Fig 2: Diverging molecular adaptations to CUS in male and female microglia. Male and female C57BL/6 mice were exposed to 14 days of chronic unpredictable stress (CUS) or handled as control (CON). A) Two hours after the final stressor frontal cortex was dissected for microglia enrichment through Percoll gradient isolation. Following enrichment microglia were sorted (FACS) based on CD11b+/CD45lo expression. B) Gating strategy for FACS is shown. C) Relative quantities of microglia (CD11b+/CD45lo) and brain macrophages (CD11b+/CD45hi). Surface levels of CSF1R (CD115) (D), CX3CR1 (E), and CD11b (F) were determined (n = 4–8/group). Following FACS, sorted microglia were used for transcriptional analyses via qRT-PCR (n = 6–8/group) (G). Bars represent the mean ± S.E.M. Means significantly different than same-sex controls based on ANOVA are denoted (*, p < 0.05). Means significantly different from opposite sex controls based on ANOVA are noted by (^, p < 0.05).

Fig 3: The gene expression signature associated with XO4+ microglia is reversible and is acquired through phagocytosis of amyloid plaques.a Schematic representing the experimental design involving addition of 2 × 104 microglia to NIAD4-stained organotypic hippocampal slice cultures (OHSCs), followed by FACS isolation of carboxyfluorescein succinimidyl ester (CFSE)-labelled replenished and CFSE- endogenous microglia that differentially phagocytose endogenous NIAD4-labelled plaques after 5 days co-culture with wild-type (WT) or 5xFAD OHSCs, created with BioRender.com. b, c k-nearest-neighbour (kNN) graph rendered using a force-directed layout (SPRING)142, coloured by single cell consensus (SC3) cluster (b), and log2-transformed ?Ct values of selected DEGs (c). Each dot represents 10 sorted cells, and data are from (d) n = 120 cells, (e) n = 240 cells, (f) n = 110 cells, (g) n = 280 cells sorted during 3 independent experiments. Replicates from independent experiments are closed circles, technical replicates are open circles. The XO4+ score is defined as the x-axis position of each sorted population on the kNN graph. The colour scales are log2(?Ct), Mafb min ?Ct = 0.0003, max ?Ct = 3.37; Cx3cr1 min ?Ct = 0.0001, max ?Ct = 9.69; Cst7 min ?Ct = 0.0002, max ?Ct = 4.56; Igf1 min ?Ct = 0.0001, max ?Ct = 1.07. d–g Experimental schematic, XO4+ score and proportion Cluster 1 and Cluster 2 membership of groups of exogenous and endogenous (d) WT microglia added into a WT OHSC, (e) WT microglia added into 5xFAD slices, recapitulating the gene expression signature associated with XO4+ microglia upon plaque phagocytosis. f XO4+ phenotype is stable in exogenous CFSE+NIAD4+ 5xFAD microglia recovered from 5xFAD slices, but (g) is lost in CFSE+ 5xFAD microglia recovered from WT slices. N.D., not detected. Data are presented as mean ± SEM.

Fig 4: Mouse tDC Phenotype Transitions from pDC-like to cDC-like(A) tSNE plot of CyTOF data as in Figure 1, colored by Cx3cr1 expression (top left). Top row: biaxial plots of CyTOF data. Black dots represent gated Cx3cr1+ Tcf4+ cells. Cells were divided based on the expression of CD11c and Ly6C and mapped to the CyTOF tSNE plot (lower left). Bottom row: flow cytometry gating strategy (see Figure S3A for the full gating strategy). One representative of two independent experiments (exp).(B) Total numbers of DC subsets in mouse spleen (n = 14) and skin-draining lymph nodes (LNs; n = 7). Shown is mean ± SD.(C) tSNE map of spleen and LNs analyzed by CyTOF, with manual annotation (left) and colored by MHC class II expression (right). One representative of two exp.(D) Surface markers analyzed in each DC subset by flow cytometry representative of n ≥ 3. Numbers indicate geometric Mean Fluorescence Intensity (gMFI) × 102.(E) Noncanonical DCs were gated as described (Bar-On et al., 2010) and overlaid in the tSNE plot of mouse spleen CyTOF data (orange dots, upper panel). Noncanonical DCs were also overlaid in our flow cytometry gating strategy described in (A) (bottom panels).(F) Frequency of noncanonical DCs within CD11chigh and CD11clow tDC gates (pie charts) and total number of tDCs and noncanonical DCs in the spleen (n = 14).

Fig 5: Drp1 Is Required for T Cell Accumulation into Draining LNs and for Infiltration and Exhaustion into Tumor Masses during Immune-Surveillance(A and B) In vitro-activated control (eFluor670-labeled) and Drp1 KO (CFSE-labeled) T cells were injected i.v. into WT recipient mice bearing 13 day-old MC38-derived tumors. After 24 hr, peripheral blood (PB), inguinal DLNs, and tumor masses (TILs) were collected, and the KO:control ratio (A, both total T cells or CD8+ T cells only) and the exogenous CD8+:CD4+ T cell ratio (B) (considering both control and KO cells) were quantified by flow cytometry (TILs, n = 4; DLNs, n = 6).(C) Size of MC38-induced s.c. tumors in +/+ cre+ control and fl/fl cre+ Drp1 conditional KO mice at the indicated times (n = 11 ctrl, 8 KO).(D) Tumor weight graphs and pictures of representative MC38-derived tumors isolated 18 days after cell injection (n = 11 ctrl, 8 KO).(E and F) Absolute number of +/+ cre+ control and fl/fl cre+ Drp1 KO CD4+ and CD8+ T cells collected from inguinal DLNs (E) or from MC38-derived tumors (TILs) (F) 18 days after s.c. tumor cell injection (n = 14 ctrl, 11 KO).(G) CD8+ or CD4+ IHC staining on 18-day-grown isolated MC38-derived tumor slices. Quantification of CD8+ and CD4+ TILs density is reported in the corresponding graph (n = 5 ctrl, 6 KO).(H) Density of dextramer+ CD8+ TILs per field, calculated by combining the percentage of recovered CD8+ dextramer+ cells by cytofluorimetric analysis with the quantification of total CD8+ TIL density by IHC (n = 5 ctrl, 5 KO).(I) Correlation between tumor size and CD8+ TIL percentage among CD45+ cells in tumor-bearing +/+ cre+ control and fl/fl cre+ Drp1 KO mice. R = -0.661, p = 0.019 (n = 5 ctrl, 7 KO).(J and K) Relative expression of CX3CR1 (negatively, intermediately, or highly expressed) (J, n = 9) or PD1 (K, n = 9) in +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+CD44+ T cells collected from DLNs or from MC38-derived tumors (TILs) 18 days after s.c. tumor cell injection.(L) Percentage of CD44+ cells among all CD8+ and of IFN?+, Tbet+, and Eomes+ cells among all CD8+CD44+ T cells isolated from DLNs or in the tumors (TILs) of +/+ cre+ control and fl/fl cre+ Drp1 KO mice 18 days after s.c. tumor cell injection and after 6 hr of in vitro T cell re-stimulation (n = 5 ctrl, 7 KO).Data are represented as mean ± SEM in (C), (E), and (J) and as dot density plots in (A), (B), (D), (F)–(H), (K), and (L). Data are from two representative of four independent experiments. Scale bars, 5 mm in (D) and 100 µm in (G). Significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S6.