Induction of the immune system response through the activation of antigen presenting cells mediated by conjugated aptamers

Identifier:  TFG:1615

Handle: https://hdl.handle.net/20.500.11797/TFG1615

Authors:  Gil Pitarch, Clàudia

Abstract:
In this study, the binding of conjugated aptamers with an antigenic peptide to dendritic cells (DCs) in order to induct their activation in a direct way was investigated. In consequence, T cells, and other type of effector cells from the immune system, would also be activated because of the binding with the activated DCs. The aim of this study was to validate the aptamer D#7, which had been already tested in other studies (Silvana Haβel, 2016). This means that, DCs must be evaluated with and without the aptamer (conjugated and non-conjugated) in order to know if DCs were being properly activated and to discard that non-specific bindings were not occurring. It also wanted to be determined if aptamers FN#9, FN#12, FN#14, FN#15 and FN#20 could be used as specific binding molecules for the development of immunotherapies against several diseases, for instance, against cancer. These aptamers were tested in two cell lines: THP1 and J774A1 cells, which are human monocytes and mice macrophages, respectively, and in two different conditions (with binding buffer or cell medium). In the D#7 validation, 5 different cell receptors (CD40, CD86, MHC-I, MHC-II and CD80) were analysed in order to determine if they were upregulated or downregulated when DCs were activated by LPS (lipopolysaccharides) or MHC-I peptide (Major Histocompatibility Complex) by using conjugated antibodies with a fluorophore. The binding of the aptamers and the antibodies was tested with flow cytometry. The validation of the D#7 aptamer was not completed because the binding of the aptamer was not reproducible and there was only one cell receptor that was upregulated (CD40) when the DCs were activated by LPS or MHC-I peptide. Consequently, the experiment could not be continued because of the incoherence of the obtained results. Concerning to the testing of the aptamers FN#9, FN#12, FN#14, FN#15 and FN#20, it was observed that aptamers FN#9 and FN#12 bound to the THP1 and J774A1 cells in both conditions. FN#20 also bound to J774A1 cells. Although, in the case of cell medium, the fluorescence of the aptamer was more intense, so, probably, more quantity of aptamer bound.1700