N-803 promotes donor NK and recipient CD8 T-cell expansion in vivo. (A) WUSTL and UMN trial schemas. Briefly, relapsed/refractory AML patients were lymphodepleted with fludarabine (25 mg/m³ × 5) on study days -6 to -2 and cyclophosphamide (60 mg/kg × 2) on study days -5 and -4. On study day -1, related, haploidentical donors were apheresed, NK cells were purified (WUSTL) or enriched (UMN) and activated with IL-12, IL-15, and IL-18 (WUSTL), IL-2 (UMN cohort 1), or N-803 (UMN cohort 2). Products were washed and infused into patients on study day 0 (NK purity for each cohort indicated as a percentage). Infused products were supported with IL-2 (WUSTL/UMN cohort 1) or N-803 (WUSTL/UMN cohort 2). (B) N-803 concentration in the PB from patients at the indicated times. (C) Donor NK cell expansion over time, as determined by flow cytometry between IL-2- (blue) and N-803 (purple) supported WUSTL patients (IL-2 n = 6; N-803 n = 7). (D) Fold reduction in cells from day 21 compared with maximal measure NK cells, typically days 8 through 14. (E) Representative overlay viSNE plot of purified donor NK cells (baseline, BL), infusion product (activated, ACT), and in vivo differentiated donor ML NK cells assessed by mass cytometry. (F-J) WUSTL patient CD8 T cells from PBMC and bone marrow (BM) were assessed by mass cytometry at the indicated days after NK cell infusion. (F-G) Summary data depicting recipient CD8 T-cell frequency (of CD45⁺ lymphocytes) in the (F) PBMC (IL-2; day 7 n = 8, day 14 n = 3; N-802 n = 6) and (G) BM (day 7 n = 4, day 14 n = 3). (H) Summary data showing percent Ki-67⁺ CD8 T cells in recipient BM at 7 and 14 days, after NK cell infusion (day 7 n = 4, day 14 n = 3). (I-J) Summary data showing absolute CD8⁺ (I) and Ki67⁺ CD8 (J) T-cell numbers in the PBMC at the indicated days after infusion (IL-2 day 7 n = 8, day 14 n = 3; N-802 n = 6). (K-N) Patients treated on UMN trials using IL-2 activated NK cells supported in vivo with IL-2 (gold) or N-803 (red) were also assessed by mass cytometry (see schema, supplemental Figure 2). Summary data depicting percent CD8 T cells from the (K) PBMC before (day 0) and the indicated days after NK cell infusion (IL-2 day 0 n = 6, day 7 n = 3, day 14 n = 6; N-802 day 0 n = 3, day 7 n = 2, day 14 n = 3), (L) BM (IL-2 n = 6; N-802 day 0 n = 3, day 14 n = 5). Summary data depicting percent CD4 T cells from the (M) PBMC and (N) BM. Summary data were analyzed using 2-way analysis of variance. Mean is depicted with error represented as standard error of the mean. P values are indicated within the graphs; no significant differences were detected in panels K-N.