Fig 2: A-SMase is activated downstream of the TLR4 receptor and translocated to the plasma membrane in response to Aβ oligomers. A A-SMase activity expressed as percentage of control measured in microglia treated with Aβ1−42 oligomers in the presence of Imipramine (10 µM) or TNFα receptor inhibitor (10 ng/mL) or TLR4 receptor inhibitor (10 µM). Bar graphs represent average ± SD of N > 3/group One-way ANOVA, Dunnett’s posthoc testing (**p < 0.01, ***p < 0.001, ****p < 0.0001). B Photomicrographs of primary microglia stimulated with or without Aβ1−42 peptide conjugated to Alexa 555 (Red) and subsequently immunolabeled with anti-Cer (Green) and anti-A-SMase (Magenta) antibodies. Nuclei were stained with DAPI. Scale bar = 20µm. C Violin plots depicting Pearson correlation coefficient between Cer and A-SMase fluorescent intensities of 4–5 photomicrographs / condition. Unpaired t-test (**p < 0.01. D IL-1α, TNFα and C1q levels measured in supernatant of primary microglia by ELISA after 18 h incubation with Aβ1-42 oligomers and of 10 µM Imipramine or 5 µM ARC39. Bar graphs represent average ± SD of N > 3/group. One-way ANOVA, Dunnett’s posthoc testing (*p < 0.05, **p < 0.01, ****p < 0.0001)

Fig 3: C1q disrupts CD8+ T cell metabolism, enabling immune evasion in resistant tumors.(A) Growth of E0771-Res1 tumor in CCR2-KO mice receiving WT or TREM2-KO monocytes, under combined therapy (n = 5). Arrowheads indicate monocyte transfers. (B) Representative flow plots showing CFSE dilution and CD25 expression in CD8+ T cell treated with activation beads and indicated concentrations of murine C1q. (C–E) Quantification of total CD8+ T cells (C), CD25+/total CD8+ T cell ratios (D), and IFN-γ production by CD8+ T cells (E). (F) Quantification of surviving E0771-OVA cells after coculture with pretreated OT-1 CD8+ T cells. (G) ELISA quantification of C1q in tumor lysates from E0771, E0771-Res1, and E0771-Res2 tumors (n = 4). (H) Immunofluorescence of MitoTracker Deep Red, C1q, C1QBP, and DAPI in CD8+ T cells. Scale bar: 1 μm. (I) Immunoblot of OPA1, DRP1, and phosphor-DRP1 (Ser616) in CD8+ T cells. (Bottom) Quantification of DRP1 levels normalized to β-ACTIN (3 independent experiments). (J) TEM images of activated CD8+ T cells treated with vehicle or C1q for 48 hours. Scale bar: 2 μm; 0.5 μm (zoomed-in). (K) Representative flow plots of MitoTracker Deep Red/Green (MDR/MG) populations. (L and M) CD25 expression (L) and IFN-γ production (M) in MDR/MGlo versus MDR/MGhi CD8+ T cells. (N) Representative plots and quantification of MDR/MGhi CD8+ T cells from E0771-Res1 tumors in WT (n = 5) or C1qa-KO mice (n = 4). (O) Growth of E0771-Res2 tumors in WT or C1qa-KO mice under vehicle or combined therapy. The values 0.2266 and 1.5 × 104 indicate represent P values for the comparison of tumor volumes between the combined paclitaxel and anti–PD-1 treatment group and the vehicle control at day 18, in wild-type and C1qa knockout mice, respectively. (P) Tumor growth of E0771-Res1 tumors in CCR2-KO mice receiving WT or C1qa-KO monocytes under combined therapy (n = 5). (Q) Flow cytometry of CD8+ T cells from tumors in P. Significance was calculated using 1-way ANOVA followed by Tukey’s test (C–G, and I); unpaired 2-tailed Student’s t test (A and L–Q). *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001.

Fig 4: The concentrations of C1q, C3, IgM, IgG3, and ICs increase in the ileal luminal content during morphine treatment.

Fig 5: C1q disrupts CD8+ T cell metabolism, enabling immune evasion in resistant tumors.(A) Growth of E0771-Res1 tumor in CCR2-KO mice receiving WT or TREM2-KO monocytes, under combined therapy (n = 5). Arrowheads indicate monocyte transfers. (B) Representative flow plots showing CFSE dilution and CD25 expression in CD8+ T cell treated with activation beads and indicated concentrations of murine C1q. (C–E) Quantification of total CD8+ T cells (C), CD25+/total CD8+ T cell ratios (D), and IFN-γ production by CD8+ T cells (E). (F) Quantification of surviving E0771-OVA cells after coculture with pretreated OT-1 CD8+ T cells. (G) ELISA quantification of C1q in tumor lysates from E0771, E0771-Res1, and E0771-Res2 tumors (n = 4). (H) Immunofluorescence of MitoTracker Deep Red, C1q, C1QBP, and DAPI in CD8+ T cells. Scale bar: 1 μm. (I) Immunoblot of OPA1, DRP1, and phosphor-DRP1 (Ser616) in CD8+ T cells. (Bottom) Quantification of DRP1 levels normalized to β-ACTIN (3 independent experiments). (J) TEM images of activated CD8+ T cells treated with vehicle or C1q for 48 hours. Scale bar: 2 μm; 0.5 μm (zoomed-in). (K) Representative flow plots of MitoTracker Deep Red/Green (MDR/MG) populations. (L and M) CD25 expression (L) and IFN-γ production (M) in MDR/MGlo versus MDR/MGhi CD8+ T cells. (N) Representative plots and quantification of MDR/MGhi CD8+ T cells from E0771-Res1 tumors in WT (n = 5) or C1qa-KO mice (n = 4). (O) Growth of E0771-Res2 tumors in WT or C1qa-KO mice under vehicle or combined therapy. The values 0.2266 and 1.5 × 104 indicate represent P values for the comparison of tumor volumes between the combined paclitaxel and anti–PD-1 treatment group and the vehicle control at day 18, in wild-type and C1qa knockout mice, respectively. (P) Tumor growth of E0771-Res1 tumors in CCR2-KO mice receiving WT or C1qa-KO monocytes under combined therapy (n = 5). (Q) Flow cytometry of CD8+ T cells from tumors in P. Significance was calculated using 1-way ANOVA followed by Tukey’s test (C–G, and I); unpaired 2-tailed Student’s t test (A and L–Q). *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001.