Analyzing the effects of laquinimod on innate and adaptive immunity in mice with experimental autoimmune encephalomyelitis

Laquinimod (LAQ), a new orally active immunomodulator, is currently under investigation for the treatment of multiple sclerosis (MS). In clinical trials LAQ considerably reduced the annualized relapse rate, disability progression and brain atrophy in MS patients, but its exact mechanism of action is not fully understood. Experimental data provide evidence for immunomodulatory effects of LAQ on antigen presenting cells and T cells, but the role of natural killer (NK) cells within the mechanism of action of LAQ has not been investigated yet. The aim of the present study was to evaluate effects of LAQ on innate and adaptive immune cells in mice with experimental autoimmune encephalomyelitis (EAE), with a special focus on NK cells. First, a detailed analysis of immune cells was performed in MOG35-55-immunized animals preventively treated with 25 mg/kg LAQ. Innate immune cells responded quickly to LAQ treatment, whereas major changes in the T cell compartment were detectable several days later. LAQ reduced the total number of splenic dendritic cells and the number of myeloid dendritic cells. In parallel, NK cells were activated by LAQ. As such LAQ increased the frequency of CD69+ NK cells and upregulated a number of activating NK cell receptors such as DNAM-1 and TACTILE. TIGIT, an inhibitory NK cell receptor, which shares the same ligand with DNAM-1 and TACTILE, was downregulated, suggesting that LAQ shifts the balance of inhibitory and activating NK cell receptors in favor of the latter. To test, whether the LAQ-induced NK cell activation improves classical NK cell effector functions, NK cells were sorted ex vivo from LAQ- or vehicle-treated animals. NK cells derived from LAQ-treated mice killed B16F10 melanoma cells in vitro more efficiently than NK cells derived from vehicle controls and produced more cytokines in response to IL-12 and IL-18 stimulation. In a triple co-culture system with 2D2 T cells and bone marrow-derived dendritic cells, T cell proliferation in response to MOG35-55 was significantly better inhibited by NK cells derived from LAQ-treated mice in a cell contact-dependent manner. Finally, LAQ efficacy was analyzed in NK cell-depleted and competent EAE animals. Th/+ mice were depleted by administration of PK-136 antibodies, immunized with MOG35-55 and preventively treated with 25 mg/kg LAQ. Treatment with LAQ markedly attenuated EAE severity in NK cell-competent Th/+ mice. This beneficial LAQ effect was however only transient in Th/+ mice depleted of NK cells. While LAQ treatment prevented the death of animals in the presence of NK cells, a considerable number of animals depleted of NK cells died despite LAQ therapy. In conclusion, this study identified NK cells as a novel direct cellular target of LAQ therapy with a central relevance for the therapeutic efficiency of the substance in EAE. These data indicate that the LAQ-mediated activation of NK cells is a crucial part of the immunomodulatory mechanism of LAQ in vivo. Most interestingly, LAQ specifically interfered with the DNAM-1/TACTILE/TIGIT pairwise receptor family on NK cells, favoring a central role of this pathway within the mechanism of action of LAQ. It remains to be investigated whether similar effects of LAQ on human NK cells can be detected in patients with MS. Since published data reveal defective NK cell function as one pathogenic factor in MS, LAQ-induced restoration of NK cell functions could be a major component of its protective mechanisms in the treatment of MS patients.