Regulatory T cells in haematopoietic stem cell transplantation

PhD ThesisGraft-versus-host disease (GvHD) remains the main complication associated with haematopoietic stem cell transplantation (HSCT). GvHD is caused by allo-reactive donor T cells mounting an attack against specific target tissues. CD4+CD25HiFoxp3+ regulatory T cells have been shown to modulate GvHD in vitro and also in vivo animal models. More recently early stage clinical trials have described the successful use of Treg to reduce the incidence of GvHD following HSCT. The aim of this study was to investigate further the suppressive mechanisms by which Treg are able to modulate GvHD and assess the influence of Treg on the beneficial graft-versus-leukaemia (GvL) effect therefore providing further insight into the use of Treg in the therapeutic management of GVHD. Data presented in this thesis demonstrates the successful isolation and expansion of a highly pure Treg population which maintained suppressive capacity throughout culture. We also confirmed that Treg retain suppressive capacity following cryopreservation resulting in reduced workload and increased consistency when used for in vitro functional studies. We also provide the first human in vitro evidence that Treg are able to prevent cutaneous GvH reaction by blocking the migration of effector T cells into the target tissues. The presence of Treg during allo-stimulation caused reduced effector cell activation, proliferation, IFNγ secretion and decreased skin homing receptor expression. Further investigation into the Treg modulation of dendritic cells demonstrated, for the first time in experimental in vitro human GvHD, that this was due to ineffective effector T cell priming in the presence of Treg caused by impairment of dendritic cell functions. Comprehensive phenotypic and functional analysis of Treg treated moDC showed their decreased antigen processing ability and allostimulatory capacity, resulting in a less severe GvH reaction in the skin explant model. Furthermore, this work has revealed that despite Treg impairing in vitro GvL mechanisms at a cellular level there was no association observed between increased Treg levels and the incidence of relapse in a small clinical cohort of HSCT patients. In conclusion this study has provided further insight into the mechanisms by which Treg are able to modulate GvHD. This would inform future clinical trials using Treg as a therapeutic alternative to current GvHD treatment and prophylaxis.The Tyneside Leukaemia Research Association (TLRA)

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function

Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro. MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro. Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro. MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent

Recombinant Acid Ceramidase Reduces Inflammation and Infection in Cystic Fibrosis.

Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems. Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection. Methods: Sphingolipids were measured using mass spectrometry in fully-differentiated cultures of primary human airway epithelial cells and co-cultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting and quantitative polymerase chain reaction were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models. Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium due to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to Pseudomonas aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in the apical membrane of cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection. Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)