Fig 2: Viral-Specific Induction of T-bet Alters Proliferation, Activation, and Transcription Factor Ratios during T Cell Differentiation(A and B) ZsGreen_T-bet reporter mice were infected with influenza or naive ZsGreen_T-bet STg cells were transferred prior to LCMV infection. Draining lymph nodes (influenza) and splenocytes (LCMV) were harvested at d4 and d8 p.i. Representative flow cytometry plots (A) and MFI of activated (d4) and NP311 (d8) and STg TFH (red) and TH1 (blue) (B) in indicated infection models.(C–J) CD4cre and Tbx21?T mice were infected with influenza or naive STg or Tbx21-/- STg cells were transferred prior to LCMV infection and analyzed at d4 and d8 p.i. Activated T cells (CD4+CD44+) and tetramer+ (NP311+ were assessed respectively for d4 and d8 p.i. influenza infection. STg cells were assessed for LCMV time points. Viral specific cells were assessed for Ki67 MFI (C and D), PD-1 MFI (E and F), CXCR3 MFI (G and H), and CD25 MFI (I and J). Representative histograms show frequency in positive gate.Data are representative of two or three independent experiments, n = 3–5 mice/group. Data are mean ± SEM.

Fig 3: DC populations have distinct capacities to stimulate TMEM cells in vitro(A) Co-culture experiment layout.(B and C) Representative flow plots and compiled data showing CD25 expression and CFSE dilution of WT TMEM cells after 3 days of culture with the indicated DCs. Percentages represent the portion of CFSElo cells expressing CD25.(D and E) Representative flow cytometry plots and compiled data from CXCR3 KO TMEM cells after 3 days of culture with the indicated DCs.(F and G) Representative histograms and compiled data of CD25 expression on WT and CXCR3 KO TMEM cells cultured with the indicated DCs. Data are normalized to the WT TMEM cells within each experiment.Data in (C) are from three independent experiments and in (E) and (G) from two independent experiments.

Fig 4: GV-971 alleviates neuroinflammation by reconditioning the gut microbiota. a Principal coordinate analysis (PCoA) of the gut microbiome composition on the operational taxonomic unit (OTU) level based on the Bray-Curtis distance for 5XFAD (Tg) mice and GV-971-treated Tg mice at 7-month old (n = 7). The shapes and colours of the points indicate samples from each individual. Coloured ellipses indicate 0.95 confidence interval (CI) ranges within each tested group. PC principal component. b Heatmap of significant gut microbiota changes represented at the genus level between 5XFAD (Tg) mice and GV-971-treated Tg mice at 7-month old (n = 7). Colours on the heatmap indicate the relative abundance of gut microbiota; red indicates bacteria that are upregulated, and blue indicates bacteria that are downregulated. Gut microbiota with significant changes were chosen using a Wilcoxon rank-sum two-tailed test with P-value that is less than 0.05 between Tg and GV-971-treated groups. f family, o order. c Changes in correlational links between the gut microbiome at the genus level (designated with numbers near the purple circles) and brain lymphocytes (other coloured circles) before (left) and after (right) oral gavage of GV-971 in 7-month-old 5XFAD (Tg) mice (n = 5–7). Lines represent either significant (P-value < 0.05) positive or negative correlation (Pearson coefficient). The size of each circle of immune cells are positively related to the links connected to this circle. The right side lists the name of each gut microbiome, also see “Materials and methods”. f family, o order, DC dendritic cells, NK natural killer cells, Neu neutrophils, B B cells, Mo/M? monocytes and macrophages. d Effect of GV-971 treatment on the frequency of brain Th1 cells in 5XFAD (Tg) mice at 7 months old (n = 5–7). Th1 cell counts (CD45highCD4+CXCR3+) are presented relative to CD45highCD4+ T cell counts detected by flow cytometry. The data are presented as mean ± standard error of the mean (mean ± SEM). *P < 0.05, ***P < 0.001, by Student’s t-test. e Effect of GV-971 treatment on the positive signal density of IBA1 immunofluorescent staining detected in hippocampal slices from 5XFAD (Tg) mice at 7-month old, reflecting activation of microglial cells (n = 4–6). The data are presented as mean ± standard error of the mean (mean ± SEM). **P < 0.01, ***P < 0.001, by one-way ANOVA (F (2, 15) = 21.94). f Effect of GV-971 treatment on levels of cytokine proteins in the brain homogenates of 5XFAD (Tg) mice at 7-month old as detected by a cytokine antibody array (n = 5–6). Colours on the heatmap indicate relative cytokine levels; red indicates cytokines that are upregulated, and blue indicates cytokines that are downregulated. g, h Effect of GV-971 on Aß-positive area (g) and tau-positive area (h) in the hippocampus of 5XFAD (Tg) mice at 7-month old, evaluated in brain slices (n = 4–7). The data are presented as mean ± standard error of the mean (mean ± SEM). For Aß analysis: *P < 0.05, ***P < 0.001 (F (2, 14) = 22.78). For tau analysis: *P < 0.05, ***P < 0.001 (F (2, 15) = 13.06) by one-way ANOVA. i Effects of GV-971 on the time out of 104 sec (s) taken to achieve 80% success (see “Materials and methods”) in a test to evaluate the discrimination learning abilities of 5XFAD (Tg) mice at 7-month old (n = 10–13). Time means the time to reach the 80% performance level (seconds); the longer it takes, the severer the cognitive impairment is (see “Materials and methods”). *P < 0.05, ***P < 0.001 by One-way ANOVA (F(2,31) = 9.751). The concentration of GV-971 was 100 mpk for all of the above results

