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. 2024 Aug 27;15:7226. doi: 10.1038/s41467-024-51411-9

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 4 — a Expression profiles of spike^- non-expressed genes in spike⁺ fibroblasts. Fibroblasts without spike mRNA transcripts from the injection-site scRNA-seq data were merged, and genes with an expressing cell ratio <0.01 were designated as the non-expressed genes in spike^- fibroblasts. The expression profiles of the spike^- non-expressed genes were evaluated in spike⁺ fibroblasts from either 2 h p.i. or 16 h p.i. samples of mRNA vaccine injection, and their expressing cell ratios are denoted on the x-axis (2 h p.i.) and the y-axis (16 h p.i.) of the scatter plot. b Ifnb1 expression (log-transformed) in each of the injection-site fibroblasts from 2 h p.i. samples of LNP or LNP+mRNA injection. Red indicates spike⁺ fibroblasts (n = 289 from LNP+mRNA injection), and gray dots represent spike^- fibroblasts (n = 1601 from LNP injection, n = 43 from LNP + mRNA injection). Data are presented as mean values ±SEM. c Representative RNA-ISH image acquired from mRNA-vaccine 2 h p.i. muscle tissue. Images were selected from biological replicates of mRNA-vaccine injected samples (n = 4). Green, blue, and red indicate detection signals for Pdgfra, Spike, and Ifnb1 transcripts, respectively. Orange arrowheads indicate co-localization of the fluorescent signals, which implies spike⁺ fibroblasts expressing Ifnb1 transcripts, and the orange arrow indicates a spike^- fibroblast not expressing Ifnb1 transcript. Scale bars indicate 10 µm. d, e Expression of (d) IFN-β transcripts (Ifnb1 gene) and (e) IFN-α transcripts (sum of the 14 IFN-α transcripts) in the injection-site scRNA-seq data from 2 h p.i. samples of LNP and LNP+mRNA. The 2 h p.i. injection site cells were split according to the treatment condition, detection of spike mRNA, and cell type identities. The y-axis represents log-transformed counts of the transcripts. f The fraction of cell types in the cells with Ifnb1 transcripts. g Measured levels of IFN-β in (top) blood and (bottom) muscle samples using ELISA. All the samples were collected 4 h after injections. Data are presented as mean values ±SEM. Statistical evaluations were conducted with two-tailed Mann–Whitney U tests (ns: nonsignificant).