Patients With Severe Multiple Sclerosis Exhibit Functionally Altered CD8+ Regulatory T Cells.

BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Studies of immune dysfunction in MS have mostly focused on CD4+ Tregs, but the role of CD8+ Tregs remains largely unexplored. We previously evidenced the suppressive properties of rat and human CD8+CD45RClow/neg Tregs from healthy individuals, expressing Forkhead box P3 (FOXP3) and acting through interferon-gamma (IFN-γ), transforming growth factor beta (TGFß), and interleukin-34 (IL-34). secretions to regulate immune responses and control diseases such as transplant rejection. To better understand CD8+CD45RClow/neg Tregs contribution to MS pathology, we further investigated their phenotype, function, and transcriptome in patients with MS. METHODS: We enrolled adults with relapsing-remitting MS and age-matched and sex-matched healthy volunteers (HVs). CD8+ T cells were segregated based on low or lack of expression of CD45RC. First, the frequency in CSF and blood, phenotype, transcriptome, and function of CD8+CD45RClow and neg were investigated according to exacerbation status and secondarily, according to clinical severity based on the MS severity score (MSSS) in patients with nonexacerbating MS. We then induced active MOG35-55 EAE in C57Bl/6 mice and performed adoptive transfer of fresh and expanded CD8+CD45RCneg Tregs to assess their ability to mitigate neuroinflammation in vivo. RESULTS: Thirty-one untreated patients with relapsing-remitting MS were compared with 40 age-matched and sex-matched HVs. We demonstrated no difference of CSF CD8+CD45RClow and CD8+CD45RCneg proportions, but blood CD8+CD45RClow frequency was lower in patients with MS exacerbation when compared with that in HVs. CD8+CD45RCneg Tregs but not CD8+CD45RClow showed higher suppressive capacities in vitro in MS patients with exacerbation than in patients without acute inflammatory attack. In vitro functional assays showed a compromised suppression capacity of CD8+CD45RClow Tregs in patients with nonexacerbating severe MS, defined by the MSSS. We then characterized murine CD8+CD45RCneg Tregs and demonstrated the potential of CD45RCneg cells to migrate to the CNS and mitigate experimental autoimmune encephalomyelitis in vivo. DISCUSSION: Altogether, these results suggest a defect in the number and function of CD8+CD45RClow Tregs during MS relapse and an association of CD8+CD45RClow Tregs dysfunction with MS severity. Thus, CD8+CD45RClow/neg T cells might bring new insights into the pathophysiology and new therapeutic approaches of MS.

Genetic engineering of human and mouse CD4+ and CD8+ Tregs using lentiviral vectors encoding chimeric antigen receptors.

The last decade has seen a significant increase of cell therapy protocols using effector T cells (Teffs) in particular, but also, more recently, non-engineered and expanded polyclonal regulatory T cells (Tregs) to control pathological immune responses such as cancer, autoimmune diseases, or transplantation rejection. However, limitations, such as stability, migration, and specificity of the cell products, have been seen. Thus, genetic engineering of these cell subsets is expected to provide the next generation of T cell therapy products. Lentiviral vectors are commonly used to modify Teffs; however, Tregs are more sensitive to mechanical stress and require specific culture conditions. Also, there is a lack of reproducible and efficient protocols to expand and genetically modify Tregs without affecting their growth and function. Due to smaller number of cells and poorer viability upon culture in vitro, mouse Tregs are more difficult to transduce and amplify in vitro than human Tregs. Here we propose a step-by-step protocol to produce both human and mouse genetically modified CD8+ and CD4+ Tregs in sufficient amounts to assess their therapeutic efficacy in humanized immunocompromised mouse models and murine models of disease and to establish pre-clinical proofs of concept. We report, for the first time, an efficient and reproducible method to isolate Tregs from human blood or mouse spleen, transduce with a lentiviral vector, and culture, in parallel, CD8+ and CD4+ Tregs while preserving their function. Beyond chimeric antigen receptor (CAR)-Treg cell therapy, this protocol will promote the development of potential new engineered T cell therapies to treat autoimmune diseases and transplantation rejection.

Erythrocyte-derived microvesicles induce arterial spasms in JAK2V617F myeloproliferative neoplasm.

Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPNs). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPNs suggests that vascular function is altered. The consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice resulting from a disturbed endothelial NO pathway and increased endothelial oxidative stress. This response was reproduced in WT mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for their effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes suppressed their effect on oxidative stress. Antioxidants such as simvastatin and N-acetyl cysteine improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPNs are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears to be a promising therapeutic strategy in this setting.

A defect in endothelial autophagy occurs in patients with non-alcoholic steatohepatitis and promotes inflammation and fibrosis.

