Fig. 2

Expansion of the NK cells following donor cell product infusion. A Longitudinal evaluation of peripheral blood mononuclear cells (PBMCs) using flow cytometry with a customized panel, single-cell CITE-seq, a multiplexed immunoassay for secreted molecules, and next-generation sequencing of donor chimerism. The number of samples used for each assay at each time point is indicated (created using Biorender). B Size of the NK cell compartment as a proportion of total lymphocytes (left) and as absolute numbers (right) at the indicated time points after donor CIML NK cell infusion. Number of evaluable samples at each time point is shown above each bar. Adjusted p-values are determined using nonparametric Anova (Kruskal-Wallis test) followed by Dunn’s multiple comparisons test. Shown are mean with SD, medians are marked by a red line. C Spearman correlation between NK cell expansion and tumor responses by RECIST v1.1 criteria. NK cell fold change was calculated by taking the ratio of NK cells as a percentage of lymphocytes, in the peripheral blood on day 14 following CIML NK cell infusion to the percentage of NK at the time of screening. The patient ID is indicated at each point. D Donor cells as a proportion of all peripheral blood mononuclear cells at the indicated time points using two different assays. Flow cytometry-based evaluation (Flow) using anti-HLA antibodies targeting recipient-donor disparity in HLA. DNA-based chimerism using next-generation sequencing (NGS) performed by CareDx was also used to evaluate donor chimerism. ≤14 (n = 7) refers to available samples collected between day + 7 and day + 14, ≥28 (n = 4 for Flow, n = 5 for NGS) refers to available samples collected between day + 28 and day + 60. E Percentage expression of interferon gamma (IFNγ) in flow cytometry-based NK cell functional assays using live donor CD3⁻CD56⁺ lymphocytes (gated with donor-specific HLA antibodies) collected at the indicated time points following CIML NK cell infusion. In one recipient there was no donor-specific HLA antibody for flow cytometry gating, but the NGS-based chimerism confirmed that the NK cells were all donor, so the percentage was calculated on total NK cells. p-value for the comparisons in D, E were calculated using Mann-Whitney U. SRN: screening time point for the trial before NK cell infusion