Fig 2: Deletion of PD-1 in the APP/PS1 mouse model aggravates plaque pathology and behavioral deficits A, BELISA of the (A) RIPA-soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for Aßx-40 (biological replicates with n = 6 for APP/PS1 and n = 5 for APP/PS1 PD-1-/-, mean ± SEM, one-way ANOVA (df = 1, F = 0.6, P = 0.45), Tukey's HSD, **P < 0.01, ***P < 0.001) and (B) of the SDS-soluble fraction (biological replicates with n = 6 for APP/PS1 and n = 5 for APP/PS1 PD-1-/-, mean ± SEM, one-way ANOVA (df = 1, F = 32.8, P = 2 × 10-5), Tukey's HSD, **P < 0.01, ***P < 0.001).CThioflavin T histochemistry from sagittal brain sections in the hippocampus (Hc), and cortex (Cx) of female APP/PS1 and APP/PS1 PD-1-/- mice. Nuclei were stained using Hoechst 33342 (bar = 500 µm).D, EMice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n = 6 for APP/PS1 and n = 9 for APP/PS1 PD-1-/-, mean ± SEM, one-way ANOVA (df = 1, F = 0.905, P = 0.90), Tukey's HSD, ***P < 0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n = 6 for APP/PS1 and n = 9 for APP/PS1 PD-1-/-, mean ± SEM, one-way ANOVA (df = 1, F = 4.08, P = 0.054), Tukey's HSD, **P < 0.01, ***P < 0.001).FWestern blot analysis of brain lysates from female APP/PS1 and APP/PS1 PD-1-/- mice for the expression of APP and APP C-terminal fragments using antibodies 6E10 (transgene expression) and CT15 (transgene and endogenous expression). Tubulin was used as a loading control.GTime needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean ± SEM of biological replicates with n = 12 for wt, n = 5 for PD-1-/-, n = 7 for APP/PS1, and n = 8 for APP/PS1 PD-1-/-, one-way ANOVA (df = 3, F = 17.72, P = 1 × 10-9), Tukey's HSD, *P < 0.05, ***P < 0.001).HAnalysis of the area under the curve (mean + SEM of biological replicates with n = 12 for wt, n = 5 for PD-1-/-, n = 12 for APP/PS1, and n = 8 for APP/PS1 PD-1-/-, ANOVA (df = 3, F = 6.98, P = 0.001), *P < 0.05 and **P < 0.01).

Fig 3: PD-1 deletion reduces phagocytosis and CD36 expression in microglia A In vitro phagocytosis of FAM-Aß1-42 by wild-type or PD-1-/- microglia for up to 6 h (mean ± SEM of a technical quadruplicate, Student's t-test, **P < 0.01 and ***P < 0.001, representative result of an n = 2 biological replicate).B In vitro phagocytosis of FAM-Aß1-42 by wild-type microglia cultured in astrocyte-conditioned medium (ACM) from wild-type or PD-L1-/- astroglial cultures (mean ± SEM of a technical quadruplicate, Student's t-test, *P < 0.05 and ***P < 0.001, representative result of an n = 2 biological replicate).CFlow cytometry analysis of microglia from wild-type and PD-1-/- mice for the expression of CD36 and CD11b.DQuantification of microglial in vivo phagocytosis in female APP/PS1 and APP/PS1 PD-1-/- mice at 8.5 months of age after intraperitoneal injection of methoxy-XO4 (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD-1-/- mice, Student's t-test (t = 2.1808, df = 6.9829), *P < 0.05).E–GRelative expression of the cell surface marker (E) CD36 (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD-1-/- mice, ANOVA (df = 1, F = 0.002, P = 0.94), Tukey's HSD, *P < 0.05, ***P < 0.001), (F) CD11b (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD-1-/- mice), and (G) CD45 on methoxy-XO4+ versus methoxy-XO4- microglia in APP/PS1 and APP/PS1 PD-1-/- mice normalized to methoxy-X04-negative cells from APP/PS1 mice (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD-1-/- mice). Data information: (D–G) Central bands represent median, boxes show interquartile range (IQR), and whiskers define the ± 1.5xIQR.

Fig 4: Behavioral analysis of wild-type, PD-1-/-, APP/PS1, and APP/PS1 PD-1-/- mice AMice were tested one day after the last trial day for 30 s in the absence of the platform in quadrant 4. The time in the quadrants was measured and averaged for quadrants 1–3.B–EEvaluation of the open field testing for (B) time spend in the center (center time) (ANOVA (F = 0.83, df = 6, P = 0.54), Bonferroni test), (C) corner time (ANOVA (F = 0.47, df = 6, P = 0.93), Bonferroni test), (D) corridor time (ANOVA (F = 1.487, df = 6, P = 0.193), Bonferroni test, *P < 0.05), and (E) distance (ANOVA (F = 0.83, df = 6, P = 0.54), Bonferroni test, *P < 0.05, **P < 0.01, and ***P < 0.001). Data information: For all panels: mean + SEM of biological replicates with n = 12 for wt, n = 5 for PD-1-/-, n = 7 for APP/PS1, and n = 5 for APP/PS1 PD-1-/-.

Fig 5: Transcriptome analysis of PD-1-/- microglia Top 5 enriched KEGG pathways of differentially expressed genes (abs(fold change) > 2, P < 0.01, Benjamini–Hochberg-adjusted) in PD-1-/- vs. wild-type microglia.Different expression of inflammasome-related genes (abs(fold change) > 1.5, P < 0.05, Benjamini– Hochberg-adjusted) in PD-1-/- vs. wild-type microglia.Different expression of complement system-related genes (abs(fold change) > 1.5, P < 0.05, Benjamini–Hochberg-adjusted) in PD-1-/- vs. wild-type microglia.