Fig 5: The gut microbiota is required for immune cell infiltration and microglial activation. a The effects of five-month oral gavage of antibiotics on the relative abundance of gut microbes in 7-month-old 5XFAD transgenic (Tg) mice (n = 5–7). ABX, a cocktail of mixed antibiotics composed of ampicillin (0.1 mg/mL), streptomycin (0.5 mg/mL) and colistin (0.1 mg/mL). Different genera of gut microbes are coloured differently, and their changes in relative abundance are presented on the barplot. The abundance of Bacteroides in Tg mice is below 0.01 and was thus not shown in both groups. b-c The effects of five-month oral gavage of antibiotics on the frequency of Th1 cells (b) and M1-type microglia (c) in the brain homogenate of 7-month-old 5XFAD transgenic (Tg) mice. Cell counts of Th1 cells (CD45highCD4+CXCR3+) are presented relative to the frequency of CD45high cells (b), while those of M1-type microglia (CD45lowCD11b+CX3CR1+Siglec-H+CD86+) are presented relative to the frequency of CD45low cells (c). Both are detected by flow cytometry, n = 7, the data are presented as mean ± standard error of the mean (mean ± SEM). d The relative abundance of gut microbes at the genus level in WT, co-housed WT and 5XFAD transgenic (Tg) mice (n = 6–7). All three groups of mice were at 7-month old. Different colours represent different genera. Co-housed WT: WT mice that were housed with Tg mice. e-f Changes in the frequency of Th1 cells (e) and M1 type microglia (f) in the brain homogenates of 7-month-old WT, co-housed WT and 5XFAD transgenic (Tg) mice (n = 4–7). Th1 cells (CD45highCD4+CXCR3+) are presented relative to the frequency of CD45high cells (e), while the frequency of M1-type microglia (CD45lowCD11b+CX3CR1+Siglec-H+CD86+) are presented relative to the frequency of CD45lowCD11b+ cells (f). Both are detected by flow cytometry. The data are presented as mean ± standard error of the mean (mean ± SEM). *P < 0.05, **P < 0.01 by Student’s t-test. g Levels of cytokine proteins in the brain homogenates of WT, co-housed WT and 5XFAD transgenic (Tg) mice at 7-month old as detected by a cytokine antibody array (n = 5–6). Colours in the heatmap indicate relative cytokine levels; red indicates cytokines that are upregulated, and blue indicates cytokines that are downregulated