Fig 2: Immunological analyses of the tumor microenvironment.B16.OVA a Percentage of dendritic cells cross-presenting OVA (SIINFEKL) in the context of MHC class I in the tumor microenvironment, b OVA-specific, c OVA-specific antigen-experienced, and d antigen-experienced T cells in the tumor microenvironment. The number of T cells was normalized by the tumor volume. Mix identifies a treatment composed of cell membrane vesicles just mixed with adenovirus without extrusion. The results are presented as mean ± s.d (n ≥ 3) and the dot plot of the single replicates. The data were analyzed with a two-way ANOVA, followed by Dunnet (a–c) or Tukey (d) post-test comparison. The levels of significance were set at the probabilities of *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B16F10 e CD11c+ dendritic cells percentage in the tumor microenvironment and f gp100 and g TRP2 pentamer-specific T-cells, identifying the CD8+ tumor-specific T-cells in the tumor microenvironment. h Antigen-experienced T-cells measured by the expression of PD-1 on T-cells. Mix identifies a treatment composed of cell membrane vesicles just mixed with adenovirus without extrusion. The data are presented as mean ± s.d (n ≥ 7), together with the dot plot of the single value. The data were analyzed with two-way ANOVA, followed by Fisher LSD post-test. The levels of significance were set at the probabilities of *p < 0.05, **p < 0.01. LL/2 i Percentage of CD11c+ dendritic cells in the tumor microenvironment in the LL/2 model. j Percentage of CD68+ macrophages. k Percentage of CD4+ and l CD8+ T-cells in the tumor microenvironment. Mix identifies a treatment composed of cell membrane vesicles just mixed with adenovirus without extrusion. The data are presented as mean ± s.d. (n = 4) and were analyzed by two-way ANOVA, followed by Dunnet’s post-test. The levels of significance were set at the probabilities of *p < 0.05 and **p < 0.01. The error bars represent s.d.

Fig 3: Knockout of Rictor, an obligate mTORC2 subunit, rescues steatohepatitis in Dyrk1bAAV-WT liver.(A–H) ORO staining in the liver of mice fed with CD (A–D), n > 8 each, or HCD (E–H), n > 6 mice each. (I–L) CD68 (red), SMA (green), F4/80 (white) costaining in the designated mice fed with HCD for 5 months to induce inflammation; n > 6 mice each. (M) Hepatic TAG normalized to total protein in the designated mice fed with CD. The data from Figure 2E are repeated to display all the controls together. n > 6 mice each, 1-way ANOVA, Tukey’s post hoc test. (N) Hepatic TAG normalized to total protein in the indicated genotypes in the mice fed with HCD; n > 6 mice each, 1-way ANOVA, Tukey’s post hoc test. (O) Quantification of CD68-positive and F4/80-positive area in the designated genotypes by ImageJ; n > 5 mice each, 1-way ANOVA, Tukey’s post hoc test. Scale bars: 150 µm. *P = 0.05, **P = 0.01, ***P = 0.001.

Fig 4: Activation of microglia in the brain white matter of SIRPa KO mice.(A) Immunofluorescence staining of coronal brain sections prepared from control (WT) or SIRPa null mutant mice (SIRPa KO) with antibodies to Iba1 (red) and CD68 (green). Merged images are shown. fi, fimbria. (B) Quantitative analysis of the number of Iba1+ (left panel) and Iba1+/CD68+ (right panel) microglia in the fimbria of WT (filled bars) and SIRPa KO mice (open bars) at 18–20 wks of age. Data are the means ± SEM (n = 6 images from three mice for each genotype). **p<0.01 (Welch’s t-test). (C, D) Immunofluorescence staining with a specific antibody for Iba1 of coronal brain sections prepared from control CD11c–EYFP Tg mice (at 30–32 wks of age) carrying a homozygous SIRPa WT allele (SIRPa WT:CD11c–EYFP, left side) or a SIRPa null mutation (SIRPa KO:CD11c–EYFP, right side). Images in panel (C) are lower-magnification images of the immunoreactivity of Iba1 (upper panels) and of the fluorescence of EYFP in the same sections (lower panels). Images were converted to grey scale and then inverted to clarify the fluorescence signal. Images in panel (D) are higher-magnification images of the immunoreactivity of Iba1 (red) and fluorescence of EYFP (green) in grey matter (ctx, cerebrum cortex; upper panels) and white matter (ec, external capsule; lower panels). cc, corpus callosum; ec, external capsule; fi, fimbria; int, internal capsule. Data are representative of at least three independent animals. Scale bars: 100 µm (A), 1 mm (C), and 50 µm (D).

Fig 5: Flow cytometry analysis of microglia in SIRPa KO mice and WT aged mice.(A) Cells isolated from the spinal cord of control WT or SIRPa KO (KO) mice at 14 wks of age were incubated with a PE-conjugated monoclonal antibody (mAb) to SIRPa, a PerCP–Cy5.5-conjugated mAb to CD45, and an FITC-conjugated mAb to CD11b. The expression of CD11b and CD45 on monocyte cells or of SIRPa on CD11b+/CD45dim/lo microglia was analysed by flow cytometry. The percentage of CD11b+/CD45dim/lo microglia among putative monocytes is indicated in each plot (left plots). The expression profiles for SIRPa on CD11b+/CD45dim/lo microglia are shown in the right panel. (B) Cells prepared as in panel (A) from WT or SIRPa KO mice at 14–21 wks of age were stained with antibodies to CD45, CD11b, and CD11c, and analysed by flow cytometry. The percentage of CD11b+/CD45dim/lo/CD11c+ microglia among total CD11b+/CD45dim/lo microglia is indicated in each plot. Quantitative data are shown in the right panel. Filled and open bars indicate WT and SIRPa KO mice, respectively. (C) Cells prepared from SIRPa KO mice were stained for CD45, CD11b and CD11c, as well as for CD14, Dectin-1, or CD68. Plots were gated on CD11b+/CD45dim/lo cells, and CD14, Dectin-1, or CD68 on CD11c–positive and -negative microglia were analysed. The expression profiles for each molecule in CD11b+/CD45dim/lo microglia are shown. (D) Cells isolated from adult (18 wks of age) or aged (104 wks of age) mice were isolated and analysed as in panel (A). (E) Cells prepared as in panel (D) from adult (16–18 wks of age) or aged (69–105 wks of age) mice were analysed as in (B). Quantitative data are shown in the right panel. Filled and open bars indicate adult and aged mice, respectively. (F) Cells prepared from aged mice were analysed as in panel (C).Data in panels (B) and (E) are the means ± SEM (n = 5 (B) and 3 (E) independent experiments). **p<0.01 (Welch’s t-test). Other data are representative of at least 3–5 independent experiments. Filled and open traces in panels (A), (C, F), and (D) indicate WT and SIRPa KO mice (A), CD11c– and CD11c+ cells (C, F), and adult and aged mice (D), respectively.