Background: Multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS), is characterized by blood-brain barrier (BBB) disruption and massive infiltration of activated immune cells. Engagement of programmed cell death-1 (PD-1) expressed on activated T cells with its ligands (PD-L1 and PD-L2) suppresses T cell responses. We recently demonstrated in MS lesions elevated PD-L1 expression by glial cells and absence of PD-1 on many infiltrating CD8 T cells. We have now investigated whether human brain endothelial cells (HBECs), which maintain the BBB, can express PD-L1 or PD-L2 and thereby modulate T cells.
Methods: We used primary cultures of HBECs isolated from non-tumoral CNS tissue either under basal or inflamed conditions. We assessed the expression of PD-L1 and PD-L2 using qPCR and flow cytometry. Human CD8 T cells were isolated from peripheral blood of healthy donors and co-cultured with HBECs. Following co-culture with HBECs, proliferation and cytokine production by human CD8 T cells were measured by flow cytometry whereas transmigration was determined using a well established in vitro model of the BBB. The functional impact of PD-L1 and PD-L2 provided by HBECs was determined using blocking antibodies. We performed immunohistochemistry for the detection of PD-L1 or PD-L2 concurrently with caveolin-1 (a cell specific marker for endothelial cells) on post-mortem human brain tissues obtained from MS patients and normal controls.
Results: Under basal culture conditions, PD-L2 is expressed on HBECs, whilst PD-L1 is not detected. Both ligands are up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 leads to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increases CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions does not express detectable PD-L1; in contrast, all blood vessels in normal brain tissues are PD-L2-positive, while only about 50% express PD-L2 in MS lesions.
Conclusions: Our observations suggest that brain endothelial cells contribute to control T cell transmigration into the CNS and immune responses via PD-L2 expression. However, such impact is impaired in MS lesions due to downregulation of endothelium PD-L2 levels.