Figure 3. T cell-specific acid sphingomyelinase (Asm) deficiency leads to reduced CD8⁺ T cell activation in vitro.

Isolated CD8⁺ T cells from Smpd1^fl/fl/Cd4^cre/+ mice (Asm/CD4cre) and Smpd1^fl/fl/Cd4^+/+ littermates (wildtype [WT]) where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. (A) mRNA expression of Asm (Smpd1) following activation was analyzed by RT-qPCR (n=4–8). (B) Ceramide levels of CD8⁺ T cells were determined by mass spectrometry (n=4). (C) Expression of CD25, CD69, and CD44 was analyzed by flow cytometry (n=6–7). Representative histograms are shown in the upper panel. (D) OVA-specific cytotoxic lymphocytes were generated and incubated with OVA-peptide 257–264 loaded CFSE^high-labeled target and unloaded CFSE^low-labeled control cells. Specific killing was evaluated by frequencies of target and control populations determined by flow cytometry (n=6–7). Representative histograms are shown in the left panel. (E) Frequencies of granzyme B-expressing CD8⁺ T cells from Asm/CD4cre mice and WT littermates without and (F) in the presence of C16 ceramide were analyzed by flow cytometry (n=4–8). Representative contour plots are shown in the left panel. Results from two to four independent experiments are depicted as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test, or Student’s t-test. (*p<0.05, **p<0.01, ****p<0.0001).

Figure 3—figure supplement 1. In vitro characterization of acid sphingomyelinase (Asm)-deficient CD4⁺ T cells.

(A) Isolated CD4⁺ T cells from Asm/CD4cre mice and wildtype (WT) littermates were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies for indicated time points. mRNA expression of Asm (Smpd1) was analyzed by RT-qPCR (n=6–8). (B) Ceramide levels of unstimulated or anti-CD3/anti-CD28 stimulated (24 hr) CD4⁺ T cells from Asm/CD4cre mice and WT littermates were determined by mass spectrometry (n=4). (C) Sorted CD4⁺CD25⁻ T cells from Asm/CD4cre and WT mice were stimulated with anti-CD3 and anti-CD28 antibodies 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=6–7). Representative dot plots are depicted in the left panel. (D) In order to differentiate naïve CD4⁺CD25⁻ T cells into Th1 cells, isolated cells were cultured in the presence of anti-CD3, anti-CD28, anti-IL-4, and IL-12 (Th1), or only stimulated with anti-CD3 and anti-CD28 (Th0). After 6 days, Th1 differentiation was assessed by IFN-γ expression (n=5–6). Representative dot plots are shown in the left panel. Results from two to three 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).