Dendritic cell-based vaccines: evaluating their anti-tumoral efficacy

• Autor(a) principal: Simões, Gonçalo Miguel Fernandes
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/40170
• topic: DC-based vaccine; Immunotherapy; Antigen presentation; Antigen-specific T cell

Dendritic cell (DC)-based antitumor vaccines have undergone significant development in the last decades and have proven to be a safe therapeutic approach. During the vaccine development process, it is important to monitor the ability of this strategy to elicit an effective immune response. An important parameter for determining their effectiveness is the assessment of their ability to (cross-)present antigens to cytotoxic T lymphocytes (CTLs). This can be assessed by several in vitro protocols, which are time-consuming, hence the need to establish a faster protocol. Therefore, in addition to optimizing the protocol currently used (mixed lymphocyte reaction - MLR) to evaluate this capability, it was also sought to implement an alternative protocol that requires less time and resources. The first aspect of optimizing the MLR assay involved replacing the culture medium used (X-VIVO 15) with a more readily available alternative (RPMI 1640). However, the results showed that RPMI 1640 medium did not increase T cell presentation and proliferation as effectively as X-VIVO 15 medium. Conversely, delaying IL-2 supplementation until day 7 of MLR contributed to a slight increase in the generation of antigen-specific CD8+ T lymphocytes (clones). Additionally, MLR restimulation with autologous peptide-loaded monocyte-derived DCs (moDCs) was also tested on days 7 and 10 of the assay. In this particular assay, a single restimulation was found to be more effective, as it resulted in higher number of clones. Furthermore, in addition to optimizing the MLR, the aim was also to replace the cytotoxicity detection method and thus increase its sensitivity. Of the two methods tested, flow cytometry was found to be the most sensitive compared to calcein-AM and the currently used lactate dehydrogenase (LDH) release assay. The alternative protocol for accelerated DCs testing focused on assessing the response of clones when co-cultured with moDCs loaded with the peptide for which these clones are specific. This response was assessed by measuring the levels of IFN-γ released by the stimulated clones. Preliminary testing demonstrated a correlation between these results and previous cytotoxicity testing data, reducing the test time to less than 48 hours. After confirming their functionality, expansion of these clones was also sought to establish a stock of pure cell population that could be used for further testing. For this purpose, in addition to an IL-2 stimulus and an anti-CD3 antibody, two types of feeder cells were also tested. Nevertheless, the use of these feeder cells proved to be suboptimal, which was reflected in a significant reduction in the purity of the clones. In sum, and since the optimization of laboratory protocols is a fundamental step in translational investigation, this study led to the optimization of certain procedures of the MLR assay by increasing its efficiency in assessing antigen presentation by moDCs. Furthermore, the increased sensitivity of the detection method potentially allows comparison of subtle differences between conditions, which was not possible with the LDH release assay. Finally, the alternative protocol tested provided promising results in significantly reduced the time and resources required for antigen presentation testing.