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: B7-H4 (VTCN1) is highly correlated with epithelial gene markers in mouse and human cells unlike other checkpoint ligands. A, In human breast cancer cell lines (CCLE), B7-H4 is the only checkpoint ligand positively correlated with epithelial markers and negatively correlated with mesenchymal markers. Data shown are Spearman correlations between genes. Significant correlations (P < 0.05) are indicated by asterisk. B, Genes downregulated during EMT are positively correlated with VTCN1 in human cell lines (CCLE). C, Murine cancer cell lines express B7-H4. D, B7-H4 is only expressed on epithelial EpCAM+ cells in murine and human cells. E, The MMTV-neu cell line with highest B7-H4 levels is comprised of phenotypically epithelial-like (abbreviated E) and mesenchymal-like (M) cells. F, In MMTV-neu cells, the CD44+ M cells do not express B7-H4 but all the EpCAM+ E cells do. G, MMTV-neu E and M cells express hallmark markers. H, Single-cell RNA sequencing of the heterogenous MMTV-neu cells confirms Vtcn1 is solely expressed in the epithelial cell population. I, A cohort of human tumors including both TNBC and ER+HER2− were stained for B7-H4, EpCAM, and CD44 by mIF. Data shown are log2 of % B7-H4+ tumor cells and include 132 samples with >1% B7-H4 expression (paired t test of transformed data).

Fig 5: 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.