Fig 2: Microglial depletion with PLX5622 alters antigen presentation and T cell reactivation in the spinal cord.Histograms showing MHC-II, CD80 and CD86 fluorescence intensity in different APCs (dendritic cells (DCs), macrophages/monocytes (MCs/MOs) and microglia (Mglia)) and in diverse tissues, as analyzed by flow cytometry at the pre-onset stage of EAE (A; dpi 8–9) (n = 5 spinal cord samples, n = 10 spleen and blood samples), and at the onset stage of EAE (B; dpi 13–14) (n = 10). C Representative images showing CD3+ lymphocytes and Ki67 proliferative-associated expression in spinal cord, at EAE onset (dpi 14) in control and PLX5622-treated mice. Histogram show the proportion of Ki67+ CD3+ T cells in relation to the total number of CD3+ T cells (n = 3). Scale bar = 25 µm. D Representative images showing the early Ki67+ CD3+ proliferating lymphocytes in the spinal cord at EAE onset (dpi 10, D). Scale bar = 30 µm. E Representative images and quantification of CD3+ lymphocytes proliferation at a timepoint coincident with the beginning of symptoms in both groups (dpi 14 for control mice and dpi 20 for PLX5622-treated mice (n = 3). Scale bar = 25 µm. Data are presented as means ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001.

Fig 3: a) Schematic illustration of the immunotherapy of orthotopic GL261 tumor by using PX and CS‐J NPs or CS‐J@CM/6 NPs. b) The TUNEL (green fluorescence) for characterizing the apoptotic cells, in which the nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue fluorescence) (scale bar: 50 µm). c) H&E staining images of brain slices from different groups of mice after being treated for 20 days (scale bar: 1 mm). d) Survival rates of mice from different groups (n = 5). e) Immunofluorescence images of CRT exposed in tumor (scale bar: 25 µm). f,g) Flow cytometry analysis of matured DCs (CD11c+CD80+CD86+) and their percentage in the CLNs of mice from different groups (n = 5).

Fig 4: A lack of autophagy significantly impacts gene expression of pro-inflammatory cytokines(A) Volcano plot of differentially expressed genes in poly(I:C)-treated Atg5KO vs. Atg5flox/flox mice.(B) Volcano plot of differentially expressed genes in poly(I:C)-treated TfebTG vs. Tfebflox/flox mice.(C) Heatmap representation of cytokines significantly modulated in poly(I:C)-treated Atg5KO mice.(D) Heatmap representation of cytokines significantly modulated in poly(I:C)-treated TfebTG mice. n = 3 mice per group.(E and F) The regulation of genes encoding for relevant M1/M2 markers represented as fold change in poly(I:C)-treated Atg5KO vs. Atg5flox/flox control mice and in (F) TfebTG vs. Tfebflox/flox control mice.(G) The number of M1 and M2 macrophages in the lungs of Atg5flox/flox and Atg5KO mice challenged with poly(I:C).(H–K) Frequency of CD11c+ CD11b+ CD64+ macrophages expressing (H) CD86, (I) CD206, (J) CD163, and (K) CD36.Data are represented as means ± SEM (two-way ANOVA). ns, not significant, *p < 0.05; **p < 0.01; ***p < 0.001.See also Figure S5.

Fig 5: Neu1-/- splenic macrophages show an aberrant M1/M2 phenotype. (A) Representative pseudocolor dot-plots of splenic macrophages (F4/80+, CD11blow/−) (left) and CD206 and CD86 expression (right). (B, C) Quantification of frequency (B) and number (C) of F4/80+ splenic macrophages from WT and Neu1-/- mice. (D) Quantification of CD86+CD206-, CD86-CD206+, double-positive (DP) (CD206+CD86+) and double-negative (DN) (CD206-CD86-) frequencies of WT and Neu1 -/- splenic macrophages. (E, F) Representative histograms and relative expression of CD86 (E) and CD206 (F) in WT (green) and Neu1 -/- (blue) splenic macrophages. Mean fluorescence intensity (MFI) was normalized relative to the average of WT mice. Bars represent mean ± SEM. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired t-test for (B, C, E, F) and two-way ANOVA with Bonferroni post-hoc test for (D) Data from n=6 from 5 independent experiments. MΦ, macrophages.