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. 2022 Apr 13;12(4):61. doi: 10.1038/s41408-022-00660-2

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© The Author(s) 2022

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A Scheme of the in vivo evaluation of a repetitive treatment with CD33-CAR-NK cells (1 × 10⁷ intravenously) combined with subcutaneous IL-2 treatment in OCI-AML2 (Luc⁺) xenograft NSG-SGM3 mice. B Total flux analysis, as well as representative BLI images of differently treated OCI-AML2 (Luc+), engrafted NSG-SGM3 mice over time (n = 7 per group). Mice received a total of three weekly doses of 1 × 10⁷ NK cells. Mice that were treated with CD33-CAR-NK cells show severely reduced leukemic burden compared to untreated mice (UT) or mice which received untransduced (UTD)-NK cells. C Total flux analysis of femurs/tibiae and spleens, as well as flow cytometry analysis of isolated cells from BMs or spleens at day 22 post AML-injection, revealed the absence of GFP-positive leukemic cells in CD33-CAR-NK treated mice as well as increased NK cell infiltration (n = 6–7 per group). Values of zero were set to 1 for total flux analysis. Median ± range. D Chimerism analysis d22 post AML-injection revealed high amounts of DNA from human NK cells without detectable DNA of AML in blood of mice that were treated with CD33-CAR-NK cells (n = 6–7 per group). Mean ± SD. E Serum analysis of blood day 3 before AML injection and day 1 post first NK cell application showed significantly increased pro-inflammatory human cytokines for mice that received CD33-CAR-NK cells (n = 6–7 per group). F Flow cytometry-based CAR-expression analysis of BM- or spleen-infiltrating NK cells in CD33-CAR-NK treated mice revealed the presence of mainly CAR-positive cells (n = 6–7 per group). Mean ± SD. G Confocal microscopy imaging demonstrated GFP-positive leukemia cells in BM of UTD-NK treated NSG-SGM3 mice while absent in mice that received CD33-CAR-NK cells. Additionally, CAR-NK cells could be detected in the BM of CD33-CAR-NK treated mice. One representative image from a total of four are shown. Statistical analysis was performed by Mann–Whitney-test (for total flux analysis) or Student’s t test (for the rest) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).