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. 2023 Apr 3;8(5):860–874. doi: 10.1038/s41564-023-01352-8

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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. 5 — a–k, T-cell subsets by scRNA-seq (2 dpc) of prime-boost-vaccinated hamsters. Frequencies and numbers of (a) CD4 (cluster 0,1,2) and CD8 (cluster 3,4) T cells, and (b) proliferating T cells (cluster 6) in blood. Bar graph with mean ± s.e.m., n = 3 for all groups, except n_mock+mock = 4. Ordinary one-way ANOVA and Tukey’s multiple comparisons test. c, Dot plots showing expression of selected genes in blood cluster 6 (T and NK subcluster analysis in Supplementary Fig. 7a). Dot size represents fraction of cells with UMI > 1, colour indicates expression. d, IFN-γ ELISpot analysis 14 d post prime vaccination. Bar graph with mean ± s.e.m., n = 6, displaying spot counts normalized to medium stimulation for each animal, individually. Dotted line, upper limit of detection (ULD). Two-way ANOVA and Tukey’s multiple comparisons test. e–g, Frequencies and numbers of (e) tissue-resident memory T cells (Trm, cluster 6), (f) activated T cells (Act T, cluster 2) and (g) proliferating T cells (prolif T cells, cluster 5 + 8) in lungs. Ordinary one-way ANOVA and Tukey’s multiple comparisons test. Bar graph with mean ± s.e.m., n = 3 for all groups, except n_mock+mock = 4. In a,b,d–g: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. h, Dot plots showing expression of selected genes in lungs, clusters 5 and 8 (T and NK subcluster analysis in Supplementary Fig. 9a). Dot size represents fraction of cells with UMI > 1, colour indicates expression. i,j, Trm gene set (refs. ^28,31) signature score in cells from selected T-cell subclusters over all groups (i) and for individual groups (j), colour indicates signature score for Trm gene set. k, Trm signature score in cells of cluster 5. Centre, median; box, 25th to 75th percentiles; and whiskers, minimum to maximum values. Circles indicate individual analysed cells in cluster 5 pooled from n = 3 for all groups, except n_mock+mock = 4 animals. l, PAGA. Nodes represent clusters, edges represent extent of cluster connection, node size corresponds to cluster cell number and line thickness is proportional to connectivity. m, Trm signature (refs. ^28,31) score as a function of diffusion pseudotime rank, with black line showing a polynomial fit of degree three.

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