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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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PD-L1 knockout in U87 cells polarizes human TAMs toward an M1 phenotype. U87 cells were treated with Cas9-g82/165 + HDR for 24 h, then human macrophages were added allowing TAM differentiation for 48 h. The co-cultures were collected and analyzed by flow cytometry. Contour plots of (A) CD11b and PD-L1, and (B) Arginase I and CD80 subpopulations are shown for Cas9-g82/165 + HDR treated cultures and the control. (C) PD-L1 knockout in U87 cells was confirmed by gating for CD45-CD11b- cells and examining PD-L1 + cells. Co-cultivation of TAMs and U87 cells treated with Cas9-g82/165 + HDR repolarized M2 TAMs into (D) CD80 + M1 TAMs. In contrast, PD-L1 knockout in U87 cells leads to (E) Arginase I + M2 depletion. 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.