Conventional dendritic cells for regeneration of immune tolerance

Abstract

Tolerogenic dendritic cells (tolDC) are a promising cell therapy for the treatment of autoimmune disease due to their ability to regenerate immune tolerance. The most commonly used cells for their generation are monocyte-derived DC (moDC). However, this approach has some limitations, including the poor migratory ability of these cells. The work presented in this thesis aims to generate a conventional DC (cDC)-based tolDC therapy, because cDC have excellent migratory capacity towards T cell areas in lymphoid tissues, where they can educate naïve CD4+ T cells. The first objective of this thesis was to select the most appropriate cDC subset for tolDC generation. Therefore, the Nanostring nCounter platform was used to compare the gene expression profiles of cultured CD34+ stem cell-derived type 1 and type 2 cDC (cDC1 and cDC2) and monocyte-derived tolerogenic DC (Mo-tolDC). This analysis revealed that cDC2 was more closely aligned than cDC1 with Mo-tolDC, with regard to the expression of tolerogenic genes, including PDL-1, PRDM1 and MRC1. In addition, both cultured and peripheral blood-derived cDC2 expressed significantly higher level of LILRB4 (ILT3 encoding gene) compared to cDC1, suggesting its contribution to the regulatory function of cDC2. Therefore, the cDC2 subset was chosen for the generation of cDC-derived tolDC. Another objective of this project was to investigate the optimal tolerogenic treatment to generate migratory cDC-derived tolDC and to assess the impact of different culture media on the phenotype and migratory ability of cDC-derived tolDC. Hence, peripheral blood cDC2 were magnetically isolated from leukocyte reduction cones (LRS) and cultured in vitro in the presence of GM-CSF with or without tolerogenic agents and LPS. Cells were phenotypically characterized by flow cytometry, and their cytokine secretion profiles were determined by Meso-Scale-Discovery (MSD) multiplexing or ELISA. In addition, CCR7-dependent migration towards CCL19 and CCL21 was assessed. Treating freshly isolated cDC2 with the active form of vitamin D3 (VitD3) and LPS (VitD3-cDC2) significantly enhanced the expression of the tolerogenic marker PDL-1. Remarkably, IL-10 secretion was also high in VitD3-cDC2, suggesting its contribution to their regulatory capacity. However, while secretion of IL-12p70 was very low, VitD3-cDC2 secreted high levels of the pro-inflammatory cytokines IL-6 and TNF-a. Prominently, CCR7 expression remained high on VitD3-cDC2, and they exhibited good migratory ability, albeit slightly reduced compared to LPS-activated cDC2. To further characterize the stimulatory capacity of VitD3-cDC2, co-culture assays with allogeneic naïve CD4+ T cells were performed. VitD3-cDC2 demonstrated lower T cell stimulatory capacity with significant reduced levels of IFN-g secretion than untreated and unstimulated cDC2. Finally, T cells that were primed with VitD3-cDC2 and rested before restimulation with LPS-treated cDC2 demonstrated an increase in their expression of CD25 and FOXP3, which could be indicative of Treg induction. In addition, I also observed higher IL-10 secretion and lower IFN-g/IL-10 ratio in T cells primed with VitD3-cDC2 after restimulation assays, suggesting a potential regulatory capacity of these cells. The findings in this thesis support the use of cDC2 as an alternative to moDC for the generation of therapeutic tolDC. Additionally, this thesis has provided the basis for future work on the generation of tol-cDC2 for treating autoimmune diseases. Further work is required to understand the mechanism(s) by which tol-cDC2 regulate T cell responses.