Figure 1. Ablation of acid sphingomyelinase (Asm) results in decreased T cell activation and enhanced tumor growth.

(A) B16-F1 melanoma cells were transplanted into Smpd1-deficient mice (Asm-KO) mice and control littermates (Asm-WT). Tumor volume was monitored daily once tumors were palpable (n=12–18). (B) Frequencies of CD4⁺ T cells, CD4⁺Foxp3⁺ Tregs, and CD8⁺ T cells within draining lymph nodes (dLN) and tumor of Asm-KO and Asm-WT mice were determined by flow cytometry. Representative contour plots (dLN) are shown in the left panel. (C) IFN-γ, and CD44 expression of CD4⁺ and CD8⁺ tumor-infiltrating lymphocytes (TILs) in tumor-bearing mice. (D) Percentages of Foxp3⁺ Tregs of CD4⁺ T cells within spleen of naive Asm-KO and Asm-WT mice were determined by flow cytometry (n=16–17). (E) Sorted CD4⁺CD25⁻ T cells from Asm-KO and Asm-WT mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 and TGF-β1 (iTreg). Respective controls (Th0) were only stimulated with anti-CD3 and anti-CD28 antibodies. After 3 days, Treg differentiation was analyzed by Foxp3 expression (n=3–4). Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p<0.05, **p<0.01, ****p<0.0001).

Figure 1—figure supplement 1. Tumor growth and T cell response in amitriptyline-treated mice.

(A) B16-F1 melanoma cells were injected into amitriptyline-treated mice, and tumor growth was monitored once tumors have been established (n=8–9). (B) CD44 and IFN-γ expression of tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Results from two independent experiments are depicted as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p<0.05, **p<0.01, ****p<0.0001).