BackgroundThe mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation.MethodsHigh-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels.ResultsA subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5+ leukocytes in blood as well, implying these cells are present prior to TEM. Moreover, imaging of inflamed CNS vessels and the associated perivascular cell infiltrates from Tie-2-eGFP-CLN-5 mice with EAE revealed leukocytes bearing the eGFP label, further supporting the hypothesis CLN-5 is transferred from endothelial cells to circulating leukocytes in vivo. Western blotting of BMEC-derived EVs, corresponding in size to both exosomes and microvesicles, and FACS analysis of plasma-derived EVs from Tie-2-eGFP-CLN-5 mice with EAE validated expression of CLN-5 by EVs of endothelial origin. Confocal imaging and FACS further revealed both PKH-67-labeled EVs from cultured BMECs and eGFP-CLN-5+ EVs from plasma of Tie-2-eGFP-CLN-5 mice with EAE can bind to leukocytes. Lastly, serial EM and 3D contour-based surface reconstruction revealed a close association of EV-like structures between the marginating leukocytes and BMECs in situ during EAE.ConclusionsDuring neuroinflammation, CLN-5+ leukoc...

Regulatory T cells (Treg) are vital for immune homeostasis and are dysfunctional in autoimmunity. Interleukin 2 (IL-2) drives the proliferation and function of Tregs via its heterotrimeric receptor (CD25/CD122/CD132). Low dose IL-2 is being evaluated for treatment of autoimmune diseases and has been shown to expand Tregs, yet it has a small selectivity window over conventional T cells (Tconv) and natural killer (NK) cells. To enhance IL-2 selectivity, mutations can be introduced to reduce its CD122/CD132 affinity thus creating a CD25 dependency for signaling through CD122/CD132 upon IL-2 facilitated CD25/CD122/CD132 trimer formation. Using structure guided approach, we introduced mutations in IL-2 that significantly decreased CD122 affinity in addition to mutations that increased CD25 affinity. Finally, we explored other mutations, orientation, and linkers to generate a potent, selective molecule with drug-like manufacturability. These structure activity relationship efforts culminated in the generation of PT101, a mutant IL-2 Fc fusion that was selective in activating and expanding Tregs. PT101 selectively induced STAT5 phosphorylation in human and non-human primate (NHP) Tregs in vitro. In humanized mice and NHPs, administration of PT101 dose-dependently and selectively expanded Treg without significant effects on other immune cell types, nor eliciting proinflammatory cytokine production. In a Phase 1a clinical trial, PT101 was well-tolerated and selectively expanded total Tregs by up to a mean maximum of 3.6-fold. There was no evidence of expansion of NK cells nor pro-inflammatory Tconv at any dose studied. Clinical trials in patients with ulcerative colitis and systemic lupus erythematosus are planned with PT101.

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