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. 2022 Mar 24;10:13. doi: 10.1186/s40364-022-00358-4

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Evaluating the antitumor effects of ITNKs in vitro and in vivo. A The bar chart shows IFN-γ secretion by T cells, ITNKs and NK cells after activation for 24 h with the indicated antibodies (anti-NKp30, anti-NKp46 and anti-CD3/CD28 at 5 μg/ml). Samples were collected from three individual donors. Data are shown as the mean ± SD; *P ≤ 0.05 and ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test. B Cytokine secretion profiles of ITNKs, T cells, and NK cells in coculture with K562 cells. ITNKs, T cells, and NK cells were incubated with K562 cells at an E:T ratio of 1:1 for 24 h. The supernatants were then harvested, and the concentrations of the indicated cytokines were measured by a multiplex immunoassay. The values shown represent the mean ± SD of 3 different donors. **P ≤ 0.01 and ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test. C Killing assays showing the percent cytotoxicity of ITNKs, NK cells and T cells against K562 cells. The data are shown as the mean ± SD; P < 0.0001 (ITNKs vs. T cells for K562 cells) and ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test. D The percent cytotoxicity of ITNKs and T cells against NALM-6 and OTK3-overexpressing NALM-6 cells in coculture. The results were obtained from three independent experiments. P < 0.0001 (ITNKs lysing OKT3⁺ NALM-6 cells vs. ITNKs lysing NALM-6 cells) and ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test. E Bioluminescence images showing the fate of K562 cells transplanted into NSI mice at the indicated time points. F Quantification of the total flux analyzed by in vivo bioluminescence imaging of luciferase activity (n = 5, per group). The results represent the mean ± SD; P = 0.0347 (ITNKs vs. T cells) and *P ≤ 0.05; two-way ANOVA with Tukey’s multiple comparisons test. G Survival analysis of mice treated with PBS (n = 10), T cells (n = 15), ITNKs (n = 15), or NK cells (n = 5); P = 0.0001 (ITNKs vs. T cells) by the log-rank Mantel-Cox test. H Tumor progression of patient-derived HCC xenografts (donor: 47-year-old female with grade IV hepatocellular carcinoma) treated with PBS, T cells, ITNKs, or NK cells (n = 5). Data are shown as the mean ± SD; P = 0.0001 (ITNKs vs. T cells) by two-way ANOVA with Tukey’s multiple comparisons test, ***P ≤ 0.001