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. 2023 Apr 12;14:2081. doi: 10.1038/s41467-023-37697-1

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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 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 — A Schedule for immunization of BALB/c mice. At day 10 after the second immunization, lung cells and splenocytes were obtained and stimulated with or without spike peptide pools (Supplementary Table 2) overnight in the presence of BFA. Surface markers (CD4, CD8, and Zombie) were stained, and cells were fixed and permeabilized. Intracellular cytokines (TNFα and IFNγ) were then stained with specific antibodies. Delta4N-DAF and Omi4N-DAF induced IFN-γ and TNF-α positive CD4+ and CD8+ T cell responses in lungs (B) and spleens (C) of BALB/c mice. Percentages of IFNγ+ or TNFα+ CD4+ and CD8+ T cells in immunized mice (n = 5 for each group) were compared. Error bars represent mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunn’s multiple comparisons test: ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant. Mouse cartoons created with BioRender.com.