Objectives: To investigate the effect of fingolimod on Tc cell activation, cytokine production and cytotoxic function.
Methods: To address this question, we utilized two in vivo models; experimental autoimmune encephalomyelitis (EAE) and a murine influenza model. Tc cell trafficking, as well as IFNγ and Granzyme B (GrB) production was assessed ex vivo by flow cytometry. To further investigate fingolimod's mechanism of action, murine splenocyte cultures were treated with both phosphorylated and unphosphorylated fingolimod, as both forms exist in vivo. Tc cell function was assessed in vitro by intracellular cytokine staining, flow cytometry and an LDH cytotoxicity assay.
Results: Fingolimod ameliorated EAE by sequestering T cells in lymph nodes, but also inhibited splenic Tc cell IFNγ and GrB expression. Influenza was exacerbated and mortality was increased, as fingolimod inhibited Tc cell activation, GrB production and lung infiltration. Remarkably, the in vitro investigations revealed that only the unphosphorylated compound was able to reduce IFNγ and GrB levels in Tc cells and inhibit their function in an LDH cytotoxicity assay. Tc cell function remained unaffected by the phosphorylated compound. Furthermore, the addition of arachidonic acid in the culture rescued Tc cell function, suggesting that fingolimod’s effect is due to inhibition of cytosolic phospholipase A2.
Conclusions: Herein, we demonstrate that fingolimod suppresses Tc cell function independently of its cell trafficking effect. This provides a novel explanation not only for the increased rate of viral infections in fingolimod-treated patients, but also for its efficacy in MS, as Tc cells have emerged as crucial mediators of MS pathogenesis.