Characterising human monocyte-derived tolerogenic dendritic cells

Abstract

Dendritic cells (DC) are responsible for eliciting and determining the fate of T cell responses. Manipulating DC to induce regulatory T cells (Tregs) and re-establish tolerance where there has been a breach in regulation has provided an attractive avenue for cellular therapies. A variety of protocols detailing in-vitro generation of tolerogenic DC (tolDC) from monocyte derived DC (moDC) are currently available, some of which have progressed through phase I clinical trials. However, methodological differences have resulted in heterogeneity between tolDC products; likely affecting which regulatory pathways are deployed. Further investigation is needed to build upon our knowledge of mechanisms of tolDC action to subsequently match them to the disease they are best suited for. To this end, my project set out to compare two different methods of tolDC generation using Vitamin D3 alone (VD3DC) or in combination with Dexamethasone (DexVD3DC). I aimed to understand the differences between the phenotype, cytokine secretion profile, and metabolic profiles of different tolDC types. In addition, their effect on CD4+ T cells in a more translational context was characterised by co-culturing the tolDC with allogeneic PBMCs from rheumatoid arthritis patients. Multiple protocols detailing generation of VD3DC, with addition of Vitamin D3 (VD3) at different timepoints, were available. Therefore, my first task was to find the optimal timepoint for VD3 addition. My results showed earlier addition of VD3 to culture was optimal. Both DexVD3DC and VD3DC showed characteristically low expression of maturation and co stimulatory molecules and produced low levels of IL-12p70, whilst VD3DC showed distinctly higher expression of regulatory molecules PDL-1 and ILT3. Moreover, when compared to DexVD3DC, VD3DC had a reduced capacity to induce T cell proliferation. To further characterise the mechanisms driving the functional effects of these tolDC types, I performed targeted transcriptomic analysis on proliferated CD4+ T cells that had been primed in co-culture with either mature DC (TMatDC), DexVD3DC (TDexVD3DC) or VD3DC (TVD3DC). Both tolDC types induced transcriptionally distinct T cells from their mature counterpart. As expected, regulatory genes such as FoxP3 and TIGIT were amongst the genes upregulated in the TDexVD3DC and TVD3DC compared to TMatDC; whilst pro-inflammatory IFNγ was downregulated. Moreover, differential expression of genes between TDexVD3DC and TVD3DC was observed, alluding to different mechanisms of action in the tolDC types. Targeted metabolomic analysis of paired cell and culture supernatants were performed using a combination of liquid chromatography and mass spectrometry. Additionally, mitochondrial function was assessed using Seahorse Extracellular Flux technology. Differential expression of metabolites involved in multiple anabolic and catabolic pathways was seen between tolDC types. Assessment of mitochondrial function indicated differential reliance on oxidative phosphorylation between tolDC types providing potential targets to explore in the future and new insights into how metabolism influences immune responses. My findings highlighted distinct characteristics of both DexVD3DC and VD3DC, in terms of phenotype, cytokine secretion and levels of intracellular and extracellular metabolites. Moreover, DexVD3DC and VD3DC have differential proliferative capacity on CD4+ T cells, in addition to inducing T cells with distinct transcriptional signatures. These differences suggest that these tolDC types do have unique mechanisms driving their functional effects, highlighting an important consideration for the future of tolDC therapy. Further exploration is required to confirm whether these pathways are essential for their tolerogenic function, to ensure stability and maximal efficacy of the tolDC for therapeutic use.