Siponimod Induces Peripheral T Cell Redistribution Without Affecting Glial Cells or Behavior in Naïve Mice

Background: Siponimod is a selective modulator of sphingosine-1-phosphate receptors 1 and 5, approved for the treatment of secondary progressive multiple sclerosis. Apart from its immunomodulatory effects, it has been proposed to exert pro-regenerative and pro-myelinating effects. However, it is unclear whether the reported neuroprotective and pro-regenerative effects of siponimod reflect direct engagement on glial cells or arise secondary to reduced neuroinflammation. Methods: We investigated the systemic and central effects of siponimod in naïve mice treated orally with 3 mg/kg for 19 consecutive days. Peripheral immune cell populations were analyzed by flow cytometry, drug exposure was quantified by liquid chromatography and high-resolution mass spectrometry, glial cell populations were assessed by immunohistochemistry, and functional outcomes were evaluated using behavioral tests. Results: Siponimod treatment induced pronounced T cell lymphopenia, characterized by reduced CD8+ T cells and increased proportions of double-negative T cells and CD4+CD25+ T cells. Lymphocyte distribution was compartment-specific, with increased splenic T cell and reduced thymic T cell counts, while lymph nodes were largely unaffected. Siponimod penetrated the CNS, with hippocampal concentrations exceeding plasma levels. Despite robust brain exposure, siponimod did not affect oligodendrocyte lineage cell density, proliferation, microglial cell density, or morphology, nor did it affect behavior in mice. Conclusions: Taken together, these findings demonstrate that under physiological conditions, the actions of siponimod lie primarily in its immunomodulatory effects, while direct effects on glial cells and behavior are absent. Furthermore, this study paves the way for studies of siponimod’s effect on glial cells and neurons in models with minimal or no blood–brain barrier damage.