Characterization of biomarkers of the generation and functionality of tolerogenic dendritic cells differentiated with vitamin d3
- Autores: Juan Navarro Barriuso
- Directores de la Tesis: Eva María Martínez Cáceres (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2019
- Idioma: español
- Materias:
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- Resumen
The last years have witnessed a breakthrough in the development of tolerance-inducing cell therapies for the treatment of autoimmune diseases such as multiple sclerosis (MS), as well as other immune-mediated pathologies. In particular, tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach in clinical trials worldwide due to their potential ability to restore immune tolerance without compromising the protective immunity of the patients, contrary to conventional and currently available treatments. However, the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic, as well as better understanding the mechanisms involved in the induction of immune tolerance.
In this regard, the main goal of this thesis was to identify and validate common transcriptomic biomarkers of the generation and functionality of three of the most common tolDC-inducing protocols, using either rapamycin (rapa-tolDC), dexamethasone (dexa-tolDC) or vitamin D3 (vitD3-tolDC) to generate them. However, after performing a microarray study using cells differentiated from healthy donors, we could not identify any differentially expressed gene in common for these three different tolDC protocols compared to both mature (mDC) and immature dendritic cell (iDC) control conditions. Nevertheless, our results revealed that dexa tolDC and vitD3 tolDC, but not rapa-tolDC, do actually share several immune regulatory and anti-inflammatory pathways towards tolerance induction.
Afterwards, and due to their potent regulatory properties, we focused on the study of vitD3-tolDC. We could validate that CYP24A1, MAP7 and MUCL1 genes were strongly up-regulated in these cells, in samples from healthy donors and MS patients. Therefore, these genes constitute robust biomarkers of the adequate generation of vitD3-tolDC, and thus can be tested as a quality control in clinical trials for MS before the administration of these cells into the patients. Furthermore, we constructed a functional network of protein interactions which evidenced that MAP7 and MUCL1 —but not CYP24A1— were involved in the modulation of many relevant immune-related pathways, such as HLA class II presentation and anti-inflammatory responses, and could have a crucial role in the tolerogenic properties of vitD3-tolDC.
Finally, in order to better understand the mechanisms triggered by vitD3-tolDC for the induction of immune tolerance, we developed a protocol to generate and study the transcriptomic profile of T CD4+ cells upon their antigen-specific interaction with vitD3-tolDC, using an RNA-seq analysis. In this regard, our results evidenced an important down-modulation of several genes involved in cell cycle and in cell responses to, mainly, pro-inflammatory immune-related stimuli. However, only JUNB gene could be identified as a potential biomarker of the functionality of vitD3-tolDC, since its expression was slightly induced in those T CD4+ cells that interacted with them.
Altogether, the results presented in this thesis describe the whole process of screening, selection and validation of transcriptomic biomarkers of the generation —and functionality— of vitD3-tolDC towards their final translation into the clinic, serving as a bridge from the bench to the bedside.
