Pre-Germinal Center Interactions with T Cells Are Natural Checkpoints to Limit Autoimmune B Cell Responses.

Interactions with Ag-specific T cells drive B cell activation and fate choices that ultimately determine the quality of high-affinity Ab responses. As such, these interactions, and especially the long-lived interactions that occur before germinal center formation, may be important checkpoints to regulate undesirable responses. Using mouse model Ag systems, we directly observed interactions between T and B cells responding to the self-antigen myelin oligodendrocyte glycoprotein (MOG) and found that they are of lower quality compared with interactions between cells responding to the model foreign Ag nitrophenyl-haptenated OVA. This was associated with reduced expression of molecules that facilitate these interactions on the B cells, but not on T cells. B cell expression of these molecules was not dictated by the T cell partner, nor could the relative lack of expression on MOG-specific (MOG-sp.) B cells be reversed by a multivalent Ag. Instead, MOG-sp. B cells were inherently less responsive to BCR stimulation than MOG-non-sp. cells. However, the phenotype of MOG-sp. B cells was not consistent with previous descriptions of autoimmune B cells that had been tolerized via regular exposure to systemically expressed self-antigen. This suggests that alternate anergy pathways may exist to limit B cell responses to tissue-restricted self-antigens.

A non-canonical role for desmoglein-2 in endothelial cells: implications for neoangiogenesis.

Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by non-desmosome-forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34(+)CD45(dim) hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34(+)CD45(dim)DSG2(+) progenitor cells are multi-potent and pro-angiogenic in vitro. Using a 'knockout-first' approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2 (lo/lo)) and observed that, in response to reduced levels of Dsg2: (i) CD31(+) ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrow-derived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell-cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.

Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-γ that regulates neoangiogenesis.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator-activated receptor (PPAR)γ. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase-tagged PPARγ-specific gene reporter by ∼12-fold, independent of the S1P receptors. More specifically, in silico docking, gene reporter, and binding assays revealed that His323 of the PPARγ ligand binding domain is important for binding to S1P. PPARγ functions when associated with coregulatory proteins, and herein we identify that peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1)ß binds to PPARγ in ECs and their progenitors (nonadherent endothelial forming cells) and that the formation of this PPARγ:PGC1ß complex is increased in response to S1P. ECs treated with S1P selectively regulated known PPARγ target genes with PGC1ß and plasminogen-activated inhibitor-1 being increased, no change to adipocyte fatty acid binding protein 2 and suppression of CD36. S1P-induced in vitro tube formation was significantly attenuated in the presence of the PPARγ antagonist GW9662, and in vivo application of GW9662 also reduced vascular development in Matrigel plugs. Interestingly, activation of PPARγ by the synthetic ligand troglitazone also reduced tube formation in vitro and in vivo. To support this, Sphk1(-/-)Sphk2(+/-) mice, with low circulating S1P levels, demonstrated a similar reduction in vascular development. Taken together, our data reveal that the transcription factor, PPARγ, is a bona fide intracellular target for S1P and thus suggest that the S1P:PPARγ:PGC1ß complex may be a useful target to manipulate neovascularization.

Systemic administration of anti-CD20 indirectly reduces B cells in the inflamed meninges in a chronic model of central nervous system autoimmunity.

Anti-CD20 B cell depleting therapies have demonstrated that B cells are important drivers of disease progress in Multiple Sclerosis, although the pathogenic mechanisms are not well understood. A population of B cells accumulates in the inflamed meninges in MS and also some chronic animal models of disease, typically adjacent to demyelinating lesions. The role of these meningeal B cells in disease is not known, nor is their susceptibility to anti-CD20 therapy. Here, we administered anti-CD20 to 2D2 IgHMOG spontaneous experimental autoimmune encephalomyelitis mice in the chronic phase of disease, after the establishment of meningeal B cell clusters. Compared to the circulation, lymph nodes, and spleen, B cell depletion from the meninges was delayed and not evident until 7d post-administration of anti-CD20. Further, we did not find evidence that anti-CD20 accessed meningeal B cells directly, but rather that depletion was indirect and the result of ongoing turnover of the meningeal population and elimination of the peripheral pool from which it is sustained.

Monovalent and trivalent VSV-based COVID-19 vaccines elicit neutralizing antibodies and CD8+ T cells against SARS-CoV-2 variants.

Recombinant vesicular stomatitis virus (rVSV) vaccines expressing spike proteins of Wuhan, Beta, and/or Delta variants of SARS-CoV-2 were generated and tested for induction of antibody and T cell immune responses following intramuscular delivery to mice. rVSV-Wuhan and rVSV-Delta vaccines and an rVSV-Trivalent (mixed rVSV-Wuhan, -Beta, -Delta) vaccine elicited potent neutralizing antibodies (nAbs) against live SARS-CoV-2 Wuhan (USAWA1), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529) viruses. Prime-boost vaccination with rVSV-Beta was less effective in this capacity. Heterologous boosting of rVSV-Wuhan with rVSV-Delta induced strong nAb responses against Delta and Omicron viruses, with the rVSV-Trivalent vaccine consistently effective in inducing nAbs against all the SARS-CoV-2 variants tested. All vaccines, including rVSV-Beta, elicited a spike-specific immunodominant CD8+ T cell response. Collectively, rVSV vaccines targeting SARS-CoV-2 variants of concern may be considered in the global fight against COVID-19.

