Abstract: Fingolimod inhibits cytotoxic T cells: A novel immunomodulatory effect of the unphosphorylated compound (The 27th Annual Meeting of the CMSC and the 5th Cooperative Meeting of the CMSC-ACTRIMS)

Saturday, June 1, 2013: 4:10 PM

Achilles Ntranos, MD , Neurology, Johns Hopkins School of Medicine, Baltimore, MD

Inna V Grishkan, BS , Neurology, Johns Hopkins School of Medicine, Baltimore, MD

Dionne P Robinson, BS , Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD

Olivia J Hall, MSc , Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD

Sabra L Klein, PhD , Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD

Peter A Calabresi, MD, FAAN , Neurology, Johns Hopkins School of Medicine, Baltimore, MD

Anne R Gocke, PhD , Neurology, Johns Hopkins School of Medicine, Baltimore, MD

Background: Fingolimod, a sphingosine-1-phosphate receptor modulator, is a multiple sclerosis (MS) therapeutic that upon phosphorylation traps CCR7+ T cells in lymph nodes. CCR7- effector T cells are spared, thus allowing effective infection clearance. Nonetheless, fingolimod-treated patients are more susceptible to viral infections, indicating an impaired cytotoxic T (Tc) cell response. Yet, fingolimod’s effect on Tc cells remains unclear.

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.

7.1 Fingolimod inhibits cytotoxic T cells: A novel immunomodulatory effect of the unphosphorylated compound