Fig 2: LC–MS/MS proteomics identified upregulated and downregulated proteins in isolated brain cells from Mcoln1−/− mice. Volcano plot of protein changes in CD200+ neurons (A), CD15+ neural stem cells (B), O4+ oligodendrocytes (C), ACSA2+ astrocytes (D) and CD11b + microglia (E) from Mcoln1−/− cerebral cortex; log10 p < 0.1, log2FC > 0.6. Left panel show corresponding top 5 upregulated and top 5 downregulated proteins in each data set.

Fig 3: Endothelial CD200 is protective against stroke. (A) Representative brain slices stained with crystal violet, showing infarct area (white) in endothelial CD200 CKO vs. flox. (B) Quantification of infarct volumes in cortex, striatum and hemisphere in (A). (C) NDS and (D) adhesive removal tests, in endothelial CD200 CKO vs. flox mice. n = 6 per group. P * <0.05.

Fig 4: CD200 expression in endothelia inCD200 CKO and flox mice at 3d after MCAO. Upper panels: CD200 and VWF are universally colocalized in CD200 flox mice. Lower panels: CD200 are scarcely colocalized with VWF in CD200 CKO mice. Arrows indicate CD200 and VWF signals that are colocalized. Of note, some CD200 signals are not colocalized with VWF, suggesting they are from CD200+ neurons. Scale bar = 50 μm.

Fig 5: Optimization of DCvax-IT for T cell-depleted tumors(A) Metagene expression for indicated signatures in different subcutaneous tumors (from GEO: GSE85509).(B) Flow cytometry analysis of CD45+ fraction from subcutaneous MC38/TC1 tumors on day 23 after injection (percentage of CD8+ of CD3+ cells, n = 6; two-tailed Student’s t test).(C) Tumor volume of TC1-tumor-bearing mice treated with anti-PD-1/CTLA4 on day 9/16 after injection (n = 6; area under curve; one-way ANOVA, Kruskal-Wallis test).(D) Survival of WT, Ripk3−/−, and Mlkl−/− TC1 cells 24/48 h after treatment (three or four repeats).(E) Cell death of WT and Mlkl−/− TC1 cells 48 h after treatment. p values depict comparison WT vs. Mlkl−/− TC1 cells (n = 3; two-way ANOVA, Sidak’s multiple comparisons test).(F) Schematic overview of the vaccine formulation process.(G and H) Functional analysis of DCs untreated or stimulated with LPS, IFNβ, or with untreated or dying TC1s (with/without IFNβ). (G) Flow cytometry of DC maturation (MHCII+ CD86+ frequency of CD11c+). p values depict comparison vs. UT DCs (n = 3; one-way ANOVA, Dunnett’s multiple comparisons test). (H) IFN-signature expression (qPCR). p values depict comparison vs. UT DCs (n = 3; one sample t test).(I) Flow cytometry of frequency of PD-L1+PD-L2+CD200+ of CD11c+ cells (moDCs alone/cocultured with untreated/dying WT/Mlkl−/− TC1 cells). p values depict comparison vs. UT moDCs (n = 4, LPS/IFNβ n = 3; one-way ANOVA, Fischer least significant difference [LSD]).(J) Flow cytometry of frequency of CD11b+F4/80+ in moDCs (alone/cocultured with untreated/dying WT/Mlkl−/− TC1 cells) or bone-marrow-derived macrophages (BMDMs). p values depict comparison vs. BMDMs (n = 3; one-way ANOVA, Dunnett’s multiple comparisons test).(K) Cytokine secretion via cytokine array. From all values, the background was subtracted. Normalization was done using moDCs + untreated cancer cells (n = 3). Here, “n” represents biological replicates and error bars represent SEM. See also Figures S2 and S3.