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. 2020 Nov 27;11:6080. doi: 10.1038/s41467-020-19486-2

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© The Author(s) 2020

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 — Isolated human CD8+ T cells were stimulated with beads that are coated with antibodies against TCR/CD3 and co-stimulatory CD28 receptors. Twenty-four hours later, beads were removed and CD8-targeted nanoparticles (NPs) containing either mRNA encoding the leukemia-specific 1928z CAR (a–e) or the HBcore18-27 TCR (f–j) were mixed into the cell suspension at a concentration of 3 µg of mRNA/10⁶ cells. a qPCR measurements of relative 1928z CAR mRNA expression over time after T cells were exposed to 1928z CAR NPs. Shown are mean values ± SD. N = 9 biologically independent samples. b Flow cytometry of T cells at the indicated time points after incubation with NPs bearing 1928z CAR-encoding mRNA. c Summary plot of in vitro gene transfer efficiencies. Shown are mean values ± SD. N = 9 biologically independent samples. d In vitro assay comparing cytotoxicity of nanoparticle-transfected vs. retrovirus-transfected T cells against Raji lymphoma cells. T cells were co-cultured with Raji tumor cells at a 5:1 ratio. We used the IncuCyte Live Cell Analysis System to quantify immune cell killing of Raji NucLight Red cells by 1928z-CAR or control (P28z-CAR)-transfected T cells over time. Data are representative of two independent experiments. Each point represents the mean ± s.e.m. pooled from two independent experiments conducted in triplicate. e ELISA measurements of IL-2 (at 24 h) and TNF-α and IFN-γ (at 48 h) secretion by transfected cells. Shown are mean values ± SD; two tailed unpaired Student’s t-test. N = 9 biologically independent samples. f qPCR measurements of relative HBcore18-27 TCR mRNA expression over time after T cells were exposed to HBcore18-27 TCR NPs. Shown are mean values ± SD. N = 9 biologically independent samples. g, h Gene transfer efficiencies. i Cell killing of HepG2-core NucLight Red cells by HBcore18-27 or control (MSLN-) TCR-transfected T cells over time. T cells were co-cultured with HepG2 tumor cells at a 5:1 ratio. N = 9 biologically independent samples. j ELISA measurements of cytokine secretion by transfected cells. Shown are mean values ± SD; two tailed unpaired Student’s t-test.