Fecal microbiota transplantation is safe and tolerable in patients with multiple sclerosis: A pilot randomized controlled trial.

Background: Patients with MS have an altered gut microbiota compared to healthy individuals, as well as elevated small intestinal permeability, which may be contributing to the development and progression of the disease. Objective: We sought to investigate if fecal microbiota transplantation was safe and tolerable in MS patients and if it could improve abnormal intestinal permeability. Methods: Nine patients with MS were recruited and provided monthly FMTs for up to six months. The primary outcome investigated was change in peripheral blood cytokine concentrations. The secondary outcomes were gut microbiota composition, intestinal permeability, and safety (assessed with EDSS and MRI). Results: The study was terminated early and was subsequently underpowered to assess whether peripheral blood cytokines were altered following FMTs. FMTs were safe in this group of patients. Two of five patients had elevated small intestinal permeability at baseline that improved to normal values following FMTs. Significant, donor-specific, beneficial alterations to the MS patient gut microbiota were observed following FMT. Conclusion: FMT was safe and tolerable in this cohort of RRMS patients, may improve elevated small intestinal permeability, and has the potential to enrich for an MS-protective microbiota. Further studies with longer follow-up and larger sample sizes are required to determine if FMT is a suitable therapy for MS.

Over-expression of sphingosine kinase-1 enhances a progenitor phenotype in human endothelial cells.

OBJECTIVES: The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de-differentiation of mature human endothelial cells toward a progenitor phenotype. METHODS: The lipid enzyme sphingosine kinase-1 was lentivirally over-expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. RESULTS: Sphingosine kinase-1 mRNA expression was induced approximately 150-fold with a resultant 20-fold increase in sphingosine kinase-1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de-differentiated phenotype with increased viability, reduced uptake of acetylated low-density lipoprotein and decreased tube formation in Matrigel in the cells over-expressing sphingosine kinase-1. CONCLUSIONS: These findings suggest that over-expression of sphingosine kinase-1 in human endothelial cells promotes, in part, their de-differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use.

Autoreactive, Low-Affinity T Cells Preferentially Drive Differentiation of Short-Lived Memory B Cells at the Expense of Germinal Center Maintenance.

B cell fate decisions within a germinal center (GC) are critical to determining the outcome of the immune response to a given antigen. Here, we characterize GC kinetics and B cell fate choices in a response to the autoantigen myelin oligodendrocyte glycoprotein (MOG) and compare the response with a standard model foreign antigen. Both antigens generate productive primary responses, as evidenced by GC development, circulating antigen-specific antibodies, and differentiation of memory B cells. However, in the MOG response, the status of the cognate T cell partner drives preferential B cell differentiation to a memory phenotype at the expense of GC maintenance, resulting in a truncated GC. Reduced plasma cell differentiation is largely independent of T cell influence. Interestingly, memory-phenotype B cells formed in the MOG GC are not long lived, resulting in a failure of the B cell response to secondary challenge.

Local Sphingosine Kinase 1 Activity Improves Islet Transplantation.

Pancreatic islet transplantation is a promising clinical treatment for type 1 diabetes, but success is limited by extensive ß-cell death in the immediate posttransplant period and impaired islet function in the longer term. Following transplantation, appropriate vascular remodeling is crucial to ensure the survival and function of engrafted islets. The sphingosine kinase (SK) pathway is an important regulator of vascular beds, but its role in the survival and function of transplanted islets is unknown. We observed that donor islets from mice deficient in SK1 (Sphk1 knockout) contain a reduced number of resident intraislet vascular endothelial cells. Furthermore, we demonstrate that the main product of SK1, sphingosine-1-phosphate, controls the migration of intraislet endothelial cells in vitro. We reveal in vivo that Sphk1 knockout islets have an impaired ability to cure diabetes compared with wild-type controls. Thus, SK1-deficient islets not only contain fewer resident vascular cells that participate in revascularization, but likely also a reduced ability to recruit new vessels into the transplanted islet. Together, our data suggest that SK1 is important for islet revascularization following transplantation and represents a novel clinical target for improving transplant outcomes.

Interleukin-3 greatly expands non-adherent endothelial forming cells with pro-angiogenic properties.

Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133(+) EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs), namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133(+) cell expansion with clear pro-angiogenic properties (in vitro and in vivo) and thus may provide clinical utility for humans in the future.