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. 2023 Sep 8;14:5546. doi: 10.1038/s41467-023-41338-y

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© The Author(s) 2023

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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Fig. 1 — a Schematic of the lentiviral vector construct with EF1α promoter followed by the leader sequence, nfP2X7 antigen binding domain, hinge region (short, medium or long), CD28 transmembrane domain, 41BB, CD3 zeta, T2A and EGFRt. b Flow cytometric analysis of CAR expression in CD4⁺/CD8⁺ compartments on the three different CAR-T cells (nfP2X7-S, nfP2X7-M and nfP2X7-L) by EGFR staining. c Specific cytotoxicity of target cells by CD8⁺ nfP2X7-CAR-T cells with three different hinge lengths (nfP2X7-S, nfP2X7-M and nfP2X7-L) compared with CD8⁺ untransduced (UT) cells. Target cell lines include: K562 (leukaemia), MDA-MB-231 (breast cancer), U87 (glioma), M21 (melanoma) and SK-ND-Z (neuroblastoma). K562 transduced to express OKT3 was included as a positive control. CAR-T cells were co-cultured with target cancer cell lines at effector: target ratios of 30:1, 10:1, 3:1 and 1:1 for 4 h. Specific cytotoxicity was measured using a chromium-51 release cytotoxicity assay. Pooled data from two independent experiments. Data are presented as mean values +/− SEM. d Cytokine release assay for nfP2X7-CAR-T cells. CD4⁺ CAR-T cells and target cells were co-cultured for 24 h and the concentration of IL-2, IFN-ɣ and TNF-α in the supernatant was measured using the Bio-Plex system. Cytokines produced by untransduced CD4⁺ cells were compared with nfP2X7-CAR-T cells harbouring different hinge lengths. Target cell lines include: K562, K562-OKT3, MDA-MB-231, U87 and SK-ND-Z. Data represent two independent experiments. Data are presented as mean values +/− SEM.