Acid ceramidase expression reduces IFNγ secretion by mouse CD4⁺ T cells and is crucial for maintaining B-cell numbers...

Acid ceramidase (Ac) is a lysosomal enzyme catalyzing the generation of sphingosine from ceramide, and Ac inhibitors are currently being investigated as potential cancer therapeutics. Yet, the role of the Ac in immune responses, particularly anti-viral immunity, is not fully understood. To investigate the impact of Ac expression on various leukocyte populations, we generated a tamoxifen-inducible global knockout mouse model for the Ac (iAc-KO). Following tamoxifen administration to healthy mice, we extracted primary and secondary lymphoid organs from iAc-KO and wild-type (wt) littermates and subsequently performed extensive flow cytometric marker analysis. In addition, we isolated CD4⁺ T cells from the spleen and lymph nodes for sphingolipid profiling and restimulated them in vitro with Dynabeads™ Mouse T-activator CD3/CD28. Intracellular cytokine expression (FACS staining) was analyzed and secreted cytokines detected in supernatants. To study cell-intrinsic effects, we established an in vitro model for iAc-KO in isolated CD4⁺ T and B cells. For CD4⁺ T cells of iAc-KO versus wt mice, we observed reduced Ac activity, an increased ceramide level, and enhanced secretion of IFNγ upon CD3/CD28 costimulation. Moreover, there was a marked reduction in B cell and plasma cell and blast numbers in iAc-KO compared to wt mice. To study cell-intrinsic effects and in line with the 3R principles, we established in vitro cell culture systems for iAc-KO in isolated B and CD4⁺ T cells. Our findings pinpoint to a key role of the Ac in mature B and antibody-secreting cells and in IFNγ secretion by CD4⁺ T cells.