Fig 2: HFD-induced obesity accelerated tumor growth in a CD4+ T cell-dependent manner. (a) Experimental schema is shown. After 9 weeks of feeding, the colon cancer cell line MC38 (4 × 106 cells) was inoculated subcutaneously on the backs of mice. Anti-CD4 antibodies were administered starting from the day before tumor inoculation, and the effect on tumor progression was observed. (b) Tumor volume of the CD- and HFD-fed groups during 3 weeks of tumor inoculation was measured. (c) Tumor volume of the CD4- and CD8-depleted and isotype groups during 3 weeks of tumor inoculation was measured. (d) the number and (e) PD-1 expression of CD8+ T cells in each organ were analyzed. (f) The percentage of CD107a expression in CD8 + T cells in tumors following CD4 depletion are shown. The data are expressed as the mean ± S.D. (n = 7 mice in each group). Two-way analysis of variance (2 × 2) was used to analyze the effects and the interaction of two factors: diet (unrelated to anti-CD4) or anti-CD4 (unrelated to diet). * p < 0.05, ** p < 0.01, *** p < 0.005. HFD, High-fat diet; CD, Control diet; PD-1, programmed death-1.

Fig 3: No change in Lamp1 levels in the brains of DKO mice after AAV treatment, except in the thalamus and pons. Immunostaining of Lamp1 (lysosome marker) in coronal brain sections (A) from cortex, hippocampus, thalamus, cerebellum, and pons of 20- and 30-week-old WT, 20-week-old DKO mice, and 20- and 30-week-old DKO mice treated with AAV, as well as DKO mice treated with AAV and Istradefylline. Images were taken at 20X magnification under identical light conditions, using different filters for DAPI and anti-Lamp-1. Sections were stained with anti-Lamp1 antibody (green; lysosome marker) and DAPI (blue; nucleus). Lamp1 intensity was quantified using ImageJ (B-F). One-way ANOVA was used to calculate p-values with GraphPad. Data are shown as means ± SEM (n = 3; *p < 0.05, **p < 0.025, ***p < 0.01, ****p < 0.001)

Fig 4: Reduction of neuroinflammation in the cortex (A-H) and cerebellum (I-P) of AAV-treated DKO mice. The expression levels of CCL2, CCL3, CCL5, Cxcl10, IL-1β, Iba1, GFAP, and Lamp1, a lysosome-associated protein, were measured in 20-week-old WT mice, 20-week-old DKO mice, and DKO mice treated with AAV at 20 and 30 weeks, with or without Istradefylline. Expression levels were assessed using the ΔCT method, and p-values were calculated with One-way ANOVA using GraphPad. Data are shown as means ± SEM (n = 3; *p < 0.05, **p < 0.025, ***p < 0.01, and ****p < 0.001)