029 – PD-L1 and PD-L2 expression in blood immune cells and cerebrospinal fluid extracellular vesicles in Multiple Sclerosis. ThereÕs room for novel therapeutic approaches.
Tommaso Croese (1) – Susanna Manenti (1) – Annamaria Finardi (1) – Roberto Furlan (1)
Institute of Experimental Neurology (INSPE), Clinical Neuroimmunology Unit, Milan, Italy (1)
Extracellular Vesicles (EVs) are small (100-1000nm) membrane particles released by all cell types. EVs have been recently identified as mediators of intercellular communication in both physiologic and pathologic conditions. In particular, myeloid EVs seem to be significantly upregulated in the cerebrospinal fluid (CSF) of patients with neuro-inflammatory disorders, such as those suffering from the most severe forms of multiple sclerosis (MS). Recent studies emphasize the role of PD-1 and its two ligands (PD-L1 and PD-L2) both in neuro-degenerative and neuro-inflammatory diseases. PD-1 is preferentially expressed on activated T cells. Cross-linking of PD-L1/ PD-L2 and PD-1 is supposed to inhibit T cell activation and favor their exhaustion and apoptosis. We assess the expression of PD-1 and its ligands on peripheral blood immune cells. Patients with MS display lower level of PD-L1 on myeloid cell compartment suggesting a defect of PD-L1 in MS that was never described before. On the other hand, PD-L2 was significantly up-regulated only on neutrophils in patients with clinical and neuro-radiological evidence of disease activity. Since other paper have already demonstrated PD-L1 expression on circulating EVs, we sought to characterize CSF derived EVs in patients with neurological disorders. After lumbar puncture, CSF was processed to isolate shedding EVs and analyzed with a specific flow-cytometer (Cytoflex LX). We observed a statistically significant increase in PD-L2 expression on EVs of myeloid origin, identified by the use of Isolectin B4 (Ib4). These results were even more clear-cut in patients with active form of MS. To evaluate whether Ib4 positive CSF-derived EVs originate from resident CNS cells (microglia) or infiltrating myeloid cells, we generated chimera mice by transplanting bone marrow from CAG-GFP to wild-type mice. Subsequently, we induced Experimental Autoimmune Encephalomyelitis (EAE) and performed cisterna magna puncture to collect CSF. As expected, the number of GFP positive EVs goes up to almost 50 percent of the total at disease peak. Combining our date from blood and CSF, we can speculate that the increase of PD-L2 positive EVs during disease activity reflects an augmented infiltration of neutrophils. This deficiency of PD-L1 and the increase of PD-L2 may serve as novel mechanisms of disease for MS and allow for establishment of new therapeutic approaches.