BACKGROUND & AIMS: Previous studies demonstrated that autophagy is protective in hepatocytes and macrophages, but detrimental in hepatic stellate cells in chronic liver diseases. The role of autophagy in liver sinusoidal endothelial cells (LSECs) in non-alcoholic steatohepatitis (NASH) is unknown. Our aim was to analyze the potential implication of autophagy in LSECs in NASH and liver fibrosis. METHODS: We analyzed autophagy in LSECs from patients using transmission electron microscopy. We determined the consequences of a deficiency in autophagy: (a) on LSEC phenotype, using primary LSECs and an LSEC line; (b) on early stages of NASH and on advanced stages of liver fibrosis, using transgenic mice deficient in autophagy specifically in endothelial cells and fed a high-fat diet or chronically treated with carbon tetrachloride, respectively. RESULTS: Patients with NASH had half as many LSECs containing autophagic vacuoles as patients without liver histological abnormalities, or with simple steatosis. LSECs from mice deficient in endothelial autophagy displayed an upregulation of genes implicated in inflammatory pathways. In the LSEC line, deficiency in autophagy enhanced inflammation (Ccl2, Ccl5, Il6 and VCAM-1 expression), features of endothelial-to-mesenchymal transition (α-Sma, Tgfb1, Col1a2 expression) and apoptosis (cleaved caspase-3). In mice fed a high-fat diet, deficiency in endothelial autophagy induced liver expression of inflammatory markers (Ccl2, Ccl5, Cd68, Vcam-1), liver cell apoptosis (cleaved caspase-3) and perisinusoidal fibrosis. Mice deficient in endothelial autophagy treated with carbon tetrachloride also developed more perisinusoidal fibrosis. CONCLUSIONS: A defect in autophagy in LSECs occurs in patients with NASH. Deficiency in endothelial autophagy promotes the development of liver inflammation, features of endothelial-to-mesenchymal transition, apoptosis and liver fibrosis in the early stages of NASH, but also favors more advanced stages of liver fibrosis. LAY SUMMARY: Autophagy is a physiological process controlling endothelial homeostasis in vascular beds outside the liver. This study demonstrates that autophagy is defective in the liver endothelial cells of patients with non-alcoholic steatohepatitis. This defect promotes liver inflammation and fibrosis at early stages of non-alcoholic steatohepatitis, but also at advanced stages of chronic liver disease.

Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice.

Polyclonal CD8+CD45RClow/- Tregs are potent regulatory cells able to control solid organ transplantation rejection and even induce tolerance. However, donor major histocompatibility complex (MHC)-specific Tregs are more potent than polyclonal Tregs in suppressing T-cell responses and preventing acute as well as chronic rejection in rodent models. The difficulty of identifying disease-relevant antigens able to stimulate Tregs has reduced the possibility of obtaining antigen-specific Tregs. To bypass this requirement and gain the advantage of antigen specificity, and thus improve the therapeutic potential of CD8+ Tregs, we stably introduced a chimeric antigen receptor (CAR) derived from a HLA-A*02 antigen-specific antibody (A2-CAR) in human CD8+ Tregs and developed a clinically compatible protocol of transduction and expansion. We demonstrated that A2-CAR CD8+ Tregs were not phenotypically altered by the process, were specifically activated, and did not exhibit cytotoxic activity toward HLA-A*02+ kidney endothelial cells (ECs). We showed that A2-CAR CD8+ Tregs were more potent suppressors of immune responses induced by HLA-A*02 mismatch than control-CAR CD8+ Tregs, both in vitro and in vivo, in models of human skin graft rejection and graft-versus-host disease (GVHD) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. We showed that integrity of human skin graft was preserved with A2-CAR CD8+ Tregs at least 100 days in vivo after administration, and that interaction between the A2-CAR CD8+ Tregs and HLA-A*02+ kidney ECs resulted in a fine-tuned and protolerogenic activation of the ECs without cytotoxicity. Together, our results demonstrated the relevance of the CAR engineering approach to develop antigen-specific CAR-CD8+ Tregs for clinical trials in transplantation, and potentially in other diseases.

Endothelial autophagic flux hampers atherosclerotic lesion development.

Blood flowing in arteries generates shear forces at the surface of the vascular endothelium that control its anti-atherogenic properties. However, due to the architecture of the vascular tree, these shear forces are heterogeneous and atherosclerotic plaques develop preferentially in areas where shear is low or disturbed. Here we review our recent study showing that elevated shear forces stimulate endothelial autophagic flux and that inactivating the endothelial macroautophagy/autophagy pathway promotes a proinflammatory, prosenescent and proapoptotic cell phenotype despite the presence of atheroprotective shear forces. Specific deficiency in endothelial autophagy in a murine model of atherosclerosis stimulates the development of atherosclerotic lesions exclusively in areas of the vasculature that are normally resistant to atherosclerosis. Our findings demonstrate that adequate endothelial autophagic flux limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence and inflammation.

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow.

It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α-induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation.

Quantitative analysis of ligand effects on bioefficacy of nanoemulsion encapsulating depigmenting active.

Efficient skin delivery of active molecules is the main challenge to overcome in order to achieve significant therapeutic efficiency of cosmetics or dermo-pharmaceutical products. Nanocarriers such as nanoemulsions have been envisaged to overcome main challenges of active solubilization, protection and transport to their site of biological action. Nonetheless, their skin permeation is still limited and a new approach is required to significantly improve bioavailability. We here explored the possibility of increasing the whitening activity of a model active, licorice, by implementing a targeting approach of nanoemulsions to melanocyte cells. Targeting requires particle surface modification with specific molecules favoring nanoemulsion/cells contact through ligand-receptor interactions. The uniqueness of our strategy is that unlike classical covalent chemical grafting, we propose a self-assembled strategy based on a selection of amphiphilic ligands able to localize at nanoemulsion droplets interface. Four ligand candidates were thus assayed in terms of formulation and in vitro biological evaluation: a palmitoyl-peptide (palmitoyl-GQPR), a lipidized hyaluronic acid (caproyl-HA) and two amphiphilic actives (polydatin and isopilosine). A functional analysis based on a cellular assay of melanin inhibition was realized. The intrinsic properties of ligand candidates were first evaluated. Then, nanoemulsions encapsulating a drug model, licorice, and targeted with the different ligand candidates were assayed. The use of caproyl-HA significantly improved bioefficacy of the encapsulated licorice, suggesting a better interaction with the cells. The improved value observed was not attributed to a synergetic action as caproyl-HA did not evidence intrinsic melanogenesis modulation activity. In this study, we demonstrated the feasibility of targeting nanoemulsion droplets without chemical covalent modification of nanoemulsion droplets to increase bioefficacy of encapsulated drugs in vitro.