Fig 2: DC-specific deletion of Lkb1 leads to specific enlargement of Treg cell pool. a Flow cytometric analysis of Treg cell frequencies among splenic CD4+ T cells from Lkb1f/f and Cd11cCreLkb1f/f mice. b Quantification of the percentage and absolute number of Treg cells in the spleen and lymph nodes (LNs) from Lkb1f/f (red dots) and Cd11cCreLkb1f/f (blue dots) mice of different ages. c, d Flow cytometric analysis (c) and quantification (d) of Ki67 expression in splenic Treg cells from Lkb1f/f and Cd11cCreLkb1f/f mice. e Flow cytometric analysis of Treg cell frequencies among CD4+ T cells in the blood, bone marrow (BM), lungs, liver, kidneys and brain of Lkb1f/f and Cd11cCreLkb1f/f mice. f Quantification of the percentages of Treg cells among CD4+ T cells in the blood, BM, lungs, liver, kidneys and brain of Lkb1f/f (red dots) and Cd11cCreLkb1f/f (blue dots) mice. g Flow cytometric analysis of CD25, CD44, CD62L, Helios, CD73, ICOS, Nrp1 and CTLA4 expression on splenic Treg cells from Lkb1f/f (red line) and Cd11cCreLkb1f/f (blue line) mice. h Suppression assay in which CFSE-labelled naïve T (Tn) cells were co-cultured with Treg cells from Lkb1f/f and Cd11cCreLkb1f/f mice at a 4:1 ratio. Each symbol (b, f) indicates an individual mouse; the results are presented as the mean ± S.E.M., *P < 0.05, **P < 0.01, ***P < 0.001, by Student's t-test (b, d, f). Data are pooled from b, d, f or are representative of a, c, e, g, h three independent experiments with similar results

Fig 3: Lkb1 conditional deletion in Treg induces the loss of Treg in the bone marrow (BM) and alters the CD4+T subpopulations. (A) The generation of Lkb1 conditional deletion in Treg mice. (B) Flow cytometric analysis of Treg cell ratios among CD4+T cells in the BM from wild‐type (WT) and knock‐out (KO) mice. (C) Statistical comparison of the proportions of Treg among CD4+T cells between WT and KO BM (n = 3). (D) Helios expression in the BM Treg from WT and KO mice. (E) The flow cytometric analysis of CD62Llow CD44high effector cells, CD62L+CD44+ central memory cells and CD62+CD44− naïve cells in the BM among CD4+T cells from WT and KO mice. (F) Statistical comparison of the proportions of effector cells, central memory cells, and naïve cells among CD4+T cells in the BM from WT and KO mice (n = 3). The results were presented as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by Student's t‐test (C, F). All results were performed with at least three independent experiments.

Fig 4: Respiratory T cell response induced by mRNA-N in mice.A Mouse experimental design and timeline (Created with BioRender.com). Two groups of C57BL/6 mice (n = 5/group) were intramuscularly vaccinated with either empty LNP (mock) or mRNA-N vaccine (1 μg) at weeks 0 and 3. Two weeks after booster dose (week 5), immune responses were analyzed. B, C Analysis of tetramer positive, N-epitope specific T cells in the lungs by flow cytometry. B Representative flow cytometry plots for N-tetramer staining of CD8 T cells from LNP and mRNA-N vaccinated mice. C Frequencies of N epitope-specific CD8 T cells in the lungs of LNP and mRNA-N vaccine group. Analysis of total and N epitope-specific T cells in the BAL. Frequencies of activated (CD44+) CD4 and CD8 T cells (D), CXCR6+ CD4 and CD8 T cells (E), and N epitope-specific CD8 T cells (F) in BAL were examined. Data were presented as median and interquartile range, and were compared by Mann-Whiteney test between the two groups. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Respiratory T cell response following different mRNA vaccination.A Mouse experimental design and timeline (Created with BioRender.com). Three groups of C57BL/6 mice (n = 5/group) were intramuscularly vaccinated with either empty LNP (mock), mRNA-S vaccine (1 μg), or mRNA-S+N vaccine (1 μg for each mRNA) at week 0 and 3. Two weeks after final dose (week 5), mice were euthanized and immune analyses were performed. Analysis of activated T cells in the lungs (B), BAL (C), and spleens (D) of mice. Expression of CD44 on CD4 and CD8 T cells was examined by flow cytometry and shown as percent CD44+ of the parental population. Expression of CXCR6 on CD4 and CD8 T cells in the lungs (E), BAL (F), and spleens (G) was examined by flow cytometry and shown as percent CXCR6+ of the parental population. H Representative flow cytometry plots for S- and N-tetramer staining of CD8 T cells from LNP, mRNA-S, and mRNA-S+N vaccinated mice. Tetramer+ (S- and N-epitope specific CD8 T cells) in the lungs (I), BAL (J), and spleens (K) of mice. Data are presented as median and interquartile range. Kruskal-Wallis test was used for statistical comparison among the three groups. *p < 0.05, **p < 0.01.