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. 2022 Feb 14;12:2417. doi: 10.1038/s41598-022-06430-1

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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 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/.

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Functional polarization of TAMs by PD-L1 knockout in U87 cells. U87 cells were treated with Cas9-g82/165 + HDR for 24 h, then human macrophages were added allowing TAM differentiation for 48 h. An ELISA assay was used to detect (A) human IL-4 secretions in the medium from co-cultured human macrophages and U87 cells with or without Cas9-g82/165 + HDR. In parallel, mouse BMMs co-cultured with U87 cells were investigated to examine PD-L1 knockout on mouse (B) TNF-α and (C) IL-4 secretions. PD-L1 deletion in U87 cells significantly upregulated TNF-α levels and reduced IL-4 secretion compared to the control. All experiments were performed in triplicate and results were normalized for direct comparisons. Data represents the mean ± SEM. Statistical significance was evaluated with a t-test. *p < 0.05, **p < 0.01.