Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Zbulj, V.
Establishment of a CD8+ T-cell activation protocol via APC and CD3/CD28 activation.
[ Diplomarbeit/Master Thesis (UNI) ] Universität Graz; 2023.
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Deutsch Alexander
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- Abstract:
- Cytotoxic CD8 T-cells are an essential component of the adaptive immune system as they facilitate a robust immune response to cells presenting a stimulating antigen. The T-cells express T-cell receptors, which can recognize and bind a specific corresponding antigen presented by the MHCI molecules on the surface of the body's native cells. This binding enables the transduction of multiple signaling pathways, which result in the differentiation of the T-cells into their active states and promote the lytic effector function of the T-cells, destroying the antigen-presenting cells. This project aimed to establish and compare two activation approaches of murine OT-1 CD8+ T-cell activation over the course of 24h and 48h, namely the activation through antigen-presenting-cells (APC) and the activation by anti-CD3/anti-CD28 antibodies (CD3/CD28). Additionally, we examined the effect of the cytokines IL-2, IL7, and IL-12 on the activation process of the T-cells. We investigated the change in T-cell phenotype throughout the four activation states (naïve, P4, central memory, and effector memory T-cells) based on the expression of cell surface markers CD44 and CD62L. We also followed the changes in the expression of activation markers CD69, CD25, PD-1, and IFN-γ. To compare the lytic abilities of the T-cells activated in the various conditions we generated an ovalbumin-expressing murine 3T3 fibroblast model and co-cultured it with the activated T-cells. We observed a sufficient activation of the CD8+ T-cells in both activation approaches at the 48h timepoint. The APC and CD3/CD28 activation delivered similar fluctuations of T-cell population states and showed consistent expression of the activation markers at both timepoints. Furthermore, the addition of IL-7 and IL-12 to the activation did not affect the differentiation of the T-cells. IL-2 proved to be essential for the generation of sufficiently activated T-cells. Additionally, we successfully generated an ovalbumin-expressing and SIINFEKL-presenting 3T3 fibroblast cell line that could be recognized and specifically lysed by the activated OT-1 T-cells. The addition of IL-7 to the APC activation additionally boosted the T-cells’ lytic abilities compared to the other activation conditions. The findings of this master thesis establish that both the APC and CD3/CD28 activation methods are viable and comparable procedures in the activation of CD8+ T-cells. Furthermore, we showed that the IL-2 cytokine has an essential role in the success of the activation, positively influencing the speed of T-cell activation and furthering their cytolytic abilities. While the addition of IL-7 and IL-12 had no observable effect on the T-cells and their effector functions over the course of our experiments, they demand further examination to elucidate the extensiveness of their role in T-cell activation. Additionally, through the functional OT-1 T-cell mediated lysis of the OVA-expressing 3T3 fibroblasts, we determined that they are a viable model for further lysis experiments involving OT-1 CD8+ T-cells.