Fig 2: TG2 is required for cell proliferation but has no effect on surface marker expression in human umbilical cord-derived MSCs. (a) Human umbilical cord-derived MSCs were induced into replicative senescence after subculture for a long-term expansion (passage number 3, 7, and 15). The expression levels of TG2, NRF2, NRF2-responsive genes, and senescence marker (p16) were assessed by western blot analysis. β-Actin was used as the loading control, and the relative intensities were normalized using the loading control. (b) MSCs were transfected with lentivirus expressing shRNA for TG2. The level of TG2 protein was assessed by western blot analysis. β-Actin was used as the loading control in the experiment. (c, d) Cell proliferation rate of wild-type and TG2-knockdowned MSCs was estimated by measuring cumulative population doubling level (CPDL) through passage 3–20 (c) and the percentage of BrdU incorporated cells with flow cytometry (d). (e, f) Representative examples of flow cytometric profiles from wild-type and TG2-knockdowned MSCs. The percentage of cells expressing positive surface markers ((e), CD73, CD90, CD105 and CD44) and negative markers ((f), CD45, CD31, and CD11b) for MSCs was shown. The results for western blots are represented from three independent experiments. The measurement of growth rate in MSCs and flow cytometry images are representative results from three independent experiments performed. Statistical significance was tested by Student's t-test (n = 3; ∗∗∗P < 0.001).

Fig 3: MCMV-B5 can stimulate and maintain epitope-specific T cell responsesMSP-1-specific B5 TCR Tg CD4 T cells (CTV+Thy1.2+) were adoptively transferred into Thy1.1 mice that were then infected with MCMV-BAC or MCMV-B5, as shown in (A) experimental schematic. Splenic T cells were gated on B5 TCR Tg (CD4+CTV−Thy1.2+) recovered at days 15 and 55 post-MCMV infection.(B) Plot and histogram show gating of divided B5 T cells, and graphs show percent and number of divided B5 T cells.(C and D) Plots and graphs show B5 Teff (CD4+Thy1.2+CTV−, CD127−CD44+) and Tmem (CD4+Thy1.2+CTV−, CD127hiCD44hi) (C) on day 15, or (D) day 55. Plots are concatenated, whereas graphs represent 3–5 animals per group. Data are representative of two experiments. Mean shown with error bars representing SEM and analyzed using one-way ANOVA followed by Tukey’s post-test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3.

Fig 4: Accumulation of tissue-resident memory CD8+ T cells in liver of DKO mice.a tSNE plots of WT and DKO liver CD8+ T cells, colored by cell clusters. Feature plots showing Cd8a, Cd44, Sell expression of all the CD8+ T cell clusters. b Expression heatmap shows top 50 differentially expressed genes of each cluster. Selected genes are indicated to the right, and complete lists of top genes are available in Supplementary Table 1. c Feature plots showing Trm up-or downregulated genes expression of all the CD8+ T cell clusters. d Percentage of CD8+ cell clusters by Sc-RNA-seq data of WT and DKO mice. e Percentage of CD8+ cell clusters by flow cytometry of DKO mice (n = 8). f Representative FACS plot showing percentage of CD69+CD62L−CD8+ T cell subset from liver of Ctrl mice or liver, spleen and draining lymph node of DKO mice. g Representative FACS plot showing percentage of CD69+CD62L−CD8+ T cell subset from peripheral, sinusoidal, portal venous and postcaval venous blood of DKO mice. h Representative FACS histogram of CXCR6, CD11a and Ly6C expression on CD8+ Tcm, Tem and Trm cell subsets. The ranged gate, the percentages of the positive population and gMFI of CD11a are shown in the figure. i Relative mRNA expression level of S1pr1 and Hobit on CD8+ Tcm, Tem and Trm cell subsets in DKO mice liver (n = 6). Representative frequency plots (j) and statistical analysis (k) of peripheral blood CD8+ T cells or liver CD8+ Tcm and Trm cells derived from host (CD45.2+) or partner (CD45.1+) DKO mice (n = 8) after parabiosis. Data in (f–h, j) are representative results of at least three independent experiments. The p values were determined by a one-way ANOVA with Tukey’s multiple comparisons test (i, k). Data are shown as Means ± SEM.

Fig 5: IL-33 deficiency alleviated SARS-CoV-2-induced pulmonary inflammation in miceWT B6 and IL-33−/− mice were infected with SARS-CoV-2 as indicated in Figure 1. B6 mice in the mock group were i.n. treated with the same volume of cell culture medium.(A) Bodyweight changes of WT and IL-33−/− mice after SARS-CoV-2 infection. Data were pooled from two independent experiments and were analyzed by repeated measures two-way ANOVA.(B) Relative viral load was measured by RT-qPCR; bar graph represents as a mean ± SD (n = 7–8/group). These data were analyzed by t tests at each time point.(C) Histological changes in the lungs of WT and IL-33−/− mice after SARS-CoV-2 infection: immune cell infiltration (black arrow), intrabronchial mucus and cell debris (blue arrows) and epithelial regeneration (red arrows).(D) Gating strategy flow cytometry data. Lymphocytes were selected from a forward scatter-A vs. side scatter-A dot plot, and single cells were subsequently selected. Then, live lymphocytes were selected by live/dead dye and CD45. Neutrophils were identified as Ly6G+ cells. Macrophages were identified as CD11b+CD64+ cells. T cells were first identified as CD3+ and further subdivided into CD4+ and CD8+ T cells. NK cells were identified as CD3− and NK1.1+. γδT cells were identified as CD3+ and γδTCR+. CD44+CD69+ population was characterized as activated cells.(E) Flow cytometric analysis of lung tissues of WT and IL-33−/− mice at days 2 and 4 post-SARS-CoV-2 infection. The numbers of neutrophils, activated NK cells, macrophages, activated CD4 T cells, activated CD8 T cells and activated γδ T cells were plotted. Bar graph represents as a mean ± SD (n = 4–5/group) (one-way ANOVA followed by Tukey’s multiple comparison test at each time point).(F) Protein levels of IL-5, IL-6, IL-12p40, IFN-g, G-CSF, CXCL1, CCL2, and CCL-11 in the serum of WT and IL-33−/− mice after SARS-CoV-2 infection. Data were analyzed by unpaired/two-tailed t tests at each time point. All results are presented as the mean ± SD, and this experiment was performed twice independently with similar trends. Statistically significant values are denoted as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.